沉默PPARγ并表达CGRP的双基因联合调控BMSCs分化预防酒精性股骨头坏死的实验研究

The alcohol-induced osteonecrosis of femoral head(ONFH) is a common disease that can lead to physical disability in high rate. The effective preventive means are poor at present. The key in pathogenesis of the ONFH have indicated that alcohol can up-regulate expression of adipogenic gene PPARγ and down-regulate expression of osteogenic gene in the bone marrow mesenchymal stem cells (BMSCs), and then increase adipogenesis and reduce osteogenesis of the cells. We have demonstrated that siRNA can prevent alcohol-induced expression of PPARγ and adipogenic differentiation, and neuropeptide CGRP gene can promote the proliferation and osteogenic activity of the cells, which showed their respective strengths and preventive role. If the union of the two aspects may get redouble effect. In order to explore the higher efficient preventive measures with double effect that could inhibit the adipogenesis and promote the osteogenesis of the cell simultaneously, the project intends to construct double gene recombinant vectors silencing PPARγ and expressing CGRP. The vectors are transfected to rabbit BMSCs in vitro. The cells will be injected into the animal femoral head in vivo. It can suppress the expression of PPARγ and efficiently inhibit adipogenic differentiation of the cells, and directly up-regulate expression of CGRP and actively promote the osteogenic differentiation simultaneously, which play a role with double effects inhibited adipogenesis and promoted osteogenesis, and then higher efficiently prevent the onset of alcohol-induced ONFH from the key aspects. This innovative idea has a more important scientific significance and application prospects.

酒精性股骨头坏死是一种致残率很高的常见病，尚无有效预防方法。其发病机制的关键是酒精上调BMSCs内成脂基因PPARγ表达，下调成骨基因表达，细胞成脂增多且成骨减少。我们已证明siRNA能阻止酒精诱导细胞PPARγ表达及成脂分化，神经肽CGRP基因可促进细胞增殖及成骨活性，各居优势，均有预防作用。若将二者联合，可能倍增功效。为了探索既可阻止细胞成脂且能同时促进其成骨的双倍高效预防方法，本项目拟构建沉默PPARγ并表达CGRP的双基因重组载体，转染兔BMSCs，并植入ONFH模型股骨头内，靶向阻断细胞内致病基因PPARγ表达，高效阻止细胞成脂分化，同时上调其CGRP表达，直接主动促进成骨分化，达到抑制成脂和促进成骨的双重联合作用，从发病机制的初始关键环节上预防酒精性ONFH的发生。这一创新思路具有更加重要的科学意义和应用前景。

酒精性股骨头坏死发病机制的初始关键环节是酒精诱导股骨头细胞成脂增多且成骨减少。为了探索高效预防ONFH新方法，本项目构建沉默PPARγ并表达CGRP的双基因重组载体，转染兔BMSCs，并植入ONFH模型兔股骨头内，探讨其预防效果。. 实验结果显示：（1）成功构建出沉默PPARγ并表达CGRP的双基因重组载体pGFP-V-RS-siPPARγ-exCGRP。（2）细胞学实验：将单基因、双基因重组载体转染兔体外BMSCs，同时给予细胞0.09mol/L酒精。双基因组中：PPARγ基因表达量最低，显著低于模型组、无关序列组、exCGRP组(P=0.000)，稍低于正常组(P>0.05)；CGRP、Runx2、Osteocalcin基因表达量最高，显著高于模型组、无关序列组、siPPARγ组、正常组(P=0.000)；细胞ALP活性、骨钙素、层粘连蛋白、胶原含量均最高，明显高于其它各组(P=0.000)。细胞ALP染色胞浆深蓝，脂滴少。（3）动物学实验：将双基因重组载体转染兔BMSCs，植入酒精性ONFH模型兔股骨头内。双基因组中：股骨头细胞PPARγ低表达，接近正常组(P>0.05)，显著低于模型组、无关序列组(P<0.05)；CGRP、Runx2、Osteocalcin表达最高，显著高于其它各组(P<0.05)。双基因组骨组织结构接近正常组，无脂肪堆积，骨小梁面积分数、空骨陷窝计数、脂肪细胞平均直径与正常组接近(P>0.05)，坏死率显著降低(P<0.01)。 体内外实验结果证实：双基因重组载体能够靶向阻断兔体外BMSCs及ONFH模型活体股骨头细胞内成脂基因PPARγ表达，阻止细胞成脂分化，并遏制了股骨头内脂肪堆积；同时显著上调细胞CGRP和成骨基因Runx2、Osteocalcin表达，增高成骨活性，直接主动促进成骨分化，增强了股骨头内骨组织修复与重建，显著降低酒精性ONFH发生率。达到了抑制成脂和促进成骨的双重联合作用，显著优于单一基因作用。. 本研究证明了双基因重组载体pGFP-V-RS-siPPARγ-exCGRP调控BMSCs分化的优势功效，能够从发病机制初始关键环节上高效预防酒精性ONFH的发生，为阐明酒精性ONFH的发病机制和高效防治提供了重要的科学理论依据，这一创新科研思路具有重要的科学意义和应用研究前景。