本文選題：HaCaT細(xì)胞 + 皮質(zhì)抑素��； 參考：《中南大學(xué)》2011年碩士論文

【摘要】：目的 檢測皮質(zhì)抑素(CST)在尋常型銀屑病病人血漿及皮膚中的表達(dá),并在表皮生長因子(EGF)誘導(dǎo)的HaCaT細(xì)胞增殖模型中觀察外源性應(yīng)用CST對HaCaT細(xì)胞增殖的影響,初步探討CST在銀屑病發(fā)病機(jī)制中的作用。 方法 1.ELISA法檢測72例尋常型銀屑病和76例正常對照組血漿中的CST的表達(dá),免疫組化方法檢測14例銀屑病病人(取自上述72例中)皮損區(qū)及正常對照皮膚中的CST的表達(dá)。 2.1×106/孔HaCaT細(xì)胞種六孔板,培養(yǎng)24小時后,細(xì)胞免疫組化方法檢測HaCaT中CST蛋白的表達(dá),RT-PCR方法檢測CST mRNA的表達(dá)。 3.按5000/孔接種HaCaT細(xì)胞至96孔板,待細(xì)胞貼壁后,換含10 ng/ml EGF的RPMI-1640培基,外源性給予不同濃度的CST (10-9-10-6M)處理含10 ng/ml EGF的HaCaT細(xì)胞24小時,MTT法檢測細(xì)胞增殖的改變,重復(fù)三次,觀察外源性CST對EGF誘導(dǎo)的HaCaT細(xì)胞增殖的影響并選取最佳皮質(zhì)抑素濃度進(jìn)行后續(xù)實(shí)驗(yàn)。 4.細(xì)胞免疫組化方法檢測無CST和EGF的HaCaT細(xì)胞組(空白組),含10 ng/ml EGF的HaCaT細(xì)胞組(對照組)及含10-6M CST和10 ng/ml EGF的HaCaT細(xì)胞組(實(shí)驗(yàn)組)三組中增殖指標(biāo)Ki-67的變化。 5.酶聯(lián)免疫吸附試驗(yàn)(ELISA法)檢測以上三組的第二信使cAMP的變化。 結(jié)果 1.銀屑病病人血漿及皮損中皮質(zhì)抑素的表達(dá)較正常對照組均顯著下降(P0.05)但銀屑病病人血漿CST表達(dá)水平與PASI積分無顯著相關(guān)性(P0.05)。 2.CST蛋白在HaCaT細(xì)胞胞漿中有表達(dá)。且CST mRNA(173 bp)在HaCaT細(xì)胞有表達(dá)。 3.不同濃度CST (10-9-10-6M)呈濃度依賴性抑制10 ng/ml的EGF誘導(dǎo)的HaCaT細(xì)胞的增殖。在10-6M時有最好的抑制效應(yīng),抑制率54.62%(P值均0.05) 4.Ki-67在10 ng/ml EGF處理的HaCaT細(xì)胞組(對照組)中表達(dá)較空白組增高,而10-6M CST處理后,Ki-67表達(dá)下調(diào)(P值均0.05)。 5.第二信使cAMP與Ki-67變化一致(P值均0.05)。 結(jié)論 (1)尋常型銀屑病患者皮損及血漿中CST較正常對照組表達(dá)降低可能參與銀屑病的發(fā)病。 (2)外源性CST可以抑制EGF誘導(dǎo)的HaCaT細(xì)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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R758.63

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 田清平;馮雪茹;龐永正;唐朝樞;劉梅林;;血漿皮質(zhì)醇激素抑制素水平與冠心病的關(guān)系[J];北京大學(xué)學(xué)報(醫(yī)學(xué)版);2009年05期

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本文編號：2092107