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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 fibronectin (Fn) and thrombopoietin (TPO) fusion gene on human bone marrow mesenchymal stem cells (MSCs). Methods The recombinant retroviral vector carrying Fn-TPO fusion gene was constructed and its gene was modified on bone marrow MSCs. The expression of Fn-TPO gene in bone marrow MSCs was observed, and the proliferation of bone marrow MSCs after gene modification was observed. Adhesion of hematopoietic cells And to secrete TPO. Cord blood CD34 ~ + cells were inoculated into the trophoblast layer formed by genetically modified bone marrow MSCs, and cultured for 7 days to observe the effect of modified bone marrow MSCs on the proliferation and colony formation ability of hematopoietic cells in vitro. Results The recombinant retroviral vector carrying Fn-TPO gene was successfully constructed and the in vitro gene modification of bone marrow MSCs was performed with this retrovirus vector. The Fn and TPO genes were able to be transcribed normally in bone marrow MSCs. (6.92 ± 0.77) × 10 ~ 4 / ml] compared with the control group [(7.18 ± 0.89) × 10 ~ 4 / ml]. There was no significant difference between the Keegan modified group and the control group The ability of hematopoietic cells were 0.188 ± 0.018 and 0.167 ± 0.017 respectively (P <0.01), and the ability of secreting TPO was (7.46 ± 0.59) ng / ml and (5.58 ± 0.37) ng / ml respectively Ability was not affected by the culture time, but affected by the cell growth status; 2 × 10 ~ 4 cord blood CD34 ~ + hematopoietic stem / progenitor cells were genetically modified bone marrow MSC combined with the necessary cytokines in vitro expansion 7 d, the number of nucleated cells (29.9 ± 2.7) × 10 ~ 4, (33.3 ± 2.8)%, 109.3 ± 4.1 / 1 × 10 ~ 4 CD34 ~ + cells , 163.7 ± 7.1 / 1 × 10 ~ 4 CD34 ~ + cells, 13.3 ± 1.5 / 1 × 10 ~ 4 CD34 ~ + cells, which were significantly increased compared with the control group (P <0.01). Conclusion Fn-TPO gene modification can enhance the adherence of bone marrow MSC to hematopoietic cells, secrete TPO and support the expansion of cord blood CD34 ~ + cells.