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本文报道用构建有人凝血因子Ⅸ cDNA的重组质粒(pCMV Ⅸ)或重组反转录病毒(XLⅨ和N2CMVⅨ)转染家兔原代培养的皮肤成纤维细胞(RSF),经过选择后将转有人Ⅸ因子cDNA的细胞包埋于胶原基质,然后进行自体或同种异体移植。腹腔移植有RSF-XLⅨ转化细胞的家兔血浆中人Ⅸ因子表达水平可达100ng/ml,表达持续5个半月;腹腔移植有RSF-pCMVⅨ细胞的家兔血浆中人Ⅸ因子水平可高达2.95ng/ml,表达至今已超过10个月,而且仍在继续检测中。在移植方法上,我们做了进一步改进,将收缩后的细胞胶原块移植改为细胞胶原液注射,使移植方法更简便有效,无需进行手术,用此法皮下移植有RSF-N2CMVⅨ转化细胞的家兔血浆中,人Ⅸ因子表达水平可高达480ng/ml,表达至今已有3个多月,其持续表达的趋势仍很乐观,为基因治疗的临床试验提供了一条更加切实可行的技术路线。
Herein, primary cultured human dermal fibroblasts (RSF) were transfected with recombinant plasmids (pCMV Ⅸ) or recombinant retroviruses (XLIX and N2CMV Ⅸ) constructed with human coagulation factor Ⅸ cDNA. After selective selection, human Ⅸ Cells of Factor cDNA are embedded in a collagen matrix and then autologous or allograft. The expression of human factor Ⅸ in rabbit plasma with RSF-XLⅨ transformed cells was up to 100ng / ml for 5 and a half months. The level of human factor Ⅸ in rabbit plasma with RSF-pCMVⅨ cells transplanted into abdominal cavity was up to 2.95ng / ml, the expression has been more than 10 months, and is still continuing testing. In the transplantation method, we made further improvements, the contraction of the cell collagen block transplantation into the cell collagen injection, the transplant method is more simple and effective, without surgery, with this method subcutaneous transplantation of RSF-N2CMV Ⅸ transformed cells In rabbit plasma, the expression level of human factor Ⅸ can be as high as 480 ng / ml, and the expression has been more than three months now. The trend of sustained expression is still optimistic, which provides a more feasible technical route for gene therapy clinical trials.