Intervertebral disc degeneration is a leading cause of lower back pain and is characterized by pathological processes such as nucleus pulposus cell apoptosis,extracellular matrix imbalance,and annulus fibrosus rupture...Intervertebral disc degeneration is a leading cause of lower back pain and is characterized by pathological processes such as nucleus pulposus cell apoptosis,extracellular matrix imbalance,and annulus fibrosus rupture.These pathological changes result in disc height loss and functional decline,potentially leading to disc herniation.This comprehensive review aimed to address the current challenges in intervertebral disc degeneration treatment by evaluating the regenerative potential of stem cell-based therapies,with a particular focus on emerging technologies such as exosomes and gene vector systems.Through mechanisms such as differentiation,paracrine effects,and immunomodulation,stem cells facilitate extracellular matrix repair and reduce nucleus pulposus cell apoptosis.Despite recent advancements,clinical applications are hindered by challenges such as hypoxic disc environments and immune rejection.By analyzing recent preclinical and clinical findings,this review provided insights into optimizing stem cell therapy to overcome these obstacles and highlighted future directions in the field.展开更多
Transfection efficiency of hydroxyapatite nanoparticles(HAnps)is relative to the particle size,morphology,surface charge,surface modifier and so on.This study prepared HAnps with doped Tb/Mg by hydrothermal synthesis ...Transfection efficiency of hydroxyapatite nanoparticles(HAnps)is relative to the particle size,morphology,surface charge,surface modifier and so on.This study prepared HAnps with doped Tb/Mg by hydrothermal synthesis method(HTSM)and investigated the effects of different Tb/Mg contents on the morphology,particle size,surface charge,composition and cellular endocytosis of HAnps.The results showed that Mg-HAnps possessed better dispersion ability than Tb-HAnps.With increasing doping content of Tb/Mg-HAnps,the granularity of Tb-HAnps increased,while that of Mg-HAnps declined.Both particle size and zeta potential of Mg-HAnps were lower than those of Tb-HAnps.7.5%Mg-doping HAnps presented relatively uniform slender rod morphology with average size of30nm,while10%Mg-doping HAnps were prone to agglomeration.Moreover,Mg-HAnps-GFP(green fluorescent protein)endocytosed by MG63cells was dotted in the perinuclear region,while Tb-HAnps were more likely to aggregate.In conclusion,as gene vectors,Mg-HAnps showed enhanced properties compared to Tb-HAnps.展开更多
Some novel lipids bearing nucleosides were designed and synthesized as gene vectors, and the structures of these compounds were characterized by UV, IR, 1HNMR, 13CNMR and elemental analysis.
Some cationic and neutral lipids bearing cholesteryl group were synthesized as gene vectors, and the structures of the compounds were characterized by IR, (HNMR)-H-1, MS and elemental analysis.
Summary: To evaluate the feasibility of using polyethyleneimine (PEI) coated magnetic iron oxide nanoparticles (polyMAG-1000) as gene vectors. The surface characteristics of the nanoparticles were observed with scanni...Summary: To evaluate the feasibility of using polyethyleneimine (PEI) coated magnetic iron oxide nanoparticles (polyMAG-1000) as gene vectors. The surface characteristics of the nanoparticles were observed with scanning electron microscopy. The ability of the nanoparticles to combine with and protect DNA was investigated at different PH values after polyMAG-1000 and DNA were combined in different ratios. The nanoparticles were tested as gene vectors with in vitro transfection models. Under the scanning electron microscope the nanoparticles were about 100 nm in diameter. The nanoparticles could bind and condense DNA under acid, neutral and alkaline conditions, and they could transfer genes into cells and express green fluorescent proteins (GFP). The transfection efficiency was highest (51 %) when the ratio of nanoparticles to DNA was 1:1 (v:w). In that ratio, the difference in transfection efficiency was marked depending on whether a magnetic field was present or not: about 10 % when it was absent but 51 % when it was present. The magnetic iron oxide nanoparticles coated with PEI may potentially be used as gene vectors.展开更多
Gene therapy has emerged as a potential new approach to treat genetic disorders by delivering therapeutic genes to target diseased tissues. However, its clinical use has been impeded by gene delivery systems. The vira...Gene therapy has emerged as a potential new approach to treat genetic disorders by delivering therapeutic genes to target diseased tissues. However, its clinical use has been impeded by gene delivery systems. The viral vectors are very efficient in delivering and expressing their carried genes, but they have safety issues in clinical use. While nonviral vectors are much safer with very low risks after careful material design, but their gene transcription efficiency is too low to be clinically used. Thus, rational design of nonviral vectors mimicking the viral vectors would be a way to break this bottleneck. This review compares side-by-side how viral/nonviral gene vectors transcend these biological barriers in terms of blood circulation, cellular uptake, endosome escape, nucleus import and gene transcription.展开更多
