胰岛素基因转染的人脐带间充质干细胞与丝素蛋白支架构建组织工程脂肪的研究

Construction of tissue engineering adipose with human umbilical cord mesenchymal stem cells transfected by insulin gene and silk fibroin scaffold in vitro

目的探讨携带重组人胰岛素基因慢病毒载体转染的人脐带间充质干细细（human um—bilical cord mesenchymal stem cells．hUCMSCs）与丝素蛋白支架在体外构建组织工程化脂肪（tissue en—gineering adipose）的可行性。方法以最适感染复数（MOI）为10重组慢病毒pLenti6．3-insulin-IRES-EGFP转染hUCMSCs组为实验组（A组），EGFP基因转染组为对照组（B组）；将两组细胞接种于丝素蛋白支架．观察细胞在支架上的生长及黏附情况；尔后行细胞一支架复合物成脂诱导。四甲基偶氮唑蓝（MTT）法检测蓖组慢病毒转染对hUCMSCs生长增殖的影响以及基因转染后的hUCMSCs在丝素蛋白支架上的活性。结果细胞支架复合物成脂诱导5～7d后，见A组支架内脂肪样细胞数量明显多于B组．差异有显著统计学意义（P=0．007，P〈0．01）；逆转录-聚合酶链式反应（RT-PCR）检测显示，A组hUCMSCs表达的成脂特异性基因PPARγ-2明显高于B组。MTT法检测结果显示，转染重组慢病毒的hUCMSCs与末转染组各刚‘问点吸光度A值比较，差异均无统计学意义（P=0．056，P〉0．05）。细胞组与细胞支架组A值比较．差异无统计学意义（P=0．066。P〉0．05）。结论转染胰岛素基因的hUCMSCs成脂分化能力明显提高，且能有效地与丝素蛋白支架在体外构建组织工程化脂肪。

Objective To study the construction of tissue engineering adipose in vitro with silk fibroin scaffold and human umbilical cord mesenchymal stem cells （hUCMSCs） transfected by recom-binant human insulin gene lentivirus. Methods hUCMSCs infected with recombinant lentiviral pLen-ti6.3-insulin IRES EGFP （Group A） by the best MOI=10 were seeded in silk fibroin scaffold; hUC-MSCs transfected by EGFP gene （Group B）was regarded as negative control; and then the cell-scaf-fold complexes were cultured in adipogenic differentiation medium. MTT test was used to detect whether recombinant lentiviral affected the growth and proliferation of hUCMSCs and the growth ac-tivity of hUCMSCs seeded in silk fibroin. Results After 5-7 days for adipogenic culture, the numbers of fat-like cells in group A were significantly more than those in group B （P =0. 007,P〈0. 01）. RT-PCR results showed that the expression of PPARγ-2 in group A was much stronger than that in group B. MTT test showed that there was no significant difference in optical density （A）at each time be- tween transfected group and nontransfected group （P=0. 056,P〉0.05）. And there was also no sig-nificant difference in optical density （A）between cell group and cell-scaffold group （P=0. 066, P〉 0.05）. Conclusions Insulin gene could obviously promote hUCMSCs getting into adipose, and carry-ing recombinant human insulin gene lentiviral vector transfection of hUCMSCs and silk fibroin seaf-folds could effectively construct tissue engineering adipose in vitro.