Neuzil D, Holzman M, Rozga J, Levine J, Moscioni A D, Demetriou A A
Department of Surgery, Vanderbilt University School of Medicine, Nashville, Tennessee 37203.
J Surg Res. 1993 Jun;54(6):631-7. doi: 10.1006/jsre.1993.1097.
The purpose of this study was to develop a method of transduction of normal skin fibroblasts with a retroviral vector expressing the human factor IX (hF-IX) gene and transplantation of the transduced cells into rats. A retroviral vector containing the hF-IX gene, as well as a selectable marker (hygromycin B resistance; HB), was used to transduce cultured normal skin (rat and human) fibroblasts. Transduced fibroblasts were selected with HB; resistant clones were propagated and assessed for expression of active hF-IX in vitro. Transduced cells secreted significant amounts of hF-IX in vitro (327.9 +/- 14.2 ng/10(6) cells) which showed measurable clotting activity, indicating terminal processing of the gene-transcribed hF-IX product by the transduced fibroblasts. Integration of the proviral gene into rat and human fibroblast genomic DNA was confirmed by polymerase chain reaction. Transduced rat fibroblasts were attached to collagen-coated microcarriers and transplanted intraperitoneally into syngeneic recipients. Transplanted microcarrier-attached cells formed aggregates in the peritoneal cavity and exhibited positive immunohistochemical staining for hF-IX up to 8 weeks following transplantation, the time of termination of the experiment. These experimental results may have significant implications in developing strategies for future clinical therapy for hemophilia B.
本研究的目的是开发一种用表达人凝血因子IX（hF-IX）基因的逆转录病毒载体转导正常皮肤成纤维细胞并将转导后的细胞移植到大鼠体内的方法。使用含有hF-IX基因以及一个选择标记（潮霉素B抗性；HB）的逆转录病毒载体转导培养的正常皮肤（大鼠和人）成纤维细胞。用HB筛选转导后的成纤维细胞；扩增抗性克隆并在体外评估活性hF-IX的表达。转导后的细胞在体外分泌大量的hF-IX（327.9±14.2 ng/10⁶个细胞），其显示出可测量的凝血活性，表明转导后的成纤维细胞对基因转录的hF-IX产物进行了终末加工。通过聚合酶链反应证实了前病毒基因整合到大鼠和人成纤维细胞基因组DNA中。将转导后的大鼠成纤维细胞附着于胶原包被的微载体上，并经腹腔移植到同基因受体中。移植的附着于微载体的细胞在腹腔内形成聚集体，并且在移植后长达8周（实验结束时）均表现出hF-IX的阳性免疫组织化学染色。这些实验结果可能对制定未来B型血友病的临床治疗策略具有重要意义。
