骨髓基质干细胞移植在兔激素性股骨头坏死模型中成骨及成血管作用的评价

Evaluation on the effect of osteogenesis and angiogenesis after transplanting mesenchymal stem cells in the rabbit model of avascular necronesis of femoral head induced by corticosteroid

目的评估在激素性股骨头坏死模型中血管内移植骨髓基质干细胞(MSCs)的成骨及成血管作用。方法日本大耳白兔20只,雌雄不限,体重2.9±0.7 kg,从兔耳缘静脉内注射马血清2 mL,从第二天起于臀肌注射甲强龙(4mg/kg),前两周每周2次,以后每周1次,共6周。造模结束后死去5只兔子,剩下15只随机分成两组,8只行右侧髂总动脉MSCs移植,作为移植组,其他7只不移植MSCs,作为对照组。从20天龄的日本大耳白兔的骨髓中提取MSCs,贴壁培养法分离、培养、传代MSCs,用BrdU标记第三代MSCs。将MSCs移植入造模组的右侧髂总动脉,5周后取左右两侧股骨头、颈部肌肉,行免疫组化BrdU和CD34双标记检测及冰冻切片免疫荧光双标记检测。并取双侧股骨头,10%甲醛固定,EDTA脱钙,切片行免疫组化BMP-2、BrdU双标记检测,并行HE染色以观察骨质变化。结果移植组股骨头、颈部周围肌肉组织BrdU、CD34双染色显示,该处肌肉组织中有大量BrdU阳性细胞,其中一部分胞浆CD34阳性,表明移植的一部分MSCs在股骨头颈部周围肌肉组织中向血管内皮细胞方向转化。冰冻切片BrdU-Cy3、CD34-FITC免疫双荧光也显示同样的结果。MSCs移植组骨陷窝中可见BrdU阳性细胞,其胞浆中有BMP-2的表达,而对照组见大量空骨陷窝,BMP-2较少表达或不表达。HE染色也显示,对照组骨陷窝内细胞核明显萎缩,接近空骨陷窝,哈弗氏管内细胞大量减少,而移植组的骨陷窝内细胞核较对照组组明显增大,哈弗氏管内细胞明显增加,但仍低于未造模未移植MSCs的正常兔子;未造模未移植的正常兔子的骨陷窝内细胞数量多,细胞核饱满,哈弗氏管内细胞多,核深染,活力正常。结论在股骨头供血血管内移植骨髓基质干细胞在局部肌肉组织中可向血管内皮细胞方向转化,在骨组织中可向成骨细胞方向转化,血管内移植骨髓基质干细胞有助于骨坏死的修复。

Objective To evaluate the effects of osteogenesis and angiogenesis after transplanting mesenchymal stem cells（MSCs） in the rabbit model of Avascular Necrosis of Femoral Head（ANFH） induced by corticosteroid.Methods Twenty Japanese rabbits are brought into the study,whose genders are unlimited and mean weight is 2.9±0.7 kg.Two milliliters of horse serum is injected from auricular veins,and then methylprednisolone（4 mg/kg） is injected from gluteous medius muscle,twice a week during the first two weeks,and later once a week.It lasts for six weeks.Five rabbits have been dead at the end of modeling.Fifteen rabbits left are randomly divided into two groups,of which eight ones are used to transplanting MSCs（transplanting group）,while other ones aren＇t transplanted MSCs（control group）.MSCs are extracted from the marrow of a Janpanese rabbit（age of 20 days）,then isolated and regenerated by adhesive culture method.The third generation of MSCs are labelled with BudU and planted into the right iliac artery in model rabbits.Five weeks later,samples of muscles around the femoral head and the femoral head itself are obtained.The expression of CD34 and BrdU is measured by immunological histochemistry and immunological fluorescent chemistry in the muscular samples.The expression of BMP-2 and BrdU is measured by immunological histochemistry in the samples of bone.HE stain is used to watch osteogenic changes.Results There are a lot of BrdU-positive cells in muscles around femoral head in transplanting group,of which many MSCs express CD34.BrdU-positive cells are also found in the lacunae of femoral head,in which BMP-2 is expressed in cellular plasm.HE stain shows that cellular nucleus are evidently shrinked and close to empty laculae in control group.Cells evidently decrease in Haverison canal.Meanwhile,cellular nucleus are much larger in transplanting group than those in contol group.Cells evidently increase in Haverison canal,but they are still less than those in normal rabbits without modeling.Conclusion 1 In the model of corticosteroid-induced ANFH,it is plausible to transplant MSCs from vessels;2 MSCs can be converted into vascular endothelial cells after transplanting,and also can be converted into osteoblasts in lacunae.