The preparation of ovarian cancer gene therapy model by the TGF β type I receptor gene

将TGF β RIwt基因插入pAxCAwt载体中，利用体外包装试剂盒GIGApacIII在大肠杆菌中扩增，并与DNA- tpc共转染到HEK293细胞中。96份样品中有18份获得第一份病毒溶液。另外，对HEC293细胞进行一次病毒感染，得到5/18个样本的二次病毒液。证实这5种病毒溶液均不同时破坏HeLa细胞。用限制性内切酶分析证实了病毒液与野生型病毒的混种，用PCR分析证实了插入物的正确性。最后，用上述方法得到了第三种病毒溶液。采用不表达TGF β RI的人卵巢癌细胞株进行感染和生长抑制分析。观察4组细胞增殖，A；pAxCAwt-TGF β RI (+), TGF β (+)， B；pAxCAwt-TGF β RI (+), TGF β (-)， C；pAxCAwt-TGF β RI（-）、TGF β(+)、D；空盘。12 D时，A组和D组的细胞数量有明显差异。另一方面，B组和c组均有一定的细胞增殖抑制作用，这可能与pAxCAwt-TGF β RI载体在大量基因表达条件下对TGF β功能的抑制作用和对非特异性蛋白合成的抑制机制有关。虽然体内实验需要越来越多地制备高滴度的病毒溶液，但我们的研究怀疑利用腺病毒引入TGF β RI基因，制作突变替代治疗卵巢癌的基因治疗模型的可能性。

TGF β RIwt gene was inserted in pAxCAwt vector, and it was amplified in E.coli using in vitro packaging kit GIGApacIII , and the cosmid DNA was co-trasfected with DNA-TPC into the HEK293 cells. The first virus soluton were obtaind 18/96 samples. In addition, the secondary virus solution were obtaind 5/18 samples by first virus infection to HEC293 cells. These 5 virus solution were confirmed that they did not destory HeLa cell, simultaneously. Also these virus solution were confirmed that the interminglement of the wild type virus was denied by using restriction enzyme analysis, and that the insert was rightly came with PCR analysis. Finary, the third virus solutions were obtaind also abouve methods. Human ovarian cancer cell lines without expressing TGF β RI were used the infection and growth suppression analysis. Cell proliferation was observed at 4 groups, A ; pAxCAwt-TGF β RI (+) , TGF β (+), B ; pAxCAwt-TGF β RI (+) , TGF β (-), C ; pAxCAwt-TGF β RI(-), TGF β (+), and D ; null dish. The clear difference between group A and D was observed in cell numbers at 12 days. On the other hands, somewhat cell proliferation suppression effect was observed in Group B and C. These were explaind by the suppression effect of TGF β function thourgh the way without TGF β RI, and non-specific protein synthesis suppresion mechanism in the condition of massive gene expression by pAxCAwt-TGF β RI vectors, respectively. Though virus solution with high titer had to be prepared more and more for the experiment of in vivo studys, our research suspected the possiblity to make a gene therapy model of ovarian cancer with mutation-replacement therapy by the TGF β RI gene introduction using adenovirus.