论文标题:马拉色菌在花斑癣色素改变中所起作用的研究
Study on the Role of Malassezia in the Color Diversity of Pityriasis Versicolor
论文作者
论文导师 刘维达,论文学位 博士,论文专业 皮肤性病学
论文单位 中国协和医科大学,点击次数 721,论文页数 67页File Size5208K
2007-05-01论文网 http://www.lw23.com/lunwen_174407/
1．针对传统吐温试验中的一些不足之处，摸索一种新型的吐温试验方法，以提高马拉色菌菌种鉴定的准确性。方法制作不同浓度梯度的吐温平板，将马拉色菌菌悬液滴加其上，37℃培养5天，对菌落孢子进行计数，通过方差分析，获得较适于马拉色菌生长的4种吐温(20、40、60和80)的浓度。利用7种马拉色菌标准株验证新型的吐温培养基。结果4种吐温最适于马拉色菌生长的浓度为2％。新型吐温培养基在对7种标准株的鉴定中得到了良好的验证。结论新型吐温培养基显著提高了马拉色菌菌种鉴定的准确性，同时也提高了马拉色菌鉴定的效率和经济性。 2．观察引起不同临床色素表现的马拉色菌与角质形成细胞共培养液对B16F10黑素瘤细胞黑素合成的影响。方法MTT法筛选不同比例的马拉色菌对角质形成细胞增殖率的影响；不同组共培养液培养B16F10细胞6天后，测定B16F10细胞增殖率、黑素含量、酪氨酸酶活性和酪氨酸酶蛋白表达。结果角质形成细胞与马拉色菌在1∶10比例以下，角质形成细胞的生长状况不受马拉色菌的影响(P＞0.05)。当比例提高至1∶20以上时，角质形成细胞的生长受到显著抑制(P＜0.01)。色沉区马拉色菌与角质形成细胞共培养液引起B16F10细胞黑素含量、酪氨酸酶活性和酪氨酸酶蛋白表达增加(P＜0.01)，细胞增殖率无明显变化(P＞0.05)。色减区马拉色菌共培养液的作用与之相反。结论马拉色菌通过与角质形成细胞相互作用，影响B16F10细胞酪氨酸酶的活性和表达，调节黑素合成。不同色素异常皮损来源的菌株其影响效果截然不同。 3．探讨引起花斑癣不同临床色素表现的马拉色菌与角质形成细胞共培养，导致与黑素合成相关的细胞因子的变化。方法用色沉和色减区分离的马拉色菌与角质形成细胞共培养，在不同时间段，收集上清液，ELISA法测定碱性成纤维细胞因子(b-FGF)、内皮素-1(ET-1)、神经生长因子-β(NGF-β)、白介素-1a(IL-1a)、白介素-6(IL-6)、肿瘤坏死因子-a(TNF-a)和干细胞因子(SCF)的动态变化。结果马拉色菌刺激角质形成细胞分泌IL-1a、IL-6、TNF-a、ET-Ⅰ增加(P＜0.01)。未检测到b-FGF、NGF-β和SCF的产生(P＞0.05)。色沉区马拉色菌刺激产生的ET-1显著高于色减区马拉色菌(P＜0.01)。结论不同马拉色菌刺激角质形成细胞分泌黑素合成相关因子的能力不同。ET-1在花斑癣色素沉着中可能起了一定作用。
1. To overcome the disadvantage of traditional Tween test, we try to explore a new kind of method for Tween test, which may improve the accuracy of classification of Malassezia species. Methods First prepare four kinds of Tween cultures of different concentration gradient, then apply the suspension of Malassezia to the surface of the culture. After cultivation in 37℃for 5 days, the colony is harvested and the spores are counted under microscope. By means of variance analysis, we get the most suitable concentration of four kinds of Tween (20, 40, 60, 80) for MaIassezia. Then the new kind of Tween culture is tested by 7 type strains of Malassezia. Results 2% is the most suitable concentration of four kinds of Tween for MaIassezia. The reliability of the new method is proved by the classification of 7 type strains of Malassezia. Conclusion The new kind of Tween culture significantly improves the accuracy of classification of Malassezia species, and it also improves the efficacy and economy of the test. 2. To observe the influence on the melanogenesis of B16F10 melanoma cells by the co-culture supematant of Keratinocytes with Malassezia isolates which cause hyper- or hypo- pigmentation. Methods The effects of Malassezia isolates with different proportions on the growth rate of keratinocytes Were assessed with MTT. 6 days after the B16F10 cells having been cultivated with the co-culture supernatants of different groups, the cell growth rate, the melanin content, the tyrosinase activity and the expression of tyrosinase protein of B16F10 cells are determined. Results When the ratio between keratinocytes and Malassezia isolates was lower than 1:10, the growth rate of keratinocytes was not affected by Malassezia (P＞0.05). When the ratio was increased above 1:20, the growth rate of keratinocytes was significantly inhibited by Malassezia (P＜0.01). The melanin content, the tyrosinase activity and the expression of tyrosinase protein of B16F10 cells are increased by the co-culture supematant of Keratinocytes with Malassezia isolates from hyper-pigmentation area (P＜0.01). The cell growth rate has no obvious changes (P＞0.05). The co-culture supernatant of Malassezia isolates from hoper-pigmentation area has the opposite effect. Results By the interaction with Keratinocytes, Malassezia isolates can have an influence on the tyrosinase activity and the expression of tyrosinase protein of B16F10 cells, and regulate the melanogenesis. 3. To investigate the co-culture of keratinocytes with MaIassezia isolates which cause the Pityriasis Versicolor with different color and to analyze the changes of cytokines associated with melanogenesis. Methods Co-culture of keratinocytes and Malassezia species were performed with isolates from hyer-and hypo-pigmentation areas of pityriasis versicoior. The supematants were collected at different time points, and the changes of basic fibroblast growth factor (b-FGF), endothelin-1 (ET-1), nerve growth factor-β(NGF-β), interleukin-la (IL-la), interleukin-6(IL-6), tumor necrosis factor-a (TNF-a), colony stimulating factors (SCF) were recorded. Three control groups were established accordingly. Results The secretions of IL-1a, IL-6, TNF-a, and ET-1 was significantly increased after the co-culture of keratinocytes and Malassezia (P＜0.01), while those of b-FGF, NGF-β, and SCF had no significant changes (P＞0.05). Compared with the isolates from the hypo-pigmentation area, ET-1 induced by isolate from hyper-pigrnentation area significantly increased (P＜0.01). Conclusion When Malassezia isolates are co-cultured with keratinocytes, the secretions of cytokines associated with melanogenesis may differ from each other. ET-1 may play certain role in the hyper-pigmentation of pityriasis versicolor.
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