原位逆转录PCR间接法检测人大肠癌组织和细胞株中CD44V6 mRNA的应用研究

Applied study of the detection of CD44V 6mRNA expression in human colorectal carcinoma and cell lines by indirect in situ RT PCR

目的探讨用原位逆转录ＰＣＲ间接法检测人类大肠癌组织及大肠癌细胞株内ＣＤ４４Ｖ６ｍＲＮＡ的表达。方法在大肠癌组织冰冻切片及ＨＴ２９和ＬｏＶｏ大肠癌细胞爬片上，先作蛋白酶Ｋ及ＤＮａｓｅ消化处理，再行逆转录、原位ＰＣＲ扩增和扩增后原位杂交检测，分析扩增产物与Ｖ６探针结合显色情况。结果ＬｏＶｏ细胞较ＨＴ２９细胞显色快而着色深，大肠癌癌灶较癌旁组织及远端正常大肠粘膜组织显色速度及强度均有显著性差异；反应液内有特异的ｃＤＮＡ扩增产物渗出；自制的原位ＰＣＲ模具性能稳定，封闭和防渗漏效果好。结论用原位逆转录ＰＣＲ间接法检测大肠癌组织及大肠癌细胞株内ＣＤ４４Ｖ６ｍＲＮＡ的表达，特异性强，定位性好，显色效果达到了光镜下分析的要求；反应液内的扩增产物还可作Ｓｏｕｔｈｅｒｎ杂交及基因分析；自制的原位ＰＣＲ模具经济、方便。

Objective To detect and localize the expression of CD44V 6 mRNA by indirect in situ RT PCR in human colorectal carcinoma and cell lines.Methods On the frozen tissue sections of human colorectal carcinoma and the slides which were paved with cell lines,the tissue and cells were digested with proteinase K and DNase,after the in situ reverse transcription and in situ PCR reactions,the amplification products in cell were detected with probe of CD44V 6mRNA by in situ hybridization.Results The blue purple′s staining of CD44V 6mRNA in the LoVo cells appeared denser and more rapid than that in the HT29 cells,which was specifically localized in cytoplasm rather than in cell nucleus.The staining, of CD44V 6mRNA tissue was denser in human colorectal carcinoma than in the normal colorectal mucosa,and there were some amplification produts in the supernatant too;The self seal glue was effective in protecting slides from drying out during the cycling process.Conclusion It is sccessful in specificity and localization to dectect the expression of CD44V 6mRNA in human colorectal carcinoma and cell lines by indirect in situ RT PCR,and the signal can be analyzed under the microscope.The amplification products in the supernatant can be investigated by Southern blot and other analysis.The self seal glue made in our laboratory is easy and economical to use and is effective for a wide variety of applications.