AIM. To study the differential gene expression profiles of target cells in primary gastric cancer and its metastatic lymph nodes using laser microdissection （LMD） in combination with cDNA microarray. METHODS： Norma...AIM. To study the differential gene expression profiles of target cells in primary gastric cancer and its metastatic lymph nodes using laser microdissection （LMD） in combination with cDNA microarray. METHODS： Normal gastric tissue samples from 30 healthy individuals, 36 cancer tissue samples from primary gastric carcinoma and lymph node metastasis tissue samples from 58 patients during gastric cancer resection were obtained using LMD in combination with cDNA microarray independently. After P27-based amplification, aRNA from 36 of 58 patients （group 1） with lymph node metastasis and metastatic tissue specimens from the remaining 22 patients （group 2） were applied to cDNA microarray. Semiquantitative reverse transcriptase polymerase chain reaction （RT-PCR） and imrnunohistochemical assay verified the results of microarray in group 2 and further identified genes differentially expressed in the progression of gastric cancer. RESULTS： The expression of 10 genes was up-regulated while the expression of 15 genes was down-regulated in 22 gastric carcinoma samples compared with that of genes in the normal controls. The results were confirmed at the level of mRNA and protein, and suggested that four genes （OPCML, RNASE1, YES1 and ACK1） could play a key role in the tumorigenesis and metastasis of gastric cancer. The expression pattern of 3 genes （OPCML, RNASE1 and YES1） was similar to tumor suppressor genes. For example, the expression level of these genes was the highest in normal gastric epithelium, which was decreased in primary carcinoma, and further decreased in metastatic lymph nodes. On the contrary, the expression pattern of gene ACK1 was similar to that of oncogene. Four genes were further identified as differentially expressed genes in the majority of the cases in the progression of gastric cancer. CONCLUSION： LMD in combination with cDNA microaro ray provides a unique support foe the identification of early expression profiles of differential genes and the expression pattern of 3 genes （OPCML, RNASE1 and YES1） associated with the progression of gastric cancer. Further study is needed to reveal the molecular mechanism of lymph node metastasis in patients with gastric cancer.展开更多
AIM: To explore the expression of differential gene expression profiles of target cell between non-invasive submucosal and invasive advanced tumor in colon carcinoma using laser microdissection (LMD) in combination wi...AIM: To explore the expression of differential gene expression profiles of target cell between non-invasive submucosal and invasive advanced tumor in colon carcinoma using laser microdissection (LMD) in combination with polypeptide analysis. METHODS: Normal colon tissue samples from 20 healthy individuals and 30 cancer tissue samples from early non-invasive colon cancer cells were obtained. The cells from these samples were used LMD independently after P27-based amplification. aRNA from advanced colon cancer cells and metastatic cancer cells of 40 cases were applied to LMD and polypeptide analysis, semiquantitative reverse transcribed polymerase chain reaction (RT-PCR) and immunohistochemical assays were used to verify the results of microarray and further identify differentially expressed genes in non-invasive early stages of colon cancer. RESULTS: Five gene expressions were changed in colon carcinoma cells compared with that of controls. Of the five genes, three genes were downregulated and two were upregulated in invasive submucosal colon carcinoma compared with non-invasive cases. The results were confirmed at the level of aRNA and gene expression. Five genes were further identified as differentially expressed genes in the majority ofcases (> 50%, 25/40) in progression of colon cancer, and their expression patterns of which were similar to tumor suppressor genes or oncogenes. CONCLUSION: This study suggested that combined use of polypeptide analysis might identify early expression profiles of five differential genes associated with the invasion of colon cancer. These results reveal that this gene may be a marker of submucosal invasion in early colon cancer.展开更多
Spatial omics technologies have become powerful methods to provide valuable insights into cells and tissues within a complex context,significantly enhancing our understanding of the intricate and multifaceted biologic...Spatial omics technologies have become powerful methods to provide valuable insights into cells and tissues within a complex context,significantly enhancing our understanding of the intricate and multifaceted biological system.With an increasing focus on spatial heterogeneity,there is a growing need for unbiased,spatially resolved omics technologies.Laser capture microdissection(LCM)is a cutting-edge method for acquiring spatial information that can quickly collect regions of interest(ROIs)from heterogeneous tissues,with resolutions ranging from single cells to cell populations.Thus,LCM has been widely used for studying the cellular and molecular mechanisms of diseases.This review focuses on the differences among four types of commonly used LCM technologies and their applications in omics and disease research.Key attributes of application cases are also highlighted,such as throughput and spatial resolution.In addition,we comprehensively discuss the existing challenges and the great potential of LCM in biomedical research,disease diagnosis,and targeted therapy from the perspective of high-throughput,multi-omics,and single-cell resolution.展开更多
