马拉色菌对培养人黑素细胞代谢及酪氨酸酶基因表达的影响
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摘要
[目的]:马拉色菌感染所致花斑癣的基本皮肤损害包括色素减退和/或色素沉着,皮肤色素改变除考虑个体差异性外,是否与不同菌种或菌株的生物学特性差异有关?本课题将①马拉色菌与角质形成细胞共同培养后的上清和②马拉色菌分别加入黑素细胞培养体系中,探讨马拉色菌对培养人黑素细胞酪氨酸酶活性、黑素含量及酪氨酸酶mRNA表达的影响。
[方法]:将人包皮表皮细胞悬液用MCDB153培养基添加佛波酯(12-O-tetradecanoyl phorbol-14-acetate,TPA)、霍乱毒素(Cholera toxin,CT)、重组人表皮细胞生长因子(recombinan human epidermal growthfactor,rhEGF)、3-异丁基-1-甲基黄嘌呤(Isobutyl methylxanthine,IBMX)等进行黑素细胞纯化培养。用多巴染色、Fontana银染和S—100蛋白免疫组化染色法鉴定培养的黑素细胞。利用氧化反应原理建立酪氨酸酶活性检测体系。将7种马拉色菌分别与角质形成细胞株(HaCat)共同培养24小时后,获得共同培养上清液,按1∶1,1∶3,1∶7(上清体积∶黑素细胞培养液体积)加入黑素细胞培养体系中,24小时后观察黑素细胞增殖情况(MTT法)、测定酪氨酸酶活性(氧化多巴反应)、黑素含量(NaOH溶解法)、酪氨酸酶mRNA的表达(实时荧光定量PCR法)以及超微结构的变化。
[结果]:1.建立了黑素细胞纯化培养体系,能在体外培养及传代5次。
[Objective]: The primary lesion of pityriasis versicolor caused by Malassezia is hypopigment and /or hyperpigment. Are the pigmentary changes connected with the biology difference of different Malassezia species in addition to considering individual difference? In this study, we use seven species of Malassezia, to react with melanocytes, by directly and indirectly, with the condition medium after co-culture with keratinocyte, to investigate the effects of Malassezia on tyrosinase activity, melanogenesis and tyrosinase gene expression of human melanocytes.[Methods]: Normal human melanocytes were isolated from healthy men and cultured with MCDB153 medium supplemented with 12-O-tetradecanoyl phorbol-14-acetate (TPA), cholera toxin (CT), recombinan human epidermal growth factor (rhEGF) and isobutyl methylxanthine (IBMX). Dopa stain, Fontana silver stain and S-100 protein histochemical stain were performed to identify the cultured melanocytes cells. The reaction assay system of tyrosinase activity was established according to the principle of oxidation. Co-culture supernatants were obtained after 24 hours of culturing keratinocytes with different Malassezia. The medium used for melanocyte experiments was 1:1, 1:3 and 1:7 (v:v) mixture of co-culture supernatants and
melanocyte growth medium. Dopa oxidization reaction and NaOH assay were used to determine the activity of tyrosinase and synthesis of melanin; MTT method was used to determine the proliferation of melanocytes; Real-Time PCR assay was used to determine the tyrosinase gene expression of human melanocytes. The ultra structural changes of melanocytes were observed by transmission electron microscopy(TEM). [Results]: 1. The melanocytes culture system is stable and melanocytes could be cultured for five passages in vitro. It is proved to be melanocytes by the dopa stain, Fontana silver stain and S-100 protein histochemical stain. 2. The reaction system of tyrosinase activity is stable and credible. The optimal reation buffer was pH 9.0, 0.25 mol/L Tris-HCl; The optimal concentration of substrate L-Dopa was 0.15% and dissolved with pH 8.0, 0.01 mol/L Tris-HCl; The sensitivity of reation was increased markedly when affiliate 0.03% H2O2 in substrate; The optimal melanocytes amount of test was ≥ 2.5 ×104, the reation fastigium was within twenty minutes and connected with the melanocytes amounts, the more of the melanocytes number, the earlier of fastigium. 3. Increased tyrosinase activity (A450 value increasing from 1.298 ± 0.046 to 1.559 ± 0.059 per minute) and melanogenesis (increasing from 6.593 ± 0.852 mg/L to 13.715 ± 0.811 mg/L) are showed in melanocytes treated 24 hours with culture supernatants of co-culture of keratinocytes with Malassezia globosa compared with non-treated melanocytes; Increased tyrosinase gene expression (the relative fluorescence intensity ratio of Real Time RT-PCR product of tyrosinase mRNA to comparison GAPDH mRNA increasing from 0.911 ± 0.562 to 35.445 ± 26.297) of human melanocytes are showed in melanocytes treated with culture supematants of co-culture of
