慢病毒颗粒包装、浓缩及对脐带血CD34^＋细胞感染

目的：建立慢病毒包装、浓缩及感染脐带血CD34＋细胞的条件方法。方法：采用第3代慢病毒系统包装病毒上清,结合超滤和超速离心两种方法对病毒进行浓缩。联合应用体外扩增培养,促进静止期细胞进入细胞周期、感染过程中促进细胞黏附和固定及重复感染等方法促进病毒感染。结果：CD34＋细胞体外培养48h,细胞表面CD34标志物表达水平没有明显改变,通过两步法浓缩后,病毒滴度可达5.06×10^7/ml,对脐带血CD34＋细胞的感染效率可达37.7%。结论：通过上述方法可以获得滴度为107/ml以上的慢病毒上清,实现对脐带血CD34＋细胞的有效感染。

Lentivirus packaging, concentration and infection of CD34~+ cells from umbilical blood

Objective:To methodologically establish the lentivirus granule packaging, concentration and infection against CD34~+ cells from umbilical blood. Methods:The lentivirus system of the 3~(rd) generation was used to produce the virus. Ultrafiltration and ultracentrifugation were employed to concentrate virus. Several treatments were used to improve virus infection including in vitro amplification culture, facilitation of rest cells into cell cycle, promotion of cell adhesion and immobilization during infection, and repeat infection methods. Results:CD34~+ cells were not obviously changed by checking the expression level of CD34 marker on the cell surface after 48 h culture. After two-step concentration, virus titer was increased up to 5.06×10~7/ml, and the infection rate against CD34~+ cells from umbilical blood was increased up to 37.7%.Conclusion:Lentivirus supernatant with over 10~7/ml titer can be obtained using the above methods. Efficient infection against CD34~+ cells from umbilical blood can be achieved.