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中药三棱及土鳖虫对多囊肾病衬里上皮细胞增殖和信号转导作用的研究
【作者】 徐成钢；
【导师】 梅长林；
【作者基本信息】 第二军医大学 ， 肾脏内科， 2001， 博士
【摘要】 本研究以FuGene6介导SV40转染原代培养的常染色体显性遗传多囊肾病 (ADPKD)囊肿衬里上皮细胞，建立囊肿衬里上皮细胞株。并以该细胞株为研 究对象，采用 Brdu掺入法、流式细胞仪检测以及Western blot等分子生物学方 法，分别观察了 EGF、酪氨酸激酶抑制剂 Compond 32、中药三棱与土鳖虫对囊 肿衬里上皮细胞株增殖的影响，以及它们对衬里上皮细胞株上皮生长因子受体 (EGFR)与有丝分裂原激活蛋白激酶(MAPK)等信号转导蛋白磷酸化的影响。厂 结果表明：①经SV40转染的原代囊肿衬里上皮细胞成为连续细胞系，并经有 限稀释法获得单克隆细胞株，其生物学特性与原代细胞无明显差异；②与末处 理组比较，EGF(2．5～20ng／ml)可刺激衬里上皮细胞株增殖(P<0.01)；③与对照 组比较，Compond 32(0.2～10umol／L)可抑击 EGF激活的衬里上皮细胞株增殖 (P＜0.01)；④与对照血清比较，三棱、士鳖虫含药血清(1％～5％能明显抑制经EGF 刺激后的衬里上皮细胞株增殖(p＜0.01)，其作用效果呈浓度依赖性；⑤Compond 32、三棱、土鳖虫含药血清能抑制囊肿衬里上皮细胞株EGFR、MAPK的磷酸化，与对照组相比较(P<0.05)。结果提示：多囊肾病囊肿衬里上皮细胞株可作 为多囊肾病发病与治疗机制研究的良好的体外实验模型；EGF等多肽生长因子 在多囊肾病发病中起促进作用，Compond 32、三棱、土鳖虫可能通过对 EGF信 ’t 号转导途径的于预而达到抑制细胞增殖的作用，它们可抑制多囊肾病的发生与 发展。
【Abstract】 Autosomal-dominant polycystic kidney disease is an inherited disorder usually manifest in adulthood and essentially characterized by the development of multiple renal cysts variably associated with extrarenal(mainly hepatic and cardiovascular) abnormalities. Traditional chinese medicine is an important method to treat ADPKID.To investigate the effect of chinese herb on autosomal dominant polycentric kidney disease(ADPKD) cystic-lining epithelial cells, we built up a culture method of primary culture of ADPKD cystic-lining epithelial cells in vitro and transfected the SV4O into primary cultured cells in order to establish a cell strain. Then, we observed the effect of epidermal growth factor(EGF), Compond 32(PTK inhibitor), Sparganium stoloniferum Buch Ham(SBH) and Eupolyphaga sinensis Walker(EW) on both phosphorylation of EGFR, mitogen-activated protein kinase(MAPK) and proliferation of cystic-lining epithelial cell sti1ain by Brdu in corporation assay, flow cytometer detect and w~鐂tern blot method. The resultS show: ~D there was no significant difference in the cell biological characteristics found between cell strain and primary cultured cells; ~ EGF with the concentration from 2.5 to 2Ong/ml had a stimulating effect on the cyst-lining epithelial cells compared with untreated control 2 98 ~ group(p < 0.01); ?Compond32(0.2?.Oumol/L) could significantly inhibit the prolifration of cyst-lining epithelial cells actived by EGF(lOng/ml) compared with treated with EGF(lOng/ml) only(p ~ 0.01); ?SBH and EW serum(1%?%) could significantly inhibit the prolifration of cyst-lining 昬pithelial cells actived by EGF(1 OngIml) compared with EGF(lOng/ml) plus untreated serum(1%~5%) group (p < 0.01);?Compond32, SBH and EW could inhibit phosphorylation of EGFR and MAPK in cyst-lining epithelial cells stimulated by EGF. The result suggest that ADPKD cyst-lining epithelial cell strain can be established by SV4O transfect, and the cell strain can be used as a kind of cell model in the research of ADPKD cellular and molecular mechamism; EGF maybe accelerate the ADPKD development; Compond 32,EW and SBH might have beneficial effects on prolong the progression of ADPKD.