摘要
目的通过组织工程和基因工程结合的方法从种子细胞、生长因子、细胞支架三方面满足老年机体骨修复的需要,为这些方法在老年机体的应用提供参考。方法体外分离、扩增、骨形态发生蛋白-2(BMP2)基因修饰MSCs,检测细胞上清液中BMP2的表达;通过检测成骨细胞标志性蛋白如碱性磷酸酶(ALP)、Ⅰ型胶原(COLⅠα1)、骨涎蛋白(BSP)、骨桥素(OPN)的表达观察老年大鼠MSCs的成骨分化能力;检测BMP2基因修饰的MSCs与磷酸三钙(TCP)复合物在裸鼠体内的异位成骨能力;将BMP2基因修饰的MSCs/TCP植入老年大鼠骨缺损部位,修复自体股骨6mm节段性骨缺损。结果BMP2基因修饰的MSCs可以有效分泌BMP2,诱导后第7天有ALP、COLⅠα1、OPN、BSP的明显表达,并可诱导裸鼠体内异位成骨。放射学及组织学检测证明BMP2转染的MSCs与TCP载体复合后可成功修复老年大鼠股骨节段性缺损。结论BMP2基因修饰的老年MSCs可以恢复成骨分化能力,在体内可以成功修复老年大鼠股骨节段性骨缺损,组织工程和基因工程方法可能成为治疗老年性骨骼疾病的新途径。
Objective To investigate whether the bone morphogenetic protein 2 (BMP-2) gene engineered mesenchymal stem cells (MSCs) can facilitate bone healing in the aged skeleton. Methods The MSCs from 24-month-old Wistar rats were isolated, multiplied, transduced with adenovirus-mediated BMP2 gene ( Adv-BMP2 ) or adenovirus-mediated βgal gene ( Adv-βgal ) ( control ). The expression of BMP2 and osteoblastic markers such as alkaline phosphatase (ALP) , type I collagen ( COL Ⅰ-α1 ), osteopontin (OPN) , bone sialoprotein (BSP) are assayed during the process of differentiation. The ectopic bone formation in nude mice are also assayed, In order to analyze the osteogenic potential in aged body,the MSCs/TCP were implanted in the 24-month-old rats to repair femoral bone defects (6 mm long).Results MSCs modified by BMP2 produced BMP-2 protein, differentiated into osteoblastic line, induced ectopic bone formation in nude mice, and successfully repaired cortical bone defects in aged rats. These Adv-BMP2 transduced MSCs had significant BMP2 expression, increased alkaline phosphatase activity,increased expression of COL Ⅰ-α1, OPN, BSP mRNA, and induced increased volume of ectopic bone in nude mice, compared with cells transduced with a control adenoviral construct. The production of new bone in femoral defects was demonstrated by radiographic examination three weeks after operation in AdvBMP2 group, and the defects achieved healing after six weeks, whereas no healing was detected in the defects in control group. Histological examination further confirmed the results from radiographic examination.Conclusion MSCs obtained from old rats can restore their osteogenic activity following human BMP-2 gene transduction and successfully repair femoral bone defects, suggesting a new way to address challenges of the aging skeleton.
出处
《中华创伤杂志》
CAS
CSCD
北大核心
2005年第8期611-616,共6页
Chinese Journal of Trauma