【摘要】：近年來,紫外線與人皮膚健康的關(guān)系一直是研究的熱點(diǎn)。紫外線是把“雙刃劍”,一方面,紫外線一直被認(rèn)為是皮膚的殺手,除了可能導(dǎo)致各種光敏性皮膚病,過多的紫外線照射也是造成皮膚光老化及暴露部位皮膚癌多發(fā)的最重要因素；另一方面,紫外線也存在有益的生物學(xué)效應(yīng),接受適當(dāng)?shù)淖贤饩�照射可以提高機(jī)體免疫力,預(yù)防某些自身免疫性疾病,促進(jìn)骨骼的生長發(fā)育,減少腫瘤的發(fā)生。這些有益的生物效應(yīng)可能與其低劑量照射誘導(dǎo)的興奮效應(yīng)存在一致的關(guān)系。適應(yīng)性反應(yīng)是輻射興奮效應(yīng)的重要內(nèi)容。我們前期實驗已證實低劑量的長波紫外線(UVA)輻射可以誘導(dǎo)培養(yǎng)皮膚細(xì)胞出現(xiàn)適應(yīng)性反應(yīng),這種適應(yīng)性反應(yīng)表現(xiàn)為單次或多次低劑量UVA預(yù)照射能減輕隨后致死劑量UVA照射對培養(yǎng)皮膚成纖維細(xì)胞形態(tài)學(xué)上的毒性反應(yīng),并使細(xì)胞凋亡的比例下降、DNA鏈斷裂減少及修復(fù)加快,但這種適應(yīng)性反應(yīng)的機(jī)制仍有待進(jìn)一步闡明。自噬(autophagy)是真核細(xì)胞中普遍存在的生命現(xiàn)象,是將細(xì)胞內(nèi)變形、衰老或損傷的蛋白質(zhì)和細(xì)胞器轉(zhuǎn)運(yùn)到溶酶體腔中消化降解的一種代謝過程。自噬有利于為此細(xì)胞和組織的動態(tài)平衡,是維持真核細(xì)胞內(nèi)環(huán)境的自我穩(wěn)定并實現(xiàn)更新的一種有效途徑,也是其適應(yīng)環(huán)境變化的一種重要的防御機(jī)制,但低劑量UVA照射誘導(dǎo)的適應(yīng)性反應(yīng)與自噬之間的相關(guān)性目前仍末見報道。本文觀察了低劑量UVA誘導(dǎo)人皮膚成纖維細(xì)胞適應(yīng)性反應(yīng)中自噬的變化,探討其在誘導(dǎo)培養(yǎng)人皮膚成纖維細(xì)胞適應(yīng)性反應(yīng)的可能作用,為全面闡明低劑量UVA誘導(dǎo)人皮膚細(xì)胞適應(yīng)性反應(yīng)的機(jī)制提供更多的實驗依據(jù)。目的：1.觀察低劑量長波紫外線誘導(dǎo)人皮膚成纖維細(xì)胞出現(xiàn)適應(yīng)性反應(yīng)中自噬的形態(tài)及數(shù)量的變化,對比不同組細(xì)胞的自噬形態(tài)及數(shù)量的差異。2.通過檢測自噬相關(guān)蛋白LC3、Beclinl來探討低劑量UVA誘導(dǎo)人皮膚成纖維細(xì)胞適應(yīng)性反應(yīng)中自噬的可能機(jī)制。3.探討自噬在誘導(dǎo)培養(yǎng)人皮膚成纖維細(xì)胞適應(yīng)性反應(yīng)的可能作用。方法：1.取18歲健康男性陰莖包皮環(huán)切術(shù)后包皮(取自廣州軍區(qū)廣州總醫(yī)院泌尿外科門診手術(shù)室)采用組織消化法培養(yǎng)人皮膚成纖維細(xì)胞。2.分組：假照射組：為正常的培養(yǎng)細(xì)胞,與照射組同樣處理后置于照射裝置下進(jìn)行假照射；低劑量照射組：(1)低劑量照射A組：每天以單次劑量分別為7.2J/cm2、14.4 J/cm2、 21.6J/cm2、28.8J/cm2、36J/cm2照射進(jìn)行照射；(2)低劑量照射B組：每天以劑量為7.2 J/cm2進(jìn)行照射,累積劑量分別為7.2J/cm2、14.4J/cm2、21.6J/cm2、28.8 J/cm2、36 J/cm2。高劑量照射組：(1)高劑量照射A組：每天以單次劑量分別為7.2J/cm2、14.4J/cm2、21.6 J/cm2、28.8 J/cm2、36 J/cm2照射后,隨后以86.4J/cm2 UVA照射細(xì)胞。(2)高劑量照射B組：每天為先以累積劑量分別為7.2 J/cm2、14.4 J/cm2、21.6 J/cm2、 28.8 J/cm2、36 J/cm2低劑量UVA預(yù)照射后,再以86.4J/cm2 UVA照射細(xì)胞。3.運(yùn)用激光共聚焦掃描顯微鏡觀察MDC染色后各組細(xì)胞內(nèi)自噬形態(tài)及數(shù)量的變化,對比不同組細(xì)胞的自噬形態(tài)及數(shù)量的差異；利用流式細(xì)胞術(shù)檢測各組自噬相關(guān)蛋白LC3、Beclinl自噬陽性表達(dá)百分比。4.統(tǒng)計學(xué)方法：用GraphPad Prism 5軟件進(jìn)行統(tǒng)計學(xué)分析,所有數(shù)據(jù)均采用均數(shù)±標(biāo)準(zhǔn)差(x±s)。方差齊性檢驗后,進(jìn)行雙因素方差分析,以P0.05為差異有統(tǒng)計學(xué)意義。結(jié)果：1.組織消化法培養(yǎng)的人皮膚成纖維細(xì)胞24h內(nèi)貼壁,剛開始貼壁時細(xì)胞多數(shù)呈圓形,隨貼壁時間延長逐漸伸展成梭形或多邊形。5-6天進(jìn)行傳代,傳代后約5天長滿。取第3-10代人皮膚成纖維細(xì)胞進(jìn)行實驗研究。2.采用激光共聚焦顯微鏡觀察,假照射組的細(xì)胞自噬體陽性表達(dá)率在1d、2d、3d、4d、5d時分別為4.333±0.577、4.333±1.528、5.000±1.155、7.000±1.000、5.333±1.155,假照射組組內(nèi)的細(xì)胞自噬體陽性表達(dá)率無顯著差異(P0.05)；低劑量照射A組的細(xì)胞自噬體陽性表達(dá)率在1d、2d、3d、4d、5d時分別為13.677±1.528、37.333±1.528、57.333±2.082、79.333±1.528、36.330±1.528,與假照射組相比較有顯著差異(P0.01)；低劑量照射B組的細(xì)胞自噬體陽性表達(dá)率在1d、2d、3d、4d、5d時分別為13.677±1.284、32.000±2.000、46.677±1.528、57.000±2.000、30.000±1.000,與假照射組相比較有顯著差異(P0.01)；低劑量照射A組和低劑量照射B組的細(xì)胞自噬體陽性表達(dá)率均隨著照射劑量的增加而增加,但在28.8 J/cm2后細(xì)胞自噬體陽性表達(dá)率出現(xiàn)減少,低劑量照射A組細(xì)胞自噬體陽性表達(dá)率比低劑量照射B組高,低劑量照射A組與低劑量照射B組相比較有顯著差異(P0.01)。高劑量照射A組的細(xì)胞自噬體陽性表達(dá)率在1d、2d、3d、4d、5d時分別為29.360±2.020、80.130±3.096、45.140±1.845、19.630±0.772、7.537±0.463,與假照射組相比較有顯著差異(P0.01)；高劑量照射B組的細(xì)胞自噬體陽性表達(dá)率在1d、2d、3d、4d、5d時分別為29.360±2.020、35.850±1.513、56.100±1.365、76.000±1.579、31.440±1.662,與假照射組相比較有顯著差異(P0.01)；高劑量照射A組在14.4 J/cm2接受86.4J/cm2照射后,細(xì)胞自噬體陽性表達(dá)率出現(xiàn)減少,高劑量照射B組細(xì)胞自噬體陽性表達(dá)率在28.8 J/cm2接受86.4J/cm2照射后出現(xiàn)減少,高劑量照射A組與高劑量照射B組相較有顯著差異(P0.01)。3.與細(xì)胞自噬體陽性表達(dá)率檢測結(jié)果相一致,低劑量照射A組和低劑量照射B組的Beclinl、LC3表達(dá)均隨著照射劑量的增加而升高,但在28.8 J/cm2后Beclinl、LC3表達(dá)水平下降,低劑量照射A組Beclinl、LC3表達(dá)水平均比低劑量照射B組高,低劑量照射A組與低劑量照射B組相比較有顯著差異(P0.01)；高劑量照射A組與高劑量照射B組相比,高劑量照射A組Beclinl、LC3表達(dá)很快達(dá)到高峰值然后迅速下調(diào),而高劑量照射B組Beclinl、LC3表達(dá)仍能慢慢隨著劑量的累積而增加高峰值才出現(xiàn)下調(diào)。高劑量照射A組與高劑量照射B組相比較有顯著差異(P0.01)。結(jié)論：1.低劑量UVA照射誘導(dǎo)人皮膚成纖維細(xì)胞出現(xiàn)適應(yīng)性反應(yīng)同時可引起人皮膚成纖維細(xì)胞自噬的改變,其變化的規(guī)律與劑量密切相關(guān),其調(diào)控與LC3、Beclinl表達(dá)有關(guān)。2.結(jié)果表明,自噬在低劑量UVA照射誘導(dǎo)人皮膚成纖維細(xì)胞出現(xiàn)適應(yīng)性反應(yīng)可能起重要作用。
