hBMP-7基因转染修复骨缺损的实验研究

The repair of bone defect by bone marrow stromal cell transferred by retroviral vector mediated hBMP-7 gene

目的探讨利用人骨形态发生蛋白7(hBMP-7)基因转染骨髓基质干细胞(BMSc)构建组织工程化骨组织并修复兔桡骨骨缺损的可行性。方法制备含hBMP-7真核细胞表达载体的逆转录病毒液PT-PLNCX2-hBMP-7并转染BMSc,使用免疫组织化学的方法检测hBMP-7在BMSc中的表达。兔桡骨1.5cm骨缺损修复实验分为4组:转染组,未转染组,单纯PLLA组和对照组,每组6个样本。分别于术后3周,6周采用大体观察、X线、组织学检测、免疫组织化学检测等方法对骨缺损修复情况进行对比。结果免疫组织化学检测显示经PT-PLNCX2-hBMP-7病毒液转染的BMSc中有较强的阳性结果出现。定量检测分析结果证实经转染的BMSc修复骨缺损的成骨速度和成骨量均优于未转染组,单纯PLLA组和对照组中均未见骨缺损得到修复。结论hBMP-7基因转染能够提高BMSc形成组织工程化骨组织和修复骨缺损的能力,可用于以BMSc为种子细胞的组织工程化骨组织的构建。

Objective To explore the ability of BMSc(Bone marrow stroma l cell) transferred by hBMP-7 gene combined with the biomaterials of PLLA(poly-L-lactide) to enhance segmental bone defect healing of the rabbit radius. Method s Human BMP-7 gene vector was reconstructed in retroviral vector. The targeted c ells of BMSc were infected with the virus granules. The protein of hBMP-7 gene i n transferred cells was determined by immunohistochemistry. The BMSc were divide d into three groups: transferred by PT-PLNCX2-hBMP-7 and PT-PLNCX2, and no trans fection. A segmental defect of 1.5 cm was created surgically in the radius of Ne w Zealand white rabbits. The defects were repaired with 5×106 BMSc transferred by PT-PLNCX2-hBMP-7 in combination with PLLA materials, and PLLA biomaterials al one, while the defect in control group was left untreated. The defect repaired c apabilities for each of the reconstructive modalities were assessed by gross obs ervation, radiographic, histological and immunohistochemistry analysis. Results The BMSc transferred by PT-PLNCX2-hBMP-7 expressed abundant human BMP-7 mRNA and protein in the cytoplasm. However, the BMSc transferred by PT-PLNCX2 was not ob served buffy granule in the cytoplasm by immunohistochemistry. By histological e xamination of 6 weeks, a bone bridge formation across the defect was observed in the transferred and un-transferred groups, however, only fibrous tissue could b e found in defect area in the PLLA and control groups. By radiographic examinati on of 6 weeks, a continuous bone bridge and more new bone formation could be see n in bone defect, whereas, the delimitation of defect in PLLA and control groups were still obvious. Furthermore, the results of the average transmittance detec tion suggested that there was 82.39 of transferred BMSc higher than 69.69 of un-transferred BMSc. Generally, gross observation,radiographic and histomorphometr ic analysis revealed a significantly greater total area of new bone formation an d amount and quality of the new bone in the defects treated with the transferred BMSc. The use of BMSc transferred by PT-PLNCX2-hBMP-7 in conjunction with PLLA materials exhibited the strongest defect repaired capacity as compared to the ot her groups. Conclusion Gene transfer with the use of autologous short-term cultu red BMSc may be used to increase the osteogenic capability of BMSc. These data e ncourage the further development of genetic approaches to enhance bone healing i n bone tissue engineering.