AIM： To examine the gene expression profile of gastric cancer （GC） by combination of laser capture microdissection （LCM） and microarray and to correlate the profiling with histological subtypes. METHODS： Using L...AIM： To examine the gene expression profile of gastric cancer （GC） by combination of laser capture microdissection （LCM） and microarray and to correlate the profiling with histological subtypes. METHODS： Using LCM, pure cancer cells were procured from 45 cancerous tissues. After procurement of about 5 000 cells, total RNA was extracted and the quality of RNA was determined before further amplification and hybridization. One microgram of amplified RNA was converted to cDNA and hybridized to cDNA microarray. RESULTS： Among 45 cases, only 21 were qualified for their RNAs. A total of 62 arrays were performed. These included 42 arrays for cancer （21 cases with dyeswab duplication） and 20 arrays for non-tumorous cells （10 cases with dye-swab duplication） with universal reference. Analyzed data showed 504 genes were differentially expressed and could distinguish cancerous and non-cancerous groups with more than 99% accuracy. Of the 504 genes, trefoil factors 1, 2, and 3 were in the list and their expression patterns were consistent with previous reports. Immunohistochemical staining of trefoil factor 1 was also consistent with the array data. Analyses of the tumor group with these 504 genes showed that there were 3 subgroups of GC that did not correspond to any current classification system, including Lauren＇s dassification. CONCLUSION： By using LCM, linear amplification of RNA, and cDNA microarray, we have identified a panel of genes that have the power to discriminate between GC and non-cancer groups. The new molecular classification and the identified novel genes in gastric carcinogenesis deserve further investigations to elucidate their clinicopathological significance.展开更多
The method of laser capture microdissection (LCM) combined with suppressive subtractive hybridization (SSH) was developed to isolate specific germ cells from human testis sections and to identify the genes expressed d...The method of laser capture microdissection (LCM) combined with suppressive subtractive hybridization (SSH) was developed to isolate specific germ cells from human testis sections and to identify the genes expressed during differentiation and development. In the present study, over 10,000 primary spermatocytes and round spermatid cells were successfully isolated by LCM. Using the cDNAs from primary spermatocytes and round spermatids, SSH cDNAs library of primary spermatocyte-specific was constructed. The average insert size of the cDNA isolated from 75 randomly picked white clones was 500 bp, ranging from 250 bp to 1.7 kb. Using the dot-blot method, a total of 421 clones were examined, resulting in the identification of 390 positive clones emitting strong signals. Partial sequence of cDNAs prepared from each clone was determined with an overall success rate of 84.4%. Genes encoding cytochrome c oxidase II and the rescue factor-humanin were most frequently expressed in primary spermatocytes, suggesting their roles involved in meiosis.展开更多
Epithelial-mesenchymal interactions（EMIs） are critical for tooth development.Molecular mechanisms mediating these interactions in root formation is not well understood.Laser capture microdissection（LCM） and subseq...Epithelial-mesenchymal interactions（EMIs） are critical for tooth development.Molecular mechanisms mediating these interactions in root formation is not well understood.Laser capture microdissection（LCM） and subsequent microarray analyses enable large scale in situ molecular and cellular studies of root formation but to date have been hindered by technical challenges of gaining intact histological sections of non-decalcified mineralized teeth or jaws with well-preserved RNA.Here,we describe a new method to overcome this obstacle that permits LCM of dental epithelia,adjacent mesenchyme,odontoblasts and cementoblasts from mouse incisors and molars during root development.Using this method,we obtained RNA samples of high quality and successfully performed microarray analyses.Robust differences in gene expression,as well as genes not previously associated with root formation,were identified.Comparison of gene expression data from microarray with real-time reverse transcriptase polymerase chain reaction（RT-PCR） supported our findings.These genes include known markers of dental epithelia,mesenchyme,cementoblasts and odontoblasts,as well as novel genes such as those in the fibulin family.In conclusion,our new approach in tissue preparation enables LCM collection of intact cells with well-preserved RNA allowing subsequent gene expression analyses using microarray and RT-PCR to define key regulators of tooth root development.展开更多
