皮质抑素在寻常型银屑病中的表达及外源性皮质抑素对EGF诱导的HaCaT细胞增殖模型的影响

发布时间：2018-07-03 03:03

  本文选题：HaCaT细胞 + 皮质抑素　； 参考：《中南大学》2011年硕士论文

【摘要】：目的 检测皮质抑素(CST)在寻常型银屑病病人血浆及皮肤中的表达,并在表皮生长因子(EGF)诱导的HaCaT细胞增殖模型中观察外源性应用CST对HaCaT细胞增殖的影响,初步探讨CST在银屑病发病机制中的作用。 方法 1.ELISA法检测72例寻常型银屑病和76例正常对照组血浆中的CST的表达,免疫组化方法检测14例银屑病病人(取自上述72例中)皮损区及正常对照皮肤中的CST的表达。 2.1×106/孔HaCaT细胞种六孔板,培养24小时后,细胞免疫组化方法检测HaCaT中CST蛋白的表达,RT-PCR方法检测CST mRNA的表达。 3.按5000/孔接种HaCaT细胞至96孔板,待细胞贴壁后,换含10 ng/ml EGF的RPMI-1640培基,外源性给予不同浓度的CST (10-9-10-6M)处理含10 ng/ml EGF的HaCaT细胞24小时,MTT法检测细胞增殖的改变,重复三次,观察外源性CST对EGF诱导的HaCaT细胞增殖的影响并选取最佳皮质抑素浓度进行后续实验。 4.细胞免疫组化方法检测无CST和EGF的HaCaT细胞组(空白组),含10 ng/ml EGF的HaCaT细胞组(对照组)及含10-6M CST和10 ng/ml EGF的HaCaT细胞组(实验组)三组中增殖指标Ki-67的变化。 5.酶联免疫吸附试验(ELISA法)检测以上三组的第二信使cAMP的变化。 结果 1.银屑病病人血浆及皮损中皮质抑素的表达较正常对照组均显著下降(P0.05)但银屑病病人血浆CST表达水平与PASI积分无显著相关性(P0.05)。 2.CST蛋白在HaCaT细胞胞浆中有表达。且CST mRNA(173 bp)在HaCaT细胞有表达。 3.不同浓度CST (10-9-10-6M)呈浓度依赖性抑制10 ng/ml的EGF诱导的HaCaT细胞的增殖。在10-6M时有最好的抑制效应,抑制率54.62%(P值均0.05) 4.Ki-67在10 ng/ml EGF处理的HaCaT细胞组(对照组)中表达较空白组增高,而10-6M CST处理后,Ki-67表达下调(P值均0.05)。 5.第二信使cAMP与Ki-67变化一致(P值均0.05)。 结论 (1)寻常型银屑病患者皮损及血浆中CST较正常对照组表达降低可能参与银屑病的发病。 (2)外源性CST可以抑制EGF诱导的HaCaT细胞增殖。
[Abstract]:Objective to detect the expression of cortiostatin (CST) in plasma and skin of patients with psoriasis vulgaris, and to observe the effect of exogenous CST on the proliferation of HaCaT cells induced by epidermal growth factor (EGF). To explore the role of CST in the pathogenesis of psoriasis. Methods 1. The expression of CST in plasma of 72 patients with psoriasis vulgaris and 76 normal controls was detected by Elisa. The expression of CST in the lesions of 14 patients with psoriasis (from above 72 cases) and normal control skin was detected by immunohistochemical method. 2. 1 脳 10 6 / well HaCaT cells were cultured for 24 hours. The expression of CST protein in HaCaT was detected by immunohistochemistry and the expression of CST mRNA was detected by RT-PCR. HaCaT cells with 10 ng/ml EGF were inoculated into 96-well plates according to 5 000 / well. After the cells adhered to the cells, the RPMI-1640 cells containing 10 ng/ml EGF were replaced by RPMI-1640. The cells with 10 ng/ml EGF were treated with different concentrations of CST (10-9-10-6 M) for 24 hours to detect the changes of cell proliferation. To observe the effect of exogenous CST on the proliferation of HaCaT cells induced by EGF and to select the best concentration of cortiostatin for further experiment. 4. The changes of Ki-67 in HaCaT cells without CST and EGF (blank group), HaCaT cell group (control group) with 10 ng/ml EGF and HaCaT cell group (experimental group) with 10-6M CST and 10 ng/ml EGF were detected by immunohistochemistry. Enzyme-linked immunosorbent assay (Elisa) was used to detect the changes of second messenger camp in the above three groups. Result 1. The expression of cortiostatin in plasma and lesions of psoriatic patients was significantly lower than that in normal controls (P0.05), but there was no significant correlation between CST expression and PASI score in psoriatic patients (P0.05). 2. CST protein was expressed in the cytoplasm of HaCaT cells. CST mRNA (173 BP) was expressed in HaCaT cells. Different concentrations of CST (10-9-10-6 M) inhibited the proliferation of HaCaT cells induced by EGF for 10 ng/ml in a concentration-dependent manner. At 10-6 M, the inhibitory rate was 54.62% (P < 0.05). The expression of Ki-67 in HaCaT cells treated with 10 ng/ml EGF (control group) was higher than that in control group, but the expression of Ki-67 was down-regulated (P < 0.05) after 10-6M CST treatment. The changes of camp and Ki-67 in the second messenger were consistent (P 0.05). Conclusion (1) the lower expression of CST in skin lesions and plasma of psoriasis vulgaris may be involved in the pathogenesis of psoriasis. (2) exogenous CST can inhibit the proliferation of HaCaT cells induced by EGF.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R758.63

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