负载胰岛素样生长因子-1的骨髓间充质干细胞与壳聚糖-胶原复合物修复大鼠骨缺损的实验研究

背景：目前已有较多研究表明将种子细胞复合于支架材料进行骨缺损的修复能够取得良好的效果，但仍需探索有效的生长因子以促进修复过程。 目的：探讨负载胰岛素样生长因子-1（insulin-like growth factor，IGF-1）的骨髓间充质干细胞与壳聚糖-胶原复合物对大鼠骨缺损的修复作用。 方法：分离大鼠骨髓间充质干细胞，流式细胞术鉴定细胞表面标志物；将骨髓间充质干细胞进行成骨诱导后检测碱性磷酸酶的活性以鉴定其成骨能力；IGF-1过表达载体转染骨髓间充质干细胞并筛选稳转细胞系，Real-time PCR以及Western blot鉴定稳转细胞系中IGF-1的表达。扩培细胞，并与壳聚糖-胶原进行复合培养。同时建立大鼠骨缺损模型，造模成功后将20只大鼠随机分为4组，每组5只。实验组植入负载IGF-1的骨髓间充质干细胞复合支架，支架修复组植入未经细胞复合的支架，干细胞复合支架修复组植入单纯骨髓间充质干细胞复合支架，空白对照组缺损处未植入任何物质。术后分别通过X射线和组织学切片观察比较各组大鼠的骨缺损修复情况。 结果：流式细胞术鉴定结果显示细胞CD44阳性率为98.19%，CD45阳性率为3.65%，CD90阳性率为97.62%。成骨诱导后碱性磷酸酶浓度升高至（11.57±0.48）U/L，显著高于未经成骨诱导的细胞（5.55±0.63）U/L（〈0.05）。Real-time PCR以及Western blot检测结果IGF-1稳定转染的细胞中IGF-1 mRNA以及蛋白的表达均显著升高（〈0.05）。X射线以及组织学观察结果表明空白组骨缺损不能自行修复，壳聚糖-胶原支架与骨髓间充质干细胞复合物植入大鼠后未见明显的免疫排斥反应，与其他组相比负载IGF-1的骨髓间充质干细胞与壳聚糖-胶原复合物组的成骨及修复效果均较为明显。 结论：骨髓间充质干细胞是较为理想的骨组织工程种子细胞，将IGF-1负载于骨髓间充质干细胞能够促进骨组织的修复。

Repair of segmental tibial defects by composite grafts of IGF-1 transfected bone marrow mesenchymal stem cells and chitosan-collagen gel in rats

Background：Many studies have shown that cell-scaffold composite is feasible in the repair of bone defects, but it is essen-tial to find effective growth factors to promote the repair process. 〈br〉 Objective：The purpose of the study is to explore the effect of insulin-like growth factor-1 （IGF-1） transfected bone marrow stem cells （BMSCs） and chitosan-collagen composite on the repair of segmental tibial defect of rat. 〈br〉 Methods：BMSCs were isolated and flow cytometry was used to detect the cell surface markers. Osteogenesis was induced and alkaline phosphatase activity was detected to identify the osteogenic capability. IGF-1 overexpression vector was trans-fected into BMSCs and stably transfected cell lines was screened. Real-time PCR and Western blot was used to detect the expression of IGF-1. Stably transfected cells were co-cultured with chitosan-collagen gel in vitro. Bone defect was estab-lished in 20 rats. They were randomly divided into four groups （n=5）：control group （nothing was implanted）, chitosan-colla-gen gel group, BMSCs with chitosan-collagen group and IGF-1 transfected BMSCs with chitosan-collagen gel group （the composite grafts were implanted into bone defects, respectively）. Radiographic examination and histologic analysis were＆amp;nbsp;used to evaluate the curative effect after operation. 〈br〉 Results：Flow cytometry showed that positive rate of CD14, CD45 and CD90 was 98.19%, 3.65%and 97.62%, respectively. The concentration of alkaline phosphatase was significantly increased after osteogenic induction （11.57＆#177;0.48]U/L vs 5.55＆#177; 0.63]U/L, P＆lt;0.05）. Real-time PCR and Western blot showed that the levels of IGF-1 mRNA and protein were increased sig-nificantly after transfection （P＆lt;0.05）. X-ray examination and histologic analysis showed that bone defect was not repaired in control group. No rejection occurred in the IGF-1 transfected BMSCs with chitosan-collagen group and new bone forma-tion was apparently superior to that in the other groups. 〈br〉 Conclusions：BMSCs are ideal seeded cells for bone tissue engineering and IGF-1 transfected BMSCs and chitosan-colla-gen gel complexes can accelerate bone formation.