骨形态发生蛋白-2和7联合基因修饰构建组织工程骨的实验研究

Construction of tissue engineered bone modified by bone morphogenetic protein-2 and bone morpho-genetic protein-7 genes

目的探讨人工合成B-磷酸三钙（B—TCP）复合骨形态发生蛋白（BMP）-2与BMP-7联合基因修饰的人鼠脂肪来源干细胞（ADSCs）构建组织工程骨的町行性。方法取SD大鼠ADSCs，传至第3代．随机分为4组：BMP-2组（Lv—BMP-2转染ADSCs），BMP-7组（Lv—BMP．7转染ADSCs），BMP-2＋BMP-7纰（Lv-BMP-2、Lv—BMP．7共转染ADSCs），对照组（未转染的ADSCs）。以1×106／30uL的密度接种于B-TCP，进行体复合培养.，采用碱性磷酸酶（ALP）定城检测细胞ALP活性，通过茜素红染色、荧光定量聚合酶链式反心榆测复合物的成骨活性，并进行体内组织工程骨的研究。结果细胞接种后0～14d，4组细胞的DNA量变化趋势一致。与对照组相比，BMP-2组、BMP-7组和BMP．2＋BMP-7组复合物体外培养14d后钙化绱节的数目明显增多，其中BMP-2＋BMP-7组钙化结节数日最多，早片状分布。BMP-2＋BMP-7组的ALP）、骨桥蛋白（OPN）、骨钙索（OC）mRNA表达量及OPN、OC的蛋白表达明显高于对照组、BMP-2组及BMP-7组，差异均有统计学意义（P〈0．05）。体内成骨结果：术后6周X线片示BMP-2＋BMP-7绀大鼠骨缺损基本愈合，而其他3组大鼠骨缺损未完全恢复；HE染色示BMP-2＋BML-7组产生了完档的皮层骨和骨髓，而其他3组仅产生了少量骨小梁、皮层骨及骨髓。结论联合应用BMP．2和BMP-7可明显促进组织工程骨的成骨效应，为应用骨纰织工程科技术修复骨缺损提供了初步实验依据。

Objective To investigate tile feasibility and advantages of constructing a novel tissue engineered bone through lentivirus transfection using [3-tricalcium phosphate （[3-TCP） and adipose （leriw＋d stem cells （AI）SCs） and modification with human bone morphogenetic protein 2 gene （BMP-2） and human bone morphogenetic protein 7 gene （BMP-7）. Methods ADSCs derived from SD rats were passaged into the third generation before they were randomly divided into 4 groups： BMP-2（1,v-BMP-2-transfected ADSCs）, BMP-7 （Ix-BMP-7-transfected ADSCs）, BMP-2＋ BMP-7 （ADSCs eotransfec, ted by Lv-BMP-2 and Lv-BMP-7） and control （non-transt^cted ADSCs） . The ADSCs at a concentration of 1 106/30 Ixl, were seeded onto the [3-TCP for compound euhure in vitro. PienGreen dsDNA assay evaluated the growth of ADSCs. The alkaline phosphatase （ALP） activity of AI）SCs was measured. Alizarin red S staining and real-time PCR were used to tesl the osteoblast activities of A1）SCs. Results After the AI）SCs were seeded onto the scaffohls for 0 tn 14 days, a similar trend was observed in the 4 groups regarding changes in the DNA amount. At 14 days after cell seeding, the BMP-2, BMP-7 anti BMP-2 ＋ BMP-7 groups had more calcifie nodules than the control group, with the highest number in the BMP-2 ＋ BMP-7 group. The ALP, mRNA and protein ex- pressions of osteopuntin anti usteucalcin in the FIMP-2 ＋ BMP-7 group were significantly higher than those in the other 3 groups （ P 〈 0. 05）. X-ray at 6 weeks postuperation showed bone defec.ts were almost healed in the BMP-2 ＋ BMP-7 group but not in the other 3 groups. HE staining showed complete cortex bone anti bone marrow in the BMP-2 ＋ BMP-7 gruup but only a little amount of bone trabecula, cortex l）one anti bone marrow in the other 3 groups. Conclusions Tissue engineered bone can be constructed using [3-TCP combined with AI）SCs co-transfect：ed with BMP-2 and BMP-7. As such tissue engineered bone has a great advantage of remarkably increased capability of osteogenesis, it can be used as a new means of treating bone detects.