microRNA调控骨髓干细胞治疗激素性股骨头坏死的实验研究

发布时间：2018-05-07 12:01

  本文选题：干细胞 + 股骨头坏死　； 参考：《北京协和医学院》2015年博士论文

【摘要】：研究背景：激素性股骨头坏死是骨科常见疾病,其致残率高,保守治疗效果欠佳。激素导致骨髓间充质干细胞成骨分化能力下降是其中的一个重要机制。课题组前期通过microRNA芯片技术,筛选出激素性股骨头坏死患者与正常健康人骨髓间充质干细胞增殖分化过程中miRNA表达谱的差异特征,并将其与正常健康人骨髓间充质干细胞经大剂量激素刺激前后的miRNA表达差异谱进行比较,找到表达变化一致的miRNAs,为microRNA-23a,并进一步构建microRNA-23a mimics和inhibitor,进一步发现上调microRNA-23a可以导致骨髓间充质干细胞成骨分化减弱,下调microRNA-23a能够促进BMSC成骨分化。利用生物信息学分析结合双荧光素酶报告基因系统预测低密度脂蛋白相关蛋白5(low density lipoprotein receptor-related protein 5, LRP-5)为miRNA-23a的靶基因,使用shRNA沉默LRP5基因后可观察到BMSC成骨分化减弱,与上调miRNA-23a类似。激素性股骨头坏死坏死目前仍没有特效药物,研究发现,间断小剂量应用甲状旁腺激素能够促进成骨细胞生成,促进干细胞向成骨方向分化。人重组甲状旁腺激素[rhPTH (1-34)]是目前唯一批准上市的具有促进骨合成的抗骨质疏松药物,除治疗骨质疏松外,在促进骨折愈合、促进脊柱融合、促进软骨修复及下颌骨坏死都显示出了良好治疗效果。激素性股骨头坏死的发病机制包括激素抑制干细胞成骨分化,我们推测,人重组甲状旁腺激素[rhPTH(1-34)]是否也可以通过促进成骨细胞生成,促进干细胞向成骨方向分化的作用来治疗股骨头坏死。研究目的：1.构建microRNA-23a mimics和inhibitor,转染大鼠骨髓间充质于细胞。将转染成功的大鼠骨髓间充质干细胞注射至大鼠激素性股骨头坏死模型体内。评价microRNA-2 3a调控BMSC对大鼠股骨头坏死的修复作用。2.研究目前唯一的骨合成类抗骨质疏松药物特立帕肽对大鼠股骨头坏死的治疗作用。研究方法：1.第一部分：全骨髓贴壁培养法分离并培养大鼠骨髓间充质干细胞并进行鉴定。采用慢病毒法携带microRNA-23a mimics,inhibitor,vector转染大鼠骨髓间充质干细胞。使用脂多糖(LPS,20μg/Kg×2)联合大剂量甲强龙(40mg/Kg×3)的方法建立大鼠激素性股骨头坏死动物模型。将miRNA-23a mimics, inhibitor, vector转染成功后的BMSC通过大鼠股骨髓腔注射的方法将干细胞悬液移植到大鼠激素性股骨头坏死模型体内。处死大鼠取大鼠股骨头行micro CT扫描及病理切片,观察股骨头坏死情况。免疫组化检测LRP-5在各组表达情况,观察股骨头坏死发生率和LRP5表达的差异。2.第二部分：首先应用脂多糖(LPS,20μg/Kg×2)联合大剂量甲强龙(40mg/Kg×3)的方法建立大鼠激素性股骨头坏死动物模型。大鼠造模成功后,实验组给予人重组甲状旁腺激素[rhPTH (1-34)] 20μg/Kg每天皮下注射,对照组给予等量生理盐水,治疗4周处死大鼠,取大鼠静脉血行血清骨转换标志物骨钙素检测,取双侧股骨头行micro CT检查,观察股骨头坏死情况及分析骨小梁参数,行病理切片观察骨坏死情况。研究结果：1.采用全骨髓贴壁培养法可快速简便的分离培养大鼠骨髓间充质干细胞,大鼠BMSC在体外可诱导成骨及成脂方向分化,流式细胞仪分析结果显示表面CD29,CD44,CD105,CD106 阳性, CD31,CD34,HLA-DR阴性。采用慢病毒法可成功转染大鼠BMSC。脂多糖(LPS,20μg/Kg×2)联合大剂量甲强龙(40mg/Kg×3)的方法可成功建立大鼠激素性股骨头坏死动物模型,造模成功率83%。Micro CT分析显示模型组股骨头出现囊变,骨小梁参数分析显示模型组骨小梁体积、骨小梁厚度等参数明显低于对照组。HE染色可观察到空骨陷窝形成、骨髓坏死、骨小梁断裂纤维组织修复。注射转染microRNA23a inhibitor BMSC组BV/TV、Tb.Th明显高于mimics组,而对照组介于两者之间,骨小梁数量及骨皮质厚度两组差异没有统计学意义。microPNA-23a mimics组股骨头坏死发生率明显高于inhibitor组(75%vs.18.2%,P=0.024),免疫组化显示microRNA-23a mimics组LRP5表达低于inhibitor组。2.人重组甲状旁腺激素[rhPTH(1-34)]20μg/Kg×28天治疗组股骨头坏死率为16.7%(2/12),对照组股骨头坏死率为75%(9/12),两组差异有统计学意义。(P0.05),骨小梁参数定量分析显示BV/TV、Tb.Th、BMD治疗组明显高于对照组。结论：1.microRNA-23a在大鼠体内能够调控BMSC成骨分化能力,从而对大鼠激素性股骨头坏死呈现不同程度的修复效应。抑制microRNA-23a,股骨头坏死发生率降低,骨小梁参数BV/TV、Tb.Th升高,LRP5表达升高,micRNA-23a表达升高则呈现相反的效应。2.人重组甲状旁腺激素[rhPTH (1-34)]20μg/Kg×28天皮下注射能够降低大鼠激素性股骨头坏死率,提高骨小梁体积等相关参数。
[Abstract]:Background: steroid induced osteonecrosis of the femoral head is a common disease in the Department of orthopedics. The rate of disability is high and the effect of conservative treatment is not good. The decrease of osteogenic differentiation of bone marrow mesenchymal stem cells is an important mechanism. In the earlier period, the microRNA chip technology was used to screen out the bone marrow of the patients with irritable necrosis of the femoral head and the normal healthy human bone marrow. The differential characteristics of miRNA expression profiles during the proliferation and differentiation of mesenchymal stem cells were compared with the miRNA expression profiles of normal healthy human bone marrow mesenchymal stem cells before and after the large dose of hormone stimulation, to find the miRNAs