他克莫司对黑素细胞和角质形成细胞的作用研究
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摘要
目的
通过检测新型免疫抑制剂他克莫司(tacrolimus)对黑素细胞(melanocyte,MC)和角质形成细胞(keratinocytes, KC)的作用,观察他克莫司对黑素细胞的直接和间接影响,寻找其作用靶点,以揭示他克莫司治疗白癜风的作用机制,从而为临床提供实验依据,使之更好地服务于临床。
方法
1.建立小鼠B16黑素瘤细胞培养体系,并根据不同的实验需要将细胞分别接种于相应的培养板,选择对数生长期的细胞进行实验。以不同浓度的他克莫司(10,102,103,104nmol/L)对小鼠B16黑素瘤细胞进行干预,每个浓度组设3个复孔,并设空白对照组。观察他克莫司对小鼠B16黑素瘤细胞的影响并检测黑素细胞增殖(MTT法)、迁移(微孔膜法)、酪氨酸酶活性(多巴氧化法)、黑素含量(NaOH裂解法)等指标。
2.建立人永生化角质形成细胞(HaCaT)培养体系,并根据不同的实验需要将细胞分别接种于相应的培养板,选择对数生长期的细胞进行实验。以不同浓度的他克莫司(10,102,103,104nmol/L)对角质形成细胞进行干预,每个浓度组设3个复孔,并设空白对照组,培养48小时后,收集上清液。采用ELISA法检测上清液中干细胞因子(SCF)和碱性成纤维细胞生长因子(bFGF)的含量,并测定角质形成细胞的增殖情况。收集角质形成细胞和小鼠B16黑素瘤细胞,提取RNA,采用实时荧光定量PCR检测角质形成细胞SCF mRNA、黑素细胞c-kit mRNA的表达水平。
3.以他克莫司作用后的角质形成细胞培养上清液作为条件化培养基对小鼠B16黑素瘤细胞进行干预,检测黑素细胞的增殖(MTT法)、酪氨酸酶活性(多巴氧化法)、黑素含量(NaOH裂解法)等指标,以观察他克莫司通过角质形成细胞对黑素细胞的间接作用。
结果
1.低浓度(10nmol/L和102nmol/L)他克莫司对黑素细胞增殖的影响不明显,高浓度(103nmol/L和104nmol/L)对增殖有抑制作用。各浓度他克莫司均可提高黑素细胞的酪氨酸酶活性并增加黑素含量。此外,他克莫司可以促进黑素细胞的迁移。
2.低浓度(10nmol/L和102nmol/L)他克莫司对角质形成细胞的增殖影响不明显,高浓度(103nmol/L和104nmol/L)对增殖有抑制作用。在适当浓度的他克莫司作用下,角质形成细胞分泌SCF的含量增加,但对bFGF含量无明显影响。他克莫司能上调角质形成细胞SCF mRNA和黑素细胞c-kit mRNA的表达。
3.不同浓度他克莫司作用后的角质形成细胞条件化培养基能明显促进黑素细胞的增殖,但酪氨酸酶活性和黑素细胞含量与对照组相比较变化不明显。
结论
他克莫司可以通过促进黑素细胞迁移、提高酪氨酸酶活性、增加黑素含量等环节直接作用于黑素细胞,产生复色；他克莫司还可以通过角质形成细胞促进黑素细胞的增殖,并促进角质形成细胞分泌细胞因子SCF进而改变黑素细胞生存的微环境,产生复色,从而揭示了他克莫司治疗白癜风的作用机制,为临床提供实验依据,使之更好地服务于临床。
Objective
To investigate the direct and indirect effects of the new topical tacrolimus on pigmentation of melanocytes.Then get the underlying mechanism of how topical tacrolimus induces repigmentation in vitiligo.Our study provide evidence for clinical work.At the same time, topical tacrolimus could be widely used in vitiligo.
Methods
1.Melanogenesis in B16 melanoma cells were seeded in different culture plates for different experiments. Cells at logarithmic phase were used in these experiments.Tacrolimus was added to cultured melanogenesis in B16 melanoma cells at 0(the control group),10,102,103,104nmol/L to investigate the direct effects on them, each group was repeated three times.The measurements included cell proliferation,cell migration,tyrosinase activtion, melanin contents.