keratinocytes with Malassezia globosa compared with non-treated melanocytes; Abundant mitochondria, endoplasmic reticulum, ribosome, and melanin granule were shown in cell plasma under TEM. 4. There is no difference of tyrosinase activity, melanogenesis, and tyrosinase gene expression when melanocytes are treated 24 hours with culture supernatants of co-culture of keratinocytes with the other six species of Malassezias compared with non-treated melanocytes. 5. There is no difference of tyrosinase activity in melanocytes treated 24 hours directly with all of the seven species Malassezia compared with non-treated melanocytes. [Conclusions]: 1. The co-culture supernatants of keratinocytes with Malassezia globosa could increase tyrosinase activity, melanogenesis, tyrosinase gene expression of melanocytes. Abundant mitochondria, endoplasmic reticulum, ribosome and melanin granule were shown in cell plasma. 2. There is no difference of tyrosinase activity, melanogenesis, and tyrosinase gene expression when melanocytes are treated 24 hours with culture supernatants of co-culture of keratinocytes with the other six species of Malassezias compared with non-treated melanocytes. 3. There is no change of tyrosinase activity in melanocytes treated 24 hours directly with Malassezia.
[方法]:将人包皮表皮细胞悬液用MCDB153培养基添加佛波酯(12-O-tetradecanoyl phorbol-14-acetate,TPA)、霍乱毒素(Cholera toxin,CT)、重组人表皮细胞生长因子(recombinan human epidermal growthfactor,rhEGF)、3-异丁基-1-甲基黄嘌呤(Isobutyl methylxanthine,IBMX)等进行黑素细胞纯化培养。用多巴染色、Fontana银染和S—100蛋白免疫组化染色法鉴定培养的黑素细胞。利用氧化反应原理建立酪氨酸酶活性检测体系。将7种马拉色菌分别与角质形成细胞株(HaCat)共同培养24小时后,获得共同培养上清液,按1∶1,1∶3,1∶7(上清体积∶黑素细胞培养液体积)加入黑素细胞培养体系中,24小时后观察黑素细胞增殖情况(MTT法)、测定酪氨酸酶活性(氧化多巴反应)、黑素含量(NaOH溶解法)、酪氨酸酶mRNA的表达(实时荧光定量PCR法)以及超微结构的变化。
[结果]:1.建立了黑素细胞纯化培养体系,能在体外培养及传代5次。
[Objective]: The primary lesion of pityriasis versicolor caused by Malassezia is hypopigment and /or hyperpigment. Are the pigmentary changes connected with the biology difference of different Malassezia species in addition to considering individual difference? In this study, we use seven species of Malassezia, to react with melanocytes, by directly and indirectly, with the condition medium after co-culture with keratinocyte, to investigate the effects of Malassezia on tyrosinase activity, melanogenesis and tyrosinase gene expression of human melanocytes.[Methods]: Normal human melanocytes were isolated from healthy men and cultured with MCDB153 medium supplemented with 12-O-tetradecanoyl phorbol-14-acetate (TPA), cholera toxin (CT), recombinan human epidermal growth factor (rhEGF) and isobutyl methylxanthine (IBMX). Dopa stain, Fontana silver stain and S-100 protein histochemical stain were performed to identify the cultured melanocytes cells. The reaction assay system of tyrosinase activity was established according to the principle of oxidation. Co-culture supernatants were obtained after 24 hours of culturing keratinocytes with different Malassezia. The medium used for melanocyte experiments was 1:1, 1:3 and 1:7 (v:v) mixture of co-culture supernatants and
melanocyte growth medium. Dopa oxidization reaction and NaOH assay were used to determine the activity of tyrosinase and synthesis of melanin; MTT method was used to determine the proliferation of melanocytes; Real-Time PCR assay was used to determine the tyrosinase gene expression of human melanocytes. The ultra structural changes of melanocytes were observed by transmission electron microscopy(TEM). [Results]: 1. The melanocytes culture system is stable and melanocytes could be cultured for five passages in vitro. It is proved to be melanocytes by the dopa stain, Fontana silver stain and S-100 protein histochemical stain. 