摘要
α-黑素细胞刺激素(α-MSH)和lpa-miR-nov-66在羊驼黑色素细胞产生黑色素过程中均起重要的调控作用,但二者之间的关系尚未报道.本研究在体外培养的羊驼黑色素细胞中通过转染lpamiR-nov-66和添加α-MSH处理,用实时定量PCR和Western印迹检测黑色素细胞内基因表达水平,ELISA法检测c AMP和cGMP的产量,RTCA实时无标记细胞功能分析黑色素细胞增殖以及紫外分光光度法检测黑色素产量,证实二者在调控羊驼黑色素细胞产生黑色素颗粒过程中的关系.结果显示,与单纯α-MSH处理相比,lpa-miR-nov-66转染结合α-MSH处理组中,小眼转录因子(MITF)和酪氨酸酶(TYR)在转录水平和翻译水平的表达均降低,而酪氨酸酶相关蛋白2(TYRP2)在转录和翻译水平的表达均升高;cGMP的产量升高,cAMP的产量下降;黑色素细胞增殖没有显著变化;黑色素细胞内黑色素产量下降.与单纯转染lpa-miR-nov-66相比,lpa-miR-nov-66转染结合α-MSH处理组中,MITF、TYR和TYRP2在转录水平和翻译水平的表达均升高;cGMP的产量下降,cAMP的产量升高;黑色素细胞增殖没有显著变化;黑色素细胞内黑色素产量升高.上述结果证明,lpa-miR-nov-66通过调控羊驼黑色素细胞中毛色形成的c AMP路径,抑制α-MSH对黑色素细胞产生黑色素的促进作用.
α-Melanocyte stimulating hormone（ α-MSH） and lpa-miR-nov-66 have been identified to regulate melanogenesis in melanocyte of alpaca. However,the interaction of these factors in the regulation of melanogenesis has not been reported. Employing an alpaca melanocyte cell line followed by the tranfection with lpa-miR-nov-66,α-MSH treatment（ in the medium supplemented with α-MSH） and lpamiR-nov-66 transfection plus α-MSH treatment,the objective of the present study was to determine the interaction between α-MSH and lpa-miR-nov-66 on melanogenesis. Real-time PCR and Western blotting were used for determining the gene and protein expression. ELISA was used for measuring cGMP and c AMP production. RTCA machine was used for monitoring the proliferation activity of melanocytes. And ultraviolet spectrometry was used for measuring the melanin production. The results showed that comparedwith α-MSH treatment, the expression levels of microphthalmia transcription factor（ MITF） and tyrosinase（ TYR） genes in melanocytes with lpa-miR-nov-66 transfection plus α-MSH treatment were lower,while the expression level of tyrosinase related protein 2（ TYRP2） was higher; the production levels of cGMP and cAMP in melanocytes with lpa-miR-nov-66 transfection plus α-MSH treatment were higher and lower,respectively; the melanin production in melanocytes with lpa-miR-nov-66 transfection plus α-MSH treatment were lower. Compared with lpa-miR-nov-66 transfection,the expression level of MITF,TYR and TYRP2 in melanocytes with lpa-miR-nov-66 transfection plus α-MSH treatment was higher; the production levels of cGMP and c AMP in melanocytes with lpa-miR-nov-66 transfection plusα-MSH treatment were lower and higher,respectively; the melanin production in melanocytes with lpamiR-nov-66 transfection plus α-MSH treatment was higher. No differences were observed in the cell proliferations among all melanocytes. These results suggested that lpa-miR-nov-66 inhibited the function of α-MSH on melanogenesis of alpaca by regulat
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2015年第11期1206-1212,共7页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家自然科学基金(No.31201868)
山西省科技攻关项目(No.20120311024-2)资助~~