Objective：To isolate ceils of cardiac conduction system （CCS） with laser capture microdissec tion （LCM） and extract and evaluate quality of small amount of RNA from ceils of CCS. Methods： Cryo star sections were...Objective：To isolate ceils of cardiac conduction system （CCS） with laser capture microdissec tion （LCM） and extract and evaluate quality of small amount of RNA from ceils of CCS. Methods： Cryo star sections were followed by H-E staining. 20 pieces of H-E stained eryostat sections were scraped and its RNA was assessed to insure that RNA didn＇t degrade in dyeing and dehydration process. Ceils of CCS were captured with LCM and quality of small amount of RNA was verified with RT-PCR. Results： Ceils of CCS isolated with LCM had clear morphology after staining. High quality RNA was extracted from LCM samples and scraped tissues; 18S rRNA and 28S rRNA were seen distinctly on gel eleetrophoresis. Low level of small amount of RNA extracted from LCM sample was below the limit of detection on gel eleetrophoresis or ultraviolet speetrophotometer. The housekeeping genes β-aetin and GAPDH were successfully amplified with small amount of RNA. Conclusion ：This study resolves the problem of acquiring material of CCS precisely that hinders gene research of CCS. It is found out that the method is easy and reliable to extract and assess the quality of small amount of RNA from mierodisseeted ceils of CCS.展开更多
Objective To compare the quality and quantity of total RNA from different source-original neurons applied in LMPC technique. Methods （ 1 ） Aglient 2100 bioanalyzer and RT-PCR were used to check the concentration and...Objective To compare the quality and quantity of total RNA from different source-original neurons applied in LMPC technique. Methods （ 1 ） Aglient 2100 bioanalyzer and RT-PCR were used to check the concentration and fragmentation of total RNA from unfixed, temporal fixed and fixed 12 h hypothalamus sections; （2）Different neurons of PVN and SON were collected by LMPC, CRH, TRH, AVP, OT mRNA level were measured by RT-PCR; （3）Labeled neurons by injecting CTB into stomach and non-labeled neurons in DMV collected by LMPC were checked for house keeping genes by RT-PCR. Results （ 1 ） Unfixed section had higher concentration and better quality of total RNA compared with fixed sections applied in LMPC ; relative short amplicons such as GAPDH, NSE, MCH and MCAR were successfully obtained from fixed and unfixed and long amplicon of GR can only be obtained from unfixed material; （2） In magnocellular PVN and SON the expressions of AVP and OT were more special than those in the parvocellular PVN. Oppositely, the expressions of CRH, TRH in the parvocellular were more special than the other two ; （3） The expressions of house keeping genes had no significant difference between labeled and non-labeled DMV neurons. Conclusion The quality and quantity of total RNA from unfixed brain tissues were better than fixed tissues applied in LMPC and the CTB tracer which may differentiate neurons had no significant effect on physiology of the neurons applied in LMPC. The results showed that the LMPC technique is suitable for the qualitative and quantitative study on individual neurons at mRNA level.展开更多
Glandular trichomes of plants produce a wide variety of secondary metabolites which are considered as major defensive chemicals. The capitate glandular trichomes of Oenothera glazioviana(Onagraceae) were collected wit...Glandular trichomes of plants produce a wide variety of secondary metabolites which are considered as major defensive chemicals. The capitate glandular trichomes of Oenothera glazioviana(Onagraceae) were collected with laser microdissection and analyzed by gas chromatography-mass spectrometry. The volatile compound 4-hydroxy-4-methylpentan-2-one(1) was identified. We found that compound 1 displays antimicrobial, insecticidal, and phytotoxic activities. These results suggest that compound 1 might function as a defensive compound in the capitate glandular trichomes of O. glazioviana against pathogens, insect herbivores, and presumably competitive plants as well.展开更多
Background In recent years the proportion of lung adenocarcinoma （adCA） which occurs in lung cancer patients has increased. Using laser capture microdissection （LCM） combined with liquid chip-mass spectrometry tec...Background In recent years the proportion of lung adenocarcinoma （adCA） which occurs in lung cancer patients has increased. Using laser capture microdissection （LCM） combined with liquid chip-mass spectrometry technology, we aimed to screen lung cancer biomarkers by studying the proteins in the tissues of adCA. Methods We used LCM and magnetic bead based weak cation exchange （MB-WCX） to separate and purify the homogeneous adCA cells and normal cells from six cases of fresh adCA and matched normal lung tissues. The proteins were analyzed and identified by matrix assisted laser desorption/ionization time-of-fight mass spectrometry （MALDI-OF-MS）. We screened for the best pattern using a radial basic function neural network algorithm. Results About 2.895x10s and 1.584x10s cells were satisfactorily obtained by LCM from six cases of fresh lung adCA and matched normal lung tissues, respectively. The homogeneities of cell population were estimated to be over 95% as determined by microscopic visualization. Comparing the differentially expressed proteins between the lung adCA and the matched normal lung group, 221 and 239 protein peaks, respectively, were found in the mass-to-charge ration （M/Z） between 800 Da and 10 000 Da. According to ttest, the expression of two protein peaks at 7521.5 M/Zand 5079.3 M/Z had the largest difference between tissues. They were more weakly expressed in the lung adCA compared to the matched normal group. The two protein peaks could accurately separate the lung adCA from the matched normal lung group by the sample distribution chart. A discriminatory pattern which can separate the lung adCA from the matched normal lung tissue consisting of three proteins at 3358.1 M/Z, 5079.3 M/Z and 7521.5 M/Z was established by a radial basic function neural network algorithm with a sensitivity of 100% and a specificity of 100%. Conclusions Differential proteins in lung adCA were screened using LCM combined with liquid chip-mass spectrometry technology, and a biomarker model was established. It is possible that this technology is going to become a powerful tool in screening and early diagnosis of lung adCA.展开更多