2.HaCaT cells were seeded in different culture plates for different experiments. HaCaT cells at logarithmic phase were used in these experiments. Tacrolimus was added to cultured HaCaT cells at 0 (the control group),10,102,103, 104 nmol/L,followed by incubation for 48h and collection of culture supernatant,each group was repeated three times.Cytokines and growth factor in culture supernatant were measured by a commercially available enzyme-linked immunosorbent assay(ELISA) kit.The measurements included stem cell factor(SCF),basic fibroblast growth factor(bFGF). Cell proliferation was also detected. Collect HaCaT cells and melanogenesis in B16 melanoma cells,then extract RNA,investigate the level of mRNA expression of SCF and c-kit by real-time SYBR Green PCR assays.
3.HaCaT cells supernatant obtained from HaCaT cells treated with various tacrolimus concentrations for 48h was added to cultured melanogenesis in B16 melanoma cells.Then investigate the.indirect effects on melanogenesis in B16 melanoma cells via HaCaT cells. The measurements included cell proliferation,tyrosinase activtion, melanin contents.
Results
1.Tacrolimus treatment increased tyrosinase activity and melanin contents, although there was an inhibitory effects on growth of melanocytes. Furthermore, cell migration were enhanced by tacrolimus treatment.
2.Tacrolimus treatment did not increase the proliferation of HaCaT cells.The concertration of SCF in HaCaT cells supernatant increased with tacrolimus treatment. And the level of mRNA expression of SCF and c-kit were significantly impacted by tacrolimus.
3.Results demonstrated that proliferation of melanogenesis in B16 melanoma cells was significantly enhanced by tacrolimus-treated HaCaT cells supernatant.But tyrosinase activity and melanin contents were not significantly impacted.
Conclusions
These findings provide evidence demonstrating direct and indirect effects of tacrolimus on pigmentation of melanocyte proliferation. Migration, tyrosinase activity,melanin contents, mRNA expression of cytokines and growth factors may provide a possible mechanism for the effect of tacrolimus in vitiligo.
通过检测新型免疫抑制剂他克莫司(tacrolimus)对黑素细胞(melanocyte,MC)和角质形成细胞(keratinocytes, KC)的作用,观察他克莫司对黑素细胞的直接和间接影响,寻找其作用靶点,以揭示他克莫司治疗白癜风的作用机制,从而为临床提供实验依据,使之更好地服务于临床。
方法
1.建立小鼠B16黑素瘤细胞培养体系,并根据不同的实验需要将细胞分别接种于相应的培养板,选择对数生长期的细胞进行实验。以不同浓度的他克莫司(10,102,103,104nmol/L)对小鼠B16黑素瘤细胞进行干预,每个浓度组设3个复孔,并设空白对照组。观察他克莫司对小鼠B16黑素瘤细胞的影响并检测黑素细胞增殖(MTT法)、迁移(微孔膜法)、酪氨酸酶活性(多巴氧化法)、黑素含量(NaOH裂解法)等指标。
2.建立人永生化角质形成细胞(HaCaT)培养体系,并根据不同的实验需要将细胞分别接种于相应的培养板,选择对数生长期的细胞进行实验。以不同浓度的他克莫司(10,102,103,104nmol/L)对角质形成细胞进行干预,每个浓度组设3个复孔,并设空白对照组,培养48小时后,收集上清液。采用ELISA法检测上清液中干细胞因子(SCF)和碱性成纤维细胞生长因子(bFGF)的含量,并测定角质形成细胞的增殖情况。收集角质形成细胞和小鼠B16黑素瘤细胞,提取RNA,采用实时荧光定量PCR检测角质形成细胞SCF mRNA、黑素细胞c-kit mRNA的表达水平。
3.以他克莫司作用后的角质形成细胞培养上清液作为条件化培养基对小鼠B16黑素瘤细胞进行干预,检测黑素细胞的增殖(MTT法)、酪氨酸酶活性(多巴氧化法)、黑素含量(NaOH裂解法)等指标,以观察他克莫司通过角质形成细胞对黑素细胞的间接作用。
结果
1.低浓度(10nmol/L和102nmol/L)他克莫司对黑素细胞增殖的影响不明显,高浓度(103nmol/L和104nmol/L)对增殖有抑制作用。各浓度他克莫司均可提高黑素细胞的酪氨酸酶活性并增加黑素含量。此外,他克莫司可以促进黑素细胞的迁移。
2.低浓度(10nmol/L和102nmol/L)他克莫司对角质形成细胞的增殖影响不明显,高浓度(103nmol/L和104nmol/L)对增殖有抑制作用。在适当浓度的他克莫司作用下,角质形成细胞分泌SCF的含量增加,但对bFGF含量无明显影响。他克莫司能上调角质形成细胞SCF mRNA和黑素细胞c-kit mRNA的表达。