which was consistent with the expression of microRNA-23a, and to construct microRNA-23a mimics and inhibitor further. Up regulation of microRNA-23a can lead to osteogenic differentiation in bone marrow mesenchymal stem cells, and down regulation of microRNA-23a to promote BMSC osteogenesis. Using bioinformatics analysis and double luciferase reporter gene system to predict low density lipoprotein related protein 5 (low density lipoprotein receptor-related protein 5, LRP-5) is miRNA-23a The target gene, after shRNA silencing the LRP5 gene, can be observed that the osteogenic differentiation of BMSC is weakened and is similar to the up regulation of miRNA-23a. There is still no special drug for the necrosis and necrosis of the femoral head. The study found that intermittent small dose of parathyroid hormone can promote osteoblast formation and promote the differentiation of stem cells into osteogenic direction. Adenohormone [rhPTH (1-34)] is the only approved anti osteoporosis drug to promote bone synthesis at present. Except for the treatment of osteoporosis, it has shown good therapeutic effect in promoting fracture healing, promoting spinal fusion, promoting cartilage repair and mandibular necrosis. The pathogenesis of steroid induced osteonecrosis includes steroid suppression. We speculate whether recombinant parathyroid hormone [rhPTH (1-34)] can also promote osteonecrosis of the femoral head by promoting osteoblast formation and promoting osteonecrosis of stem cells to osteogenesis. Purpose: 1. to construct microRNA-23a mimics and inhibitor, and to transfer the bone marrow mesenchymal to cells. Rat bone marrow mesenchymal stem cells injected into the rat model of steroid necrosis of the femoral head. Evaluation of the effect of microRNA-2 3A on the repair of the femoral head necrosis in rats..2. research is the only therapeutic effect of the bone synthetic anti osteoporosis drug stranmpeptide on the necrosis of the femoral head in rats. 1. part 1: the first part: the whole bone marrow The rat bone marrow mesenchymal stem cells were isolated and cultured with adherent culture method. The rat bone marrow mesenchymal stem cells were transfected with microRNA-23a mimics, inhibitor and vector with lentivirus method. The rat model of steroid femoral head necrosis was established by using lipopolysaccharide (LPS, 20 u g/Kg x 2) combined with large dose of methylprednisolone (40mg/Kg x 3). After transfection of miRNA-23a mimics, inhibitor and vector, BMSC was injected into the rat femoral head necrosis model in rat femoral bone marrow cavity. The rat femoral head was killed by micro CT scan and pathological section, and the necrosis of the femoral head was observed. Immunohistochemistry was used to detect the expression of LRP-5 in each group. The difference between the incidence of osteonecrosis of the femoral head and the difference of LRP5 expression was observed in.2. second part: first, a rat model of steroid necrosis of the femoral head was established by using lipopolysaccharide (LPS, 20 mu g/Kg x 2) combined with large dose of methylprednisolone (40mg/Kg * 3). After the rat model was successful, the experimental group was given a recombinant parathyroid hormone [rhPTH (1-34)] 20 u g/Kg each. In the control group, the control group was given the same amount of normal saline, and the rats were sacrificed for 4 weeks, and the blood serum bone conversion markers were detected for 4 weeks. The bilateral femoral head was examined by the osteonecrosis of the femoral head and the parameters of the bone trabecula were observed. The results of pathological section were observed. 