Our previous studies have successfully grafted biotin and galactose onto chitosan(CS)and synthesized biotin modified galactosylated chitosan(Bio-GC).The optimum N/P ratio of Bio-GC and plasmid DNA was 3:1.At this N/P ...Our previous studies have successfully grafted biotin and galactose onto chitosan(CS)and synthesized biotin modified galactosylated chitosan(Bio-GC).The optimum N/P ratio of Bio-GC and plasmid DNA was 3:1.At this N/P ratio,the transfection efficiency in the hepatoma cells was the highest with a slow release effect.Bio-GC nanomaterials exhibit the protective effect of preventing the gene from nuclease degradation,and can target the transfection into hepatoma cells by combination with galactose and biotin receptors.The transfection rate was inhibited by the competition of galactose and biotin.Bio-GC nanomaterials were imported into cells’cytoplasm by their receptors,followed by the imported exogenous gene transfected into the cells.Bio-GC nanomaterials can also cause inhibitory activity in the hepatoma cells in the model of orthotopic liver transplantation in mice,by carrying the gene through the blood to the hepatoma tissue.Taken together,bio-GC nanomaterials act as gene vectors with the activity of protecting the gene from DNase degradation,improving the rate of transfection in hepatoma cells,and transporting the gene into the cytoplasm in vitro and in vivo.Therefore,they are efficient hepatoma-targeting gene carriers.展开更多
DNA delivery is a core technology for gene structure and function research as well as clinical settings. The ability to safely and efficiently targeted transfer foreign DNA into cells is a fundamental goal in biotechn...DNA delivery is a core technology for gene structure and function research as well as clinical settings. The ability to safely and efficiently targeted transfer foreign DNA into cells is a fundamental goal in biotechnology. With the development of nanobiotechnology, nanoparticle gene vectors brought about new hope to reach the goal. In our research, silica nanoparticles (SiNP) were synthesized first in a microemulsion system polyoxyethylene nonylphenyl ether (OP-10)/cyclohexane/ammonium hydroxide, at the same time the effects of SiNP size and its distribution were elucidated by orthogonal analysis; then poly-L-lysine (PLL) was linked on the surface of SiNP by nanoparticle surface energy and electrostatically binding; lastly a novel complex nanomate-rial-poly-L-lysine-silica nanoparticles (PLL-SiNP) wasprepared. The analysis of plasmid DNA binding and DNase I enzymatic degradation discovered that PLL-SiNP could bind DNA, and protect it against enzymatic degradation. Cell transfection showed that展开更多
BACKGROUND: Gene therapy as part of modern molecu-lar medicine holds great promise for the treatment of hepa-tocellular carcinoma (HCC) and has the potential to bringa revolutionary era to cancer treatment. For the pa...BACKGROUND: Gene therapy as part of modern molecu-lar medicine holds great promise for the treatment of hepa-tocellular carcinoma (HCC) and has the potential to bringa revolutionary era to cancer treatment. For the past de-cade various viral and non-viral vectors have been engi-neered for improved liver gene therapy.DATA RESOURCES: An English-language literature searchusing MEDLINE (2004), Index Medicus (2004) and biblio-graphic reviews of books and review articles. Liver-directedgene transfer vectors and their history and recent clinicalapplications.RESULTS: The ultimate goal of liver-directed gene therapyfor HCC is the stable expression of a therapeutic transgenein a significant proportion of hepatocytes. The design of avector system providing efficient and stable gene engraft-ment and expression in human hepatocytes is still a chal-lenging issue. The advantages and disadvantages of the ge-netically engineered vector of viral or non-viral origin arediscussed with respect to their essential relevance.CONCLUSION: Liver gene therapy has a long way to goand efficient and innocuous liver-directed gene transfer vec-tors are therefore urgently required.展开更多
Objective： The aim of the study was to observe the transfection efficacy of hepatitis B virus envelope （HBVE） and evaluate its ability as a gene transfer vector for liver cancer cells. Methods： To obtain HBVE, the...Objective： The aim of the study was to observe the transfection efficacy of hepatitis B virus envelope （HBVE） and evaluate its ability as a gene transfer vector for liver cancer cells. Methods： To obtain HBVE, the supematant fluid of HepG 2.2.15 cells was mixed with a PEG8000 solution for concentration and was inactivated by β-propiolactone. The acquired HBVE was used to pack plRES2-EGFP to test its package ability. Then, we examined its quantity and quality with ELISA, PCR, SDS-PAGE and electron microscopy. The plRES2-EGFP was packed with HBVE and obtained the product HBVE-GFP. The plRES2-EGFP was packed with liposome and obtained the product liposome-GFP. HBVE-GFP and liposome-GFP were used to transfer HepG 2 cells to study the transfection efficiency. HBVE-GFP was used to transfer HepG 2, A549, HeLa and FB cells to study the targeting ability. The green fluorescent protein （GFP） expression was observed under a fluorescent microscope. The rate of GFP positive cells was determined by flow cytometry. Results： 1. The acquired HBVE could retain the surface protein HBsAg ＋ pre S1 ＋ pre S2 and had no virus DNA. It had good package ability for plRES2-EGFP. 2. Transfection efficiency： The GFP could be observed in both the liposome group and HBVE group under the fluorescent microscope. But the HBVE group had a higher fluorescent intensity than liposome group. The transfection rate of liposome group was 49.97% ＋ 2.37% while the HBVE group was 70.65% ＋ 3.15% and the fluorescent intensity of the HBVE group was 3-4 times （P = 0.000） for liposome group with the determination of flow cytometry. 3. Targeting ability： The GFP could be observed in the four groups under the fluorescent microscope. The HepG 2 group had the highest fluorescent intensity among the four groups. The transfection rate of HepG 2 group was 71.35% ＋ 0.03% which was highly expressed than other groups （P = 0.000） and the fluorescent intensity of the HepG 2 group was 2-3 times （P = 0.000） for the other 3 groups with the determination of flow cytometry. Conclusion： HBVE can be constructed successfully with the methods of PEG8000 and β-propiolactone from the supernatant fluid of HepG 22.15 cells. The HBVE can be a candidate gene transfer vector for liver cancer cells.展开更多