Bupleuri Radix has been widely used in traditional Chinese medicine.In the current herbal market,the species Bupleurum marginatum Wall.ex DC.is the main source of Bupleuri Radix.Although Bupleuri Radix from the roots ...Bupleuri Radix has been widely used in traditional Chinese medicine.In the current herbal market,the species Bupleurum marginatum Wall.ex DC.is the main source of Bupleuri Radix.Although Bupleuri Radix from the roots of B.marginatum grown wild in the North West of Hubei province has higher quality compared with those from other regions according to the previous investigations,the exhaustive exploitation driven by increasing demand has drastically reduced the wild resource.As a result,germplasm evaluation and quality resource exploration are important for the sustainable utilization and cultivation of B.marginatum.A preliminary study indicated differences in the tissue structure of B.marginatum grown in different areas of North Western Hubei province.In the current study,various tissues of the roots of B.marginatum grown in different areas of North Western Hubei were subjected to laser microdissection and analyzed by microscopy and ultra-high performance liquid chromatography quadrupole time-of-flight mass spectrometry(UHPLC–Q-TOF-MS).The results show that wild plants from Maqiao Town,Baokang County contain the most saikosaponins distributed mainly in cork,cortex and phloem.This study provides key chemical information for evaluating the quality of B.marginatum roots.展开更多
Glandular trichomes produce a wide variety of secondary metabolites that are considered as major defensive chemicals against herbivore attack.The morphology and secondary metabolites of the peltate glandular trichomes...Glandular trichomes produce a wide variety of secondary metabolites that are considered as major defensive chemicals against herbivore attack.The morphology and secondary metabolites of the peltate glandular trichomes of a lianoid Labiatae,Colquhounia seguinii Vaniot,were investigated.Three new clerodane diterpenoids,seguiniilactones A-C（1-3）,were identified through precise trichome collection with laser microdissection,metabolic analysis with ultra performance liquid chromatography-tandem mass spectrometer,target compound isolation with classical phytochemical techniques,structure elucidation with spectroscopic methods.All compounds showed significant antifeedant activity against a generalist plant-feeding insect Spodoptera exigua.Seguiniilactone A（1） was approximately 17-fold more potent than the commercial neem oil.a-Substituted α,β-unsaturated γ-lactone functionality was found to be crucial for strong antifeedant activity of this class of compounds.Quantitative results indicated that the levels of these compounds in the peltate glandular trichomes and leaves were sufficiently high to deter the feeding by generalist insects.Moderate antifungal activity was observed for seguiniilactone C（3） against six predominant fungal species isolated from the diseased leaves of C seguinii,while seguiniilactones A and B were generally inactive.These findings suggested that seguiniilactones A-C might be specialized secondary metabolites in peltate glandular trichomes for the plant defense against insect herbivores and pathogens.展开更多
Oligosaccharide Mass Profiling （OLIMP） allows a fast and sensitive assessment of cell wall polymer structure when coupled with Matrix Assisted Laser Desorption Ionisation Time Of Flight Mass Spectrometry （MALDI-TOF...Oligosaccharide Mass Profiling （OLIMP） allows a fast and sensitive assessment of cell wall polymer structure when coupled with Matrix Assisted Laser Desorption Ionisation Time Of Flight Mass Spectrometry （MALDI-TOF MS）. The short time required for sample preparation and analysis makes possible the study of a wide range of plant organs, revealing a high degree of heterogeneity in the substitution pattern of wall polymers such as the cross-linking glycan xyloglucan and the pectic polysaccharide homogalacturonan. The high sensitivity of MALDI-TOF allows the use of small amounts of samples, thus making it possible to investigate the wall structure of single cell types when material is collected by such methods as laser micro-dissection. As an example, the analysis of the xyloglucan structure in the leaf cell types outer epidermis layer, entire epidermis cell layer, palisade mesophyll cells, and vascular bundles were investigated. OLIMP is amenable to in situ wall analysis, where wall polymers are analyzed on unprepared plant tissue itself without first isolating cell walls. In addition, OLIMP enables analysis of wall polymers in Golgi-enriched fractions, the location of nascent matrix polysaccharide biosynthesis, enabling separation of the processes of wall biosynthesis versus post-deposition apoplastic metabolism. These new tools will make possible a semi-quantitative analysis of the cell wall at an unprecedented level.展开更多
基金Supported by the Natural Science Foundation of Shanghai, No02ZB14072