1. the results of the study were all bone marrow adherence. Rat bone marrow mesenchymal stem cells could be isolated and cultured quickly and easily. The rat BMSC could induce osteogenesis and lipid differentiation in vitro. Flow cytometry showed that the surface CD29, CD44, CD105, CD106 positive, CD31, CD34, HLA-DR negative. The rat BMSC. lipopolysaccharide (LPS, 20 mu g/Kg x 2) could be successfully transfected by the lentivirus method. The method of dose methylprednisolone (40mg/Kg x 3) could successfully establish the rat model of steroid femoral head necrosis. The success rate of the model group 83%.Micro CT analysis showed that the femoral head appeared cystic change in the model group. The bone trabecular parameter analysis showed that the size of the bone trabecular bone in the model group and the bone small Liang Houdu parameters were obviously lower than the control group.HE staining to observe the empty lacunae. Osteonecrosis, bone trabecular fracture and fibrous tissue repair. MicroRNA23a inhibitor BMSC group BV/TV, Tb.Th was significantly higher than group mimics, while the control group was between the two groups, the number of trabecular bone and the thickness of bone cortex of two groups were not statistically significant, the incidence of necrosis of the femoral head in.MicroPNA-23a mimics group was significantly higher than that of the inhibitor group (75%vs). .18.2%, P=0.024), immunohistochemical staining showed that the expression of LRP5 in group microRNA-23a mimics was lower than that of inhibitor group.2. human recombinant parathyroid hormone [rhPTH (1-34)]20 micron g/Kg x 28 days, the necrosis rate of femoral head was 16.7% (2/12), and the necrosis rate of the femoral head in the control group was 75% (9/12), and the quantitative analysis of the bone trabecular parameters showed that the two groups were statistically significant. TV, Tb.Th, BMD treatment group was significantly higher than the control group. Conclusion: 1.microRNA-23a can regulate the osteogenic differentiation ability of BMSC in rats, thus to the hormone induced osteonecrosis in rats to different degree of repair effect. Inhibition of microRNA-23a, the incidence of necrosis of the femoral head decreased, bone trabecular parameters BV/TV, Tb.Th, LRP5 expression increases, micRNA-23a, micRNA-23a. The increase in expression showed the opposite effect of.2. human recombinant parathyroid hormone [rhPTH (1-34)]20 mu g/Kg for 28 days, which could reduce the rate of steroid necrosis of the femoral head and improve the volume of trabecular bone.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R681.8
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本文编号：1856836