摘要
第一部分：人纯化ET-1通过调节RhoA和Rac1活性对人黑素细胞树突生成的影响
目的：探讨311nm UVB对人角质形成细胞分泌内皮素(ET)-1的影响及人纯化ET-1通过调节RhoA和Rac1活性对人黑素细胞树突生成的作用。
方法：采用二苯基四氮唑溴盐(MTT)法检测311nm UVB对人角质形成细胞存活率的影响。应用ELISA法检测311nm UVB照射人角质形成细胞后收集的培养上清液中白细胞介素(IL)-1a、ET-1和碱性成纤维细胞生长因子(bFGF)的浓度。采用相差显微镜观察人纯化ET-1对人黑素细胞树突生成的影响。应用pull down方法检测人纯化ET-1对人黑素细胞GTP-RhoA和GTP-Rac1蛋白的表达情况。
结果：在25和50mJ／cm~2剂量下，311nm UVB照射后24h对人角质形成细胞的存活率分别为102.55±1.82％和100.57±1.61％，与对照组比较无统计学差异；在100和200mJ／cm~2时，存活率分别下降至91.38±2.41％和80.56±3.31％，与对照组比较明显下降，有统计学意义(P＜0.01)。在25和50mJ／cm~2剂量时，人角质形成细胞培养上清液中IL—1a和ET-1的浓度与对照组比较明显升高(P＜0.05)，其中ET-1在25mJ／cm~2时达到38.85±1.52pg／mL。未经ET-1处理的人黑素细胞树突多为双极或三极，而经ET-1处理的细胞胞体增大，树突明显增多，多于3个树突(不包括3个)以上的细胞(63.67±5.51％)较未处理细胞(28.67±3.06％)明显增多(P＜0.01)。Pull down实验显示人黑素细胞GTP-Rac1的表达在ET-1处理后逐渐升高，10 min时达到处理前的4倍，随后逐渐下调，但处理60 min时仍高于处理前水平；而GTP-RhoA表达开始就明显下降，10 min时降到处理前的1／3，之后有所回升，在处理60 min时几乎达到处理前水平。
结论：311nm UVB具有促进人角质形成细胞分泌ET-1及ET-1可通过活化Rac1和抑制RhoA双重途径促进人黑素细胞树突生成的作用。
第二部分：311nm UVB通过活化Rac1对B16黑素瘤细胞F-actin和树突生成的影响
目的：探讨311nm UVB通过活化Rac1对B16黑素瘤细胞F-actin和树突生成的影响。
方法：采用MTT法检测311nm UVB对B16黑素瘤细胞存活率的影响。应用相差显微镜和激光共聚焦显微镜分别观察311nm UVB对B16黑素瘤细胞的形态学变化和细胞骨架蛋白F-actin的影响。应用pull down方法检测311nmUVB照射前及照射后15 min、30 min、60 min和120 min B16黑素瘤细胞GTP-RhoA和GTP-Rac1蛋白的表达情况。
结果：在25、50和100mJ／cm~2剂量下，311nm UVB照射后24h对B16黑素瘤细胞的存活率与对照组比较无明显差异(P＞0.05)；在200和300mJ／cm~2时，B16黑素瘤细胞的存活率明显下降，存活率分别为87.75±2.66％和79.40±2.31％(P＜0.01)。311nm UVB100 mJ／cm~2照射24h后，B16黑素瘤细胞呈胞体增大近似球状，树突明显增多似树枝，与未照光细胞比较明显增多((P＜0.01)，而未照光细胞树突仅表现为双极或三极。激光共聚焦显微镜显示，B16黑素瘤细胞在饥饿24h而未经311nm UVB照射时，细胞内骨架蛋白张力纤维纹理清晰，而经311nm UVB 100 mJ／cm~2照射30min和60min后细胞骨架蛋白张力纤维解聚，纹理欠清晰，并逐渐加重，在照射6h时张力纤维纹理模糊，呈团块状。Pull down实验显示，与照射前比较311nm UVB100 mJ／cm~2照射后15 min和30 min B16黑素瘤细胞GTP-Rac1蛋白的表达逐渐升高，尤其在30 min时表达是照射前的2倍之多，随后略有下降，但在照射后60 min和120 min表达仍高于对照组；而GTP-RhoA蛋白在照射后略有下降，而后逐渐升高，在120min时升高到照射前的1.6倍。
结论：311nm UVB可通过活化Rac1诱导B16鼠黑素瘤细胞内张力纤维F-actin的解聚，进而促进B16黑素瘤细胞胞体增大和树突增多等形态学的改变。
第三部分：熊果苷和淫羊藿苷对人黑素细胞树突生成及共培养中黑素小体转运的影响
目的：探讨熊果苷和淫羊藿苷对人黑素细胞树突生成及细胞共培养中黑素小体转运的影响。
方法：MTT法检测熊果苷和淫羊藿苷对人黑素细胞和角质形成细胞共培养细胞存活率的影响。相差显微镜观察熊果苷和淫羊藿苷对人黑素细胞树突生成的影响。pull down方法检测熊果苷对人黑素细胞GTP-RhoA和GTP-Rac1蛋白的表达情况。应用流式细胞术检测熊果苷和淫羊藿苷对共培养细胞中黑素小体转运的影响。
结果：熊果苷在药物浓度320-640 mmol／L之间时抑制细胞增殖作用明显(P＜0.05)，在40-160 mmol／L时，抑制作用不明显；淫羊藿苷具有促细胞生长的作用，但当药物浓度在120-240mmol／L之间时，促增殖作用下降。经熊果苷(160mmol／L)处理后，黑素细胞树突退缩，树突平均长度为110.67±7.37μm，与对照组(267±10.15μm)比较有显著性差异(P＜0.01)；而经淫羊藿苷处理后，黑素细胞树突长度虽略增加(285±10.54μm)，但与对照组比较无统计学意义。熊果苷(160mmol／L)处理15 min后黑素细胞GTP-RhoA蛋白表达增多，30 min时达到高峰，60 min时表达出现下降，但在120 min表达仍高于对照组；GTP-Rac1蛋白的表达在15 min、30 min、60 min和120 min与处理前比较无显著性改变。熊果苷和淫羊藿苷对黑素细胞和角质形成细胞共培养中黑素小体转运的抑制作用随药物浓度的增加而逐渐增强，以熊果苷的抑制作用明显，在160mmol／L其抑制作用与1mmol／L的烟酰胺作用相当，抑制率为37.15±3.87％。
结论：熊果苷可通过活化RhoA诱导黑素细胞树突退缩，进而抑制黑素小体的转运。
PartⅠ: Effects of human purified ET-1 on the dendricityvia RhoA and Rac1 in cultured human melanocytes
