内皮素3对不同毛色绵羊黑色素细胞的影响

【目的】探索内皮素3(EDN3)对来源于不同毛色体外培养的黑色素细胞产生黑色素作用机制及差异的影响。【方法】体外培养不同毛色皮肤来源的黑色素细胞,通过MTT法检测不同细胞在EDN3影响下的增殖率,分别提取两种细胞的总RNA和总蛋白,总RNA经反转录合成c DNA,采用实时荧光定量PCR方法检测EDN3对两种细胞内EDNRB、NRas及TYR在m RNA水平相对表达量的影响,总蛋白通过Western blot方法检测EDN3对两种细胞内EDNRB、NRas及TYR在蛋白水平的表达量是否发生变化,结果均使用SPSS19.0软件进行差异显著性分析。【结果】MTT法测得EDN3对黑白两种来源的黑色素细胞的增殖均产生促进作用,实时荧光定量PCR结果显示添加EDN3的白色来源细胞内EDNRB m RNA的相对表达量是正常细胞组的1.7992倍,NRas是正常细胞组的1.8536倍差异极显著(P0.01),但TYR m RNA的相对表达量未发生明显变化;添加EDN3的黑色来源细胞内EDNRB m RNA的相对表达量是正常细胞组的2.2512倍,NRas是正常细胞组的1.3859倍,TYR是正常细胞组的15.5710倍差异极显著(P0.01);Western blot结果显示添加EDN3的白色来源细胞内EDNRB蛋白表达量是正常细胞组的3.0827倍差异极显著(P0.01),NRas是正常细胞组的1.2936倍差异显著(P0.05),TYR蛋白表达量未发生明显变化;添加EDN3的黑色来源细胞内EDNRB蛋白表达量是正常细胞组的3.9800倍差异极显著(P0.01),NRas是正常细胞组的1.3658倍差异显著(P0.05),TYR是正常细胞组的1.8498倍差异显著(P0.05)。【结论】EDN3促进白色来源的黑色素细胞增殖但对色素合成的限速酶TYR未产生促进作用;EDN3促进黑色来源的黑色素细胞增殖并可能促进黑色素生成。

Effect of EDN3 on of Sheep Skin Melanocytes with Different Coat Colors in Vitro

【Objective】 The objective of this study is to explore the effects of EDN3 in vitro on sheep melanocytes with different coat colors and to find the mechanisms of production of melanin and their differences. 【Method】 Melanocytes from black and white coat colors were cultured in vitro, and MTT assay was used to detect different cell proliferation rates by affected EDN3. After extraction of total RNA and total protein of both cells, the total RNA was reversely transcribed to cDNA, and then the effect of EDN3 on mRNA relative expression of EDNRB, NRas and TYR was analyzed by RT-PCR, and Western blot was performed to ensure whether EDN3 had influence on the protein expression of EDNRB, NRas and TYR. Significant difference was analyzed by using statistical method with software SPSS19.0. 【Result】 MTT assay showed that EDN3 promoted cells proliferation from both black and white coat colors. RT-PCR revealed that compared with the control group, the relative expressions of EDNRB and NRas mRMA in EDN3 group from white coat color were significantly increased by 1.7992 folds and 1.8536 folds respectively (P<0.01), while TRY mRNA had no significant difference. In cells from black coat color, the expressions of EDNRB, NRas and TYR were all induced significantly with multiples of 2.2512, 1.3859 and 15.5710 respectively (P<0.01). The result of Western blot in EDN3 group was consistent with RT-PCR, the EDNRB protein expression from white coat color was 3.0827 times of control group (P<0.01), and the NRas protein expression was 1.2936 fold of control group (P<0.05), while TYR had no change. The protein expressions in cells from black coat color of EDNRB, NRas and TYR were observably added up to 3.9800 time (P<0.01), 1.3658 and 1.8498 times (P<0.05), respectively. 【Conclusion】 EDN3 promoted melanocytes proliferation from both black and white coat colors, while had no effect on TYR which is a rate-limiting enzyme of pigment synthesis in cells from white coat colors, and might play a role in producing melanin in cells isolated from black coat color.