透明质酸水凝胶包封对携带BMP-2基因的腺病毒转染骨髓间充质干细胞增殖活性及其表达的影响

Bone marrow-derived mesenchymal stem cells transfected with adenovirus vector containing bone morphogenetic protein 2 encapsulated by hyaluronic acid hydrogel and its effect on proliferation and expression of target protein

目的:探讨携带骨形态发生蛋白2(bone morphogenetic protein 2,BMP-2)基因的腺病毒转染骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)后应用透明质酸水凝胶(hyaluronic acid hydrogel,HAH)包封对BMSCs增殖能力及其表达目标蛋白BMP-2的影响。方法:体外分离、提取、鉴定BMSCs,并将提前构建好的Ad-BMP-2转染BMSCs并使用HAH进行包封,激光共聚焦显微镜下观察Ad-BMP-2转染的BMSCs在HAH包封下的三维形态。实验分为4组(各组培养条件无差异):A组(未转染BMP-2的BMSCs);B组(HAH+未转染BMP-2的BMSCs);C组(转染BMP-2的BMSCs);D组(HAH+转染BMP-2的BMSCs)。分别一周内不同时相点(1d、2d、3d……7d)使用CCK-8法检测各组OD值来评价细胞增殖活性;ELISA法检测各组BMP-2的量(pg/ml)来评价目标蛋白的表达状况。结果 :激光共聚焦显微镜三维重建可观察到BMSCs可在HAH中任意平面铺展,有充足的生长空间。BMSCs的增殖活性检测显示,随着培养时间延长,细胞增殖活性逐渐增强,A组、C组4d后的OD值较1d时明显增大(P<0.05);B组和D组5d后的OD值较1d时明显增大(P<0.05);至7d时,A组OD值为1.283±0.061,B组为1.844±0.075;C组为1.570±0.099;D组为1.976±0.142,包封HAH的B、D组OD值均优于没有包封HAH的A、C组(P<0.05)。BMP-2的表达在A、B组较弱,不同时间点间无显著性差异(P>0.05);C、D组的BMP-2表达各时间点均明显高于A、B组(P<0.05);从4d时起,C、D两组的BMP-2表达显著增加(与1d时比较P<0.05),且D组明显优于C组,差异有显著性(P<0.05)。结论 :三维环境下(即包封HAH后),Ad-BMP-2转染的BMSCs不仅有较强的增殖活性;而且对BMP-2的表达具有一定的促进作用。

Objectives： To observe the proliferative ability of bone marrow-derived mesenchymal stem cells （BMSCs） transfected with the adenovirus vector containing bone morphogenetic protein 2（BMP-2） in the threedimensional condition of hyaluronic acid hydrogel（HAH） and its effect on the expresseion of BMP-2 originated from BMSCs. Methods： BMSCs were isolated, extracted, identified in vitro and transfeeted with the constructed adenovirus vector containing BMP-2（Ad-BMP-2） in advance, then BMSCs were encapsulated by HAH; the three-dimensional shape of Ad-BMP-2 BMSCs in HAH was observed by laser scanning confocal microscope. Four groups in the experiments （all groups no difference in culture conditions） were classified as followings： group A, untransfected BMSCs; group B, HAH＋untransfeeted BMSCs; group C, transfected BMSCs; group D, HAH＋transfected BMSCs. CCK-8 method was used to detect each OD value for evaluating cell proliferation of transfected cells encapsulated by HAH at different time point（ld, 2d, 3d 7d）. ELISA method was used to detect the quantity of each BMP-2 expression （pg/ml） . Results： In HAH by used confocal laser 3d reconstruction of BMSCs morphology could be observed that BMSCs rendered arbitrary planar spreading with plenty of growth space. BMSCs proliferation activitis detection showed the extension of incubation time as, cell proliferation activities increased, OD value of group A and group C after 4d was obviously higher than that in ld（P〈0.05）; OD value of group B and group D after 5d was obviously higher than that in ld（P〈0.05）. At 7d, the OD value of group A was 1.283±0.061, group B was 1.844±0.075, group C was 1.570±0.099, group D was 1.976±0.142 and encapsulated HAH OD value of group B and group D were better than that in group A and group C without encapsulated HAH（P〈0.05）. Weak expression of BMP-2 were in group A and group B, there were no significant difference between different time points （P〉0.05）. At various time points, the expression of BMP-2 in both group C and group D were obviously higher than that in group A and group B （P〈0.05）. Since 4d, group C and group D of BMP-2 expression significantly increased（compared to ld, P〈 0.05）; the expression of BMP-2 in group D was significantly better than that in group C, and the difference between the two group was statistically significant （P〈0.05）. Conclusions： Ad-BMP-2 BMSCs not only have strong proliferationpotency, but also can promote the expression of BMP-2 in three-dimensional environment （encapsulated by HAH）.