本文關(guān)鍵詞： 紫外線 皮膚成纖維細(xì)胞 角質(zhì)形成細(xì)胞 自噬 凋亡　出處：《北京協(xié)和醫(yī)學(xué)院》2012年博士論文　論文類(lèi)型：學(xué)位論文

【摘要】：探討中波紫外線(ultraviolet B, UVB)照射人皮膚成纖維細(xì)胞誘導(dǎo)的自噬對(duì)細(xì)胞凋亡活性的影響。通過(guò)單丹磺酰戊二胺(Monodansylcadaverine, MDC)染色和免疫熒光標(biāo)記微管相關(guān)蛋白1輕鏈3(light chain3, LC3)的方法,確定不同劑量3-甲基腺嘌呤(3-methyladenine3-MA)對(duì)自噬的抑制作用。UVB照射后立即使用0.5mmol/L3-MA孵育細(xì)胞4h作為自噬的抑制方法。Hoechst和碘化丙啶(propidium iodide, PI)染色結(jié)合Annexin V-異硫氰酸熒光素(fluorescein isothiocyanate, FITC)和PI標(biāo)記細(xì)胞后流式細(xì)胞術(shù)作為凋亡的分析方法。0.5mmol/L3-MA能明顯抑制人皮膚成纖維細(xì)胞自噬活性(饑餓誘導(dǎo)組自噬細(xì)胞陽(yáng)性百分?jǐn)?shù)為63.037%±5.876%,3-MA孵育后下降至34.425%±5.183%)。0.5mmol/L3-MA對(duì)細(xì)胞活性影響最小。50mJ/cm2照射劑量下3-MA孵育較未孵育細(xì)胞強(qiáng)Hoechst和強(qiáng)PI雙染細(xì)胞增多；100mJ/cm2照射劑量下3-MA孵育較未孵育細(xì)胞強(qiáng)Hoechst和強(qiáng)PI雙染細(xì)胞減少。在50mJ/cm2照射劑量下,抑制自噬的細(xì)胞的中晚期凋亡細(xì)胞百分比(10.933±0.839)較未抑制細(xì)胞(7.267±0.473)上升,兩者之間差異具有統(tǒng)計(jì)學(xué)意義(t=5.197,P=0.05)；而在100mJ/cm2照射劑量下,抑制自噬的細(xì)胞的中晚期凋亡水平(7.100±0.781)較未抑制細(xì)胞(10.133±0.681)下降,兩者之間差異具有統(tǒng)計(jì)學(xué)意義(t=6.286, P0.05)。UVB誘導(dǎo)的人皮膚成纖維細(xì)胞自噬在50mJ/cm2照射劑量下通過(guò)抑制凋亡對(duì)細(xì)胞起到保護(hù)作用,而在100mJ/cm2照射劑量下發(fā)生的較高水平白噬可能誘導(dǎo)了自噬性細(xì)胞死亡。 觀察慢性紫外線損傷對(duì)小鼠皮膚角質(zhì)形成細(xì)胞凋亡和自噬交互調(diào)控元件Bax、 Bcl-2、Beclin-1及半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)的調(diào)控效應(yīng)。采用模擬日光的UV對(duì)實(shí)驗(yàn)小鼠進(jìn)行照射,設(shè)立對(duì)照。照射從最小紅斑量(UVB0.07J/cm2,長(zhǎng)波紫外線(ultraviolet A, UVA)0.7J/cm2)開(kāi)始,1次/d,每周增加0.5個(gè)紅斑量,每周照射5天,共9周,UVB總劑量達(dá)9.45J/cm2, UVA總劑量達(dá)94.5J/cm2,應(yīng)用免疫組化法分別對(duì)實(shí)驗(yàn)前后(W0和W9) Bax、Bcl-2、Beclin-1和Caspase-3的表達(dá)進(jìn)行檢測(cè),表達(dá)強(qiáng)度以免疫反應(yīng)強(qiáng)度分布指數(shù)(immunoreactivity intensity distribution index, IRIDI)表示。在損傷組,照光(?)(?) Beclin-1、Caspase-3、Bax、Bcl-2的表達(dá)計(jì)分均值(范圍)分別為0.30(0-2)、0.25(0-2)、0.35(0-2)、0.25(0-1),照光后計(jì)分分別為2.70(2-6)、3.30(2-9)、3.35(2-6)、0.25(0-1)。損傷組小鼠表皮角質(zhì)形成細(xì)胞中Beclin-1、Caspase-3、Bax蛋白表達(dá)顯著升高,且差異有統(tǒng)計(jì)學(xué)意義(P均0.05),Bcl-2蛋白表達(dá)變化不明顯,且無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。損傷組W9時(shí)Beclin-1和Bax的表達(dá)明顯高于Bcl-2(P均0.05)。在同時(shí)期正常飼養(yǎng)小鼠Beclin-1、Caspase-3. Bax、Bcl-2的均值(范圍)在W0為0.20(0-1)、0.20(0-1)、0.30(0-1)、0.20(0-1),W9則為0.30(0-1)、0.20(0-1)、0.30(0-1)、0.10(0-1)。分析發(fā)現(xiàn)Beclin-1、Caspase-3、Bax、Bcl-2表達(dá)無(wú)統(tǒng)計(jì)學(xué)意義上的表達(dá)差異(P0.05)。慢性UV損傷對(duì)小鼠皮膚角質(zhì)形成細(xì)胞中凋亡和自噬的交互調(diào)控元件Beclin-1和Bax具有上調(diào)表達(dá)的作用,而對(duì)另一元件Bcl-2表達(dá)調(diào)控效應(yīng)不顯著。這一系列的調(diào)控效應(yīng)可能參與了凋亡和自噬在慢性UV皮膚損傷中的交互調(diào)控。 