盆底组织成纤维细胞Mfn2表达与盆腔器官脱垂的机制研究

The most of studies on pelvic organ prolapse(POP) focus on the extracellular matrix, for example, the lower expression of collagen in tissue, but not the abnormalities of cells that secrete the pro-collagen(e.g. fibroblasts). Mitofusin2(Mfn2) is a protein that mediate the fusion of mitochondria, participate signal transduction, proliferation and apoptosis of cells.Mfn2 is also associated with cell aging. So it is very important to indicate how Mfn2 affect the function of fibroblast. Our previous study showed that in the postmenopausal women, the expressions of procollagen 1A1/1A2/3A1 in pelvic floor fibroblast of POP were lower than that in patients without POP, but the expression of Mfn2 of POP was higher than that of non-POP patients. So hypotheses that Mfn2 may affect the proliferation and apoptosis and procollagen secretion of fibroblast by regulating mitochondria fusion and signal transduction of mitochondria was emerged. Therefore, we prepare to culture the fibroblast of pelvic floor tissue of POP and non-POP patients, at the same time, eukaryotic expression vectors of Mfn2 will be constructed to overexpress and inhibit the expression of Mfn2 in fibroblast, then the proliferation and apoptosis and procollagen 1A1/1A2/3A1 secretion of fibroblast and mitochondrial morphology will be detected. We aim at the pathogenesis of POP by the research in vivo & in vitro and the overall level & cellular level. This study will provide a theoretical basis for clinical prevention and treatment in POP.

目前对盆腔器官脱垂（POP）的实验室研究多集中在细胞外基质，如组织中胶原含量变化方面，而对产生这些胶原的成纤维细胞的功能变化研究很少。线粒体融合蛋白2（Mfn2）可介导线粒体融合，参与细胞信号转导、调节细胞增殖及凋亡等过程，并与细胞老化相关，因此，明确Mfn2如何影响成纤维细胞的功能意义重大。我们的前期研究发现，POP患者盆底组织成纤维细胞中I、III型前胶原蛋白表达显著低于非POP患者，而Mfn2表达显著增高，推测Mfn2通过调节线粒体融合或信号转导，影响成纤维细胞的增殖和凋亡以及前胶原蛋白合成。因此，我们拟培养POP和非POP患者盆底组织成纤维细胞，同时构建Mfn2真核表达载体，在成纤维细胞中过表达和抑制表达Mfn2，检测成纤维细胞增殖和凋亡以及I、III型前胶原蛋白表达量及线粒体形态，以证实Mfn2对成纤维细胞的影响。通过在体和离体、整体和细胞不同水平的研究探讨POP的发病机制。

目前对盆腔器官脱垂（Pelvic organ prolapse，POP）的研究集中在细胞外基质，很少有人关注产生细胞外基质的成纤维细胞。线粒体融合蛋白2（Mitofusin 2，Mfn2）作为一种和老化相关的蛋白，和老化相关疾病--POP的关系鲜有报道。我们既往体内试验证实POP患者骶韧带成纤维细胞Mfn2表达升高，在此基础上，我们通过培养POP和非POP患者盆底组织成纤维细胞，检测两组细胞中Mfn2和前胶原蛋白的表达差异。同时构建慢病毒表达载体，在成纤维细胞中过表达和抑制表达Mfn2，检测成纤维细胞增殖和凋亡以及I、III型前胶原蛋白表达量，结果为POP组体外培养的成纤维细胞Mfn2表达升高，而I，III型前胶原蛋白表达下降，在非POP组成纤维细胞中转染慢病毒过表达载体后，Mfn2表达上升，而前胶原蛋白表达下降，且成纤维细胞的增殖被抑制，凋亡增加。而在POP组抑制Mfn2表达后得到了相反的结果，从而通过细胞水平的研究进一步揭示了Mfn2在POP发生发展过程中作用。另外，老化相关基因--线粒体 DNA 4977缺失情况与POP的关系研究甚少。本课题组通过研究POP患者子宫骶韧带组织和mtDNA4977缺失的变化，证实DNA4977缺失在POP患者中显著增加。为揭示POP的发病机制提供了理论依据。