文摘AIM. To study the differential gene expression profiles of target cells in primary gastric cancer and its metastatic lymph nodes using laser microdissection （LMD） in combination with cDNA microarray. METHODS： Normal gastric tissue samples from 30 healthy individuals, 36 cancer tissue samples from primary gastric carcinoma and lymph node metastasis tissue samples from 58 patients during gastric cancer resection were obtained using LMD in combination with cDNA microarray independently. After P27-based amplification, aRNA from 36 of 58 patients （group 1） with lymph node metastasis and metastatic tissue specimens from the remaining 22 patients （group 2） were applied to cDNA microarray. Semiquantitative reverse transcriptase polymerase chain reaction （RT-PCR） and imrnunohistochemical assay verified the results of microarray in group 2 and further identified genes differentially expressed in the progression of gastric cancer. RESULTS： The expression of 10 genes was up-regulated while the expression of 15 genes was down-regulated in 22 gastric carcinoma samples compared with that of genes in the normal controls. The results were confirmed at the level of mRNA and protein, and suggested that four genes （OPCML, RNASE1, YES1 and ACK1） could play a key role in the tumorigenesis and metastasis of gastric cancer. The expression pattern of 3 genes （OPCML, RNASE1 and YES1） was similar to tumor suppressor genes. For example, the expression level of these genes was the highest in normal gastric epithelium, which was decreased in primary carcinoma, and further decreased in metastatic lymph nodes. On the contrary, the expression pattern of gene ACK1 was similar to that of oncogene. Four genes were further identified as differentially expressed genes in the majority of the cases in the progression of gastric cancer. CONCLUSION： LMD in combination with cDNA microaro ray provides a unique support foe the identification of early expression profiles of differential genes and the expression pattern of 3 genes （OPCML, RNASE1 and YES1） associated with the progression of gastric cancer. Further study is needed to reveal the molecular mechanism of lymph node metastasis in patients with gastric cancer.
基金The Natural Science Foundation of Shanghai, No. 04ZB14072
文摘AIM: To explore the expression of differential gene expression profiles of target cell between non-invasive submucosal and invasive advanced tumor in colon carcinoma using laser microdissection (LMD) in combination with polypeptide analysis. METHODS: Normal colon tissue samples from 20 healthy individuals and 30 cancer tissue samples from early non-invasive colon cancer cells were obtained. The cells from these samples were used LMD independently after P27-based amplification. aRNA from advanced colon cancer cells and metastatic cancer cells of 40 cases were applied to LMD and polypeptide analysis, semiquantitative reverse transcribed polymerase chain reaction (RT-PCR) and immunohistochemical assays were used to verify the results of microarray and further identify differentially expressed genes in non-invasive early stages of colon cancer. RESULTS: Five gene expressions were changed in colon carcinoma cells compared with that of controls. Of the five genes, three genes were downregulated and two were upregulated in invasive submucosal colon carcinoma compared with non-invasive cases. The results were confirmed at the level of aRNA and gene expression. Five genes were further identified as differentially expressed genes in the majority ofcases (> 50%, 25/40) in progression of colon cancer, and their expression patterns of which were similar to tumor suppressor genes or oncogenes. CONCLUSION: This study suggested that combined use of polypeptide analysis might identify early expression profiles of five differential genes associated with the invasion of colon cancer. These results reveal that this gene may be a marker of submucosal invasion in early colon cancer.
基金supported by the National Natural Science Foundation of China(81973701 and 82204772)the Natural Science Foundation of Zhejiang Province(LZ20H290002)+2 种基金the Innovation Team and Talents Cultivation Program of National Administration of Traditional Chinese Medicine(ZYYCXTD-D-202002)the China Postdoctoral Science Foundation(2022M712811)Westlake Laboratory(Westlake Laboratory of Life Sciences and Biomedicine).
文摘Spatial omics technologies have become powerful methods to provide valuable insights into cells and tissues within a complex context,significantly enhancing our understanding of the intricate and multifaceted biological system.With an increasing focus on spatial heterogeneity,there is a growing need for unbiased,spatially resolved omics technologies.Laser capture microdissection(LCM)is a cutting-edge method for acquiring spatial information that can quickly collect regions of interest(ROIs)from heterogeneous tissues,with resolutions ranging from single cells to cell populations.Thus,LCM has been widely used for studying the cellular and molecular mechanisms of diseases.This review focuses on the differences among four types of commonly used LCM technologies and their applications in omics and disease research.Key attributes of application cases are also highlighted,such as throughput and spatial resolution.In addition,we comprehensively discuss the existing challenges and the great potential of LCM in biomedical research,disease diagnosis,and targeted therapy from the perspective of high-throughput,multi-omics,and single-cell resolution.
