【摘要】：目的 在基因及蛋白質(zhì)水平上研究白細(xì)胞介素-6,白細(xì)胞介素-4對人角質(zhì)形成細(xì)胞:HaCaT細(xì)胞合成結(jié)合珠蛋白的影響。 探討腫瘤壞死因子α及干擾素γ對HaCaT細(xì)胞合成結(jié)合珠蛋白的影響。 探討免疫抑制劑地塞米松及甲氨喋呤對HaCaT細(xì)胞合成結(jié)合珠蛋白的影響。 材料和方法 1.細(xì)胞培養(yǎng) 將HaCaT細(xì)胞接種于六孔板內(nèi),在含有10%胎牛血清(fetal calf serum,FCS),1mM丙酮酸鹽,2mM谷氨酰胺,青霉素(100μg/mL),鏈霉素(100μg/mL)的1640培養(yǎng)液中,5% CO_2,37℃條件下培養(yǎng)。細(xì)胞形成單層后加入含有不同刺激因子的,含有或不含1μM地塞米松的,不含有胎牛血清的1640培養(yǎng)液。所用刺激因子如下:白細(xì)胞介素-6(interleukin-6,50ng/mL),白細(xì)胞介素-4(interleukin-4,20ng/mL),腫瘤壞死因子α(tumor necrosis factor α,20ng/mL),干擾素γ(interferon γ,20ng/ml),地塞米松(dexamethasone,10μM,100μM,1000μM)及甲氨喋呤(methotrexate,50ng/ml,500ng/ml,5000ng/ml)。分別作用12,24,48小時(shí)后,收集細(xì)胞及細(xì)胞上清,用于結(jié)合珠蛋白(Haptoglobin,Hp)的檢測。 2.實(shí)驗(yàn)方法 (1) 半定量RT-PCR ① 細(xì)胞總RNA的提取:Trizol RNA提取試劑,由3×10~6細(xì)胞中提取總RNA。
[Abstract]:Objective to study the effects of interleukin-6 and interleukin-4 on the synthesis of globin in human keratinocytes: HaCaT cells at the gene and protein levels. To investigate the effect of tumor necrosis factor 偽 and interferon 緯 on the synthesis of globin in HaCaT cells. To investigate the effects of immunosuppressants dexamethasone and methotrexate on the synthesis of globin in HaCaT cells. Materials and methods 1. HaCaT cells were inoculated into six well plates in cell culture. The cells containing 10% fetal bovine serum (fetal calf serum,FCS, 1mM pyruvate, 2mM glutamine, penicillin (100 渭 g/mL) were cultured in vitro. Streptomycin (100 渭 g/mL) was cultured in 1640 medium at 5% CO_2,37 鈩，

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