摘要
目的研究淫羊藿苷在骨坏死微环境中对犬骨髓间充质干细胞(BMSCs)的影响,佐证淫羊藿苷可以增强BMSCs治疗股骨头无菌性坏死的疗效。方法12月龄清洁级健康犬,雌雄不限,体重约20Kg。利用液氮冷冻法制作犬股骨头坏死模型,造模后2周于髂嵴上穿刺抽取骨髓10 mL,利用犬单个核细胞分离液分离单核层细胞,常规传代培养犬BMSCs。同时处死实验犬,取出坏死的股骨头,无菌条件下切成小块,放入10%FBS的DMEM培养基中培养,每隔3天换液1次,并收集坏死骨培养液。取第3代细胞进行实验,更换含有5%坏死骨培养液的条件培养基,培养基中淫羊藿苷终浓度分别为2.58、4.18、6.77、10.95、17.72μg/mL。增殖分析采用MTT法,于成骨性诱导培养第7、14 d测碱性磷酸酶(Alkaline Phosphatase,ALP)活性,第8天进行ALP染色,第14 d进行茜素红染色及钙化结节计数。结果原代培养细胞呈典型成纤维细胞样形态;坏死骨培养液可促进BMSCs增殖,淫羊藿苷可抑制BMSCs增殖,但能显著促进其向成骨性分化,表现为提高BMSCs的ALP活性及ALP染色阳性率,增加钙化结节数量。结论以坏死骨培养液法模拟骨坏死微环境的方式可行,淫羊藿苷在骨坏死微环境中同样可促进犬BMSCs的成骨性分化,说明淫羊藿苷可能通过某种途径改善BMSCs治疗股骨头坏死的疗效。
Objective The effect of bone marrow mesenchymal stem cells(BMSCs)in dogs in the local environment of femoral head necrosis was investigated to indirectly demonstrate the efficacy of stem cells in the treatment of aseptic necrosis of the femoral head.Methods Several healthy 12-month-old dogs of clean grade were chosen,which sex were unlimited and weight were about15-20 kg.We established the animal model of osteonecrosis of canine bilateral femoral heads using liquid nitrogen freezing method.Two weeks later,10 ml bone marrow samples were collected from the canine model,and mononuclear cells were isolated and collected with bone marrow cell kit.Cells were cultured with DMEM containing 10%fetal bovine serum.Cut the femoral heads of model into small pieces at the same time,then cultured them in the same medium.Changed the medium every two days,then gathered medium in which necrotic bone have been cultured.The second generation of cells were cultured in DMEM which contain5%necrotic liquid and 10%fetal bovine serum.The final concentration of icariin were 2.58,4.18,6.77,10.95,17.72μg/mL respectively.We used MTT to evaluate the proliferation of cells,the Alkaline phosphatase activity were measured at the seventh and eighth day under the condition of osteogenic induction culture,then alizarin red stain were proceeded and calcified tubercle were counted at the fourteenth day.Results The types of the primary culture cells were similar to the types of fibroblast.Necrotic liquid could promote the proliferation of BMSCs,but had no effect on the differentiation of BMSCs,however icariin could inhibited the proliferation of BMSCs,but the Alkaline phosphatase activity and the number of calcified tubercle were advanced steeply.Conclusion It is a feasible way to mimic the osteonecrosis microenvironment by using necrotic liquid method.The icariin could promote the differentiation of BMSCs into osteogenesis under the osteonecrosis microenvironment,which suggested icariin may as a adjuvant drug to improve the curative effects of BMSCs
作者
仇志强
廖琦
蔡风
周斌
王萌
QIU ZhiQiang;LIAO Qi;CAI Feng(Department of Orthopedics,the Second Affiliated Hospital of Nanchang University,Nanchang Jiangxi,330006,P.R.China)
出处
《江西医药》
CAS
2022年第10期1344-1348,共5页
Jiangxi Medical Journal
基金
江西省科技厅项目,编号20181BBG78056
江西省中医药科技项目,编号2017B062
江西省卫生计生委科技计划,编号20185234。
关键词
骨坏死
淫羊藿苷
骨髓间充质干细胞
钙化
碱性磷酸酶
Osteonecrosis
bone marrow mesenchyme stem cell
Differentiate
Proliferate
Icariin
