电击法转移含人凝血因子IX基因重组质粒的影响因素

Effects on the transformation of plasmids carrying hFIX cDNA by electroporation

反转录病毒载体在基因治疗中可能会产生野生型病毒颗粒而引起安全性问题，本文研究含ＦＩＸｃＤＮＡ重组质粒基因治疗血友病Ｂ的可能，构建了不具反转录病毒载体结构的两重组质粒ｐＳＣＩＸＴＮ和ｐＣＩＸＴＮ，前者合ＳＶ４０早期启动子和ｈＣＭＶ启动子共同控制的ＦＩＸｃＤＮＡ，后者仅含ｈＣＭＶ启动于控制的ＦＩＸｃＤＮＡ，它们都含有ＴＫ启动于驱动的ｎｅｏ基因，通过电击法将基因转移到ＰＡ３１７和ＨＴ１０８０细胞，在ＨＴ１０８０细胞中的ＦＩＸ表达量分别为２２０、２１２ｎｇ／（１０６细胞·２４ｈ）通过与ｐＣＭＶＩＸ共转染靶细胞，可以增加人ＩＸ因子表达１．５～３．５倍，显示了用质粒载体转染靶细胞在血友病Ｂ基因治疗中是一条潜在可行的途径．本项研究用自制的电击仪转移ＰＡ３１７和ＨＴ１０８０等细胞，最高的转移效率达１０－３，并探讨了细胞种类，ＤＮＡ量，ＤＮＡ结构，电压，脉冲时间与转移效率及表达量的关系．

The safety of retroviral mediated transfer system in gene therapy should be seriously considered in order to exclude completely the probable production of the recombinant wild type retrovirus. The feasibility of recombinant plasmids carrying hFIX cDNA in the gene therapy of hemophilia B was studied. Two plasmids,pSCIXTN and pCIXTN,were constructed,the former carrying hFIX cDNA driven by SV40 early promoter and hCMV promoter,and the later carrying hFIX cDNA driven only by hCMV promoter. Both of them were equipped with the neo gene driven by TK promoter as selective gene. These two plasmids were transformed to PA317 and HT1080 cells by electroporation and expressed in HT cells on levels of 220 and 212ng (106cells·24h),respectively. When co-transformed with pCMVIX,the expression level can further be increased by 1. 5-3. 5 times. These results suggest the feasibility of the present plasmid system. Meanwhile,this study use the handmade electroporation apparatus to explore the effects of cell types, DNA concentration,DNA structure, voltage,pulse time,and etc.on transfer efficiency. The maximum transfer efficiency reached is 1 ×10-3 in the system.