黑芝麻水提液对鼠黑素瘤细胞黑色素生成及相关基因表达的影响
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摘要
白发的发生主要是由于毛囊中黑素细胞酪氨酸酶的活性降低或丧失,合成黑色素的功能减退,使毛干中缺乏黑色素而引起。传统医学治疗白发的常用中药有何首乌、黑芝麻等,目前国内对于何首乌疗效的研究较多,关于黑芝麻在白发治疗方面的研究很少。为了研究黑芝麻在治疗白发方面的疗效和作用机制,本课题采用水提法提取黑芝麻有效成分,以鼠B16F1黑素瘤细胞为模型,运用生物化学,分子生物学和免疫学方法,在细胞水平和分子水平上,研究黑芝麻水提液对黑素瘤细胞增殖、酪氨酸酶活性、黑素生成量及黑素合成途径相关基因表达的影响,并探讨黑芝麻促进黑色素生成的机理。
黑芝麻水提液能够使B16F1黑素瘤细胞的增殖率和活性有所提高(P>0.05);促进B16F1黑素瘤细胞酪氨酸酶活性显著增强(P<0.05),黑色素生成量显著增加。在分子水平上,RT-PCR和Western结果显示,黑芝麻水提物能够显著促进小眼畸形相关转录因子(MITF)的转录和翻译,其主要下游基因酪氨酸酶(TYR)的转录和翻译也呈现同步增强,并且随着处理浓度越大,增强效果越明显;酪氨酸酶相关蛋白-1(TRP-1)和酪氨酸酶相关蛋白-2(TRP-2)的表达无显著变化(P>0.05)。
本课题证实黑芝麻水提液在体外能显著增加黑素瘤细胞酪氨酸酶的活性,有效刺激B16F1黑素瘤细胞中黑素合成,实验结果表明这种效果可能是通过促进MITF基因的表达,调控TYR基因在mRNA和蛋白质水平上增强表达来完成。
White hair is caused by the lack of melanin in hair, which results from loss of tyrosinase activity in melanoma cells of Hair follicle. Semen Sesami Nigrum is a traditional medicine of curing white hair. To study the effect and mechanism of Semen Sesami Nigrum on white hair cure, we treated murine B16F1 Melanoma cells with 3 different concentrations of water extract of Semen Sesami Nigrum (WES). The effects of WES on cell viability, tyrosinase activity and melanogenesis of B16 murine melanoma cells were studied using Biochemical Molecular Biological and Immunological methods to clarify the mechanism of its stimulating effect on melanogenesis.
B16F1 cells were incubated with three different concentrations of WES for 72h and then cell viability, tyrosinase activity, melanin content were respectively measured.The protein and mRNA expression of melanogenesis key enzymes were investigated by western Blotting and RT-PCR.
In the range of experimental dose, WES increased B16 cell viability (P>0.05).,and also increased the activity of tyrosinase and melanin content significantly in a dose-dependent manner (P<0.05). The increasing mRNA and protein expression of TYR and MITF were also observed in a dose-dependent manner (P<0.05). No effect was observed on either mRNA or protein expression of TRP-1 and TRP-2.
WES could Significantly increase the tyrosinase activity, and enhance the subsequent process of melanogenesis in B16 melanoma cells, which may be attributed to the increasing expression of MITF, which Controls the Transcription of TYR gene on mRNA and protein levels.
黑芝麻水提液能够使B16F1黑素瘤细胞的增殖率和活性有所提高(P>0.05);促进B16F1黑素瘤细胞酪氨酸酶活性显著增强(P<0.05),黑色素生成量显著增加。在分子水平上,RT-PCR和Western结果显示,黑芝麻水提物能够显著促进小眼畸形相关转录因子(MITF)的转录和翻译,其主要下游基因酪氨酸酶(TYR)的转录和翻译也呈现同步增强,并且随着处理浓度越大,增强效果越明显;酪氨酸酶相关蛋白-1(TRP-1)和酪氨酸酶相关蛋白-2(TRP-2)的表达无显著变化(P>0.05)。
本课题证实黑芝麻水提液在体外能显著增加黑素瘤细胞酪氨酸酶的活性,有效刺激B16F1黑素瘤细胞中黑素合成,实验结果表明这种效果可能是通过促进MITF基因的表达,调控TYR基因在mRNA和蛋白质水平上增强表达来完成。
White hair is caused by the lack of melanin in hair, which results from loss of tyrosinase activity in melanoma cells of Hair follicle. Semen Sesami Nigrum is a traditional medicine of curing white hair. To study the effect and mechanism of Semen Sesami Nigrum on white hair cure, we treated murine B16F1 Melanoma cells with 3 different concentrations of water extract of Semen Sesami Nigrum (WES). The effects of WES on cell viability, tyrosinase activity and melanogenesis of B16 murine melanoma cells were studied using Biochemical Molecular Biological and Immunological methods to clarify the mechanism of its stimulating effect on melanogenesis.
