【摘要】：目的探討Wnt3a對小鼠肝前體細(xì)胞發(fā)生上皮-間質(zhì)轉(zhuǎn)化的影響。方法將表達(dá)Wnt3a的腺病毒Ad-GFPWnt3a轉(zhuǎn)入小鼠肝前體細(xì)胞中,與空白對照組相比,觀察其形態(tài)變化。細(xì)胞劃痕實(shí)驗(yàn)和細(xì)胞遷移實(shí)驗(yàn)觀察其對小鼠肝前體細(xì)胞遷移能力的影響。實(shí)時熒光定量Real-time PCR和Western blot分別檢測小鼠肝前體細(xì)胞中上皮標(biāo)志物和間質(zhì)標(biāo)志物的表達(dá)改變。結(jié)果鏡下觀察,高表達(dá)Wnt3a的小鼠肝前體細(xì)胞由不規(guī)則多邊形變?yōu)殚L梭形。細(xì)胞劃痕實(shí)驗(yàn)結(jié)果顯示,Ad-Wnt3a組細(xì)胞48h遷移距離為(0.53±0.05)mm,Ad-GFP組細(xì)胞48 h遷移距離為(0.33±0.02)mm,相對對照組,其遷移距離增加,差異具有統(tǒng)計學(xué)意義(P0.05)。細(xì)胞遷移實(shí)驗(yàn)結(jié)果顯示,Ad-GFP組24 h穿過小孔的細(xì)胞為(10.33±2.08)個,而Ad-Wnt3a組穿過的細(xì)胞為(62.00±3.60)個,相對對照組,其穿過小孔的細(xì)胞數(shù)量增加,差異具有統(tǒng)計學(xué)意義(P0.01)。RT-PCR和Western blot結(jié)果顯示,間質(zhì)標(biāo)志物N-cadherin、vimentin和snail的mRNA和蛋白水平表達(dá)上調(diào),相反上皮標(biāo)志物E-cadherin和CK-18的mRNA水平和蛋白水平表達(dá)下調(diào),差異具有統(tǒng)計學(xué)意義(P0.05)。結(jié)論 Wnt3a能夠促使小鼠肝前體細(xì)胞發(fā)生上皮-間質(zhì)轉(zhuǎn)化,提示其可能參與了肝纖維化的發(fā)展進(jìn)程。
[Abstract]:Objective to investigate the effect of Wnt3a on epithelial-interstitial transformation of mouse hepatic precursor cells. Methods adenovirus Ad-GFPWnt3a expressing Wnt3a was transferred into mouse liver precursor cells and compared with the blank control group, the morphological changes were observed. Cell scratch test and cell migration test were used to observe their effects on the migration ability of mouse liver precursor cells. Real-time fluorescent quantitative Real-time PCR and Western blot were used to detect the expression of epithelial markers and interstitial markers in mouse liver precursor cells. Results under microscope, the mouse liver precursor cells with high expression of Wnt3a changed from irregular polygons to fusiform. The results of cell scratch test showed that the 48-hour migration distance of cells in Ad-Wnt3a group was (0.53 鹵0.05) mm, and the migration distance of cells in mm,Ad-GFP group was (0.33 鹵0.02) mm, which was higher than that in control group. The difference was statistically significant (P0.05). The results of cell migration test showed that the number of cells passing through the pores in Ad-GFP group was (10.33 鹵2.08), and that in Ad-Wnt3a group was (62.00 鹵3.60). Compared with the control group, the number of cells traversing through micropores increased, and the number of cells passing through micropores in Ad-Wnt3a group was significantly higher than that in control group (P < 0.05). The results of RT-PCR and Western blot showed that the expression of mRNA and protein were up-regulated in stroma markers such as N-cadherin, vimentin and snail. On the contrary, the levels of mRNA and protein of epithelial markers E-cadherin and CK-18 were down-regulated, the difference was statistically significant (P0.05). Conclusion Wnt3a can promote epithelial-interstitial transformation of hepatic precursor cells in mice, suggesting that it may be involved in the development of hepatic fibrosis.
【作者單位】： 重慶醫(yī)科大學(xué)分子醫(yī)學(xué)與腫瘤研究中心;重慶醫(yī)科大學(xué)生物化學(xué)與分子生物學(xué)教研室;重慶醫(yī)科大學(xué)病理生理學(xué)教研室;
【基金】：國家自然科學(xué)基金(81071770,81201679)~~
【分類號】：R329

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