基金National Science Council, NSC-91-3112-B002-007, Taipei, Taiwan, China
文摘AIM： To examine the gene expression profile of gastric cancer （GC） by combination of laser capture microdissection （LCM） and microarray and to correlate the profiling with histological subtypes. METHODS： Using LCM, pure cancer cells were procured from 45 cancerous tissues. After procurement of about 5 000 cells, total RNA was extracted and the quality of RNA was determined before further amplification and hybridization. One microgram of amplified RNA was converted to cDNA and hybridized to cDNA microarray. RESULTS： Among 45 cases, only 21 were qualified for their RNAs. A total of 62 arrays were performed. These included 42 arrays for cancer （21 cases with dyeswab duplication） and 20 arrays for non-tumorous cells （10 cases with dye-swab duplication） with universal reference. Analyzed data showed 504 genes were differentially expressed and could distinguish cancerous and non-cancerous groups with more than 99% accuracy. Of the 504 genes, trefoil factors 1, 2, and 3 were in the list and their expression patterns were consistent with previous reports. Immunohistochemical staining of trefoil factor 1 was also consistent with the array data. Analyses of the tumor group with these 504 genes showed that there were 3 subgroups of GC that did not correspond to any current classification system, including Lauren＇s dassification. CONCLUSION： By using LCM, linear amplification of RNA, and cDNA microarray, we have identified a panel of genes that have the power to discriminate between GC and non-cancer groups. The new molecular classification and the identified novel genes in gastric carcinogenesis deserve further investigations to elucidate their clinicopathological significance.
文摘The method of laser capture microdissection (LCM) combined with suppressive subtractive hybridization (SSH) was developed to isolate specific germ cells from human testis sections and to identify the genes expressed during differentiation and development. In the present study, over 10,000 primary spermatocytes and round spermatid cells were successfully isolated by LCM. Using the cDNAs from primary spermatocytes and round spermatids, SSH cDNAs library of primary spermatocyte-specific was constructed. The average insert size of the cDNA isolated from 75 randomly picked white clones was 500 bp, ranging from 250 bp to 1.7 kb. Using the dot-blot method, a total of 421 clones were examined, resulting in the identification of 390 positive clones emitting strong signals. Partial sequence of cDNAs prepared from each clone was determined with an overall success rate of 84.4%. Genes encoding cytochrome c oxidase II and the rescue factor-humanin were most frequently expressed in primary spermatocytes, suggesting their roles involved in meiosis.
基金supported by NIH grant no.DE15109 to Dr Martha Somermana grant from the State Key Laboratory of Oral Diseases in Chengdu,China to Dr Hai Zhang
文摘Epithelial-mesenchymal interactions（EMIs） are critical for tooth development.Molecular mechanisms mediating these interactions in root formation is not well understood.Laser capture microdissection（LCM） and subsequent microarray analyses enable large scale in situ molecular and cellular studies of root formation but to date have been hindered by technical challenges of gaining intact histological sections of non-decalcified mineralized teeth or jaws with well-preserved RNA.Here,we describe a new method to overcome this obstacle that permits LCM of dental epithelia,adjacent mesenchyme,odontoblasts and cementoblasts from mouse incisors and molars during root development.Using this method,we obtained RNA samples of high quality and successfully performed microarray analyses.Robust differences in gene expression,as well as genes not previously associated with root formation,were identified.Comparison of gene expression data from microarray with real-time reverse transcriptase polymerase chain reaction（RT-PCR） supported our findings.These genes include known markers of dental epithelia,mesenchyme,cementoblasts and odontoblasts,as well as novel genes such as those in the fibulin family.In conclusion,our new approach in tissue preparation enables LCM collection of intact cells with well-preserved RNA allowing subsequent gene expression analyses using microarray and RT-PCR to define key regulators of tooth root development.
