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目的 寻找人绒毛膜上皮癌 (JAR)与孕早期绒毛组织间的差异基因。 方法 采用 m RNA差异显示法 (m RNA differential display PCR,DD- PCR) ,即分别提取正常孕早期绒毛及绒毛膜上皮癌的总 RNA ,在oligod T1 5 作用下分别合成相应的 c DNA ,并以其为模板利用试剂盒中的 2 0对引物组合和α- 32 Pd ATP,Taq酶等进行PCR,将两者的 PCR产物在 6 %变性聚丙烯酰胺凝胶 (PAG)上电泳 ,放射自显影后寻找两者间的差异片段 ,回收差异片段进行二次 PCR,对二次 PCR产物先采用反杂交法初步筛选后再用 Northern Blot鉴定。 结果 从 PAG上切下 32条差异条带 ,经反杂交初步筛选出 11个阳性片段 ,对 11个阳性片段分别做 Northern Blot鉴定 ,其中 1个片段(T2 6 )的基因长度约 1.2 kb,其在绒毛膜上皮癌中的表达远高于正常绒毛组织。 结论 T2 6基因片段可能是绒癌相关基因
Objective To search for the difference genes between human choriocarcinoma (JAR) and first trimester chorionic villi. Methods The total RNA of normal early-stage chorionic villi and choriocarcinoma was extracted by m RNA differential display PCR (DD-PCR). The corresponding cDNAs were synthesized by oligod T1 5, It was used as a template to perform PCR using 20 primer pairs and α-32 Pd ATP and Taq enzyme in the kit. The PCR products of both were electrophoresed on a 6% denatured polyacrylamide gel (PAG) After looking for the difference between the two fragments, the recovery of the difference fragments for the second PCR, the second PCR products were first used by the anti-hybrid method of screening and then Northern Blot identification. Results Thirty-two differentially expressed bands were excised from PAG, and 11 positive clones were screened by reverse hybridization. Northern Blot was performed on 11 positive clones. The length of one fragment (T2 6) was 1.2 kb, which was In choriocarcinoma, the expression is much higher than normal villi. Conclusion T2 6 gene fragment may be choriocarcinoma related genes