建立人胰腺星状细胞(hPSC)系研究维生素D及其受体通路在酒精性慢性胰腺炎抑制hPSC介导胰腺纤维化的作用和机制

The alcohol chronic pancreatitis (ACP) has been causing great harm to human health, but no effective therapies exist. The pathogenesis of ACP has so far not been clarified clearly. Previously ours studies indicated that activated rat pancreatic stellate cells (PSC) synthesized almost all of the extracellular matrix of the pancreas and transforming growth factor β1(TGF-β1) played a key role in pancreatic fibrogenesis. Recent international studies show that vitamin D (VD) via its receptor (VDR) inhibits mice pancreatic fibrosis. However, the inhibitory effect of VD-VDR signaling pathway on human pancreatic fibrosis has not yet been clarified. Recently primary hPSCs were polycloned and immortalized by using our rat PSC line technique. In this study we will create a stable activated immortalized hPSC line by screening cell clones and characterization of the selected cell line. We will determine the effect of VD on the suppression of TGF-β1-induced fibrogenic gene expression, measure VD-VDR signal transduction and gene regulation. The key factors and fibrotic markers related to VDR signaling pathway in hPSCs will be determined from patients with ACP to clarify the relationship beteen their contents and the degrees of pancreatic fibrosis. The deficient status of vitamin D and the effect of exogenous VD in lowering serum LPS levels and fibrosis marker contents will also be evaluated. This study will provide an experimental basis for anti-pancreatic fibrosis of vitamin D in human ACP.

酒精性慢性胰腺炎(ACP)对人们危害极大，无有效疗法，发病机制尚不明确。我们先前的研究显示大鼠胰腺星状细胞（PSC）合成胰腺几乎全部的细胞外基质， TGF-β1在PSC介导胰腺纤维化发挥关键作用。近来国外研究表明维生素D(VD)通过其受体(VDR)通路可抑制小鼠胰腺纤维化。然而VD-VDR通路在ACP抑制人胰腺纤维化的作用及机制研究亟待进行。最近，采用我们建立大鼠PSC系技术成功实现了原代人PSC(hPSC)的多克隆转化和永生化。本研究拟进行细胞克隆筛选和鉴定，建立稳定的hPSC系；研究VD抑制TGF-β1诱导hPSC致纤维化作用，明确VD-VDR信号传导和基因调控模式；检测ACP患者PSC内VDR通路的关键因子和纤维化标志物，明确其与胰腺纤维化的关系；明确ACP患者VD的缺乏状态及外源性VD降低ACP患者血清LPS水平和纤维化标志物的作用，为VD通过抗胰腺纤维化而防治ACP提供实验依据。

(1) 人胰腺星状细胞（PSCs）在慢性胰腺炎(CP)的纤维化过程中起着重要作用。然而，原代PSCs在体外的使用寿命较短，严重影响了胰腺纤维化细胞发生学的研究。我们采用RSV启动子/增强子驱动SV40 T抗原在原代活化人PSCs中的表达成功建立了稳定的永生化人PSC系(HP-1)。HP-1细胞在接受TGF-β1刺激后MMP-1/2 mRNAs或蛋白的表达以及蛋白质组学方面与激活的人PSCs相似，该细胞还具有高的转染活性。因此，HP-1细胞可以可作为PSC介导的胰腺纤维化研究的有效工具。. (2) ACP患者血清维生素D（VD）水平及其与胰腺纤维化的关系尚不清楚。采用串联质谱（LC-MS/MS）法检测38例ACP患者和30例健康对照者血清VD3水平，ELISA法检测TGF-β1、Col 1α1水平。双免荧光标记法检测ACP患者维生素D受体(VDR)阳性PSC的数量，分析其与胰腺纤维化关系，4D液相色谱-质谱联用(4D-LFQ)法检测和评价VD拮抗TGF-β1诱导PSC致纤维化蛋白表达谱。结果显示： ACP患者血清VD3水平均降低，VD3降低与TGF-β1和Col 1α1升高呈负相关，VDR阳性PSC数与ACP胰腺纤维化程度正相关，VD可有效抑制TGF-β1作用下PSC介导的22种致纤维化蛋白的表达。 本研究提示ACP患者VD降低是胰腺纤维化的易患因素，VD可靶向PSC抑制胰腺纤维化的发生。. (3) 白细胞介素-6(IL-6)参与TGF-β1的产生和大鼠PSC的激活。然而，IL-6促进人PSC激活和Col1的产生的机制尚不清楚。本结果显示： IL-6可促进PSC内α-SMA或Col1a1蛋白的产生，TGF-β1抗体可阻断IL-6诱导PSC内Smad2/3磷酸化和α-SMA和Col1 mRNA转录。TGF-β1 干扰RNA可拮抗α-SMA和Col1a1 mRNA表达。该研究表明IL-6通过上调TGF-β1/Smad2/3途径参与PSC的激活和Col1合成。. (4)内毒素脂多糖（LPS）在酒精性胰腺炎发生的作用不完全清楚。我们采用Lieber-DeCarli酒精(ALC)液体饮食饲喂Sprague-Dawley大鼠10周，最后3周用或不用每周1次LPS挑战。用大鼠巨噬细胞(MφS)和PSCs(RP-2细胞系）进行体外研究。结果表明： LPS通过旁分泌和自分泌