[Abstract]:In recent years, the relationship between ultraviolet radiation and human skin health has been a research hotspot. Ultraviolet radiation is a "double-edged sword". On the one hand, ultraviolet radiation has been regarded as a killer of skin. In addition to possibly causing various photosensitive skin diseases, excessive ultraviolet radiation is also the most important factor causing skin photoaging and skin cancer in exposed areas. On the other hand, ultraviolet radiation also has beneficial biological effects. Appropriate ultraviolet irradiation can improve the immunity of the body, prevent some autoimmune diseases, promote bone growth and development, and reduce the incidence of tumors. Adaptive response is an important part of the excitatory effect of radiation. Previous studies have shown that low-dose long-wave ultraviolet radiation (UVA) can induce adaptive response of cultured skin cells. This adaptive response is manifested by single or multiple low-dose UVA pre-irradiation that can reduce the subsequent lethal dose of UVA radiation on cultured skin fibroblast fineness. Autophagy is a ubiquitous life phenomenon in eukaryotic cells, which transports deformed, senescent or damaged proteins and organelles to lysosomes. Autophagy is a metabolic process of intracavitary digestion and degradation. It is an effective way to maintain the homeostasis and renewal of eukaryotic cells. It is also an important defense mechanism for eukaryotic cells to adapt to environmental changes. In this paper, we observed the changes of autophagy in the adaptive response of human skin fibroblasts induced by low dose UVA, and discussed its possible role in inducing the adaptive response of cultured human skin fibroblasts. AIM: 1. To observe the morphological and quantitative changes of autophagy in the adaptive response of human skin fibroblasts induced by low-dose long-wave ultraviolet radiation, and to compare the morphological and quantitative differences of autophagy in different groups. 2. To explore the autophagy in the adaptive response of human skin fibroblasts induced by low-dose UVA by detecting autophagy-related protein LC3 and Beclinl. To explore the possible mechanism of phagocytosis. 3. To explore the possible role of autophagy in inducing adaptive response of cultured human skin fibroblasts. Methods: 1. Human skin fibroblasts were cultured by tissue digestion method after penile circumcision in 18-year-old healthy men. Group A was irradiated with a single dose of 7.2J/cm2, 14.4J/cm2, 21.6J/cm2, 28.8J/cm2, 36J/cm2, respectively. Group B was irradiated with a low dose of 7.2J/cm2 daily. The cumulative doses were 7.2J/cm2, 14.4J/cm2, 21.6J/cm2, 28.8J/cm2, 36 J/cm2. High dose group: (1) High dose group A: Single dose was 7.2J/cm2, 14.4J/cm2, 21.6J/cm2, 28.8J/cm2, 36 J/cm2, and then 86.4J/cm2 UVA was used to irradiate the cells. (2) High dose group B: The cumulative doses were 7.2J/cm2, 14.4J/cm2, 21.6J/cm2, 28.8J/cm2, 36 J/cm2, respectively. The amount of autophagy was 7.2 J/cm2,14.4 J/cm2,21.6 J/cm2,28.8 J/cm2,36 J/cm2 after low-dose UVA pre-irradiation, and then 86.4 J/cm2 UVA was used to irradiate the cells. Autophagy-related protein LC3 and Beclinl autophagy positive expression percentage were detected by operation. 