肝细胞生长因子对ADPKD囊肿衬里上皮细胞合成细胞外基质、基质金属蛋白酶及其抑制剂的作用

目的；观察重组人肝细胞生长因子（rhHGF)对常染色体显性遗传性多囊肾病（ADPKD)囊肿衬里上皮细胞合成细胞外项质（ECM），基质金属蛋白酶及其抑制剂的作用。方法：采用放免法测定细胞合成层粘连蛋白（LN）和Ⅲ型前胶原氨基末端肽（PⅢNP），采用ELISA法检测细胞合成Ⅳ型胶原(ColⅣ），采用RT－PCR方法测定细胞表达转化生长因子β1（TGFβ1），基质金属蛋白酶2（MMP－2），金属蛋白酶组织抑制剂1（TIMP－1），金属蛋白酶组织抑制剂2（TIMP－2）mRNA的情况，用蛋白质印迹方法检测细胞上清液中MMP－2蛋白表达，明胶酶谱法检测MMP－2活性。结果：rhHGF及rhHGF 抗TGFβ1抗体组ADPKD囊肿衬里上皮细胞合成LN，ColⅣ显著增多，但两组比较无显著差异。对照组，rhHGF组，rhHGF 抗HGF抗体组及rhHGF 抗TGFβ1抗体组各间合成PⅢNP值均无显著差异。对照组，rhHGF组，rhHGF＋抗HGF抗体组各组比较TGFβ1mRNA表达无显著差异。与对照比较，rhHGF显著上调细胞MMP－2 mRNA和细胞上清液中MMP－2蛋白表达及MMP－2活性，显著下调细胞TIMP－1，TIMP－2 mRNA表达。结论：HGF促进了ADPKD囊肿衬里上皮细胞合成LN和ColⅣ，增加MMP－2表达，抑制TIMP－1，TIMP－2的表达。这些改变可能参与了ADPKD囊肿的发生及发展。

Effects of hepatocyte growth factor on synthesis of extracellular matrix and matrix metalloproteinases and tissue inhibitor of metalloproteinases in autosomal dominant polycystic kidney disease cyst-lining epithelial cells

Objective:To study the effects of recombinant human hepatocyte growth factor (rhHGF) on the synthesis of extracellular matrix (ECM) and matrix metalloproteinases and tissue inhibitor of metalloproteinases in autosomal dominant polycystic kidney disease (ADPKD) cyst-lining epithelial cells.Methods:The synthesis of laminin (LN) and aminoterminal propeptide of type Ⅲ procollagen (PⅢNP) in ADPKD cyst-lining epithelial cells stimulated by rhHGF was examined with radioimmunoassay.The synthesis of type Ⅳ collagen (ColⅣ) was analyzed with ELISA.The mRNA expression of transforming growth factor β1 (TGFβ1),matrix metalloproteinases-2 (MMP-2),tissue inhibitor of metalloproteinases-1 (TIMP-1) and TIMP-2 in rhHGF stimulated cyst-lining epithelial cells were detected by RT-PCR.MMP-2 protein expression was examined with Western blotting and MMP-2 activity was analyzed by gelatin zymography in supernatant of cyst-lining epithelial cells stimulated by rhHGF.Results:In rhHGF group and rhHGF+ anti-TGFβ1 antibody group,the synthesis of LN and ColⅣ were markedly increased.There was no significant difference in the synthesis of LN and ColⅣ between the 2 groups.Among control group,rhHGF group,rhHGF+ anti-HGF antibody group and rhHGF+ anti-TGFβ1 antibody group,no significant difference in the synthesis of PⅢNP was found.No significant difference was found in the expression level of TGFβ1 mRNA in cyst-lining epithelial cells among control group,rhHGF group and rhHGF+anti-HGF antibody group.Compared with control group,MMP-2 mRNA expression in ADPKD cyst-lining epithelial cells was significantly increased and TIMP-1 mRNA and TIMP-2 mRNA expression were significantly decreased in rhHGF group.Furthermore,MMP-2 protein expression and MMP-2 activity in supernatant of cyst-lining epithelial cells also greatly increased.Conclusion:HGF stimulates the synthesis of LN and ColⅣ.HGF up-regulates MMP-2 expression while HGF down-regulates TIMP-1 and TIMP-2 expression in ADPKD cyst-lining epithelial cells.All these changes may involve in the initiation and progression of ADPKD cysts.