低氧诱导骨髓基质干细胞移植治疗激素性股骨头坏死
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摘要
背景:现阶段已经对骨髓基质干细胞移植治疗股骨头坏死进行了探索,但是存在治疗效果不理想等问题。目的:通过对骨髓基质干细胞进行低氧预处理,观察其治疗兔激素性股骨头坏死的效果。方法:抽取新西兰兔髂骨骨髓进行骨髓基质干细胞分离、纯化和培养,放入低氧培养箱(氧气体积分数为2%)中48h进行低氧预处理,采用CCK-8实验、流式细胞术、ELISA检测低氧对细胞活性、细胞凋亡以及分泌细胞因子水平的影响。将40只新西兰兔随机分为对照组、模型组、干细胞组和低氧处理干细胞组,每组10只,后3组建立激素性股骨头坏死模型,后2组在股骨头部位移植正常培养或低氧预处理的骨髓基质干细胞,移植治疗6周后取出股骨头组织,进行大体观察和苏木精-伊红染色。结果与结论:(1)低氧处理后骨髓基质干细胞活力显著增加,细胞凋亡显著降低,释放至上清液中的血管内皮生长因子、肝细胞生长因子和胰岛素样生长因子1水平显著增加;(2)模型组兔股骨头表面软骨破坏、塌陷,表面颜色暗黑、充血,骨质变脆,干细胞组股骨头表面颜色变浅,充血范围减小,低氧处理干细胞组股骨头表面光滑、平整,股骨头颜色浅淡,无明显充血现象,接近于正常;(3)对照组骨小梁结构完整,破骨细胞均匀分散;模型组骨小梁消失明显,出现空骨陷窝,可见大量破骨细胞;干细胞组骨小梁模糊不清,破骨细胞仍密集排布;低氧处理干细胞组骨小梁结构保存较完整,破骨细胞积聚减少;(4)结果表明,低氧预处理的骨髓基质干细胞移植对激素性股骨头坏死具有更好的治疗效果。
BACKGROUND: Bone marrow stromal stem cell transplantation has been explored for the treatment of femoral head necrosis at present, but there are some problems to be solved such as unsatisfactory therapeutic effect. OBJECTIVE: To observe the effect of hypoxic preconditioning of bone marrow stromal stem cells on steroid-induced femoral head necrosis in rabbits. METHODS: Rabbit bone marrow stromal cells were isolated, purified and cultured. They were cultured in a hypoxic incubator(2% O_2) for 48 hours. Cell counting kit-8, flow cytometry, and ELISA assay were used to detect cell viability, apoptosis and secretion of cytokines, respectively. Forty New Zealand rabbits were randomly divided into control group, model group, stem cell group and hypoxia-treated stem cell group, 10 in each group. Steroid-induced femoral head necrosis model was established in the latter three groups. Bone marrow stromal stem cells that were normally cultured or induced by hypoxia were transplanted in the latter two groups. After 6 weeks of transplantation, femoral head tissue from each rabbit was taken out for general observation and hematoxylin-eosin staining. RESULTS AND CONCLUSION:(1) After hypoxic preconditioning, the viability of bone marrow stromal stem cells increased significantly, while the cell apoptosis decreased considerably. The levels of cytokines such vascular endothelial growth factor, growth factor and insulin-like growth factor 1 released into the supernatant increased markedly.(2) The surface of the femoral head of the model group was damaged and collapsed. The surface of the femoral head of the model group was dark in color and congested, and the bone became brittle. The surface of the femoral head of the stem cell group was light-colored, and the congestion alleviated. The surface of the femoral head of the hypoxia-treated stem cell group was smooth, flat and light-colored, with no obvious congestion. In this group, the femoral head was close to the normal bone of the control group.(3) In the control group, the trabecular structure was intact and osteoclasts were evenly dispersed. In the model group, the trabeculae disappeared obviously, and empty bone lacunae appeared. A large number of osteoclasts were observed. In the stem cell group, the trabeculae were blurred and the osteoclasts were still densely arranged. In the hypoxia-treated stem cell group, the trabecular structure remained intact and the accumulation of osteoclasts decreased. These results suggest that the transplantation of bone marrow stromal stem cells after hypoxic preconditioning has a better effect on the treatment of steroid-induced avascular necrosis of the femoral head.