4. Statistical method: GraphPad Prism 5 software was used for statistical analysis. All data were analyzed by means of mean (+) standard deviation (x (+) s). After homogeneity test of variance, two-way ANOVA was performed, and the difference was statistically significant with P 0.05. Results: 1. The cultured human skin fibroblasts adhered to the wall within 24 hours. Most of the cells were round when they first adhered to the wall. The cells gradually expanded into spindle or polygon with the prolongation of adherence time. After passage, they grew up about 5 days. The human skin fibroblasts of the 3rd to 10th generations were taken for experimental study. 2. The cells in the sham irradiation group were observed by laser confocal microscope. The positive rate of phage expression was 4.333+0.577,4.333+1.528,5.000+1.155,7.000+1.000,5.333+1.155 at day 1,2,3,4 and 5,respectively. There was no significant difference in the positive rate of autophagy expression in sham irradiation group (P 0.05); the positive rate of autophagy expression in low dose irradiation group A was 13.677+1.528,37.333+1.528 at day 1,4,5 and 5 respectively. The positive rates of autophagy in low dose irradiation group B were 13.677 [1.284], 32.000 [2.000], 46.677 [1.528], 57.000 [2.000] and 30.000 [1.000] at 1 day, 2 days, 3 days, 4 days and 5 days respectively (P 0.01). The positive expression rate of autophage in group A and group B increased with the increase of irradiation dose, but decreased after 28.8 J/cm 2. The positive expression rate of autophage in group A was higher than that in group B after low dose irradiation. The positive rates of autophagy in group A were 29.360 (+ 2.020), 80.130 (+ 3.096), 45.140 (+ 1.845), 19.630 (+ 0.772) and 7.537 (+ 0.463) on the 1st, 2nd, 3rd, 4th and 5th day, respectively, which were significantly different from those in sham irradiation group A (P 0.01). Compared with sham irradiation group, the positive expression rate of autophagy decreased in group A after 14.4 J/cm2 irradiation and 86.4 J/cm2 irradiation. The positive expression rate of autophagy in group B was 28.8 J/cm2 irradiation and 86.4 J/cm2 irradiation respectively. The expression of Beclinl and LC3 in low dose group A and low dose group B increased with the increase of irradiation dose, but the expression of Beclinl and LC3 decreased after 28.8 J/cm 2. The expression levels of Beclinl and LC3 in low dose group A were higher than those in low dose group B, and there was significant difference between low dose group A and low dose group B (P 0.01). Conclusion: 1. Low dose UVA irradiation can induce the adaptive response of human skin fibroblasts and induce the change of autophagy of human skin fibroblasts. The results suggest that autophagy may play an important role in the adaptive response of human skin fibroblasts induced by low dose UVA irradiation.
【學(xué)位授予單位】：廣州中醫(yī)藥大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R758.1

