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The effects of co-culture system of human epidermal keratinocytes(HEKs)and Malassezia on melanin synthesis in human epidermal melanocytes under broadband UVB exposureObjective:To establish a co-culture model of Malassezia.globosa and primary human epidermal keratinocytes(HEKs),and to observe the proliferation,tyrosinase activity,the synthesis of tyrosinase and some important melanin synthesis pathway proteins of primary human epidermal melanocytes(HEMs)effected by the co-culture system with or without broadband UVB exposure.Methods:The effects of different concentrations of Malassezia.globosa(CBS7966)on the proliferation rate of HEKs was detected by CCK8 method and real-time cell analysis(RTCA system).Then the co-culture of HEKs and Malassezia.globosa was established.The experiment groups are:(1)co-culture under 0 mJ/cm2 UVB light irradiation;(2)co-culture under 15 mJ/cm2 UVB light irradiation;(3)co-culture under 75 mJ/cm2 UVB light irradiation;(4)HEKs under 0 mJ/cm2 UVB light irradiation;(5)HEKs under 15 mJ/cm2 UVB light irradiation;(6)HEKs under 75 mJ/cm2 UVB light irradiation.The cultural supernatants of the above groups were collected to raise HEMs,and a positive control group was also established with 10umol/1 8-mop added.The proliferation curve of HEMs was monitored with a RTCA system.Tyrosinase activity of HEMs was detected by dopa oxidation method.Transwell chamber co-culture method was used to co-culture the above groups with HEMs.The synthesis of tyrosine protease(TYR),tyrosine-related protease 1(TRP1),MITF,phosphorylated ERK,and phosphorylated CREB1 was detected by western-blot.Results:The proliferation activity of HEKs was not effected when co-cultured with malassezia in a ratio of 1:1,but was significantly inhibited in a ratio or higher than 2.5:1 after a certain period of time(p<0.05).there was no significant effect on proliferation,tyrosinase activity,protein synthesis of TYR,TRP1,MITF,phosphorylated ERK and phosphorylated CREB1 of HEMs without UVB exposure(p>0.05).After low dose(15 mJ/cm2)UVB irradiation:Malassezia.globosa inhibited the HEKs supermatant-inducing-proliferation of HEMs(p<0.05);HEKs promoted TRP1 protein synthesis in HEMs cells(p<0.05),while Malassezia globosa had no significant effect on the promotion process;co-culture group and HEKs culture group had no significant effect on TYR activity,protein synthesis of TYR,MITF,phosphorylated ERK and phosphorylated CREB1 in HEMs cells(p>0.05).After high-dose(75 mJ/cm2)UVB irradiation:HEKs culture supernatant inhibited the proliferation of HEMs(p<0.05),while M.globosa had no significant effect on the inhibitory process;Malassezia.globosa inhibited the promotion effect of HEKs culture supernatants on the tyrosinase activity,protein synthesis of TYR,TRP1,MITF,phosphorylated ERK,and phosphorylation CREB1 in HEMs.Conclusion:Under UVB exposure,Malassezia.globosa inhibits the the promotion effect of HEKs culture supernatants on the proliferation,tyrosinase activity,melanin-synthesis-protein in HEMs,therefore may inhibit the hyperpigmentation caused by UVB exposure.Chapter ? The effect of Malassezia on the synthesis and secretion of melanin synthesis-related cytokines in human keratinocytes under broadband UVB exposurePart1 The effect of Malassezia on the synthesis and secretion of melanin synthesis-related cytokines in HEKs under broadband UVB exposureObjective:To observe the effect of Malassezia.globosa on the secretion of some melanin synthesis-related cytokines in HEKs cells with or without broadband UVB exposure.Methods:The experiment groups are:(1)HEKs under 0 mJ/cm2 UVB irradiation;(2)HEKs under 15 mJ/cm2 UVB irradiation;(3)HEKs under 75 mJ/cm2 UVB irradiation;(4)HEKs co-cultured with Malassezia.globosa under 0 mJ/cm2 UVB irradiation;(5)HEKs co-cultured with Malassezia.globosa under 15 mJ/cm2 UVB irradiation;(6)HEKs co-cultured with Malassezia.globosa under 75 mJ/cm2 UVB irradiation.IL-la,IL-6,TNF-?,SCF,GM-CSF,b-FGF,EDN1,?