2. The reaction system of tyrosinase activity is stable and credible. The optimal reation buffer was pH 9.0, 0.25 mol/L Tris-HCl; The optimal concentration of substrate L-Dopa was 0.15% and dissolved with pH 8.0, 0.01 mol/L Tris-HCl; The sensitivity of reation was increased markedly when affiliate 0.03% H2O2 in substrate; The optimal melanocytes amount of test was ≥ 2.5 ×104, the reation fastigium was within twenty minutes and connected with the melanocytes amounts, the more of the melanocytes number, the earlier of fastigium. 3. Increased tyrosinase activity (A450 value increasing from 1.298 ± 0.046 to 1.559 ± 0.059 per minute) and melanogenesis (increasing from 6.593 ± 0.852 mg/L to 13.715 ± 0.811 mg/L) are showed in melanocytes treated 24 hours with culture supernatants of co-culture of keratinocytes with Malassezia globosa compared with non-treated melanocytes; Increased tyrosinase gene expression (the relative fluorescence intensity ratio of Real Time RT-PCR product of tyrosinase mRNA to comparison GAPDH mRNA increasing from 0.911 ± 0.562 to 35.445 ± 26.297) of human melanocytes are showed in melanocytes treated with culture supematants of co-culture of
keratinocytes with Malassezia globosa compared with non-treated melanocytes; Abundant mitochondria, endoplasmic reticulum, ribosome, and melanin granule were shown in cell plasma under TEM. 4. There is no difference of tyrosinase activity, melanogenesis, and tyrosinase gene expression when melanocytes are treated 24 hours with culture supernatants of co-culture of keratinocytes with the other six species of Malassezias compared with non-treated melanocytes. 5. There is no difference of tyrosinase activity in melanocytes treated 24 hours directly with all of the seven species Malassezia compared with non-treated melanocytes. [Conclusions]: 1. The co-culture supernatants of keratinocytes with Malassezia globosa could increase tyrosinase activity, melanogenesis, tyrosinase gene expression of melanocytes. Abundant mitochondria, endoplasmic reticulum, ribosome and melanin granule were shown in cell plasma. 2. There is no difference of tyrosinase activity, melanogenesis, and tyrosinase gene expression when melanocytes are treated 24 hours with culture supernatants of co-culture of keratinocytes with the other six species of Malassezias compared with non-treated melanocytes. 3. There is no change of tyrosinase activity in melanocytes treated 24 hours directly with Malassezia.
引文
1. Gueho E, Midgley G, Guillot J. The genus Malassezia with description of four new species. Antonie Van Leeuwenhoek 1996; 69: 337.
2. Guillot J, Gueho E. The diversity of Malassezia yeasts confirmed by rRNA sequence and nuclear DNA comparisons. Antonie van Leeuwenhoek 1995; 67(3): 297-314.
3. Sugita T, Tajima M, Takashima M. A new yeast, Malassezia yamatoensis, isolated from a patient with seborrheic dermatitis and its distribution in patients and healthy subjects. Microbiol Immunol 2004; 48(8): 579-83.
4. Mirhendi H, Makimura K, Zomorodian K, et al. A simple PCR-RFLP method for identification and differentiation of 11 Malassezia species. J Microbiol Methods 2005; 61(2): 281-4.
5. Tsuji T, Karasek M. A procedure for the isolation of primary culture of melanocyte from newborn and adult human skin. J Invest Dermal 1983; 81: 179.
6.赵辨,黄秋玲,毕志刚,等.人正常黑素细胞体外纯培养及其生物学鉴定.临床皮肤科杂志 1991;20(5):226—228.
7.项蕾红,郑志忠,祝绿川,等.黑素细胞体外纯培养及生物学特性鉴定.临床皮肤科杂志 2001;30(3):166—167.
8.丁国斌,陈壁,汤朝武,等.不同培养条件及时相对黑素细胞生物学特性的影响.中国临床康复 2002;24(6):36665—3666.
9. Guillot G, Gueho E, Lesourd M, et al. Identification of Malassezia species: a practical approach. J Mycol Med 1996; 6: 103-110.
10. Mayser P, Haze P, Papavassilis C, et al. Differentiation of Malassezia species: selectivity of Cremophor EL, castor oil and ricinoleic acid for M. furfur. Br J Dermatol 1997; 137(2): 208-213.
