AGEs对子宫脱垂盆底成纤维细胞的作用和机制的研究

Advanced Glycated End-products (AGEs) and its receptor (RAGE) participated wildly in the metabolism of collagen in human body, involved in the conditions such as Diabetes and skin aging. It is shown that AGEs increased in pelvic of POP patients with the decreasing of collagen and weakening of strength for pelvic connective tissues, AGEs also might boost the apoptosis of fibroblast and might inhibit the synthesis of collagen. Aiming to clarify the impact of AGEs on the fibroblast of POP and its mechanisms study as following: 1, Fibroblasts of POP, normal control and the RAGE inhibited groups will be primary cultured and identified, then the AGEs impact on behaviors (apoptosis, contractility, Attachment ) of fibroblasts will be detected; 2,the product about the metabolisms of collagen, such as collagen 1,3,5, elastin， MMP-1，2，8，9，13， TIMP-1，TGF-B3, will be detected by WB and Rt-PCR after AGEs activation; 3, the possible cell signaling pathway of AGEs-RAGE-MAPK-NF-kB will be screened, and other pathway also will be investigated; 4, After re-damage and re-repair are simulated by new animal model, for which the absorbable mesh of Biodesign were implanted in abdominal wall manual defect in SD rats, the effect of AGEs on fibroblast cell behaviors and collagen metabolism will be verified again.

晚期糖化终末产物（AGEs）及受体RAGE广泛地影响了成纤维细胞的功能和胶原纤维的代谢平衡，涉糖尿病等情况。研究显示子宫脱垂盆底组织AGEs↑，胶原纤维↓；AGEs↑可能加速盆底成纤维细胞的凋亡和抑制胶原纤维的合成。为明确AGEs对脱垂盆底成纤维细胞的作用和机制，拟研究如下：一、检测AGEs对脱垂组、对照组、RAGE受体阻断组盆底成纤维细胞细胞行为的影响（增殖与凋亡、粘附和收缩能力）；二，利用WB、RT- PCR检测AGEs对成纤维细胞胶原纤维合成和分解代谢的影响与区别，即1、3、5型胶原，弹性蛋白，1、2、8、9、13型MMP，TIMP-1，TGF-B3的表达；三，检验AGEs-RAGE-MAPK-NK-kB通路影响胶原纤维的信号机制，并探讨其他可能的信号通路；四，利用SIS生物网片植入大鼠腹壁人工缺损,模拟结缔组织再损伤与再修复，验证AGEs-RAGE通路对成纤维细胞及胶原纤维的影响。

老年蛋白糖化终末产物（Advanced Glycated End-products, AGEs）和其受体 RAGE结合后，参与胶原的代谢。本项目探讨了AGEs及其受体与POP的关系和相关机制。首先，将盆底成纤维细胞进行原代培养、鉴定，采用SRB法、CCK-8等方法检测AGEs对成纤维细胞增殖、凋亡、黏附、收缩功能的影响，了解AGEs对于成纤维细胞合成和降解胶原纤维的变化，发现POP成纤维细胞更容易出现调亡，合成分泌I型胶原纤维下降，MMP-1上升。其次，采用反向蛋白芯片筛查，发现MAPK、AKT、PI3K等信号分子发生变化；再用WB、PCR的方法检测AGEs作用POP成纤维细胞信号通路的变化。分别用SiRNA、SB203580、PDTC阻断RAGE、MAPK-p38、NF-kB-p65信号分子，发现阻断RAGE后，其下游的P38，NF-kB被阻断。再其次用同样的方法验证AGEs、AKT信号通路，并且将成纤维细胞与人脐带间充质干细胞（UMSCS）共培养，发现UMSCS和减弱AGEs对成纤维细胞的抑制作用，而且可以通过AGES-RAGE-AKT-PI3K/PTEN信号途径。最后用雌性大鼠造模，形成腹壁缺损，然后植入可降解SIS网片和不可吸收聚丙烯网片，观察缺损部位修复情况和局部组织AGEs与修复之间的关系，发现SIS网片仿真度好、柔软，但出现膨出较其他组增多；而且检测到修复部位collagen I含量最低、AGEs 含量最高。因此，AGEs容易使POP成纤维细胞凋亡，更容易使collagenI下降，MMP-1上升，并通过激活P-p38、P-p65，或者AKT、PI3K来完成。在动物模型，AGEs可抑制损伤局部胶原纤维的合成、加速降解，妨碍胶原的代谢。本研究为了解POP提供了理论依据，即AGE-RAGE通路通过抑制胶原纤维合成、促进胶原纤维的降解，进一步促进了POP的发生、发展。