3.不同浓度他克莫司作用后的角质形成细胞条件化培养基能明显促进黑素细胞的增殖,但酪氨酸酶活性和黑素细胞含量与对照组相比较变化不明显。
结论
他克莫司可以通过促进黑素细胞迁移、提高酪氨酸酶活性、增加黑素含量等环节直接作用于黑素细胞,产生复色；他克莫司还可以通过角质形成细胞促进黑素细胞的增殖,并促进角质形成细胞分泌细胞因子SCF进而改变黑素细胞生存的微环境,产生复色,从而揭示了他克莫司治疗白癜风的作用机制,为临床提供实验依据,使之更好地服务于临床。
Objective
To investigate the direct and indirect effects of the new topical tacrolimus on pigmentation of melanocytes.Then get the underlying mechanism of how topical tacrolimus induces repigmentation in vitiligo.Our study provide evidence for clinical work.At the same time, topical tacrolimus could be widely used in vitiligo.
Methods
1.Melanogenesis in B16 melanoma cells were seeded in different culture plates for different experiments. Cells at logarithmic phase were used in these experiments.Tacrolimus was added to cultured melanogenesis in B16 melanoma cells at 0(the control group),10,102,103,104nmol/L to investigate the direct effects on them, each group was repeated three times.The measurements included cell proliferation,cell migration,tyrosinase activtion, melanin contents.
2.HaCaT cells were seeded in different culture plates for different experiments. HaCaT cells at logarithmic phase were used in these experiments. Tacrolimus was added to cultured HaCaT cells at 0 (the control group),10,102,103, 104 nmol/L,followed by incubation for 48h and collection of culture supernatant,each group was repeated three times.Cytokines and growth factor in culture supernatant were measured by a commercially available enzyme-linked immunosorbent assay(ELISA) kit.The measurements included stem cell factor(SCF),basic fibroblast growth factor(bFGF). Cell proliferation was also detected. Collect HaCaT cells and melanogenesis in B16 melanoma cells,then extract RNA,investigate the level of mRNA expression of SCF and c-kit by real-time SYBR Green PCR assays.
3.HaCaT cells supernatant obtained from HaCaT cells treated with various tacrolimus concentrations for 48h was added to cultured melanogenesis in B16 melanoma cells.Then investigate the.indirect effects on melanogenesis in B16 melanoma cells via HaCaT cells. The measurements included cell proliferation,tyrosinase activtion, melanin contents.
Results
1.Tacrolimus treatment increased tyrosinase activity and melanin contents, although there was an inhibitory effects on growth of melanocytes. Furthermore, cell migration were enhanced by tacrolimus treatment.
2.Tacrolimus treatment did not increase the proliferation of HaCaT cells.The concertration of SCF in HaCaT cells supernatant increased with tacrolimus treatment. And the level of mRNA expression of SCF and c-kit were significantly impacted by tacrolimus.
3.Results demonstrated that proliferation of melanogenesis in B16 melanoma cells was significantly enhanced by tacrolimus-treated HaCaT cells supernatant.But tyrosinase activity and melanin contents were not significantly impacted.
Conclusions
These findings provide evidence demonstrating direct and indirect effects of tacrolimus on pigmentation of melanocyte proliferation. Migration, tyrosinase activity,melanin contents, mRNA expression of cytokines and growth factors may provide a possible mechanism for the effect of tacrolimus in vitiligo.
引文
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