B16F1 cells were incubated with three different concentrations of WES for 72h and then cell viability, tyrosinase activity, melanin content were respectively measured.The protein and mRNA expression of melanogenesis key enzymes were investigated by western Blotting and RT-PCR.
In the range of experimental dose, WES increased B16 cell viability (P>0.05).,and also increased the activity of tyrosinase and melanin content significantly in a dose-dependent manner (P<0.05). The increasing mRNA and protein expression of TYR and MITF were also observed in a dose-dependent manner (P<0.05). No effect was observed on either mRNA or protein expression of TRP-1 and TRP-2.
WES could Significantly increase the tyrosinase activity, and enhance the subsequent process of melanogenesis in B16 melanoma cells, which may be attributed to the increasing expression of MITF, which Controls the Transcription of TYR gene on mRNA and protein levels.
引文
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[2]慕立义.植物化学保护研究方法.北京:中国农业出版社, 1994, 80-81
[3]吴磊,范卫新.毛囊色素.国际皮肤性病学杂志, 2006, 32(3): 171
[4]朱铁君,赵广,张书元.色素性皮肤病.北京:北京医科大学中国协和医科大学联合出版社, 1996, 11
[5] Smith B, Selhy P, Southgate J, et al. Detection of melanoma cells in peripheral blood by means of reverse transcriptase and polymerase chain reaction. Lancet, 1991, 338(8777): 1227-l229
[6] Kubo I, Yokokawa Y, Kinst Hori. Tyrosinase inhibitors form bolivian medicinal plants. Nat Prod, 1995, 58: 739
[7] Kim H.J., Cho Y.D., Leem K.H., et al. Effects of ephedrac herba on melanogenesis and gene expression profiles using cDNA microarray in B16 melanocytes. Phytotherapy Research, 2006, 20(9): 748-754
[8] Jake Gittlen. Molecular mechanisms controlling human melanoma Progression and metastasis. Pathobiology.1997, 65(6): 311-320
[9] Nurty V., Bouchard B., Mathew S. Assigment of the human TYR RP(brow) locus to chromosome region gp23 by nonradioactive in situ hybridization. Genomics, 1992, 13: 227-229
[10] Kwan S.G.. Enhancement of antioxidase and phase enzyme by oral feeding of green tea Polyphenols in drinking water to SKH-1 hairless mice. Cancer Res. 1992, 52(14): 4050-4052
[11] Palumbo A, NaPlitano A, Demartino L, et al. Sepicific incorporation of 2-thiouracil into biological melanins. Bioehem Biophas Acta, 1994, 1200(3): 271-276
[12] Kim D.S., Park S.H., Kwon S.B., et al. Sphingosylphosphorylcholine-induced ERK activation inhibits melanin synthesis in human melanocytes. Pigment Cell Res, 2006, 19(2): 146-153
[13]朱铁君,赵广,张书元.色素性皮肤病.北京:北京医科大学中国协和医科大学联合出版社, 1996, 11.