研究UVB照射對(duì)體外培養(yǎng)的HaCaT細(xì)胞自噬的調(diào)控效應(yīng)。使用由低到高三種劑量照射HaCaT細(xì)胞后繼續(xù)培養(yǎng)4小時(shí),使用透射電鏡進(jìn)行細(xì)胞超微結(jié)構(gòu)分析,并使用蛋白免疫印跡方法進(jìn)行LC3-Ⅰ→Ⅱ型轉(zhuǎn)換分析。結(jié)果發(fā)現(xiàn)小劑量和中等劑量的UVB照射后,細(xì)胞內(nèi)可出現(xiàn)自噬體,并且在小劑量照射后更為多見(jiàn)；然而,當(dāng)接受了高劑量照射后,細(xì)胞中不能發(fā)現(xiàn)此類(lèi)的結(jié)構(gòu)。小劑量UVB照射后,LC3-Ⅱ的蛋白表達(dá)量升高；中等劑量UVB照射對(duì)LC3-Ⅱ的蛋白表達(dá)量沒(méi)有產(chǎn)生顯著的變化；而高劑量的UVB照射產(chǎn)生了對(duì)LC3-Ⅱ的蛋白表達(dá)顯著下調(diào)。研究結(jié)果提示小劑量UVB單次照射對(duì)角質(zhì)形成細(xì)胞具有誘導(dǎo)自噬的效應(yīng),而高劑量的UVB單次照射產(chǎn)生了抑制自噬的效應(yīng)。
[Abstract]:Study of ultraviolet (ultraviolet B, UVB) the effect of fiber cells induced by irradiation of human skin autophagy on cell apoptosis activity. Through single dansyl amyl amine two (Monodansylcadaverine, MDC) staining and immunofluorescence staining of microtubule associated protein 1 light chain 3 (light chain3 LC3) method to determine the different doses of 3- methyl adenine (3-methyladenine3-MA) on autophagy inhibition.UVB immediately after irradiation with 0.5mmol/L3-MA incubated 4H cells as the suppression method of autophagy of.Hoechst and propidium iodide (propidium iodide PI) Annexin V- staining with fluorescein isothiocyanate (fluorescein isothiocyanate, FITC) and PI labeled cells after flow cytometry as the analysis method of.0.5mmol/L3-MA apoptosis can inhibit human skin fibroblast activity (autophagy induced by starvation in percentage of positive cells was 63.037% + 5.876% group of autophagy, 3-MA decreased to 34.4 after incubation 25% + 5.183%).50mJ/cm2 minimum radiation dose of 3-MA for the effect of.0.5mmol/L3-MA on cell activity was less incubated with strong Hoechst and PI double staining cells increased; 100mJ/cm2 dose 3-MA incubation is incubated with strong Hoechst and strong PI staining in 50mJ/cm2 cells decreased. Irradiation dose, apoptosis the percentage of inhibition of autophagy cells (10.933 + 0.839) is not inhibited cell (7.267 + 0.473) increased, with a significant difference (t=5.197, P=0.05); and in the 100mJ/cm2 dose, in the late stage of apoptosis inhibiting autophagy cells (7.100 + 0.781) is not inhibited cell (10.133 + 0.681) decreased, with a significant difference (t=6.286, P0.05) of human skin.UVB induced autophagy into fiber cells by inhibiting apoptosis and protect the cells in the 50mJ/cm2 radiation dose, and 100mJ/cm2 irradiation High levels of white phagocytosis at a dose may induce autophagic cell death.
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