激光微切和巢式逆转录聚合酶链反应联合检测单个肝细胞的基因表达

目的 探讨检测单个肝细胞基因表达的方法。方法应用激光微切的方法从冰冻切片上将单个肝细胞切下,提取总RNA,将RNA逆转录成cDNA,采用巢式逆转录聚合酶链反应(RT-PCR)检测mRNA的表达。结果在显微镜下用紫外激光微切机,将单个肝细胞成功切下,提取RNA后,逆转录成cDNA,巢式RT-PCR的扩增产物在琼脂糖凝胶上清晰可见,其表达与细胞数量成正比。结论联合应用激光微切和巢式RT-PCR可以检测单个肝细胞的基因表达。

联合应用激光微切和实时定量RT-PCR检测单个胰岛的基因表达

在显微镜下用紫外激光微切机将单个胰岛从胰腺切片中切下,提取RNA后逆转录成cDNA并在实时定量RT-PCR中得到有效的扩增,其表达量与细胞数量成正比。

Laser Capture Microdissection Combined with RNA in vitro Linear Amplification Detecting the Relevant Genes of Bladder Cancer

Objective： Laser capture microdisection has become indispensable to the analysis of the difference of gene expression between human bladder transitional cell and bladder transitional cell carcinoma （BTCC）. However, to obtain sufficient RNA from laser-capture microdissected cells is quite difficult. The study was designed to determinc a feasible technical routine to isolate transitional cells from bladder membrane, separate carcinoma cclls from stromal cells and to amplify the RNA isolated from laser-capture microdissected cells. Methods： Bladder transitional cell were obtained from frozen sections of bladder membrane applying LCM, by the same token, BTCC cells from frozen sections of BTCC tissue. Then RNA was extracted and linearly amplified in vitro. The expression levels of β-actin in primary total RNA and amplified RNA were detected using RT-PCR. Results： That RNA integrity was good after LCM was confirmed by control experiment Ⅰ; By control experiment Ⅱ, the correlation between the number of LCM-shooting and RNA quantity undcr arranged conditions was preliminarily confirmed. About 0.5-2.5kb RNA fragments were obtained after RNA amplification and β-actin levels were integral. Conclusion： Laser capture microdissection combined with RNA linear amplification in vitro can be successfully applied to obtain pure objective cells for research. The integrity of the amplified RNA is good and can be employed in further research.

Vascular endothelial growth factor 165b expression in stromal cells and colorectal cancer

AIM:To characterize the implications of vascular endothelial growth factor(VEGF)-A in stromal cells and colorectal cancer and the expression of VEGF-A splice variants.METHODS:VEGF-A expression in tumor and stromal cells from 165 consecutive patients with colorectal cancer was examined by immunohistochemistry.The association between VEGF-A expression status and clinicopathological factors was investigated.Twenty freshfrozen samples were obtained for laser capture microdissection to analyze the splice variants of VEGF-A.RESULTS:VEGF-A was expressed in 53.9% and 42.4% of tumor and stromal cells,respectively.VEGF-A expression in tumor cells(t-VEGF-A) was associated with advanced clinical stage(stage 0,1/9;stage 1,2/16;stage 2,32/55;stage 3,38/66;stage 4,16/19,P < 0.0001).VEGF-A expression in stromal cells(s-VEGF-A) increased in the earlier clinical stage(stage 0,7/9;stage 1,6/16;stage 2,33/55;stage 3,22/66;stage 4,5/19;P = 0.004).Multivariate analyses for risk factors of recurrence showed that only s-VEGF-A expression was an independent risk factor for recurrence(relative risk 0.309,95% confidence interval 0.141-0.676,P = 0.0033).The five-year disease-free survival(DFS) rates of t-VEGF-A-positive and-negative cases were 51.4% and 62.9%,respectively.There was no significant difference in t-VEGF-A expression status.The five-year DFS rates of s-VEGF-A-positive and-negative cases were 73.8% and 39.9%,respectively.s-VEGFA-positive cases had significantly better survival than s-VEGF-A-negative cases(P = 0.0005).Splice variant analysis revealed that t-VEGF-A was mainly composed of VEGF165 and that s-VEGF-A included both VEGF165 and VEGF165b.In cases with no venous invasion(v0),the level of VEGF165b mRNA was significantly higher(v0 204.5 ± 122.7,v1 32.5 ± 36.7,v2 2.1 ± 1.7,P = 0.03).The microvessel density tended to be lower in cases with higher VEGF165b mRNA levels.CONCLUSION:s-VEGF-A appears be a good prognostic factor for colorectal cancer and includes VEGF165 and VEGF165b.

Laser capture microdissection and genetic analysis of carbon-labeled Kupffer cells

AIM： To develop a method of labeling and microdissecting mouse Kupffer cells within an extraordinarily short period of time using laser capture microdissection （LCM）. METHODS： Tissues are complex structures comprised of a heterogeneous population of interconnected cells. LCM offers a method of isolating a single cell type from specific regions of a tissue section. LCM is an essential approach used in conjunction with molecular analysis to study the functional interaction of cells in their native tissue environment. The process of labeling and acquiring cells by LCM prior to mRNA isolation can be elaborate, thereby subjecting the RNA to considerable degradation. Kupffer cell labeling is achieved by injecting India ink intravenously, thus circumventing the need for in vitro staining. The significance of this novel approach was validated using a cholestatic liver injury model. RESULTS： mRNA extracted from the microdissected cell population displayed marked increases in colonystimulating factor-1 receptor and Kupffer cell receptor message expression, which demonstrated Kupffer cell enrichment. Gene expression by Kupffer ceils derived from bile-duct-ligated, versus sham-operated, mice was compared. Microarray analysis revealed a significant （2.5-fold, q value 〈 10） change in 493 genes. Based on this fold-change and a standardized PubMed search, 10 genes were identified that were relevant to the ability of Kupffer cells to suppress liver injury. CONCLUSION; The methodology outlined herein provides an approach to isolating high quality RNA from Kupffer cells, without altering the tissue integrity.