[14]李韶勇,孙命,曲娜,等.黑色素的合成及其常见抑制剂的作用机理.天津师范大学学报(自然科学版), 2002, 22(1): 17-21
[15]刘甲斐,仇雪梅.黑色素及其相关基因的研究进展.生物技术通报, 2007, 4: 55-58
[16] Nakamura K, Ozaki A, Akutsu T, et al. Molecular mechanisms controlling human melanoma Progression. Molecular Genetics and Genomics, 2001,(256): 687-693
[17] Kinura S, Kawakami T, Kawa Y, et al. Bcl-2 reduced and fast activated by the inhibition of stem cell factor/KIT signaling in murine melanocyte precursors. Invest Dematol, 2005, 24(1): 229-234
[18] Jake Gittlen. Molecular mechanisms controlling human melanoma Progression and metastasis. Pathobiology, 1997, 65(6): 311-320
[19] Boissy RE, Sakai C, Zhao H, et al. Human tyrosinase related protein-(lTRP-l)does not function as a DHICA oxidase activity in contrast to murine TRP-1. Exp Dermatol, 1998, 7(4): 198-204
[20] Lominski A, Tobin D.J., Shibahara S, et al. Melanin Pigmentation in mammalian skin and its hormonal regulation. Physiol Rev, 2004, 84(4): 1155-228
[21]谭城,朱文元,鲁严.白癜风皮损黑素细胞HMB-45、酪氨酸酶及其相关蛋白1的免疫组化研究.临床皮肤科杂志, 2003, 32(7): 376-378
[22]赖来展等.黑芝麻的营养功能及产品开发.广东农业科学, 1997 , 5-25
[23] Tobin DJ. et al. Biochemistry of human skin-our brain on the outside. Chemsoe Rev, 1996, 9: 304-310
[24] Richards KA, Fukai K, Oiso N, et al. A novel KIT mutation results in Piebaldism with progressive depigmentation. J Am Aead Dermatol, 2001, 44(2): 288-292
[25]章一近,许爱娥.白癜风相关基因研究进展.国外医学皮肤性病学分册, 2004, 30(2): 91-94.
[26] Winder A, Kobayashi T, Tsukamoto K, et al. The tyrosinase gene family-interactions of melanogenic proteins to regulate melanogenesis. Cell Mol Biol Res. 1994, 40: 613-626
[27] Jimbow K. Biological role of tyrosinase-related protein and its relevance to pigment disorders (vitiligo vulgaris). J Dermatol, 1999, 26(11): 734-737
[28]李春英,高天文,李廷慧,等.人酪氨酸酶相关蛋白1(TRP-1)编码基因反义核酸对人黑素细胞及恶性黑素瘤细胞影响的研究[A]. 2003中国中西医结合皮肤性病学术会议论文汇编[C], 2003
[29] Sarangarajan R, ZhaoY, Babeoek G, et al. Mutant alleles at the brown locus encoding tyrosinase related Protein-(lTRP-l)affect proliferation of mouse melanocytes in culture. Pigment Cell Res. 2000, 13(5): 337-344
[30] Amae S, Yasumoto K, Takeda K, et al. Identification of a composite enhancer of the human tyrosinase related protein 2/DOPA chrome tautomerase gene. Biochim BioPhys Aeta. 2000, 1492(2-3): 505-508
[31] Itoh T, Furuiehi Y. Hot-water extracts from adzuki beans (Vigna angularis) stimulates not only melanogenesis in cultured mouse B16 melanoma cells but also pigmentation of hair color in C3H mice. Biosci Biotechnol Biochem. 2005, 69(5): 873-882
[32] Taieb A. Intrinsic and extrinsic pathomechanisms in vitiligo. Pigment Cell Res.2000, 13(SuPP18): 41-47
[33] Kim D.S., Park S.H., Kwon S.B., et al. SphingosylphosPhoryleholine-induced ERK activation inhibits melanin synthesis in human melanocytes. Pigment Cell Res, 2006, 19(2): 146-153
[34] Tobin D.J.. Biochemistry of human skin-our brain on the outside. Chemsoe Rev. 2006, 35(I): 52-67
[35]刘之力,涂彩霞,任风,等. 56味中药乙醇水提物对酪氨酸酶活性影响及动物致色素作用的研究[J].中华皮肤科杂志, 2001, 34(4): 284-285
[36]吴可克,徐秋,陈丽风,等.八味中药水和乙醇水提物对酪氨酸酶的激活作用[J].大连轻工业学院学报, 2000, 19(1): 21-24
[37]马慧军,朱文元,王大光,等.中药单体胡椒碱促进表皮黑素细胞黑素合成的实验研究[J].临床皮肤科杂志, 2005, 34(1): 14-17
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