血友病A基因治疗的初步实验研究

Preliminary experimental research on gene therapy for hemophilia A

目的 制备血友病A的治疗基因 ,从invivo途径观察人凝血因子Ⅷ (hFⅧ )在动物体内的表达。方法 将B区缺失 (76 0～ 16 39位氨基酸 )的hFⅧcDNA克隆至真核细胞表达载体pRC/RSV ,与转染试剂DOTAP Cholesterolliposome混合 ,制备治疗基因pRC/RSV hFⅧBD DOTAP Cholesterol。肌肉注射给BALB/c小鼠后 ,在第 2 ,10 ,2 0 ,30 ,40 ,5 0天取小鼠的血液以及心、肝、脾、肺、肾、骨骼肌等组织。检测血浆中的hFⅧ抗原和抗体 ,用PCR和RT PCR方法检测hFⅧBDcDNA在小鼠各组织基因组中的分布及其转录情况 ,免疫组织化学染色检测各组织中hFⅧ的表达。结果 在注射结束后的第 48小时 ,小鼠血浆和组织中即有hFⅧ的表达 ,第 10天血浆中的hFⅧ抗原水平最高 ,达 17.5 5ng/ml,以后逐渐下降。血浆中的hFⅧ抗体在注射结束后的第 10天出现 ,以后缓慢升高 ,第 5 0天时达 37.0 6U/ml。PCR、RT PCR检测和免疫组织化学染色显示小鼠各组织中均有hFⅧBDcDNA的存在、转录和表达 ,但随着时间推移逐渐减少。脾、肺、肾脏中hFⅧBDcDNA的转录和表达时间均长于心脏、肝脏和骨骼肌。结论 由pRC/RSV hFⅧBD与DOTAP Cholesterolliposome结合而制备的治疗基因pRC/RSV hFⅧBD DOTAP Cholesterol能够通过invivo途径在动物体内很好地表达hFⅧ ,这为?

Objective To accomplish a kind of therapeutic gene for hemophilia A, and observe the expression of human factor Ⅷ (hFⅧ) in vivo. Methods Human clotting factor Ⅷ cDNA with B-domain deleted(△760aa～1639aa ) was inserted into vector pRC/RSV to form pRC/RSV-hFⅧBD, which conjugated with in vivo liposome transfection reagent (DOTAP-Cholesterol) to accomplish a kind of therapeutic gene, pRC/RSV-hFⅧBD-DOTAP-Cholesterol. Mice were injected with pRC/RSV-hFⅧBD-DOTAP-Cholesterol i.m. and sacrificed 48 hours, 10 days, 20 days, 30 days, 40 days and 50 days later, respectively. Tissues such as heart, liver, spleen, lung, kidney and muscle were harvested, the distribution and transcription as well as expression of hFⅧBD cDNA were detected by means of PCR, RT-PCR and immunohistochemistry techniques. In addition, the antigen and antibody of hFⅧ in plasma were measured. Results There was high expression of hFⅧ in plasma and tissues at the 48 th hour after injection. On day 10, antigen level of hFⅧ in plasma reached its peak,17.55?ng/ml, and gradually reduced later. The antibody of hFⅧ in plasma emerged on day 10 after injection, and increased and gradually reached 37 06 U/ml on day 50 after injection. PCR, RT-PCR and immunohistochemistry showed that hFⅧBD cDNA and its transcription as well as expression existed in all kinds of tissues, and lasted longer in spleen, lungs and kidneys than in heart, liver and muscle. Conclusion Therapeutic gene, pRC/RSV-hFⅧBD-DOTAP-Cholesterol, produced by combination of pRC/RSV-hFⅧBD and DOTAP-Cholesterol liposome can express human FⅧ successfully in vivo, which lays an experimental foundation for curing hemophilia A by gene-drug in clinic.