Objective:To construct a novel non-viral vector loaded with growth and differentiation factor-5(GDF-5) plasmid using chitosan,hyaluronic acid,and chondroitin sulfate for osteoarthritis (OA)gene therapy.Methods: Nano-m...Objective:To construct a novel non-viral vector loaded with growth and differentiation factor-5(GDF-5) plasmid using chitosan,hyaluronic acid,and chondroitin sulfate for osteoarthritis (OA)gene therapy.Methods: Nano-microspheres (NMPs)were prepared by mixing chitosan,hyaluronic acid,and chondreitin sulfate.GDF-5 plasmid was encapsulated in the NMPs through electrostatic adsorption.The basic characteristics of the NMPs were observed,and then they were co-cultured with chondrocytes to observe their effects on extracellular matrix (ECM) protein expression.Finally,NMPs loaded with GDF-5were inje.cted into the articular cavities of rabbits to observe their therapeutic effects on OA in vivo.Results:NMPs exhibited good physicochemical properties and low cytotoxicity.Their average diameter was (0.61±0.20)μm,and encapsulation efficiency was (38.19±0.36)%.According to Cell Counting Kit-8(CCK-8)assay,relative cell viability was 75%-99%when the total weight of NMPs was less than 560μg. Transfection efficiency was (62.0±2.1)% in a liposome group,and (60.0±1.8)% in the NMP group.There was no sig- nificant difference between the two groups (P>0.05).Immunohistochemical staining results suggested that NMPs can successfully transfect chondrocytes and stimulate ECM protein expression in vitro.Compared with the control groups, the NMP group significantly promoted the expression of chondrocyte ECM in vivo (P<0.05),as shown by analysis of the biochemical composition of chondrocyte ECM.When NMPs were injected into OA model rabbits,the expression of ECM proteins in chondrocytes was significantly promoted and the progression of OA was slowed down.Conclusions: Based on these data,we think that these NMPs with excellent physicochemical and biological properties could be promising non-viral vectors for OA gene therapy.展开更多
Objective To observe the effect of growth differentiation factor-5 (GDF-5) on the growth and anabolic metabolism of articular chondrocytes. Methods The articular chondrocytes isolated from rats were treated with vario...Objective To observe the effect of growth differentiation factor-5 (GDF-5) on the growth and anabolic metabolism of articular chondrocytes. Methods The articular chondrocytes isolated from rats were treated with various concentrations of rmGDF-5, and the growth of chondrocytes measured by MTT assay, the cellular cartilage matrices formation detected sulfated glycosaminoglycan by Alcian blue staining and type Ⅱcollagen by RT-PCR. Results After 7 days culture, MTT assay showed that GDF-5 enhanced the growth of chondrocytes in a dose-dependent manner, RT-PCR showed that GDF-5 clearly induced the synthesis of type Ⅱ collagen because of the col2a1 mRNA band more and more strong in a dose-dependent. Chondrocytes were cultured with GDF-5 for 14 days, the intensity of Alcian blue staining was greatly enhanced, especially, at a high concentration of 1000ng/mL, and GDF-5 enhanced the accumulation of the Alcian blue-stainable material in a concentration-dependent manner and in a does-dependent manner. Conclusion GDF-5 enhanced the growth of mature articular chondrocytes, and stimulated the cellular cartilage matrices formation in mono-layer culture.展开更多
BACKGROUND Krüppel-like factor-5(KLF5)is a zinc-finger transcription factor related to tumor progression.However,the relationship between KLF5 and lung cancer remains to be identified.AIM To investigate the clini...BACKGROUND Krüppel-like factor-5(KLF5)is a zinc-finger transcription factor related to tumor progression.However,the relationship between KLF5 and lung cancer remains to be identified.AIM To investigate the clinical value of KLF5 and interference with KLF5 mRNA transcription on the effects of biological behaviors in lung squamous-cell carcinoma(LUSC).METHODS Lung KLF5 mRNA data were extracted from bioinformatics databases.Blood and tissues from a cohort of patients with benign or malignant lung diseases were collected with ethical committee consent to validate KLF5 expression via multiplex immunofluorescence and immunohistochemistry,Western blot,Enzyme Linked Immunosorbent Assay or quantitative polymerase chain reaction.Furthermore,KLF5 mRNA was silenced in lung A549 cells to validate biological behaviors in vitro and nude mouse xenograft growth in vivo,respectively.RESULTS A cohort of bioinformatics databases revealed high KLF5 mRNA expression in LUSC(P<0.001)but lower KLF5 mRNA expression in lung adenocarcinoma.Upregulated KLF5 in the lung or sera of patients with lung cancer(P<0.001)were confirmed that related to poor differentiation,lymph node or distant metastasis.Furthermore,the incidence of KLF5 levels greater than 500 ng/mL in LUSC patients was 86.7%,which was significantly greater(P<0.001)than that in cases with benign lung diseases(13.3%)or healthy controls.Functionally,silencing KLF5 mRNA with a specific shRNA significantly suppressed A549 cell proliferation,decreased cell migration,increased the ratio of G2 phase cells in vitro,and inhibited the growth of nude mouse xenografts in vivo.CONCLUSION KLF5 is a novel diagnostic biomarker or potential therapeutic target for LUSC.展开更多