In microarray-based cancer classification, gene selection is an important issue owing to the large number of variables and small number of samples as well as its non-linearity. It is difficult to get satisfying result...In microarray-based cancer classification, gene selection is an important issue owing to the large number of variables and small number of samples as well as its non-linearity. It is difficult to get satisfying results by using conventional linear sta- tistical methods. Recursive feature elimination based on support vector machine (SVM RFE) is an effective algorithm for gene selection and cancer classification, which are integrated into a consistent framework. In this paper, we propose a new method to select parameters of the aforementioned algorithm implemented with Gaussian kernel SVMs as better alternatives to the common practice of selecting the apparently best parameters by using a genetic algorithm to search for a couple of optimal parameter. Fast implementation issues for this method are also discussed for pragmatic reasons. The proposed method was tested on two repre- sentative hereditary breast cancer and acute leukaemia datasets. The experimental results indicate that the proposed method per- forms well in selecting genes and achieves high classification accuracies with these genes.展开更多
Gene therapy appears as a promising strategy to treatincurable diseases. In particular, combined gene therapy has shown improved therapeutic efficiency. Internal ribosome entry sites(IRESs), RNA elements naturally pre...Gene therapy appears as a promising strategy to treatincurable diseases. In particular, combined gene therapy has shown improved therapeutic efficiency. Internal ribosome entry sites(IRESs), RNA elements naturally present in the 5' untranslated regions of a few m RNAs, constitute a powerful tool to co-express several genes of interest. IRESs are translational enhancers allowing the translational machinery to start protein synthesis by internal initiation. This feature allowed the design of multi-cistronic vectors expressing several genes from a single m RNA. IRESs exhibit tissue specificity, and drive translation in stress conditions when the global cell translation is blocked, which renders them useful for gene transfer in hypoxic conditions occurring in ischemic diseases and cancer. IRES-based viral and non viral vectors have been used successfully in preclinical and clinical assays of combined gene therapy and resulted in therapeutic benefits for various pathologies including cancers, cardiovascular diseases and degenerative diseases.展开更多
In order to lay a foundation for researching the function of Rosa rugose (R. rugosa) RrGlu gene, the RrGlu gene was amplified from the styles of R. rugosa “Tanghong”, a gene expression vector named PBI121-RrGlu was ...In order to lay a foundation for researching the function of Rosa rugose (R. rugosa) RrGlu gene, the RrGlu gene was amplified from the styles of R. rugosa “Tanghong”, a gene expression vector named PBI121-RrGlu was constructed and the vector was introduced into tobacco with the agrobacterium-mediated method. PCR results showed that the RrGlu gene was integrated into the tobacco genome.展开更多
To study the therapeutic effects of herpes simplex virus thymidine kinase gene transferred by the EBV based expression vector on experimental hepatocellular carcinoma, pDR2 TK gene was delivered into human hepatoc...To study the therapeutic effects of herpes simplex virus thymidine kinase gene transferred by the EBV based expression vector on experimental hepatocellular carcinoma, pDR2 TK gene was delivered into human hepatocellular carcinoma cell line SMMC 7721 by using liposome mediated transfection technique,and then gene expression was detected by RT PCR, and the killing effects were examined through MTT method. In the nude mice hepatoma model,the antitumor effects of pDR2 TK /GCV system was evaluated in terms of tumor growth. MTT results showed that the pDR2 TK /GCV had cytotoxic effect and about 70 % SMMC 7721 cells were killed when GCV was at 1000 μmol/L. In vivo experiment showed that the tumor size in nude mice with transferred pDR2 TK gene was significantly smaller than that in control group . On the 10th day the tumor in 3 mice (60 %) disappeared completely after GCV treatment. It is concluded that the pDR2 TK/GCV system has marked killing effects on the experimental hepatocellular carcinoma.展开更多