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3 唐煥文;莊志雄;何云;;hPARP-1蛋白缺陷細(xì)胞適應(yīng)性反應(yīng)研究[A];中國毒理學(xué)會——第一屆全國中青年學(xué)者科技論壇論文（摘要）集[C];2004年

4 潘燕;袁德曉;邵春林;;鎘誘導(dǎo)的細(xì)胞適應(yīng)性反應(yīng)及DNA損傷修復(fù)在其中的作用[A];中國毒理學(xué)會放射毒理專業(yè)委員會第七次、中國毒理學(xué)會免疫毒理專業(yè)委員會第五次、中國環(huán)境誘變劑學(xué)會致突專業(yè)委員會第二次、中國環(huán)境誘變劑學(xué)會致畸專業(yè)委員會第二次、中國環(huán)境誘變劑學(xué)會致癌專業(yè)委員會第二次全國學(xué)術(shù)會議論文匯編[C];2008年

5 劉建軍;黃海燕;莊志雄;袁建輝;陽帆;李習(xí)藝;;三氯乙烯誘導(dǎo)L-02肝細(xì)胞適應(yīng)性反應(yīng)蛋白質(zhì)組學(xué)改變[A];第五屆廣東省環(huán)境誘變劑學(xué)會暨第三屆廣東省預(yù)防醫(yī)學(xué)會衛(wèi)生毒理專業(yè)委員會學(xué)術(shù)交流會論文集[C];2006年

