含CTLA4Ig基因的逆转录病毒感染人骨髓间充质干细胞的实验研究

An Experimental Study of Retroviral Vector Containing Gene of CTLA4Ig Infecting Human Mesenchymal Stem Cells

目的制备含CTLA4Ig基因的逆转录病毒包装细胞并感染人骨髓间充质干细胞(MSCs),为后续实验作准备。方法采用Percoll密度梯度离心分离人骨髓MSCs。用含有CTLA4Ig基因的逆病毒感染第1代人骨髓MSCs,应用G418筛选出抗性细胞群(MSCs-C),应用RT-PCR检测其CTLA4IgmRNA表达,免疫细胞化学检测其CTLA4Ig蛋白质表达。结果Percoll(1.073g/mL)分离培养的第3代MSCs94.59%CD105表达阳性,97.58%CD34表达阴性。转染细胞(MSCs-C)RT-PCR证明CTLA4Ig基因被成功导入MSCs中并转录;免疫细胞化学结果显示绝大多数MSCs-C细胞表达CTLA4Ig蛋白,其表达于细胞浆中。结论CTLA4Ig基因可以通过逆转录病毒成功导入人骨髓MSCs中,这种基因修饰的细胞可以表达目的基因及目的蛋白,可能通过目的蛋白的分泌达到降低免疫原性和增强免疫抑制的作用。

Objective To transduce the human cytotoxic T lymphocyte associated antigen 4-Ig gene （CTLA4Ig） into human mesenchymal stem cells （MSCs） for further study. Methods MSCs were isolated from bone marrow of the iliac crest of voluntary normal adults by a modificative procedure of Pittenger. Culture-expanded MSCs at passage one were transduced by a retroviral vector containing the gene of CTLA4Ig and screened by G418. Then CTLA4Ig mRNA （by RT-PCR） and protein expression （by immunocytochemistry and western- blotting） of MSCs-C were inspected. Results The isolated cultured MSCs were CD105 positive and CD34 negative （94. 6% and 97.6% respectively at passages 3） by flow cytometric analysis of expressed surface antigens. MSCs-C expressed CTLA4Ig mRNA and CTLA4Ig protein. Conclusion CTLA4Ig gene is transduced in MSCs successfully. Transduced cells express CTLA4Ig mRNA and CTLA4Ig protein. The target protein maybe depress their immunogenicity and increase immune suppression.