OBJECTIVE： In the present study, we systemically evaluated the ability of two bioactive compounds from traditional Chinese medicine, celastrol and pristimerin, to enhance recombinant adeno-associated virus （rAAV） s...OBJECTIVE： In the present study, we systemically evaluated the ability of two bioactive compounds from traditional Chinese medicine, celastrol and pristimerin, to enhance recombinant adeno-associated virus （rAAV） serotype vector-mediated transgene expression both in human cell lines in vitro, and in murine hepatocytes in vivo. METHODS： Human cell lines were infected with rAAV vectors with either mock treatment or treatment with celastrol or pristimerin. The transgene expression, percentage of nuclear translocated viral genomes and the ubiquitination of intracellular proteins were investigated post-treatment. In addition, nonobese diabetic/severe combined immunodeficient gamma （NSG） mice were tail vain-injected with rAAV vectors and co-administered with either dimethyl sulfoxide, celastrol, pristimerin or a positive control, bortezomib. The transgene expression in liver was detected and compared over time. RESULTS： We observed that treatment with pristimerin, at as low as 1 IJmol/L concentration, significantly enhanced rAAV2 vector-mediated transgene expression in vitro, and intraperitoneal co- administration with pristimerin at 4 mg/（kg.d） for 3 d dramatically facilitated viral transduction in murine hepatocytes in vivo. The transduction efficiency of the tyrosine-mutant rAAV2 vectors as well as that of rAAV8 vectors carrying oversized transgene cassette was also augmented significantly by pristimerin. The underlying molecular mechanisms by which pristimerin mediated the observed increase in the transduction efficiency of rAAV vectors include both inhibition of proteasomal degradation of the intracellular proteins and enhanced nuclear translocation of the vector genomes. CONCLUSION： These studies suggest the potential beneficial use of pristimerin and pristimerincontaining herb extract in future liver-targeted gene therapy with rAAV vectors.展开更多
A full-length cDNA sequence of xyloglucan endotransglycosylase gene （XET）, abundantly expressed in the cambium of Anthocephalus chinensis was cloned by conserved PCR, rapid-amplification of cDNA ends and by chromoso...A full-length cDNA sequence of xyloglucan endotransglycosylase gene （XET）, abundantly expressed in the cambium of Anthocephalus chinensis was cloned by conserved PCR, rapid-amplification of cDNA ends and by chromosome walking. Analytical results of the DNA sequence show that a 912 bp complete open reading frame （ORF） encoded a 303-amino acid protein was in the 1205 bp full cDNA sequence. The deduced amino acid sequence of AcXET, which contained the conserved specific EIDFE catalytic site sequence to XETs was homologous to the other known XET proteins. In order to study the gene function of AcXET and obtain transgenic plants, a plant expression vector pBIAcXET was constructed by recombinating the AcXET fragment from the cloning vector pMD19AcXET and the binary vector pBI121 between the XbaI and SmaI sites. The fragment ofAcXET gene was inserted between the CaMV 35S promotor and the coding region of the GUS gene in pBI121. The identification results show that the plant expression binary vector pBIAcXET was constructed successfully. These results lay the foundation for studying the molecular mechanism ofAcXET gene during wood formation.展开更多
Safe and effective gene therapy approaches require targeted tissue-specific transfer of a therapeutic transgene.Besides traditional approaches, such as transcriptional and transductional targeting, micro RNA-dependent...Safe and effective gene therapy approaches require targeted tissue-specific transfer of a therapeutic transgene.Besides traditional approaches, such as transcriptional and transductional targeting, micro RNA-dependent posttranscriptional suppression of transgene expression has been emerging as powerful new technology to increase the specificity of vector-mediated transgene expression. Micro RNAs are small non-coding RNAs and often expressed in a tissue-, lineage-, activation- or differentiation-specific pattern. They typically regulate gene expression by binding to imperfectly complementary sequences in the 3' untranslated region(UTR) of the m RNA. To control exogenous transgene expression, tandem repeats of artificial micro RNA target sites are usually incorporated into the 3' UTR of the transgene expression cassette, leading to subsequent degradation of transgene m RNA in cel s expressing the corresponding micro RNA. This targeting strategy, first shown for lentiviral vectors in antigen presenting cells, has now been used for tissue-specific expression of vector-encoded therapeutic transgenes, to reduce immune response against the transgene, to control virus tropism for oncolytic virotherapy, to increase safety of live attenuated virus vaccines and to identify and select cell subsets for pluripotent stem cell therapies, respectively. This review provides an introduction into the technical mechanism underlying micro RNA-regulation, highlights new developments in this field and gives an overview of applications of micro RNA-regulated viral vectors for cardiac, suicide gene cancer and hematopoietic stem cell therapy, as well as for treatment of neurological and eye diseases.展开更多
The upstream regulatory region of a seed specific gene was isolated from the genomic DNA of Brassica napus by PCR amplification. The cloned fragment contained 1755 nucleotides, and shared a sequence homology of 99.6%...The upstream regulatory region of a seed specific gene was isolated from the genomic DNA of Brassica napus by PCR amplification. The cloned fragment contained 1755 nucleotides, and shared a sequence homology of 99.6% with the reported data. The coding region of oleic acid desaturase gene was then cloned from Arabidopsis thaliana. The sequencing analysis indicated that the sequence of the PCR product was just the same as reported before. In addition, the plant expression vector harboring the seed specific promoter and trans Fad2 gene was constructed.展开更多
基金Supported by Henan Province Key Research and Development Program,No.231111311000Henan Provincial Science and Technology Research Project,No.232102310411+2 种基金Henan Province Medical Science and Technology Key Project,No.LHGJ20220566 and No.LHGJ20240365Henan Province Medical Education Research Project,No.WJLX2023079Zhengzhou Medical and Health Technology Innovation Guidance Program,No.2024YLZDJH022.
