p53、CDK4基因引物扩增的cDNA-Biotin探针与4株克隆瘤细胞原位杂交  

IN SITU HYBRIDIZATION OF CLONED TUMOR CELLS WITH cDNA-BIOTIN PROBES AMPLIFIED BY p53,CDK4 PRIMERS

作　　者：牛彦平[1] 王秀芳[2] 徐增年[2] 吕占军[2] 

机构地区：[1]河北医科大学第二医院康复科,河北石家庄050000 [2]河北医科大学实验动物学部,河北石家庄050017

出　　处：《河北医科大学学报》2004年第2期65-67,90,共4页Journal of Hebei Medical University

基　　金：国家"九五"攻关课题 ( 96 A2 3 0 6 0 3 )

摘　　要：目的用p5 3、CDK4基因引物扩增的生物素标记的互补DNA(cDNA Biotin)探针 ,检测S2D9、H2D8、E2G8及L3E1 1克隆瘤细胞株中 p5 3、CDK4相关基因表达的差异。方法提取S2D9、H2D8、E2G8及L3E1 1克隆细胞总RNA ,分别用p5 3、CDK4基因引物经反转录聚合酶链反应 (reversetranscriptionpolymerasechainreaction ,RT PCR)扩增。扩增产物用 6 %聚丙烯酰胺凝胶电泳 ,经银染显色后 ,切下 4个克隆细胞间有差异的条带 ,回收DNA再次扩增 ,扩增产物经纯化后用生物素标记 ,制成生物素标记的cDNA探针 ,再与 4株克隆瘤细胞涂片 ,做探针原位杂交。结果 4个克隆瘤细胞株的RNA与 2种引物RT PCR后 ,获得 2 2条可以进行克隆的cDNA ,取其中 1 1条经生物素标记成cDNA探针。这些探针多数与不同瘤细胞株杂交显示较明显差异。结论这些自制的cDNAObjectiveThe cDNA Biotin probes amplified by p53, CDK4 gene primers were used to examine the expressing differences of p53,CDK4 related genes in S2D9, H2D8,E2G8 and L3E11 cloned tumor cells. MethodsThe total RNAs, extracted from S2D9, H2D8, E2G8 and L3E11 cloned tumor cells respectively, were amplified by RT PCR using p53, CDK4 gene primers. After running on 6% polyacrylamide gel, the DNA bands on gel were displayed by silver stain method. The different displayed DNA bands were visually recovered from the polyacrylamide gel and reamplified by PCR. The reamplified products were purified and labeled by biotin (cDNA Biotin probes). The hybridization in situ to the four cloned tumor cells was done with the cDNA Biotin probes. ResultsTwenty two cDNAs were acquired after amplifying with RT PCR. After labeled by biotin, 11 of the 22 cDNAs were made into cDNA probes. Distinct differences were found among S2D9, H2D8, E2G8 and L3E11 cloned tumor cells when examining these cells with these cDNA Biotin probes.ConclusionThese homemade cDNA Biotin probes can be used as reagents to control cloned tumor cells' quality.

关 键 词：克隆细胞 分子探针 原位杂交 

分 类 号：Q344.13[生物学—遗传学]