松弛素对TGF-β诱导的内皮细胞间质化的抑制作用

The inhibition effect of relaxin for TGF-β induced endothelial to mesenchymal transition in vitro

目的 :探讨松弛素(RLX)对TGF-β诱导的内皮细胞间质化现象的影响。方法:采用体外分离的人脐静脉内皮细胞(HUVECs)作为体外细胞模型,通过TGF-β10 ng/mL诱导内皮细胞间质化,100 ng/mL和200 ng/mL RLX分别预处理,再与TGF-β联合培养48 h。光镜下观察细胞形态学改变,CCK-8实验和transwell实验检测细胞增殖和迁移能力,免疫荧光单双染技术观察vimentin和CD31的表达变化,蛋白免疫印迹技术观察各实验组vimentin、CD31、ve-cadherin和α-SMA的蛋白表达。结果:阴性对照组内皮细胞呈铺路石样生长,TGF-β诱导组细胞形态向梭形转化,呈扩散迁移趋势。与阴性对照组相比,TGF-β诱导组细胞增殖、迁移能力明显增强;而RLX可抑制TGF-β诱导的增殖和迁移。免疫荧光单染结果显示,与阴性对照组相比,TGF-β诱导组内皮细胞特异性蛋白CD31表达下调而间质细胞标志性蛋白vimentin明显上升;与TGF-β组相比,RLX联合TGF-β处理组CD31表达上调而vimentin表达下调。免疫荧光双染结果显示,阴性对照组以内皮性质的细胞为主,仅存在少量的CD31和vimentin双阳性细胞;TGF-β诱导组双阳性细胞明显增多;与TGF-β组相比,RLX联合TGF-β处理组间质效应的双阳性细胞下调,而内皮效应的双阳性细胞增多。蛋白免疫印迹结果显示,与阴性对照组相比,TGF-β诱导组内皮细胞特异性蛋白CD31(0.36±0.076 vs 0.88±0.086)和ve-cadherin(0.54±0.046 vs 1.09±0.13)表达下调而间质细胞标志性蛋白vimentin(0.72±0.102 vs 0.21±0.081)和α-SMA(0.88±0.084 vs 0.24±0.046)明显上升(P<0.05);与TGF-β组相比,RLX联合TGF-β处理组CD31(0.67±0.09、0.59±0.12 vs 0.36±0.076)和ve-cadherin(0.85±0.09、0.72±0.064 vs 0.54±0.046)表达上调,而vimentin(0.56±0.011、0.48±0.06 vs 0.72±0.102)和α-SMA(0.65±0.081、0.54±0.032 vs 0.88±0.084)表达下降(P<0.05),且呈剂量依赖性。结论:RLX可抑制TGF-β诱导的内皮细胞向间质化细胞转化。

Objective： To investgate the effect of relaxin which inducing endothelial to mesenchymal transi-tion in vitro. Methods： Endothelial cells were isolated from human umbilical vein endothelial cells （HUVECs）. TGF- β 10 ng/mL was added to medium for inducing endothelial to mesenchymal transition. Pretreatment relaxin 100 ng/mL and 200 ng/mL was added to medium, then co-cultured with TGF- β 10 ng/mL for 48 h. The changes of morphology were observed under an inverted phase contrast microscope. CCK-8 assay was used to evaluate the cell proliferation as well as Transwell tested cell migration. The expression of CD31, vimentin, ve-cadherin and α- SMA were determined by immunofluoresence staining and western blot analysis. The co-expression of CD31 with vimentin and VWF with α -SMA were also determined by immunofluoresence. Results： A paving stone-like growth was observed in control group while TGF- β induced more spreading and migrating cells which was called EMT. However, this transition could be prevented by relaxin. Compared with the control group, the proliferation and the mobility were increased by TGF- β （P〈0.05）, but declined when combining with relaxin compared to adding TGF- 1 only （P〈0.05）. Compared with the control group, the specific protein of endothelial CD31 （0.36 ± 0.076 VS 0.88 ±0.086） and ve-cadherin （0.54± 0.046 VS 1.09 ± 0.13） were significantly decreased when adding TGF-β, but the specific protein of mesenchymal vimentin （0.72 ± 0.102 VS 0.21 ± 0.081） and α-SMA （0.88 5：0.084 VS 0.24 ± 0.046） were significantly increased. However, relaxin could inhibit the phenotype switch. Compared with the TGF- β inducing group, CD31 （0.67± 0.09, 0.59 ± 0.12 VS 0.36 5： 0.076） and ve-cadherin（0.85 ± 0.09, 0.72 5：0.064 VS 0.54 ± 0.046） were significantly increased, but vimentin （0.56 ±0.011, 0.48 ±0.06 VS 0.72 ±0.102） and α-SMA （0.65 ± 0.081, 0.54±0.032 VS 0.88 5± 0.084） were significantly decreased when combining with relaxin. Besides, it exhibited dose-dependent. Last but not least, the number of double positive cells was largely increased in TGF- β inducing group compared to the control group. And com- pared with the TGF- β group, double positive cells performing mesenchymal are declined while performing endothelial ones are increased in the combined group. Conclusion： Relaxin exhibits the inhibition effect for TGF- ~ induced endothelial to mesenchymal transition in vitro.