文摘Intervertebral disc degeneration is a leading cause of lower back pain and is characterized by pathological processes such as nucleus pulposus cell apoptosis,extracellular matrix imbalance,and annulus fibrosus rupture.These pathological changes result in disc height loss and functional decline,potentially leading to disc herniation.This comprehensive review aimed to address the current challenges in intervertebral disc degeneration treatment by evaluating the regenerative potential of stem cell-based therapies,with a particular focus on emerging technologies such as exosomes and gene vector systems.Through mechanisms such as differentiation,paracrine effects,and immunomodulation,stem cells facilitate extracellular matrix repair and reduce nucleus pulposus cell apoptosis.Despite recent advancements,clinical applications are hindered by challenges such as hypoxic disc environments and immune rejection.By analyzing recent preclinical and clinical findings,this review provided insights into optimizing stem cell therapy to overcome these obstacles and highlighted future directions in the field.
基金Project(2015WK3012) supported by the Hunan Provincial Science and Technology Department Project,ChinaProject(81571021) supported by the National Natural Science Foundation of China+2 种基金Project(225) supported by the High Level Health Personnel in Hunan Province,ChinaProject(621020094) supported by the State Key Laboratory of Powder Metallurgy of Central South University,ChinaProject(20160301) supported by New Talent Project of the Third Xiangya Hospital of Central South University,China
文摘Transfection efficiency of hydroxyapatite nanoparticles(HAnps)is relative to the particle size,morphology,surface charge,surface modifier and so on.This study prepared HAnps with doped Tb/Mg by hydrothermal synthesis method(HTSM)and investigated the effects of different Tb/Mg contents on the morphology,particle size,surface charge,composition and cellular endocytosis of HAnps.The results showed that Mg-HAnps possessed better dispersion ability than Tb-HAnps.With increasing doping content of Tb/Mg-HAnps,the granularity of Tb-HAnps increased,while that of Mg-HAnps declined.Both particle size and zeta potential of Mg-HAnps were lower than those of Tb-HAnps.7.5%Mg-doping HAnps presented relatively uniform slender rod morphology with average size of30nm,while10%Mg-doping HAnps were prone to agglomeration.Moreover,Mg-HAnps-GFP(green fluorescent protein)endocytosed by MG63cells was dotted in the perinuclear region,while Tb-HAnps were more likely to aggregate.In conclusion,as gene vectors,Mg-HAnps showed enhanced properties compared to Tb-HAnps.
基金This research was supported by the National Natural Science Foundation of China.
文摘Some novel lipids bearing nucleosides were designed and synthesized as gene vectors, and the structures of these compounds were characterized by UV, IR, 1HNMR, 13CNMR and elemental analysis.
文摘Some cationic and neutral lipids bearing cholesteryl group were synthesized as gene vectors, and the structures of the compounds were characterized by IR, (HNMR)-H-1, MS and elemental analysis.
文摘Summary: To evaluate the feasibility of using polyethyleneimine (PEI) coated magnetic iron oxide nanoparticles (polyMAG-1000) as gene vectors. The surface characteristics of the nanoparticles were observed with scanning electron microscopy. The ability of the nanoparticles to combine with and protect DNA was investigated at different PH values after polyMAG-1000 and DNA were combined in different ratios. The nanoparticles were tested as gene vectors with in vitro transfection models. Under the scanning electron microscope the nanoparticles were about 100 nm in diameter. The nanoparticles could bind and condense DNA under acid, neutral and alkaline conditions, and they could transfer genes into cells and express green fluorescent proteins (GFP). The transfection efficiency was highest (51 %) when the ratio of nanoparticles to DNA was 1:1 (v:w). In that ratio, the difference in transfection efficiency was marked depending on whether a magnetic field was present or not: about 10 % when it was absent but 51 % when it was present. The magnetic iron oxide nanoparticles coated with PEI may potentially be used as gene vectors.
