活骨灌注液介导microRNAs调节BMSCs定向分化治疗股骨头坏死的机制研究

Lipid metabolism disorder is one of the main reasons of osteonecrosis of the femoral head (ONFH). It has been reported that suppression of bone marrow stem cells (BMSCs) differentiation into adipocytes could relieve ONFH, miRNAs could regulate the differentiation of BMSCs and the expression of miRNAs changed in ONFH.Our studies demonstrated that intra-articular hip injection of Huogu injection could relieve ONFH and played a role of bone protection by increasing the number of bone cells and reduce the number of fat cells. Furthermore, we also demonstrated that the expression of miR196a5p and miR34c5p were up-regulated in the group of Huogu injection. In this project, we will further study that Huogu injection regulate the expression of miRNAs to conservatively treat of ONFH by inhibition the adipogenic differentiation of BMSCs and promoting the osteogenic differentiation of BMSCs in cell and animal model through lentivirus interference, electron microscopy and other techniques. What more, the chip technology will be used to screen miRNAs, which regulate the osteogenic/adipogenic differentiation of BMSCs. Based on the study that Huogu injection regulate the osteogenic/adipogenic differentiation of BMSCs,this project will further approve the theorety that nourishing kidney and activating blood will promote the osteogenic differentiation of BMSCs , reveal the mechanism of BMSCs differentiation in Huogu injection by miRNA,and elucidate the high effective and initial therapeutic targets for Treatment of ONFH with Treatment of ONFH Huogu injection.Based on the study of regulation of miRNA，this project will elucidate the mechanism of preventing and reating ONFH from upstream target.

脂质代谢紊乱是股骨头坏死(ONFH)的主要原因之一。研究显示：抑制骨髓干细胞(BMSCs)成脂分化能缓解ONFH；miRNAs调控BMSCs分化；ONFH的miRNAs表达变化。活骨灌注液髋关节灌注缓解ONFH，发挥“保头”治疗作用；并增加骨细胞数，减少脂肪细胞数，骨髓组织miR196a5p和miR34c5p表达上调。 本项目将采用细胞及动物模型运用慢病毒干扰、电镜等技术，进一步研究活骨灌注液调节miRNAs表达抑制BMSCs成脂分化，促进BMSCs成骨分化发挥保头治疗作用；采用芯片技术筛选调控BMSCs成脂/成骨分化miRNAs。通过活骨灌注液对BMSCs成骨、成脂分化的研究,进一步证实“补肾活血法”促进BMSCs的成骨分化理论依据；从miRNA角度揭示活骨灌注液调节BMSCs分化的机制，明确活骨灌注液治疗股骨头坏死高效与初始靶点，通过miRNA调控达到从上游靶点防治股骨头坏死的目的。

保头治疗是激素性股骨头坏死（SIONFH）目前研究的热点。miRNAs 调控 BMSCs的增殖及定向分化在SIONFH中发挥重要的作用，前期证实活骨灌注液髋关节腔灌注对SIONFH具有治疗作用，而对miRNAs的研究对明确药物的治疗的上游的靶点有着重要意义。.筛选miRNAs后，获取BMSCs鉴定后，构建miR-195-5p、miR-34c-5p的过表达/干扰慢病毒载体感染BMSCs，对miR-195-5p、miR-34c-5p的功能进行验证；利用生物信息学的方法预测miR-195-5p与Axin2靶基因的靶位点，miR-34c-5p与MDM4靶基因的靶位点，双荧光素酶实验进行验证。结果：miR-195-5p促进BMSCs细胞成骨分化，miR-34c-5p促进BMSCs细胞成脂分化。.筛选出2-6浓度的活骨灌注液对BMSCs成骨分化的诱导、成脂分化的抑制作用最强。并对活骨灌注液调控BMSCs细胞成骨成脂相关miRNA的筛选。.通过慢病毒构建技术对miR-195-5p的靶基因Axin2以及miR-34c-5p靶基因MDM4进行过表达、干扰，并对其进行功能研究，结果发现miR-195-5p干扰慢病毒以及Axin2过表达慢病毒能够抑制活骨灌注液的成骨分化诱导作用， miR-34c-5p过表达慢病毒以及MDM4干扰慢病毒能够抑制活骨灌注液的成脂分化抑制作用。.发现活骨灌注液通过调控miR-195-5p、miR-34c-5p缓解大鼠股骨头坏死疾病进程，提示miR-195-5p、miR-34c-5p可能为治疗股骨头坏死潜在调节分子，并发现对活骨灌注液中中药单体对治疗股骨头坏死的潜在通路。.课题为全面揭示活骨灌注液调控BMSCs细胞成骨/成脂分化的分子机制奠定了有力的实验基础，为活骨灌注液合理应用及相关靶点应用于临床转化医学奠定理论基础，提供了后续研究新的思路，具有十分重要的理论意义和应用价值。