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目的 探究骨形态发生蛋白(BMP)-2基因修饰的骨髓间充质干细胞(BMSCs)复合纳米羟基磷灰石(n HA)/聚酰胺66(PA66)对大鼠股骨骨不连的治疗作用。方法 分离大鼠BMSCs，并分为支架(sent)+BMSCs组、sent+BMSCs+rh BMP-2组、sent+BMSCs+Ad-BMP-2组;将人重组BMP-2(rh BMP-2)或腺病毒感染BMP-2(Ad-BMP-2)载体转染BMSCs并装载n HA/PA66支架材料。流式细胞仪检测BMSCs表达特定表面标志物，通过MTT法检测细胞增殖情况，ELISA检测相关生物活性因子，包括血小板衍生生长因子(PDGF)、转化生长因子-β(TGF-β)、血管内皮生长因子(VEGF)、成纤维细胞生长因子(FGF)、骨钙素(OCN)、骨连接蛋白(ON)，碱性磷酸酶(ALP)。扫描电子显微镜(SEM)观察细胞生长和黏附情况。在SD大鼠的萎缩性骨不连模型中，将sent+BMSCs+rh BMP-2或sent+BMSCs+Ad-BMP-2复合体植入缺损区域，分为rh BMP-2组及Ad-BMP-2组，并通过X射线和MicroCT评估治疗效果。结果 n HA/PA66支架表面光滑且多孔，BMSCs良好黏附。流式细胞术显示在BMSCs中高表达CD29和CD90，低表达CD45和CD34。MTT显示细胞在72 h后迅速增殖。与sent+BMSCs组及sent+BMSCs+rhBMP-2组相比，sent+BMSCs+Ad-BMP-2组ALP活性、PDGF、TGF-β、VEGF、FGF、OCN、ON表达均上调(P<0.05)。术后12周Ad-BMP-2组大鼠支架复合物已完全被新生皮质骨包裹，且表面光滑完整。X射线显示Ad-BMP-2组复合物已完全被新生皮质骨包裹，恢复程度优于rh BMP-2组。MicroCT结果显示，与rh BMP-2组相比，Ad-BMP-2组术后12周大鼠骨体积分数、骨小梁厚度、骨小梁数目、骨矿物质密度及骨矿物质含量均升高(P<0.05)，骨小梁分离水平降低(P<0.05)。结论 Ad-BMP-2转染BMSCs复合n HA/PA66支架材料可以更有效地表达生物活性，为BMSCs提供持续和良好的增殖分化微环境，有利于促进局部成骨活性，且有利于骨缺损修复。
Abstract：Objective To investigate the therapeutic effect of bone marrow mesenchymal stem cells(BMSCs) modified by bone morphogenetic protein(BMP)-2 gene combined with nano-hydroxyapatite(nHA)/polyamide 66(PA66) on femoral nonunion in rats. Methods Rat BMSCs were isolated and divided into the stent + BMSCs group,stent + BMSCs + rhBMP-2 group,and stent + BMSCs + Ad-BMP-2 group; human recombinant BMP-2(rhBMP-2) or adenovirus-infected BMP-2(Ad-BMP-2) vector was transfected into BMSCs and loaded onto nHA/PA66 stent materials. Flow cytometry was used to detect that BMSCs expressed specific surface markers. Cell proliferation was detected by MTT assay,related bioactive factors [platelet-derived growth factor(PDGF),transforming growth factor-β(TGF-β),vascular endothelial growth factor(VEGF),fibroblast growth factor(FGF),osteocalcin(OCN),osteonectin(ON) ] were detected by ELISA,alkaline phosphatase(ALP) activity was detected,and cell growth and adhesion were observed by scanning electron microscopy(SEM). In the atrophic nonunion model of SD rats,the stent + BMSCs + rh BMP-2 or stent + BMSCs + Ad-BMP-2 complex was implanted into the defect area,which was divided into the rh BMP-2 group and the Ad-BMP-2 group,and the therapeutic effect was evaluated by X-ray and MicroCT. Results The surface of the n HA/PA66 stent was smooth and porous,and BMSCs adhered well. Flow cytometry showed high expression of CD29 and CD90 and low expression of CD45 and CD34 in BMSCs. MTT showed that the cells proliferated rapidly after 72 h. Compared with the stent + BMSCs group and the stent + BMSCs + rhBMP-2 group,the ALP activity,the expressions of PDGF,TGF-β,VEGF,FGF,OCN,and ON in the stent + BMSCs + Ad-BMP-2 group were significantly up-regulated(P < 0. 05). 12 weeks after surgery,the stent complex in the Ad-BMP-2 group of rats was completely wrapped by newly formed cortical bone,and the surface was smooth and intact. X-ray showed that the complex in the Ad-BMP-2 group was completely wrapped by newly formed cortical bone,and the recovery degree was better than that in the rhBMP-2 group. Micro-CT results showed that compared with the rhBMP-2 group,the bone volume fraction,trabecular thickness,trabecular number,bone mineral density,and bone mineral content in the Ad-BMP-2 group all increased 12 weeks after surgery(P <0. 05),and the trabecular separation level decreased(P <0. 05). Conclusion Ad-BMP-2-transfected BMSCs combined with n HA/PA66 stent material can express bioactivity more effectively,provide a continuous and good microenvironment for the proliferation and differentiation of BMSCs,which is beneficial to promoting local osteogenic activity and bone defect repair.
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基本信息:
DOI：10.19405/j.cnki.issn1000-1492.2025.09.005
中图分类号:R318.08;R683
引用信息:
[1]黄永,马建文,李玉,等.BMP-2基因修饰的骨髓间充质干细胞复合纳米羟基磷灰石/聚酰胺66植入体改善大鼠萎缩性骨不连[J].安徽医科大学学报,2025,60(09):1599-1605.DOI:10.19405/j.cnki.issn1000-1492.2025.09.005.
基金信息:
青海省自然科学基金项目(编号:2022-ZJ-968Q)~~