基金supported by the Distinguished Young Scholars(No.50888001)National Natural Science Foundation of China(No.21090352)+1 种基金National Basic Research Program(973 Program,2009CB526403)Program for Changjiang Scholars and Innovative Research Team in University of China and Fundamental Research Funds for Central Universities(No.2010QNA4022)
文摘Gene therapy has emerged as a potential new approach to treat genetic disorders by delivering therapeutic genes to target diseased tissues. However, its clinical use has been impeded by gene delivery systems. The viral vectors are very efficient in delivering and expressing their carried genes, but they have safety issues in clinical use. While nonviral vectors are much safer with very low risks after careful material design, but their gene transcription efficiency is too low to be clinically used. Thus, rational design of nonviral vectors mimicking the viral vectors would be a way to break this bottleneck. This review compares side-by-side how viral/nonviral gene vectors transcend these biological barriers in terms of blood circulation, cellular uptake, endosome escape, nucleus import and gene transcription.
基金Funded by the Scientific Research Project of Shanghai Municipal Health Commission(No.201940430)。
文摘Our previous studies have successfully grafted biotin and galactose onto chitosan(CS)and synthesized biotin modified galactosylated chitosan(Bio-GC).The optimum N/P ratio of Bio-GC and plasmid DNA was 3:1.At this N/P ratio,the transfection efficiency in the hepatoma cells was the highest with a slow release effect.Bio-GC nanomaterials exhibit the protective effect of preventing the gene from nuclease degradation,and can target the transfection into hepatoma cells by combination with galactose and biotin receptors.The transfection rate was inhibited by the competition of galactose and biotin.Bio-GC nanomaterials were imported into cells’cytoplasm by their receptors,followed by the imported exogenous gene transfected into the cells.Bio-GC nanomaterials can also cause inhibitory activity in the hepatoma cells in the model of orthotopic liver transplantation in mice,by carrying the gene through the blood to the hepatoma tissue.Taken together,bio-GC nanomaterials act as gene vectors with the activity of protecting the gene from DNase degradation,improving the rate of transfection in hepatoma cells,and transporting the gene into the cytoplasm in vitro and in vivo.Therefore,they are efficient hepatoma-targeting gene carriers.
基金This work was supported by the National High Technology R&D Project of China (Grant No. 102-10-01-05)the Special Funds for Major State Basic Research of China (Grant No. 1998051008)the Key Program Funds of the Ministry of Education (Grant No. 2000-15
文摘DNA delivery is a core technology for gene structure and function research as well as clinical settings. The ability to safely and efficiently targeted transfer foreign DNA into cells is a fundamental goal in biotechnology. With the development of nanobiotechnology, nanoparticle gene vectors brought about new hope to reach the goal. In our research, silica nanoparticles (SiNP) were synthesized first in a microemulsion system polyoxyethylene nonylphenyl ether (OP-10)/cyclohexane/ammonium hydroxide, at the same time the effects of SiNP size and its distribution were elucidated by orthogonal analysis; then poly-L-lysine (PLL) was linked on the surface of SiNP by nanoparticle surface energy and electrostatically binding; lastly a novel complex nanomate-rial-poly-L-lysine-silica nanoparticles (PLL-SiNP) wasprepared. The analysis of plasmid DNA binding and DNase I enzymatic degradation discovered that PLL-SiNP could bind DNA, and protect it against enzymatic degradation. Cell transfection showed that
基金This study was supported by a grant from the National Natural Science Foun-dation of China (No. 30170925).
文摘BACKGROUND: Gene therapy as part of modern molecu-lar medicine holds great promise for the treatment of hepa-tocellular carcinoma (HCC) and has the potential to bringa revolutionary era to cancer treatment. For the past de-cade various viral and non-viral vectors have been engi-neered for improved liver gene therapy.DATA RESOURCES: An English-language literature searchusing MEDLINE (2004), Index Medicus (2004) and biblio-graphic reviews of books and review articles. Liver-directedgene transfer vectors and their history and recent clinicalapplications.RESULTS: The ultimate goal of liver-directed gene therapyfor HCC is the stable expression of a therapeutic transgenein a significant proportion of hepatocytes. The design of avector system providing efficient and stable gene engraft-ment and expression in human hepatocytes is still a chal-lenging issue. The advantages and disadvantages of the ge-netically engineered vector of viral or non-viral origin arediscussed with respect to their essential relevance.CONCLUSION: Liver gene therapy has a long way to goand efficient and innocuous liver-directed gene transfer vec-tors are therefore urgently required.