文摘Objective：To isolate ceils of cardiac conduction system （CCS） with laser capture microdissec tion （LCM） and extract and evaluate quality of small amount of RNA from ceils of CCS. Methods： Cryo star sections were followed by H-E staining. 20 pieces of H-E stained eryostat sections were scraped and its RNA was assessed to insure that RNA didn＇t degrade in dyeing and dehydration process. Ceils of CCS were captured with LCM and quality of small amount of RNA was verified with RT-PCR. Results： Ceils of CCS isolated with LCM had clear morphology after staining. High quality RNA was extracted from LCM samples and scraped tissues; 18S rRNA and 28S rRNA were seen distinctly on gel eleetrophoresis. Low level of small amount of RNA extracted from LCM sample was below the limit of detection on gel eleetrophoresis or ultraviolet speetrophotometer. The housekeeping genes β-aetin and GAPDH were successfully amplified with small amount of RNA. Conclusion ：This study resolves the problem of acquiring material of CCS precisely that hinders gene research of CCS. It is found out that the method is easy and reliable to extract and assess the quality of small amount of RNA from mierodisseeted ceils of CCS.
文摘Objective To compare the quality and quantity of total RNA from different source-original neurons applied in LMPC technique. Methods （ 1 ） Aglient 2100 bioanalyzer and RT-PCR were used to check the concentration and fragmentation of total RNA from unfixed, temporal fixed and fixed 12 h hypothalamus sections; （2）Different neurons of PVN and SON were collected by LMPC, CRH, TRH, AVP, OT mRNA level were measured by RT-PCR; （3）Labeled neurons by injecting CTB into stomach and non-labeled neurons in DMV collected by LMPC were checked for house keeping genes by RT-PCR. Results （ 1 ） Unfixed section had higher concentration and better quality of total RNA compared with fixed sections applied in LMPC ; relative short amplicons such as GAPDH, NSE, MCH and MCAR were successfully obtained from fixed and unfixed and long amplicon of GR can only be obtained from unfixed material; （2） In magnocellular PVN and SON the expressions of AVP and OT were more special than those in the parvocellular PVN. Oppositely, the expressions of CRH, TRH in the parvocellular were more special than the other two ; （3） The expressions of house keeping genes had no significant difference between labeled and non-labeled DMV neurons. Conclusion The quality and quantity of total RNA from unfixed brain tissues were better than fixed tissues applied in LMPC and the CTB tracer which may differentiate neurons had no significant effect on physiology of the neurons applied in LMPC. The results showed that the LMPC technique is suitable for the qualitative and quantitative study on individual neurons at mRNA level.
基金supported financially by the National Science Fund for Distinguished Young Scholars(31525005)the NSFC-Yunnan Joint Fund (U1202263)+1 种基金the National Basic Research Program of China (973 Program) on Biological Control of Key Crop Pathogenic Nematodes (2013CB127505)the "Hundred Talents Program" of the Chinese Academy of Sciences (awarded to S.-H. Li)
文摘Glandular trichomes of plants produce a wide variety of secondary metabolites which are considered as major defensive chemicals. The capitate glandular trichomes of Oenothera glazioviana(Onagraceae) were collected with laser microdissection and analyzed by gas chromatography-mass spectrometry. The volatile compound 4-hydroxy-4-methylpentan-2-one(1) was identified. We found that compound 1 displays antimicrobial, insecticidal, and phytotoxic activities. These results suggest that compound 1 might function as a defensive compound in the capitate glandular trichomes of O. glazioviana against pathogens, insect herbivores, and presumably competitive plants as well.
基金This work was supported by grants from the National Natural Science Foundation of China （No. 30570795） and Program for New Century Excellent Talents in University （No. NECT-06-0845） and the Program in Science and Technology of Xi＇an, Shaanxi Province （No. SF08009（1））.
文摘Background In recent years the proportion of lung adenocarcinoma （adCA） which occurs in lung cancer patients has increased. Using laser capture microdissection （LCM） combined with liquid chip-mass spectrometry technology, we aimed to screen lung cancer biomarkers by studying the proteins in the tissues of adCA. Methods We used LCM and magnetic bead based weak cation exchange （MB-WCX） to separate and purify the homogeneous adCA cells and normal cells from six cases of fresh adCA and matched normal lung tissues. The proteins were analyzed and identified by matrix assisted laser desorption/ionization time-of-fight mass spectrometry （MALDI-OF-MS）. We screened for the best pattern using a radial basic function neural network algorithm. Results About 2.895x10s and 1.584x10s cells were satisfactorily obtained by LCM from six cases of fresh lung adCA and matched normal lung tissues, respectively. The homogeneities of cell population were estimated to be over 95% as determined by microscopic visualization. Comparing the differentially expressed proteins between the lung adCA and the matched normal lung group, 221 and 239 protein peaks, respectively, were found in the mass-to-charge ration （M/Z） between 800 Da and 10 000 Da. According to ttest, the expression of two protein peaks at 7521.5 M/Zand 5079.3 M/Z had the largest difference between tissues. They were more weakly expressed in the lung adCA compared to the matched normal group. The two protein peaks could accurately separate the lung adCA from the matched normal lung group by the sample distribution chart. A discriminatory pattern which can separate the lung adCA from the matched normal lung tissue consisting of three proteins at 3358.1 M/Z, 5079.3 M/Z and 7521.5 M/Z was established by a radial basic function neural network algorithm with a sensitivity of 100% and a specificity of 100%. Conclusions Differential proteins in lung adCA were screened using LCM combined with liquid chip-mass spectrometry technology, and a biomarker model was established. It is possible that this technology is going to become a powerful tool in screening and early diagnosis of lung adCA.
