VEGF-165基因修饰的MSCs诱导分化后治疗兔股骨头缺血性坏死的实验研究

Treatment of Avascular Necrosis of the Femoral Head (ANFH) with Induced Differentiation VEGF-165 Gene Modified Mesenchymal Stem Cells in Rabbits

目的:探讨VEGF-165基因联合单纯骨髓间充质干细胞(marrow mesenchymal stem cells,MSCs)治疗激素性股骨头坏死的可行性。方法:1)体外分离、培养兔MSCs,纯化并鉴定兔MSCs;免疫荧光法检测细胞表面标志;传代培养后以1μgPcDNA3.1-hVEGF165∶3μl阳离子脂质体Lipofectamine的比例转染,通过ELISA检测转染后细胞中外源性VEGF的表达。2)测定在正常条件培养和成骨条件培养下,转染后细胞上清中碱性磷酸酶、骨钙素的水平。3)实验动物连续三天大剂量(40mg/kg)臀部肌肉注射甲泼尼龙琥珀酸钠,通过MRI和组织病理学检查,建立动物模型。4)随机分成4组,每组14只。经皮穿刺于右侧股骨头,(1)生理盐水组,(2)单纯MSCs组,(3)hVEGF165基因转染MSCs组,(4)诱导分化后的hGVEF165基因转染MSCs组。8周后,组织病理学检查成骨和细胞成活情况。结果:1)hVEGF165基因转染的MSCs能成功分泌VEGF蛋白。2)成骨条件培养下,基因转染组细胞碱性磷酸酶和骨钙素的分泌量明显高于未转染组(P<0.05);而在正常条件培养下,基因转染组细胞碱性磷酸酶和骨钙素的表达分泌量较低。3)成功建立ANFH模型。4)组织学评分:生理盐水组1.8±0.72;单纯MSCs组8.54±0.37;hVEGF165基因转染MSCs组11.96±0.26;诱导分化后的hGVEF165基因转染MSCs组13.7±0.43,各组间差异有显著性(P<0.05)。诱导分化转基因干细胞组与单纯干细胞组和生理盐水组之间差异非常显著(P<0.01)。结论:诱导分化后的hVEGF165基因转染MSCs组成骨能力更好,修复股骨头缺血性坏死的能力也是最强。

Objective： To approach the possibility of combination of VEGF-165 gene and bone marrow mesenchymal stem cells （MSCs） transplantation for steroid-induced osteonecrosis of femoral head. Methods： 1 ） MSCs were isolated from rabbits, and purification of rabbit MSCs, identification of cell surface markers were done by immunofluorescence assay, And the cultured MSCs were transferred with the composite of PcDNA3.1-hVEGF165 and lipofectamine regent in the ratio of 1/3. The expression of exogenous VEGF gene was investigated by ELISA. 2） The secretion of alkaline phosphatase （ALP） and osteocalcin （OCN） of the gene transferred MSCs were determined under normal conditional medium and osteogenic conditional medium. 3 ） After the injection of methylprednisolone （40 mg/kg） for 3 days, establishment of eorticosteroid induced avaseular necrosis of femoral head models of rabbit was done by MRI and pathogenic histology. 4） They were randomized into 4 groups （14 in each group）, received puncture of the femoral head. （1） balanced saline group, （2） MSCs group, （ 3 ） VEGF-165 MSCs group, （4） induced differentiation VEGF-165 MSCs group. Affter 8 weeks, bone formation and survival of transplantated cells were assessed by pathological examination. Results： 1 ） The transferred MSCs by hVEGF-165 can secret the protein of VEGF. 2） Compared with un-transfected group, the secretion of ALP and OCN of the gene transfered MSCs were significantly higher under osteogenie conditional medium （ P 〈 0. 05 ）. But under normal conditional medium, the secretion level of ALP and OCN of the gene transferred BMSCs were lower. 3 ） Successful ANFH models of rabbit were establihed. 4） Histological assay： balanced saline group1.8 ±0. 72; MSCs group8.54 ±0. 37; VEGF-165 MSCs groupll. 96 ±0. 26; induced differentiation VEGF-165 MSCs groupl3.7 ±0. 45. There were statistical significance between each group （P 〈0. 05）. The gene transfered MSCs were significantly higher than MSCs group and VEGF-165 MSCs group （P 〈0. 01 ）. Conclusion： Under osteogenic condition, the osteogenic activity of the transferred MSCs by hVEGFI65 is improved. Repair ANFH capacity of induced differentiation VEGF-165 MSCs is the best.