基金Supported by a grant from the National Natural Sciences Foundation of China No 30100189
文摘Objective： The aim of the study was to observe the transfection efficacy of hepatitis B virus envelope （HBVE） and evaluate its ability as a gene transfer vector for liver cancer cells. Methods： To obtain HBVE, the supematant fluid of HepG 2.2.15 cells was mixed with a PEG8000 solution for concentration and was inactivated by β-propiolactone. The acquired HBVE was used to pack plRES2-EGFP to test its package ability. Then, we examined its quantity and quality with ELISA, PCR, SDS-PAGE and electron microscopy. The plRES2-EGFP was packed with HBVE and obtained the product HBVE-GFP. The plRES2-EGFP was packed with liposome and obtained the product liposome-GFP. HBVE-GFP and liposome-GFP were used to transfer HepG 2 cells to study the transfection efficiency. HBVE-GFP was used to transfer HepG 2, A549, HeLa and FB cells to study the targeting ability. The green fluorescent protein （GFP） expression was observed under a fluorescent microscope. The rate of GFP positive cells was determined by flow cytometry. Results： 1. The acquired HBVE could retain the surface protein HBsAg ＋ pre S1 ＋ pre S2 and had no virus DNA. It had good package ability for plRES2-EGFP. 2. Transfection efficiency： The GFP could be observed in both the liposome group and HBVE group under the fluorescent microscope. But the HBVE group had a higher fluorescent intensity than liposome group. The transfection rate of liposome group was 49.97% ＋ 2.37% while the HBVE group was 70.65% ＋ 3.15% and the fluorescent intensity of the HBVE group was 3-4 times （P = 0.000） for liposome group with the determination of flow cytometry. 3. Targeting ability： The GFP could be observed in the four groups under the fluorescent microscope. The HepG 2 group had the highest fluorescent intensity among the four groups. The transfection rate of HepG 2 group was 71.35% ＋ 0.03% which was highly expressed than other groups （P = 0.000） and the fluorescent intensity of the HepG 2 group was 2-3 times （P = 0.000） for the other 3 groups with the determination of flow cytometry. Conclusion： HBVE can be constructed successfully with the methods of PEG8000 and β-propiolactone from the supernatant fluid of HepG 22.15 cells. The HBVE can be a candidate gene transfer vector for liver cancer cells.
基金Project supported by the National Basic Research Program (973) of China (No. 2002CB312200) and the Center for Bioinformatics Pro-gram Grant of Harvard Center of Neurodegeneration and Repair,Harvard Medical School, Harvard University, Boston, USA
文摘In microarray-based cancer classification, gene selection is an important issue owing to the large number of variables and small number of samples as well as its non-linearity. It is difficult to get satisfying results by using conventional linear sta- tistical methods. Recursive feature elimination based on support vector machine (SVM RFE) is an effective algorithm for gene selection and cancer classification, which are integrated into a consistent framework. In this paper, we propose a new method to select parameters of the aforementioned algorithm implemented with Gaussian kernel SVMs as better alternatives to the common practice of selecting the apparently best parameters by using a genetic algorithm to search for a couple of optimal parameter. Fast implementation issues for this method are also discussed for pragmatic reasons. The proposed method was tested on two repre- sentative hereditary breast cancer and acute leukaemia datasets. The experimental results indicate that the proposed method per- forms well in selecting genes and achieves high classification accuracies with these genes.
文摘Gene therapy appears as a promising strategy to treatincurable diseases. In particular, combined gene therapy has shown improved therapeutic efficiency. Internal ribosome entry sites(IRESs), RNA elements naturally present in the 5' untranslated regions of a few m RNAs, constitute a powerful tool to co-express several genes of interest. IRESs are translational enhancers allowing the translational machinery to start protein synthesis by internal initiation. This feature allowed the design of multi-cistronic vectors expressing several genes from a single m RNA. IRESs exhibit tissue specificity, and drive translation in stress conditions when the global cell translation is blocked, which renders them useful for gene transfer in hypoxic conditions occurring in ischemic diseases and cancer. IRES-based viral and non viral vectors have been used successfully in preclinical and clinical assays of combined gene therapy and resulted in therapeutic benefits for various pathologies including cancers, cardiovascular diseases and degenerative diseases.
文摘In order to lay a foundation for researching the function of Rosa rugose (R. rugosa) RrGlu gene, the RrGlu gene was amplified from the styles of R. rugosa “Tanghong”, a gene expression vector named PBI121-RrGlu was constructed and the vector was introduced into tobacco with the agrobacterium-mediated method. PCR results showed that the RrGlu gene was integrated into the tobacco genome.
文摘To study the therapeutic effects of herpes simplex virus thymidine kinase gene transferred by the EBV based expression vector on experimental hepatocellular carcinoma, pDR2 TK gene was delivered into human hepatocellular carcinoma cell line SMMC 7721 by using liposome mediated transfection technique,and then gene expression was detected by RT PCR, and the killing effects were examined through MTT method. In the nude mice hepatoma model,the antitumor effects of pDR2 TK /GCV system was evaluated in terms of tumor growth. MTT results showed that the pDR2 TK /GCV had cytotoxic effect and about 70 % SMMC 7721 cells were killed when GCV was at 1000 μmol/L. In vivo experiment showed that the tumor size in nude mice with transferred pDR2 TK gene was significantly smaller than that in control group . On the 10th day the tumor in 3 mice (60 %) disappeared completely after GCV treatment. It is concluded that the pDR2 TK/GCV system has marked killing effects on the experimental hepatocellular carcinoma.