Objective To investigate the endothelin (ET) -1 level secreted bykeratinocytes after 311nm narrow-band UVB irradiation and the effectof human purified ET-1 on the dendricity by regulating the activityof RhoA and Rac1 in cultured human melanocytes.
Methods MTT test was performed to detect the keratinocyticviability at various doses of 311nm UVB using. ELISA method was usedto detect the IL-la、ET-1 and bFGF level secreted by humankeratinocytes after 311nm UVB irradiation. The dendricity in humanmelanocytes were observed by phase contrast microscope. Pull downassay was performed to detect the activity of GTP-RhoA and GTP-Rac1in human melanocytes before ET-1 treatment and after treatment withET-1 for 5 min, 10 min, 30 min and 60 min respectively.
Results The results of cell viability revealed that 311nm UVB 25and 50 mJ/cm~2 irradiation had little influence on the cell viability,as compared to the control group. However, 311nm UVB 100 and 200mJ/cm~2irradiation significantly reduced the survival rate of cultruedhuman keratinocytes (P＜0.01).The concentrations of IL-la and ET-1were significantly elevated in supernatants of cultured human keratinocytes irradiated with 311nm narrow-band UVB 25 and 50mJ/cm~2,compared with control groups. The number of dendrites considerablyincreased, more than 3 dendrites(63.67±5.51%) in human melanocytestreated with ET-1 50pg/mL, compared to control cells only with twoor three dendrites. Pull down analysis revealed that GTP-Rac1 proteinsignificantly increased by 3 times after treatment with ET-1 50pg/mLfor 10 minutes, then decreased a little but still remained elevatedafter treatment for 30 min and 60 min, compared to control cells.The expression of GTP -RhoA fell into one-third after treatment withET-1 50pg/mL for 10 min, then became elevated and almost reached thebaseline level after treatment for 60 min.
Conclusion ET-1 can promote dendrite formation via dual pathwaysof activating Rac1 and surpressing RhoA in human melanocytes.
PartⅡ: Effects of 311nm UVB on F-actin rearrangement anddendricity via activating Racl in B16 murine melanoma cells
Objective To investigate the effects of 311nm UVB irradiation onF-actin rearrangement and dendrite formation by regulating theactivity of Rac1 in B16 mouse melanoma cells.
Methods MTT method was used to detect the cell viability at variousdoses of 311nm UVB irradiation on B16 mouse melanoma cells. Themorphological changes in B16 melanoma cells were observed by phasecontrast microscope and the cell cytoskeleton F-actin microfilamentwas observed by laser scanning confocal microscope(LSCM). Pull downassay was performed to detect the activity of GTP-RhoA and GTP-Rac1in B16 melanoma cells before UVB irradiation (0 min) and at 15 min, 30 min, 60 min and 120 min after irradiation respectively.