BACKGROUND: Bone marrow stromal stem cell transplantation has been explored for the treatment of femoral head necrosis at present, but there are some problems to be solved such as unsatisfactory therapeutic effect. OBJECTIVE: To observe the effect of hypoxic preconditioning of bone marrow stromal stem cells on steroid-induced femoral head necrosis in rabbits. METHODS: Rabbit bone marrow stromal cells were isolated, purified and cultured. They were cultured in a hypoxic incubator(2% O_2) for 48 hours. Cell counting kit-8, flow cytometry, and ELISA assay were used to detect cell viability, apoptosis and secretion of cytokines, respectively. Forty New Zealand rabbits were randomly divided into control group, model group, stem cell group and hypoxia-treated stem cell group, 10 in each group. Steroid-induced femoral head necrosis model was established in the latter three groups. Bone marrow stromal stem cells that were normally cultured or induced by hypoxia were transplanted in the latter two groups. After 6 weeks of transplantation, femoral head tissue from each rabbit was taken out for general observation and hematoxylin-eosin staining. RESULTS AND CONCLUSION:(1) After hypoxic preconditioning, the viability of bone marrow stromal stem cells increased significantly, while the cell apoptosis decreased considerably. The levels of cytokines such vascular endothelial growth factor, growth factor and insulin-like growth factor 1 released into the supernatant increased markedly.(2) The surface of the femoral head of the model group was damaged and collapsed. The surface of the femoral head of the model group was dark in color and congested, and the bone became brittle. The surface of the femoral head of the stem cell group was light-colored, and the congestion alleviated. The surface of the femoral head of the hypoxia-treated stem cell group was smooth, flat and light-colored, with no obvious congestion. In this group, the femoral head was close to the normal bone of the control group.(3) In the control group, the trabecular structure was intact and osteoclasts were evenly dispersed. In the model group, the trabeculae disappeared obviously, and empty bone lacunae appeared. A large number of osteoclasts were observed. In the stem cell group, the trabeculae were blurred and the osteoclasts were still densely arranged. In the hypoxia-treated stem cell group, the trabecular structure remained intact and the accumulation of osteoclasts decreased. These results suggest that the transplantation of bone marrow stromal stem cells after hypoxic preconditioning has a better effect on the treatment of steroid-induced avascular necrosis of the femoral head.
引文
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[29]郝一文.氧浓度对人骨髓造血干/祖细胞生物功能的影响[J].中国实验血液学杂志,2010,18(4):997-1001.
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[31]康少平,刘淑艳,李永升.激素性股骨头坏死患者骨髓间充质干细胞体外增殖分化能力的研究[J].中国细胞生物学学报,2015,37(11):1490-1496.
[32]Seedorf G,Metoxen AJ,Rock R,et al.Hepatocyte growth factor as a downstream mediator of vascular endothelial growth factor-dependent preservation of growth in the developing lung.Am J Physiol Lung Cell Mol Physiol.2016;310(11):L1098-1110.
[33]Poudel SB,Bhattarai G,Kook SH,et al.Recombinant human IGF-1produced by transgenic plant cell suspension culture enhances new bone formation in calvarial defects.Growth Horm IGF Res.2017;36:1-10.
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[2]农峰,唐毓金,赵凯丽.激素性股骨头坏死与相关单核苷酸基因多态性关系的研究进展[J].中华实用诊断与治疗杂志,2017,31(7):722-725.
[3]刘耀升,刘蜀彬.早中期股骨头坏死保留关节的手术治疗进展[J].中华损伤与修复杂志(电子版),2012,7(5):528-531.
[4]张思迪.回顾性研究骨瓣移植联合多孔钽棒治疗激素性股骨头坏死的疗效[D].大连:大连医科大学,2018.
[5]叶钧联,梁发树,冯骥.细针钻孔减压术治疗早期股骨头坏死[J].中国骨科临床与基础研究杂志,2015,7(4):225-229.
[6]李霏霰,吴齐英,李新志,等.髓心减压术联合自体骨髓干细胞移植治疗老年缺血性股骨头坏死的疗效[J].中国老年学杂志,2016,36(22):5671-5672.
[7]Padegimas EM,Osei DA.Evaluation and treatment of osteoporotic distal radius fracture in the elderly patient.Curr Rev Musculoskelet Med.2013;6(2):205.
[8]Kubo T,Ueshima K,Saito M,et al.Clinical and basic research on steroid-induced osteonecrosis of the femoral head in Japan.J Orthop Sci.2016;21(4):407-413.
[9]张晓晶,李盛龙,王喆.Pannexin1通道蛋白对微张力刺激下骨髓间质干细胞成骨分化的影响[J].中华骨科杂志,2017,37(1):44-51.
[10]路博丞,冷向阳,张鹏程,等.鹿茸多肽联合GDNF基因修饰的雪旺细胞对人骨髓间质干细胞体外增殖的影响[J].吉林大学学报(医学版),2017,43(6):1125-1129.
[11]孟丽,丁从珠,陆邦超,等.脐带间质干细胞对骨关节炎的治疗作用及机制研究[J].中华老年医学杂志,2018,37(1):79-83.