基金Project supported by the National Natural Science Foundation of China(No.81201407)the Bureau of Science&Technology and Intellectual Property Nanchong City(Nos.NSMC20170203 and NSMC20170310)+1 种基金the Health and Family Planning Commission of Sichuan Province(No.17JP496)the Research Projects of North Sichuan Medical College(No.CBY13-A-ZD09),China
文摘Objective:To construct a novel non-viral vector loaded with growth and differentiation factor-5(GDF-5) plasmid using chitosan,hyaluronic acid,and chondroitin sulfate for osteoarthritis (OA)gene therapy.Methods: Nano-microspheres (NMPs)were prepared by mixing chitosan,hyaluronic acid,and chondreitin sulfate.GDF-5 plasmid was encapsulated in the NMPs through electrostatic adsorption.The basic characteristics of the NMPs were observed,and then they were co-cultured with chondrocytes to observe their effects on extracellular matrix (ECM) protein expression.Finally,NMPs loaded with GDF-5were inje.cted into the articular cavities of rabbits to observe their therapeutic effects on OA in vivo.Results:NMPs exhibited good physicochemical properties and low cytotoxicity.Their average diameter was (0.61±0.20)μm,and encapsulation efficiency was (38.19±0.36)%.According to Cell Counting Kit-8(CCK-8)assay,relative cell viability was 75%-99%when the total weight of NMPs was less than 560μg. Transfection efficiency was (62.0±2.1)% in a liposome group,and (60.0±1.8)% in the NMP group.There was no sig- nificant difference between the two groups (P>0.05).Immunohistochemical staining results suggested that NMPs can successfully transfect chondrocytes and stimulate ECM protein expression in vitro.Compared with the control groups, the NMP group significantly promoted the expression of chondrocyte ECM in vivo (P<0.05),as shown by analysis of the biochemical composition of chondrocyte ECM.When NMPs were injected into OA model rabbits,the expression of ECM proteins in chondrocytes was significantly promoted and the progression of OA was slowed down.Conclusions: Based on these data,we think that these NMPs with excellent physicochemical and biological properties could be promising non-viral vectors for OA gene therapy.
文摘Objective To observe the effect of growth differentiation factor-5 (GDF-5) on the growth and anabolic metabolism of articular chondrocytes. Methods The articular chondrocytes isolated from rats were treated with various concentrations of rmGDF-5, and the growth of chondrocytes measured by MTT assay, the cellular cartilage matrices formation detected sulfated glycosaminoglycan by Alcian blue staining and type Ⅱcollagen by RT-PCR. Results After 7 days culture, MTT assay showed that GDF-5 enhanced the growth of chondrocytes in a dose-dependent manner, RT-PCR showed that GDF-5 clearly induced the synthesis of type Ⅱ collagen because of the col2a1 mRNA band more and more strong in a dose-dependent. Chondrocytes were cultured with GDF-5 for 14 days, the intensity of Alcian blue staining was greatly enhanced, especially, at a high concentration of 1000ng/mL, and GDF-5 enhanced the accumulation of the Alcian blue-stainable material in a concentration-dependent manner and in a does-dependent manner. Conclusion GDF-5 enhanced the growth of mature articular chondrocytes, and stimulated the cellular cartilage matrices formation in mono-layer culture.
基金Supported by Jiangsu Commission of Health of China,No.M2020096.
文摘BACKGROUND Krüppel-like factor-5(KLF5)is a zinc-finger transcription factor related to tumor progression.However,the relationship between KLF5 and lung cancer remains to be identified.AIM To investigate the clinical value of KLF5 and interference with KLF5 mRNA transcription on the effects of biological behaviors in lung squamous-cell carcinoma(LUSC).METHODS Lung KLF5 mRNA data were extracted from bioinformatics databases.Blood and tissues from a cohort of patients with benign or malignant lung diseases were collected with ethical committee consent to validate KLF5 expression via multiplex immunofluorescence and immunohistochemistry,Western blot,Enzyme Linked Immunosorbent Assay or quantitative polymerase chain reaction.Furthermore,KLF5 mRNA was silenced in lung A549 cells to validate biological behaviors in vitro and nude mouse xenograft growth in vivo,respectively.RESULTS A cohort of bioinformatics databases revealed high KLF5 mRNA expression in LUSC(P<0.001)but lower KLF5 mRNA expression in lung adenocarcinoma.Upregulated KLF5 in the lung or sera of patients with lung cancer(P<0.001)were confirmed that related to poor differentiation,lymph node or distant metastasis.Furthermore,the incidence of KLF5 levels greater than 500 ng/mL in LUSC patients was 86.7%,which was significantly greater(P<0.001)than that in cases with benign lung diseases(13.3%)or healthy controls.Functionally,silencing KLF5 mRNA with a specific shRNA significantly suppressed A549 cell proliferation,decreased cell migration,increased the ratio of G2 phase cells in vitro,and inhibited the growth of nude mouse xenografts in vivo.CONCLUSION KLF5 is a novel diagnostic biomarker or potential therapeutic target for LUSC.