6 劉仲榮;楊慧蘭;;低劑量長波紫外線誘導(dǎo)人皮膚成纖維細(xì)胞適應(yīng)性反應(yīng)中ROS的變化觀察[A];美麗人生 和諧世界——中華醫(yī)學(xué)會第七次全國醫(yī)學(xué)美學(xué)與美容學(xué)術(shù)年會、中華醫(yī)學(xué)會醫(yī)學(xué)美學(xué)與美容學(xué)分會20周年慶典暨第三屆兩岸四地美容醫(yī)學(xué)學(xué)術(shù)論壇論文匯編[C];2010年

7 劉光偉;李鵬武;劉淑春;龔守良;;低劑量電離輻射誘導(dǎo)小鼠睪丸生精細(xì)胞凋亡的適應(yīng)性反應(yīng)[A];“加入WTO和科學(xué)技術(shù)與吉林經(jīng)濟(jì)發(fā)展——機(jī)遇·挑戰(zhàn)·責(zé)任”吉林省第二屆科學(xué)技術(shù)學(xué)術(shù)年會論文集（下）[C];2002年

8 衛(wèi)秦芝;徐雷;姚朗;莊志雄;;應(yīng)用熒光DDRT—PCR初探低劑量的ECPs誘導(dǎo)細(xì)胞適應(yīng)性反應(yīng)的機(jī)理[A];中國毒理學(xué)會第二屆全國中青年學(xué)者科技論壇會議論文集[C];2007年

9 袁德曉;潘燕;張江虹;邵春林;;低劑量鎘暴露誘導(dǎo)Hmy2.CIR細(xì)胞對電離輻射的適應(yīng)性反應(yīng)[A];中國毒理學(xué)會第五次全國學(xué)術(shù)大會論文集[C];2009年

10 蘇旭;劉建香;鞠桂芝;劉樹錚;;低劑量輻射誘導(dǎo)TCR/CD3、CD4和CD8表達(dá)的適應(yīng)性反應(yīng)[A];中華醫(yī)學(xué)會放射醫(yī)學(xué)與防護(hù)學(xué)分會第三次全國中青年學(xué)術(shù)交流會論文匯編[C];2001年

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