[12]Croft AP,Przyborski SA.Mesenchymal stem cells expressing neural antigens instruct a neurogenic cell fate on neural stem cells.Exp Neurol.2009;216(2):329-341.
[13]Brazelton TR,Rossi FM,Keshet GI,et al.From marrow to brain:expression of neuronal phenotypes in adult mice.Science.2000;290(5497):1775-1779.
[14]赵程锦,冯阳阳,周煌虎.髓芯减压植骨术结合富血小板血浆治疗股骨头缺血性坏死的疗效及术后并发症临床观察[J].中国临床研究,2017,30(7):878-882.
[15]Ando W,Yamamoto K,Koyama T,et al.Radiologic and Clinical Features of Misdiagnosed Idiopathic Osteonecrosis of the Femoral Head.Orthopedics.2017;40(1):e117-e123.
[16]付强,闰世杰,王江泳,等.髓芯减压联合自体骨髓间充质干细胞治疗45例股骨头无菌性坏死的临床疗效分析[J].现代生物医学进展,2013,13(25):4925-4928.
[17]Ouyang Q,Wu J,Jiang Z,et al.Microarray Expression Profile of Circular RNAs in Peripheral Blood Mononuclear Cells from Rheumatoid Arthritis Patients.Cell Physiol Biochem.2017;42(2):651-659.
[18]Meena DK,Thalanki S,Sharma SB.Wrist fusion through centralisation of the ulna for recurrent giant cell tumour of the distal radius.J Orthop Surg(Hong Kong).2016;24(1):84-87.
[19]康少平,刘淑艳,李永升,等.低氧增强骨髓间充质干细胞增殖维持分化潜能[J].中国比较医学杂志,2017,27(7):70-74.
[20]江树连,朱磊,李文华,等.破瘀通络法对激素性股骨头坏死BMP-2表达影响[J].中医药临床杂志,2017,29(7):1042-1044.
[21]冯志尉,李伟.PPARγ与激素性股骨头坏死的研究进展[J].西部医学,2017,29(7):1032-1036.
[22]Thirkannad SM,Burgess RC.A technique for using the Ilizarov fixator for primary centralization in radial clubhand.Tech Hand Up Extrem Surg.2008;12(2):71-78.
[23]吕辉.活血生骨汤联合健骨生丸治疗中老年激素性股骨头坏死41例[J].中医研究,2017,30(5):32-34.
[24]张龙,韦标方.激素性股骨头坏死发病机制的研究进展[J].风湿病与关节炎,2018,7(3):73-76.
[25]邓爽,彭昊.激素性股骨头坏死发生机制的新认识[J].中国医药导报,2017,14(34):29-32.
[26]Wall LB,Ezaki M,Oishi SN.Management of congenital radial longitudinal deficiency:controversies and current concepts.Plast Reconstr Surg.2013;132(1):122-128.
[27]赵德伟.激素性股骨头坏死的防治新主张[J].中华医学杂志,2017,97(41):3201-3203.
[28]王继涛,阮红峰,付方达,等.改良型激素性股骨头坏死小鼠模型的建立与评价[J]中国中医急症,2017,26(9):1525-1528.
[29]郝一文.氧浓度对人骨髓造血干/祖细胞生物功能的影响[J].中国实验血液学杂志,2010,18(4):997-1001.
[30]王天胜,滕寿发,张英霞,等.骨髓基质干细胞移植在激素性股骨头坏死治疗中的作用机制研究[J].临床和实验医学杂志,2015,14(1):10-12.
[31]康少平,刘淑艳,李永升.激素性股骨头坏死患者骨髓间充质干细胞体外增殖分化能力的研究[J].中国细胞生物学学报,2015,37(11):1490-1496.
[32]Seedorf G,Metoxen AJ,Rock R,et al.Hepatocyte growth factor as a downstream mediator of vascular endothelial growth factor-dependent preservation of growth in the developing lung.Am J Physiol Lung Cell Mol Physiol.2016;310(11):L1098-1110.
[33]Poudel SB,Bhattarai G,Kook SH,et al.Recombinant human IGF-1produced by transgenic plant cell suspension culture enhances new bone formation in calvarial defects.Growth Horm IGF Res.2017;36:1-10.
[34]任玉萍.利用IGF-1基因敲除鼠乳腺癌模型研究IGF-1对血管生成的影响[J].中国普外基础与临床杂志,2008,15(11):820-824.
[35]张倩,陈胜广,吴国亭,等.阿卡波糖对糖耐量减低大鼠FFA、IGF-1及大血管的影响[J].同济大学学报(医学版),2009,30(4):32-35.
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