调控Smad7基因对瘢痕疙瘩角质形成细胞上皮-间质转化的影响

Effects of Regulating Smad7 Gene on Epithelial-Mesenchymal Transition in Keloid Keratinocyte

目的 阐述Smad7对瘢痕疙瘩角质形成细胞上皮-间质转化(epithelial-mesenchymal transition,EMT)的影响.方法 培养瘢痕疙瘩角质形成细胞(KK细胞)和正常皮肤角质形成细胞(NK细胞),构建Smad7过表达慢病毒载体及Smad7干扰慢病毒载体,筛选最佳过表达和干扰慢病毒以及对照载体分别感染NK和KK细胞,嘌呤霉素筛选稳定表达细胞株,将培养细胞分为8组:NK-Control(正常培养的NK细胞);NK-NC(对照慢病毒筛选的NK细胞);NK-shSmad7(干扰慢病毒筛选的NK细胞);NK-mSmad7(过表达慢病毒筛选的NK细胞);KK-Control(正常培养的KK细胞);KK-NC(对照慢病毒筛选的KK细胞);KK-shSmad7(干扰慢病毒筛选的KK细胞);KK-mSmad7(过表达慢病毒筛选的KK细胞).CCK-8法观察细胞的增殖情况,流式细胞仪检测细胞的凋亡,Transwell小室检测细胞的迁移能力,Western blot检测上皮-间质转化的关键蛋白(N-cadherin和Occludin)表达情况.结果 成功构建Smad7干扰慢病毒载体和Smad7过表达慢病毒载体.干扰Smad7可促进NK细胞和KK细胞增殖和迁移能力,并抑制KK细胞的凋亡,但对NK细胞的凋亡无明显影响(P>0.05);过表达Smad7可抑制NK细胞和KK细胞的增殖和迁移能力,并促进其凋亡.干扰慢病毒感染后,与NC组比较,NK细胞和KK细胞Occludin蛋白表达减弱(P<0.01),KK细胞N-cadherin蛋白表达增多(P<0.01),但NK细胞N-cadherin蛋白表达无明显变化(P>0.05);过表达慢病毒感染后,与NC组比较,NK和KK细胞Occludin蛋白表达增多(P<0.05),NK细胞的N-cadherin蛋白表达降低(P<0.05),但KK细胞N-cadherin蛋白表达无明显变化(P>0.05).结论 Smad7基因的调节可以影响正常皮肤角质形成细胞和瘢痕疙瘩角质形成细胞中的EMT,进而调控细胞的增殖、迁移、凋亡的能力.Smad7基因的调节对瘢痕疙瘩角质形成细胞中EMT的影响大于对正常皮肤角质形成细胞中EMT的影响.

Objective The effect of Smad7 on epithelial-Mesenchymal Transition(EMT)of keloid keratinocytes was studied.Methods Culture formed keloid cutin cells(KK)and normal skin cutin cell(NK cells),built the Smad7 too slow virus slow virus vector and Smad7 interference expression vector,screening the best expression and interfering with the slow virus infection NK and KK cells respectively,and contrast carrier puro screening stable expression cell lines,stem cells can be divided into 8 groups:NK-Control(normal training of NK cells);NK-NC(NK cells screened against lentivirus);NK-shSmad7(NK cells that interfere with lentivirus screening);NK-mSmad7(NK cells screened for overexpression of lentivirus);KK-control(normal cultured KK cells);KK-NC(KK cells screened against lentivirus);KK-shSmad7(KK cells that interfere with lentivirus screening);KK-mSmad7(KK cells screened for overexpression of lentivirus).Cell proliferation was observed by the CCK-8 method,cell apoptosis was detected by flow cytometry,cell migration ability was detected by Transwell chamber,and expression of key proteins(N-cadherin and Occludin)in epithelium-interstitial transform was detected by Western blot.Results The Smad7 interfering lentivirus vector and Smad7 overexpressing lentivirus vector were successfully constructed.Interference with Smad7 can promote NK cell and KK cell proliferation and migration,and inhibit KK cell apoptosis,but it has no significant effect on NK cell apoptosis(P>0.05).Overexpression of Smad7 inhibited the proliferation and migration of NK cells and KK cells,and promoted their apoptosis.After interfering with lentivirus infection,NK cells and KK cells showed decreased expression of Occludin protein compared with NC group(P<0.01),increased N-cadherin protein expression in KK cells(P<0.01),but there was no significant change in N-cadherin protein expression in NK cells(P>0.05);After lentivirus overexpression,NK and KK cells showed increased expression of Occludin protein(P<0.05),the expression of N-cadherin protein in NK cells decreased(P<0.05),but there was no significant change in N-cadherin protein expression in KK cells(P>0.05).Conclusion The regulation of Smad7 gene can affect the EMT in normal skin keratinocytes and keloid keratinocytes,and further regulate the ability of cell proliferation,migration and apoptosis.The effect of Smad7 gene regulation on EMT in keloid keratinocytes was greater than that on normal skin keratinocytes.