基金This work was supported by the Hong Kong Research Grants Council(GRF)(HKBU-263412).
文摘Bupleuri Radix has been widely used in traditional Chinese medicine.In the current herbal market,the species Bupleurum marginatum Wall.ex DC.is the main source of Bupleuri Radix.Although Bupleuri Radix from the roots of B.marginatum grown wild in the North West of Hubei province has higher quality compared with those from other regions according to the previous investigations,the exhaustive exploitation driven by increasing demand has drastically reduced the wild resource.As a result,germplasm evaluation and quality resource exploration are important for the sustainable utilization and cultivation of B.marginatum.A preliminary study indicated differences in the tissue structure of B.marginatum grown in different areas of North Western Hubei province.In the current study,various tissues of the roots of B.marginatum grown in different areas of North Western Hubei were subjected to laser microdissection and analyzed by microscopy and ultra-high performance liquid chromatography quadrupole time-of-flight mass spectrometry(UHPLC–Q-TOF-MS).The results show that wild plants from Maqiao Town,Baokang County contain the most saikosaponins distributed mainly in cork,cortex and phloem.This study provides key chemical information for evaluating the quality of B.marginatum roots.
基金supported financially by the NSFC-Yunnan Joint Fund(U1202263)the National Basic Research Program of China(973 Program) on Biological Control of Key Crop Pathogenic Nematodes(2013CB127505)+1 种基金the National Natural Science Foundation of China(31070320,31470395 and 31100222)the "Hundred Talents Program" of the Chinese Academy of Sciences(awarded to SH Li)
文摘Glandular trichomes produce a wide variety of secondary metabolites that are considered as major defensive chemicals against herbivore attack.The morphology and secondary metabolites of the peltate glandular trichomes of a lianoid Labiatae,Colquhounia seguinii Vaniot,were investigated.Three new clerodane diterpenoids,seguiniilactones A-C（1-3）,were identified through precise trichome collection with laser microdissection,metabolic analysis with ultra performance liquid chromatography-tandem mass spectrometer,target compound isolation with classical phytochemical techniques,structure elucidation with spectroscopic methods.All compounds showed significant antifeedant activity against a generalist plant-feeding insect Spodoptera exigua.Seguiniilactone A（1） was approximately 17-fold more potent than the commercial neem oil.a-Substituted α,β-unsaturated γ-lactone functionality was found to be crucial for strong antifeedant activity of this class of compounds.Quantitative results indicated that the levels of these compounds in the peltate glandular trichomes and leaves were sufficiently high to deter the feeding by generalist insects.Moderate antifungal activity was observed for seguiniilactone C（3） against six predominant fungal species isolated from the diseased leaves of C seguinii,while seguiniilactones A and B were generally inactive.These findings suggested that seguiniilactones A-C might be specialized secondary metabolites in peltate glandular trichomes for the plant defense against insect herbivores and pathogens.
文摘Oligosaccharide Mass Profiling （OLIMP） allows a fast and sensitive assessment of cell wall polymer structure when coupled with Matrix Assisted Laser Desorption Ionisation Time Of Flight Mass Spectrometry （MALDI-TOF MS）. The short time required for sample preparation and analysis makes possible the study of a wide range of plant organs, revealing a high degree of heterogeneity in the substitution pattern of wall polymers such as the cross-linking glycan xyloglucan and the pectic polysaccharide homogalacturonan. The high sensitivity of MALDI-TOF allows the use of small amounts of samples, thus making it possible to investigate the wall structure of single cell types when material is collected by such methods as laser micro-dissection. As an example, the analysis of the xyloglucan structure in the leaf cell types outer epidermis layer, entire epidermis cell layer, palisade mesophyll cells, and vascular bundles were investigated. OLIMP is amenable to in situ wall analysis, where wall polymers are analyzed on unprepared plant tissue itself without first isolating cell walls. In addition, OLIMP enables analysis of wall polymers in Golgi-enriched fractions, the location of nascent matrix polysaccharide biosynthesis, enabling separation of the processes of wall biosynthesis versus post-deposition apoplastic metabolism. These new tools will make possible a semi-quantitative analysis of the cell wall at an unprecedented level.