11.熊琳,冉玉平,周光平,等.马拉色菌的分类及其在几种皮肤病的分布情况中华皮肤科杂志 2000;33(5):318-320.
12.刘晓力,李克,方建华.细胞培养用血清的实验研究.免疫学杂志1989;5(2):70-71.
13.忻亚娟.动物细胞培养技术的进展.浙江省医学科学院学报 2001;12(4):25-28.
14.章静波,张世馥,黄东阳.组织和细胞培养技术.人民卫生出版社 2002:109-125.
15. Eisinger M, Marco O. Selective proliferation of normal human melanocyte in vitro in the presence of phorbol ester and cholera toxin. Natl Acad Sci USA 1982; 79: 2018-2020.
16.冉玉平,周光平,坪井良治,等.糠秕孢子菌与培养人角朊细胞的相互作用.中华皮肤科杂志 1997;30(5):294-298.
17. Watanabe S, Kano R, Sato H, et al. The effects of Malassezia yeasts on cytokine production by human keratinocytes. J Invest Dermatol 2001; 116: 769-773.
18. Baroni A, Perfetto B, Paoletti L, et al. Malassezia furfur invasinveness in a keratinocyte cell line(HaCat): effects on cytoskeleton and on adhesion molecule and cytokine expression. Arch Dermatol Res 2001; 293(8): 414-419.
2. Guillot J, Gueho E. The diversity of Malassezia yeasts confirmed by rRNA sequence and nuclear DNA comparisons. Antonie van Leeuwenhoek 1995; 67(3): 297-314.
3. Sugita T, Tajima M, Takashima M. A new yeast, Malassezia yamatoensis, isolated from a patient with seborrheic dermatitis and its distribution in patients and healthy subjects. Microbiol Immunol 2004; 48(8): 579-83.
4. Mirhendi H, Makimura K, Zomorodian K, et al. A simple PCR-RFLP method for identification and differentiation of 11 Malassezia species. J Microbiol Methods 2005; 61(2): 281-4.
5. Tsuji T, Karasek M. A procedure for the isolation of primary culture of melanocyte from newborn and adult human skin. J Invest Dermal 1983; 81: 179.
6.赵辨,黄秋玲,毕志刚,等.人正常黑素细胞体外纯培养及其生物学鉴定.临床皮肤科杂志 1991;20(5):226—228.
7.项蕾红,郑志忠,祝绿川,等.黑素细胞体外纯培养及生物学特性鉴定.临床皮肤科杂志 2001;30(3):166—167.
8.丁国斌,陈壁,汤朝武,等.不同培养条件及时相对黑素细胞生物学特性的影响.中国临床康复 2002;24(6):36665—3666.
9. Guillot G, Gueho E, Lesourd M, et al. Identification of Malassezia species: a practical approach. J Mycol Med 1996; 6: 103-110.
10. Mayser P, Haze P, Papavassilis C, et al. Differentiation of Malassezia species: selectivity of Cremophor EL, castor oil and ricinoleic acid for M. furfur. Br J Dermatol 1997; 137(2): 208-213.
11.熊琳,冉玉平,周光平,等.马拉色菌的分类及其在几种皮肤病的分布情况中华皮肤科杂志 2000;33(5):318-320.
12.刘晓力,李克,方建华.细胞培养用血清的实验研究.免疫学杂志1989;5(2):70-71.
13.忻亚娟.动物细胞培养技术的进展.浙江省医学科学院学报 2001;12(4):25-28.
14.章静波,张世馥,黄东阳.组织和细胞培养技术.人民卫生出版社 2002:109-125.
15. Eisinger M, Marco O. Selective proliferation of normal human melanocyte in vitro in the presence of phorbol ester and cholera toxin. Natl Acad Sci USA 1982; 79: 2018-2020.
16.冉玉平,周光平,坪井良治,等.糠秕孢子菌与培养人角朊细胞的相互作用.中华皮肤科杂志 1997;30(5):294-298.
17. Watanabe S, Kano R, Sato H, et al. The effects of Malassezia yeasts on cytokine production by human keratinocytes. J Invest Dermatol 2001; 116: 769-773.
18. Baroni A, Perfetto B, Paoletti L, et al. Malassezia furfur invasinveness in a keratinocyte cell line(HaCat): effects on cytoskeleton and on adhesion molecule and cytokine expression. Arch Dermatol Res 2001; 293(8): 414-419.