Fn、TPO基因修饰对人骨髓间充质干细胞的影响

Effect of fibronectin-thrombopoietin gene modification on human bone marrow mesenchymal stem cells

目的观察纤维连接蛋白(Fn)、血小板生成素(TPO)融合基因修饰对人骨髓间充质干细胞(MSC)的影响。方法构建携带 Fn-TPO 融合基因的重组逆转录病毒载体,并以其对骨髓 MSC进行基因修饰;观察 Fn-TPO 基因在骨髓 MSC 中的表达,以及基因修饰后骨髓 MSC 的体外增殖、黏附造血细胞和分泌 TPO 的能力;并将脐血 CD34^+细胞接种到基因修饰后骨髓 MSC 形成的滋养层,培养7d 观察修饰后骨髓 MSC 对造血细胞体外扩增和集落形成能力的影响。结果成功构建携带 Fn-TPO基因的重组逆转录病毒载体且以该逆转录病毒载体对骨髓 MSC 进行体外基因修饰;Fn、TPO 基因在骨髓 MSC 内能够正常转录;基凶修饰后的骨髓 MSC 体外增殖能力[(6.92±0.77)×10~4/ml]与对照组[(7.18±0.89)×10~4/ml]比较差异无统计学意义(P>0.05);基冈修饰组和对照组黏附造血细胞能力分别为0.188±0.018和0.167±0.017(P<0.01),分泌 TPO 能力分别为(7.46±0.59)ng/ml 和(5.58±0.37)ng/ml(P<0.01),细胞分泌 TPO 的能力不受培养时间的影响,但受细胞生长状态影响;2×10~4脐血 CD34^+造血干/祖细胞经基因修饰后骨髓 MSC 联合必要细胞因子体外扩增7 d,有核细胞数、CD34^+细胞比例、BFU-E、CFU-GM 及 CFU-GEMM 分别为(29.9±2.7)×10~4、(33.3±2.8)%、109.3±4.1/1×10~4 CD34^+细胞、163.7±7.1/1×10~4 CD34^+细胞、13.3±1.5/1×10~4 CD34^+细胞,较对照组明显增加(P<0.01)。结论 Fn-TPO 基因修饰能够增强骨髓 MSC 黏附造血细胞、分泌 TPO 及支持脐血 CD34^+细胞扩增的能力。

Objective To observe the effect of Fn-TPO gene modification on human bone marrow mesenchymal stem cells （MSCs）. Methods Retroviral vector containing Fn-TPO gene was constructed and bone marrow MSCs was modified by this vector. The transcription of Fn-TPO gene in MSCs was observed. The proliferation capacities, hematopoietic cells adhering capacities and TPO secretion capacities of gene modified MSCs were assayed respectively. Cord blood CD34^＋ cells were seeded on the gene modified MSCs layers and several essential growth factors were added. After co-culturing in vitro for 7 days, the number of CD34 ^＋ cells and their colony forming capacities were assayed by flow cytometry and semisolid culture assay. Results Retroviral vector containing Fn-TPO gene was successfully constructed and bone marrow MSCs were modified by this vector. Fn-TPO gene was expressed by bone marrow MSCs after gene modification. The viability of MSCs had no significant difference between pre- and post-gene-modification [ （ 7. 18 ± 0. 89 ） 10^4/ml vs （6.92 ± 0.77 ） 10^4/ml, P 〉 0.05 ]. The hematopoietic cells adhering ability of gene modified bone marrow MSCs was reinforced（0. 188 ±0.018 vs 0. 167 ±0.017, P 〈0.01 ）. The concentration of TPO in the MSCs culture supernatant raised from （5.58 ±0.37） ng/ml to （7.46 ±0.59） ng/ml （P〈0.01） and did not significantly decline in a short-time peroid, but influenced by the growth status of MSCs. After co-culturing with gene modified MSCs for 7 days, the absolute number of nucleted cells, the percentage of CD34^＋ cells and the colony numbers of BFU-E, CFU-GM, CFU-GEMM were （29.9± 2.7 ） × 10^4, （33.3 ± 2.8） % , 109.3± 4.1, 163.7 ± 7.1, 13.3 ± 1.5, respectively, being significantly higher than that co-cultured with non-modifled MSCs. Conclusions Fn-TPO gene modification can improve the capacity of human bone marrow MSCs for hematopoietic cells adhering, TPO secretion and cord blood CD34^＋ cells amplification.