TNFR Ⅰ-IgGFc融合基因重组腺病毒表达载体的构建及其在骨髓基质干细胞的表达

Construction of adenoviral vector encoding human TNFR Ⅰ-IgGFc cDNA and its expression in bone marrow stromal stem cells

目的构建携带人肿瘤坏死因子受体Ⅰ-胞外区和IgGFc融合基因的重组腺病毒载体（Ad—TNFR Ⅰ-IgGFc），体外转染兔骨髓基质干细胞（BMSCs），探讨转TNFR Ⅰ-IgGFc基因的BMSCs治疗类风湿关节炎的可行性。方法将TNFR Ⅰ-I如Fc基因插入腺病毒穿梭质粒pDC316，与辅助质粒pBHGlox△E1，3Cre共转染HEK293细胞，重组产生Ad—TNFR Ⅰ-IgGFc，PCR鉴定毒种正确后，进行扩增、纯化和滴度测定，转染培养至第2代BMSCs，利用RT—PCR、原位杂交、免疫组化方法，检测转染后细胞中TNFR Ⅰ-IgGFc的转录和表达。结果成功构建了Ad-TNFR Ⅰ-IgGFc，感染性滴度达3×10^10 TCID50／mL，转染后BMSCs在mRNA和蛋白水平均有TNFR Ⅰ-IgGFc表达。结论构建的Ad—TNFR Ⅰ-IgGFc可有效转染BMSCs，并在其中高效表达，为将表达TNFR Ⅰ-IgGFc基因的BMSCs用于类风湿关节炎治疗的实验研究提供了基础。

Objective To study the feasibility of local treatment of rheumatoid arthritis （RA） with bone marrow stromal stem cells （BMSCs） which are transfected with recombinant adenovirus vectors and express the extra-cellular region of TNF receptor I-IgGFc （TNFR Ⅰ-IgGFc） fusion protein. Methods TNFR Ⅰ-IgGFc gene was subcloned into the adenovirus shuttle plasmid pDC316. The products were co-transfected into HEK293 cell line with helper plasmid pBHGloxAE1, 3Cre. The recombinant adenovirus （Ad-TNFR Ⅰ-IgGFc） was produced by homologous recombination of the above 2 plasmids in HEK293 cells. After identification with PCR, Ad-TNFR Ⅰ-IgGFc was amplified and purified; its titer was measured by TCID50 assay. The transcription and expression of TNFR Ⅰ-IgGFe gene in the transfeeted BMSCs were detected by RT-PCR, in situ hybridization and immunological histoehemistry. Results Ad-TNFR Ⅰ-IgGFe was successfully constructed with the titer of 3 ×10^10 TCID 50/mL. The expression of TNFR Ⅰ-IgGFc mRNA and protein was confirmed in the transfected BMSCs. Conclusions Ad-TNFR Ⅰ-IgGFc can transfect BMSCs and express TNFR Ⅰ-IgGFc protein with high efficacy. Local treatment of RA is possible with BMSCs expressing TNFR Ⅰ-IgGFc.