-MSH,NT-3,PGE2 and COX-2 of above cultural supernatants of HEKs were detected by ELISA,and mRNA expression of the above cytokines were detected by RT-PCR.Results:Without UVB exposure,HEKs with Malassezia.globosa increased the protein and mRNA levels of EDN1,PGE2 and IL-la.Under UVB exposure,the expression of protein and mRNA of IL-1?,IL-6,TNF-?,SCF,GM-CSF,b-FGF,EDN1.PGE2 were increased and the expression of mRNA of COX-2,NT-3,and POMC were increased.Under 15mJ/cm2 UVB irradiation conditions,Malassezia.globosa couldfurther stimulate the expression of the above cytokines except TNF-?.Under 75 mJ/cm2 UVB irradiation,Malassezia.globosa further stimulated the expression of protein and mRNA of IL-la,IL-6,but inhibited the expression of protein and mRNA of TNF-a,EDN1,and mRNA of COX-2,POMC and NT-3.Conclusion:Without UVB or Under low-dose UVB exposure,Malassezia.globosa can stimulate some melanin-synthesis-promoting cytokines secreted by HEKs.Under high-dose UVB exposure,Malassezia.globosa can inhibit some melanin-synthesis-promoting cytokines while stimulate some melanin-synthesis-inhibiting cytokines secreted by HEKs.Part2 The effect of Malassezia on the secretion of melanin synthesis-related cytokines in hacat cells under broadband UVB exposureObjective:To establish a co-culture model of Malassezia.globosa and hacat cells,and to observe the effect of Malassezia.globosa on the secretion of some melanin synthesis-related cytokines in hacat cells with or without UVB exposure.Methods:The effect of different concentrations of Malassezia.globosa on the proliferation rate of hacat cells was detected by CCK8 method and RTCA systems.and a co-culture model was established with a best ratio of hacat cells and Malassezia.globosa.The expression of IL-la,IL-6,TNF-a,b-FGF,ET-1,SCF,PGE2 in the supernatants of the co-culture system and of the hacat cells culture group were dectected by ELISA method at different doses(0 mJ/cm2,15 mJ/cm2,75 mJ/cm2)of broadband UVB exposure.Results:When hacat and Malassezia were co-cultured at a ratio of 1:1,the proliferation of hacat cells was promoted within 48 h(p<0.05).When the ratio was higher than 1:1,there was no significant effect on the proliferation of hacat cells within 72 h(p>0.05).Under certain doses of UVB exposure,the concentrations of IL-la,IL-6,TNF-a,ET-1,and PGE2 cytokines secreted by the hacat cells were significantly increased(p<0.05),whereas there was no significant change of the amount b-FGF and SCF secreting(p>0.05).Under 15 mJ/cm2 UVB irradiation,Malassezia.globosa promoted the secretion of IL-la protein(p<0.05)and inhibited the secretion of IL-6,ET-1 and TNF-a protein(p<0.05).b-FGF and SCF had no significant effect(p>0.05).Under the irradiation condition of 75mJ/cm2UVB,Malassezia had an inhibitory effect on the secretion of IL-6 and TNF-a protein(p<0.05),but had no significant effect on other cytokines(p>0.05).Conclusion:Through interaction with hacat cells,Malassezia globosa has different effects on melanin-sythesis-related cytokines secreted by hacat cells under different dose UVB exposure.Under 15mJ/cm2 UVB exposure,ET-1 protein secretion is inhibited by Malassezia globosa,which may inihibit the hyperpigmentation caused by ultraviolet exposure.But Follow-up test is needed to confirm it.Chapter ? Characteristics of ITS and specific region genotypes of 64 strains of Malassezia from different sourcesObjective:To analyze and sequence the ITS,b3 and b6 sequences of 64 strains of pityrosporum from different local sources.Methods:64 clinical strains of Malassezia isolated from four regions of Nanjing,Sichuan,Shanghai and Jiangxi were collected,DNA was extracted,and ITS,b3 and b6 regions were amplified by PCR and PCR products were purified.Sequence analysis was performed after sequencing.Mega was used to build evolutionary tree.Results and conclusion:Through cloning,sequencing and sequence analysis of the Pityrosporum ITS,b3 and b6 regions,it was found that Pityrosporum from Nanjing was the highest of haplotype and nucleotide diversity.Pityrosporum has no obvious correlation with regionality and genotype.There is no clear correlation between the strain genotype and the site of infection,but genotype difference between the same pityrosporum strain is relatively large,such as M.furfur seperated from Nanjing.We have found that the diversity of strains in different regions is different,which may be related to the influence of selective pressure.Phylogenetic evolution shows that there are also large differences in the same species of strains.Through the study of ITS,specific sequences b3 and b6,it was found that the specific sequence b6 is more suitable for studying the polymorphism of pityrosporum than ITS and b3. |
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