Beta-TC-6细胞的免疫组化及分子生物学属性分析

Immunohistochemical and molecular analysis of biological properties of Beta-TC-6 cells

目的探讨Beta-TC-6细胞免疫组化及分子生物学基本属性及基本鉴定指标。方法 (1)用针对小鼠细胞种属特异且敏感的cox-I基因引物,以Beta-TC-6细胞的基因组DNA为模板行PCR及后续琼脂糖凝胶电泳;(2)将Beta-TC-6细胞制备成细胞蜡块,用免疫组化S-P法检测细胞蜡块胰岛素标记物(INS)和神经内分泌细胞特异性分子标记物突触素(Syn)、嗜铬素(Cg A)及神经特异性烯醇化酶(NSE)的表达,并检测细胞增殖指标Ki67和P53。(3)用计算机图像分析技术测试活细胞面积及细胞蜡块中细胞的大小。结果 (1)cox-I基因引物经PCR扩增及琼脂糖凝胶电泳后在目的泳道上得到单一目的条带;(2)免疫组化检测:INS、Syn、Cg A、Ki67及P53均表达阳性,Ki67的阳性指数为99%,NSE表达呈阴性;(3)体外培养状态下活细胞的面积均值为696.59μm2,细胞蜡块切片中细胞及核的面积分别为60.54μm2、32.17μm2,核浆比1.32。结论 cox-I基因引物PCR扩增并联合免疫组化检测指标INS、Syn、Cg A、Ki67及P53可对BetaTC-6细胞属性进行甄别。

Objective To explore the basic immunohistochemical and molecular biological properties andbasic appraisal indicators of insulinoma beta-TC-6 cells. Methods（ 1） From reference,we outsourced one pair of species-specific primers targeted to genomic DNA of cultured mouse cells. The primer was specific and sensitive to cox-I. PCR amplification with the primers,and the genomic DNAs isolated from the tested cell line and agarose gel electrophoresis of the PCR products were conducted.（ 2） We prepared paraffin sections of the tested cell line to detect the expression of pancreatic neuroendocrine tumor specific molecular markers—Syn,NSE,Cg A,Insulin,Ki67 and P53 by S-P method of immunohistochemistry.（ 3） We determined the areas of alive cells and cells in the paraffin sections by image analysis.Results（ 1） The Cox-1 gene species-specific and highly sensitive primers were used,and a single target band was obtained in the target track by PCR amplification with these primers and agarose gel electrophoresis.（ 2） The immunohistochemical analysis detected positive expression of pancreatic neuroendocrine tumor specific molecular markers—Syn,Cg A,insulin,Ki67 and P53,with a 99% positive expression of Ki67,and negative expression of NSE.（ 3） The cell morphology test results of beta-TC-6 showed that the mean area of alive cells was 696. 59 μm2,the mean area of the cells and nuclei in the paraffin sections were 60. 54 and 32. 17 μm2,respectively,and the nuclaer-cytoplasmic ratio was 1. 32.Conclusions A PCR-based method of the cox-I gene amplification combined with immunohistochemical analysis of INS,Syn,Cg A,Ki67 and P53 can be used to identify the species and identity of beta-TC-6cells.