Results The results revealed that 311nm UVB 25、50 and 100 mJ/cm~2irradiation had little influence on the cell viability, compared tothe control group. However, 311nm UVB 200 and 300mJ/cm~2 irradiationsignificantly reduced the survival rate of B16 melanoma cells(P＜0.01) .The morphological changes in B16 melanoma cells wereconsiderably evident with gobular cell body and increased dendriteslike tree branch at 24 hours following irradiation with 311nm UVB100mJ/cm~2 (P＜0.01) , compared to non- irradiated cells with two orthree dendrites. LSCM revealed that F-actin appeared organized withnumerous clear stress fibers crossing the cytoplasm innon-irradiated cells. However, these stress fibers became obscureas actin was disassembled after 311nm UVB 100 mJ/cm~2 irradiation intime-dependent manner. This event could be observed as early as 30min following irradiation and became more evident and showed punctatespots at 6 h. Pull down analysis revealed that GTP-Racl proteinsignificantly increased at 15 min and reached twice at 30 minfollowing 311nm UVB 100 mJ/cm~2 irradiation, then decreased a littlebut still remained elevated at 60 min and 120 min, compared to controlcells. However, GTP-RhoA changed a little during 30 min following311nm UVB 100 mJ/cm~2 irradiation, then became elevated at 60 min andreached 1.6 times at 120 min, compared with control cells.
Conclusion 311nm UVB can promote dendrite formation via activatingRacl in B16 mouse melanoma cells.
PartⅢEffects of arbutin and lcariin on dendricity inmelanocytes and melanin transfer in co-cultrue of melanocytes and keratinocytes
Objective To investigate the effects of arbutin and lcariin ondendricity in human melanocytes and melanin transportation inco-cultrue of human melanocytes and keratinocytes.
Methods MTT method was used to detect the cell viability inco-cultrue of human melanocytes and keratinocytes after treatmentwith arbutin and lcariin . The morphology in human melanocytes wasobserved by phase contrast microscope and pull down assay wasperformed to detect the activity of GTP-RhoA and GTP-Racl in humanmelanocytes before treatment with arbutin (0 min)and after treatmentfor 15 min, 30 min, 60 min and 120 min respectively. Flow cytometrywas performed to detect melanosome transfer in co-cultrue modelofhuman melanocytes and keratinocytes after treatment with arbutin andlcariin.
Results MTT revealed that arbutin at the concetration of 320-640mmol/L significantly reduced the cell viability and had littleinfluence at 40-160mmol/L, as compared to the control group. Lcariinsinificantly increased the cell viability, however , the cellviability reduced at high concetration of lcariin (120-240mmol/L) .Melanocytes treated with arbutin (160mmol/L) for 48h showed dendritelength was 110.67±7.37μm, compared to untreated cells with267±10.15μm. Meanwhile melanocytes treated with lcariin exibitedlittle dendrite change. GTP-RhoA protein increased in some degreeafter treatment with arbutin for 15 min and spiked by 30 min , thendecreased a little. However, GTP-Racl showed no change aftertreatment for 15 min, 30 min, 60 min and 120 min. Melanosome transferwas inhibited in co-cultrue model in a concentration-dependentmanner. Arbutin in concentration of 160mmol/L gave 37.15±3.87% inhibition of melanosome transfer in the co-culture model.
Conclusion Arbutin could inhibit dendrite elogation by activatingRhoA in melanocytes, resulting in the surpression of melanin transferin cocultrue of melanocytes and keratinocytes.
引文
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1.S Etienne-Manneville, A. Hall. Rho GTPases in cell biology[J].Nature, 2002,420(12):629-635.
2. Hall A, Nobes CD. Rho GTPases: molecular switches that control the organization and dynamics of the actin cytoskeleton[J]. Philos Trans R Soc Lond B Biol Sci, 2000,355(1399): 965-970.
3. K. Burridge, K. Wennerberg. Rho and Rac take center stage(J). Cell,2004,116(2):167-179.
4. Gerald Karp. Cell and moleculr biology:concepts and experiments(B), 3nd ed. New York: John Wiley &Sons Inc.,2002:333-392.
5. G. Scott, Rac and Rho: The story behind melanocyte dendrite formation(J).Pigment Cell Res.,2002,15(5):322-330.
6. Lowery D, Clauser K, Hjerrild M et al. Proteomic screen defines the Polo-box domain interactome and identifies Rock2 as a Plk1 substrate(J). EMBO J, 2007,26(9):2262-2273.
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