携带人胰岛素基因腺病毒载体转染人脐带间充质干细胞及其成脂分化

Adipogenic differentiation of human umbilical cord mesenchymal stem cells transfected by insulin gene-modified adenovirus vector

背景:胰岛素是成脂诱导剂的重要组成成分。然而胰岛素存在半衰期,会随着体内代谢不断灭活及降解,植入体内的细胞或材料无法像体外那样以更换培养液来达到目的。实验拟通过转染胰岛素基因,使转染后的干细胞稳定分泌胰岛素,促进其成脂分化。目的:探讨携带人胰岛素基因腺病毒载体转染人脐带间充质干细胞后对其成脂分化能力的影响。方法:取第3代人脐带间充质干细胞,以感染复数为20转染腺病毒载体。实验分为4组:对照组为第4代人脐带间充质干细胞;实验组1为腺病毒转染的第4代人脐带间充质干细胞;实验组2为第4代人脐带间充质干细胞+成脂诱导液;实验组3为腺病毒转染的第4代人脐带间充质干细胞+成脂诱导液。结果与结论:转染48h后实验组1和实验组3的人脐带间充质干细胞在荧光显微镜下显现弱荧光,72h后荧光较强。经脂肪诱导培养液培养14d后,实验组1,2,3油红O染色后显微镜下呈红色,对照组未见脂滴形成,实验组1可见一些细小脂滴形成;实验组3与实验组2相比,脂滴大且多,定量分析显示,实验组3油红O染色阳性的面积和吸光度大于实验组2(P<0.05)。由此说明携带人胰岛素基因腺病毒载体转染人脐带间充质干细胞后可促进其成脂能力。

BACKGROUND： Insulin is an important component of adipogenic inducers. Insulin has the half-life, which can continuously inactivate and degrade with the in vivo metabolism, and the cells and the materials implanted in the body cannot replace the culture medium to achieve the purpose like in in vitro environment. Transfection of insulin gene can make the transfected stem cells secret the insulin stably which can promote the adipogenic differentiation. OBJECTIVE： To investigate the adipogenic differentiation potential of human umbilical cord mesenchymal stem cells transfected by insulin gene-modified adenovirus vector. METHODS： Passage 4 human umbilical cord mesenchymal stem cells were collected and transfected with adenovirus vector with the multiplicity of infection of 20. The experiment was divided into four groups. Passage 4 human umbilical cord mesenchymal stern cells were included in the control group. Passage 4 human umbilical cord mesenchymal stem cells transfected with adenovirus vector were included in experimental group 1, passage 4 human umbilical cord mesenchymal stern cells ＋ adipogenic liquid were used in the experimental group 2, and passage 4 human umbilical cord mesenchymal stem cells transfected with adenovirus vector ＋ adipogenic liquid were used in experimental group 3. RESULTS AND CONCLUSION： After transfected for 48 hours, weak fluorescence in human umbilical cord mesenchymal stem cells was observed in experimental groups1 and 3 through the fluorescence microscope and strong fluorescence appeared after transfected for 72 hours After cultured with adipose-inducing culture medium for 14 days, the cells in the experimental groups 1,2, 3 were red under microscope after oil red O staining, no lipid droplets were observed in the control group; the lipid droplets in the experimental group 3 were larger and more than those in the experimental group 2. Quantitative analysis showed the positive area of oil red O staining and the absorbance value in the experimental group 3 were greater than those in the experimental group 2 （P 〈 0.05）. The results show that the human umbilical cord mesenchymal stem cells transfected by insulin gene-modified adenovirus vector can promote cell adipogenic differentiation.