【作者】 张丹；

【导师】 余学锋；

【作者基本信息】 华中科技大学 ， 内科内分泌， 2012， 博士

【摘要】 目的：基质细胞衍生因子-1 (Stromal cell-derived factor 1,SDF-1)是一种具有多种生物学功能的细胞因子,该因子能够介导细胞归巢,肿瘤转移和急性损伤中的组织再生,但SDF-1在慢性肾脏疾病如糖尿病肾病中的作用至今未得到广泛研究。系膜细胞周围的细胞外基质大量沉积是糖尿病肾病的主要病理生理机制之一,为了研究SDF-1在糖尿病肾病中的作用,我们使用高浓度葡萄糖和转化生长因子β1 (transforming growth factorβ1, TGF-β1)干预大鼠系膜细胞,使系膜细胞外基质增生,并观察了SDF-1对大鼠系膜细胞外基质的影响及其机制。方法：体外培养大鼠系膜细胞(HBZY-1),并用real-time RT-PCR方法检测了大鼠系膜细胞中的SDF-1α, CXCR4,纤维粘连蛋白(fibronectin, FN)的mRNA表达；高浓度葡萄糖,TGF-β1, SDF-1α干预大鼠系膜细胞指定时间后,使用real-time RT-PCR方法检测细胞CXCR4, FNmRNA的表达变化；使用TGF-β1、SDF-1α、磷脂酰肌醇-3-激酶(phosphatidylinositol-3-kinase, PI3K)特异性抑制剂LY294002和CXCR4特异性阻断剂AMD3100等,共同干预大鼠系膜细胞,并使用real-time RT-PCR方法检测FN mRNA表达的变化,以及免疫印迹技术Western Blot方法检测磷酸化Akt (phospho-Akt, pAkt),磷酸化Smad3 (phospho-Smad3, pSmad3)和FN蛋白的表达。结果：大鼠系膜细胞表达SDF-1α,CXCR4和FN的mRNA,并且TGF-β1和高浓度葡萄糖能够上调细胞中CXCR4mRNA的表达：TGF-β1和高浓度葡萄糖干预大鼠系膜细胞72小时后,系膜细胞的FN表达增高,同时加入SDF-1α干预细胞后,SDF-1α能够抑制TGF-β1和高浓度葡萄糖升高FN的效应；SDF-1α能够激活P13K/Akt途径,增加pAkt的蛋白表达,同时使用TGF-β1, SDF-1α以及LY294002干预大鼠系膜细胞时,SDF-1α对FN升高的抑制作用能够被LY294002完全阻断；在TGF-β1干预下,使用CXCR4阻断剂AMD3100只能部分阻断SDF-1α对大鼠系膜细胞FN积蓄的抑制作用；而在高浓度葡萄糖干预下,LY294002和AMD3100均能够完全阻断SDF-1α对FN的抑制作用。单独使用SDF-1α干预大鼠系膜细胞时,SDF-1α对细胞的FN mRNA表达没有影响。但是,单独使用LY294002干预大鼠系膜细胞时,细胞的FNmRNA表达升高,而AMD3100单独干预系膜细胞并不影响细胞FN mRNA的表达。结论：TGF-β1和高浓度葡萄糖均能够上调大鼠系膜细胞CXCR4和FN的表达,并且SDF-1α能够通过与CXCR4结合激活PI3K/Akt通路,从而降低TGF-β1和高浓度葡萄糖所诱导的大鼠系膜细胞的FN表达升高,这种可能的作用机制给糖尿病肾病的研究和治疗带来了新的思路和方向。更多还原

【Abstract】 Aims Stromal cell-derived factor 1 (SDF-1) is a chemokine with multiple roles in cell homing, tumor metastasis, angiogenesis and tissue regeneration in acute injuries, whereas the role of SDF-1 in diabetic nephropathy has not been illustrated distinctly. We hypothesized that SDF-la may have an effect on mesangial extracellular matrix (ECM) accumulations in mesangial cells exposed to TGF-β1 or high glucose in vitro and studied the mechanisms.Methods Rat mesangial cells (HBZY-1) were cultured in vitro and we tested the mRNA expressions of fibronectin (FN), CXCR4, SDF-1αin mesangial cells by RT-PCR; After incubated in high concentration of glucose or TGF-(31 and SDF-la for indicated times, we determined the expressions of CXCR4, FNmRNA in mesangial cells by real-time PCR and western blotting; We interferred the mesangial cells with PI3K inhibitor LY294002 or CXCR4 blocker AMD3100 at the same time, and determined the expressions of CXCR4, FNmRNA in mesangial cells by real-time PCR and FN, pSmad3, pAkt by western blotting.Results SDF-la, CXCR4, and FN mRNA were expressed in rat mesangial cells. Both TGF-β1 and high glucose raised the mRNA expressions of CXCR4 and FN in rat mesangial cells. After exposed to TGF-β1 and high concentration of glucose for 72h, the expression of FN was increased in mesangial cells, which can be reduced by SDF-1αwhen cells were incubated with SDF-la at the same time. SDF-la can activate the PI3K/Akt pathway, upregulate the pAkt expression, In mesangial cells exposed to TGF-β1 or high concentration of glucose and SDF-1α, the reduction of SDF-1αon elevated FN was blocked by LY294002. Meanwhile the CXCR4 blocker AMD3100 only partly inhibited the role of SDF-1α. In cells exposed to high concentration of glucose, CXCR4 blocker AMD3100 and LY294002 blocked the reduction of SDF-1αon the extracellular matrix accumulations. In mesangial cells incubated with SDF-1αalone, the expression of FNmRNA had no change. In mesangial cells incubated with LY294002 or AMD3100 alone, the expression of FNmRNA was elevated.Conclusions SDF-laameliorates TGF-β1/high glucose induced ECM accumulations in mesangial cells, which may mediated through PI3K/Akt pathway. This novel mechanism may bring a new insight into our understandings of diabetic nephropathy.更多还原