基金supported in part by the Alex's Lemonade Foundation,and Bankhead-Coley Cancer Research Program,Florida Department of Health(to CL), Public Health Service grants R01 HL-097088 and R21 EB-015684 from the National Institutes of Health,and an institutional grant from the Children's Miracle Network (to AS,CL and GA)supported in part by the state-sponsored program for Graduate Students from China Scholarship Council,Government of China, and the National Natural Science Foundation of China (No.30730114)
文摘OBJECTIVE： In the present study, we systemically evaluated the ability of two bioactive compounds from traditional Chinese medicine, celastrol and pristimerin, to enhance recombinant adeno-associated virus （rAAV） serotype vector-mediated transgene expression both in human cell lines in vitro, and in murine hepatocytes in vivo. METHODS： Human cell lines were infected with rAAV vectors with either mock treatment or treatment with celastrol or pristimerin. The transgene expression, percentage of nuclear translocated viral genomes and the ubiquitination of intracellular proteins were investigated post-treatment. In addition, nonobese diabetic/severe combined immunodeficient gamma （NSG） mice were tail vain-injected with rAAV vectors and co-administered with either dimethyl sulfoxide, celastrol, pristimerin or a positive control, bortezomib. The transgene expression in liver was detected and compared over time. RESULTS： We observed that treatment with pristimerin, at as low as 1 IJmol/L concentration, significantly enhanced rAAV2 vector-mediated transgene expression in vitro, and intraperitoneal co- administration with pristimerin at 4 mg/（kg.d） for 3 d dramatically facilitated viral transduction in murine hepatocytes in vivo. The transduction efficiency of the tyrosine-mutant rAAV2 vectors as well as that of rAAV8 vectors carrying oversized transgene cassette was also augmented significantly by pristimerin. The underlying molecular mechanisms by which pristimerin mediated the observed increase in the transduction efficiency of rAAV vectors include both inhibition of proteasomal degradation of the intracellular proteins and enhanced nuclear translocation of the vector genomes. CONCLUSION： These studies suggest the potential beneficial use of pristimerin and pristimerincontaining herb extract in future liver-targeted gene therapy with rAAV vectors.
基金supported by the National Natural Science Foundation of China (Grant No. 30901158)the Key Project of Chinese Ministry of Education (Grant No. 104243)
文摘A full-length cDNA sequence of xyloglucan endotransglycosylase gene （XET）, abundantly expressed in the cambium of Anthocephalus chinensis was cloned by conserved PCR, rapid-amplification of cDNA ends and by chromosome walking. Analytical results of the DNA sequence show that a 912 bp complete open reading frame （ORF） encoded a 303-amino acid protein was in the 1205 bp full cDNA sequence. The deduced amino acid sequence of AcXET, which contained the conserved specific EIDFE catalytic site sequence to XETs was homologous to the other known XET proteins. In order to study the gene function of AcXET and obtain transgenic plants, a plant expression vector pBIAcXET was constructed by recombinating the AcXET fragment from the cloning vector pMD19AcXET and the binary vector pBI121 between the XbaI and SmaI sites. The fragment ofAcXET gene was inserted between the CaMV 35S promotor and the coding region of the GUS gene in pBI121. The identification results show that the plant expression binary vector pBIAcXET was constructed successfully. These results lay the foundation for studying the molecular mechanism ofAcXET gene during wood formation.
基金Supported by The Deutsche Forschungsgemeinschaft,Nos.FE785/2-2 and FE785/4-1the Bundesministerium für Bildung und Entwicklung,No.031A331
文摘Safe and effective gene therapy approaches require targeted tissue-specific transfer of a therapeutic transgene.Besides traditional approaches, such as transcriptional and transductional targeting, micro RNA-dependent posttranscriptional suppression of transgene expression has been emerging as powerful new technology to increase the specificity of vector-mediated transgene expression. Micro RNAs are small non-coding RNAs and often expressed in a tissue-, lineage-, activation- or differentiation-specific pattern. They typically regulate gene expression by binding to imperfectly complementary sequences in the 3' untranslated region(UTR) of the m RNA. To control exogenous transgene expression, tandem repeats of artificial micro RNA target sites are usually incorporated into the 3' UTR of the transgene expression cassette, leading to subsequent degradation of transgene m RNA in cel s expressing the corresponding micro RNA. This targeting strategy, first shown for lentiviral vectors in antigen presenting cells, has now been used for tissue-specific expression of vector-encoded therapeutic transgenes, to reduce immune response against the transgene, to control virus tropism for oncolytic virotherapy, to increase safety of live attenuated virus vaccines and to identify and select cell subsets for pluripotent stem cell therapies, respectively. This review provides an introduction into the technical mechanism underlying micro RNA-regulation, highlights new developments in this field and gives an overview of applications of micro RNA-regulated viral vectors for cardiac, suicide gene cancer and hematopoietic stem cell therapy, as well as for treatment of neurological and eye diseases.
文摘The upstream regulatory region of a seed specific gene was isolated from the genomic DNA of Brassica napus by PCR amplification. The cloned fragment contained 1755 nucleotides, and shared a sequence homology of 99.6% with the reported data. The coding region of oleic acid desaturase gene was then cloned from Arabidopsis thaliana. The sequencing analysis indicated that the sequence of the PCR product was just the same as reported before. In addition, the plant expression vector harboring the seed specific promoter and trans Fad2 gene was constructed.
