【摘要】：目的:觀察紅光照射人角質(zhì)形成細(xì)胞株(Ha Ca T)后,細(xì)胞內(nèi)活性氧(ROS)濃度的變化對細(xì)胞增殖活性的影響。方法:建立人角質(zhì)形成細(xì)胞體外培養(yǎng)模型,分別用0.082J/cm2(10S)、0.164J/cm2(20S)、0.245J/cm2(30S)、0.491J/cm2(60S)、1.472J/cm2(180S)、2.453J/cm2(300S)、4.91 J/cm2(600S)、9.81 J/cm2(1200S)8個不同能量密度波長為633nm的紅光照射人角質(zhì)形成細(xì)胞株(Ha Ca T),對照組不照光,DCFH-DA(活性氧檢測試劑盒)孵育1小時后用酶標(biāo)儀分別于孵育后0h、0.5h、1h、2h測定熒光值,反映細(xì)胞內(nèi)ROS的濃度變化;用相同的照射條件照射人角質(zhì)形成細(xì)胞株,每8小時照射1次,照射6次48小時后CCK-8(細(xì)胞增殖試劑)孵育4h,酶標(biāo)儀進行熒光值測定,反映細(xì)胞增殖情況,根據(jù)細(xì)胞內(nèi)由DCFH-DA探針孵育后檢測的熒光結(jié)果,設(shè)計對照組、0.082J/cm2(10S)組、0.491 J/cm2(60S)組、2.453J/cm2(300S)組、9.81 J/cm2(1200S)組、陽性探針檢測組6個組用紅色線粒體示蹤劑Mito-Tracker染色線粒體,同時每個組細(xì)胞內(nèi)孵育活性氧探針,在共聚焦顯微鏡下觀察ROS的來源和線粒體的關(guān)系。結(jié)果:DCFH-DA孵育1小時后0h,0.5h,1h,2h四個時相點測定熒光值,能量密度在4.91J/cm2及以下實驗組,熒光值均有不同程度的增高,其中2.453 J/cm2能量密度組,1h時相點熒光值升高最明顯,和對照組相比,具有明顯的統(tǒng)計學(xué)意義,能量密度在4.91J/cm2以上的實驗組,熒光值升高呈下降趨勢,和對照組相比,沒有明顯的統(tǒng)計學(xué)意義。CCK-8孵育4小時后測定熒光值,4.91J/cm2以下實驗組熒光值增加明顯,其中2.453 J/cm2能量密度組最明顯,和對照組相比具有明顯的統(tǒng)計學(xué)意義,能量密度在4.91J/cm2以上的實驗組,熒光值降低,和對照組相比,沒有明顯的統(tǒng)計學(xué)意義。共聚焦顯微鏡下顯示ROS產(chǎn)生情況與熒光值表現(xiàn)一致,且各組與線粒體重合。結(jié)論:633nm低能量紅光照射對人角質(zhì)形成細(xì)胞增殖具有促進作用,該促進作用與紅光照射刺激人角質(zhì)形成細(xì)胞線粒體產(chǎn)生ROS的增加密切相關(guān)。
[Abstract]:Aim: to observe the effect of the concentration of reactive oxygen species (ROS) on the proliferation of human keratinocytes (Ha Ca T) after irradiation with red light. Methods: human keratinocytes were cultured in vitro and cultured with 0.082J/cm2 (10s), 0.164J/cm2 (20s), 0.245J/cm2 (30s), 0.491J/cm2 (60s), 1.472J/cm2 (180s), 2.453J/cm2 (300s), respectively. 4.91 J/cm2 (600s), 9.81 J/cm2 (1200s), 8 different energy density wavelengths of red light were used to irradiate human keratinocyte line (Ha Ca T), and no light was observed in the control group. DCFH-DA (reactive oxygen species detection kit) was incubated for 1 hour and the fluorescence values were measured at 0 h, 0.5 h, 1 h, 2 h after incubation with enzyme labeling apparatus, respectively, to reflect the change of intracellular ROS concentration. Human keratinocytes were irradiated with the same irradiation condition, once every 8 hours and 48 hours after 6 times of irradiation, CCK-8 (cell proliferation reagent) was incubated for 4 hours. The fluorescence value of enzyme labeling instrument was measured to reflect the proliferation of human keratinocytes. According to the fluorescent results after incubation with DCFH-DA probe, we designed the control group, 0.082J/cm2 (10s) group, 0.491 J/cm2 (60s) group, 2.453J/cm2 (300s) group, 9.81 J/cm2 (1200s) group, and designed the control group, 0.082J/cm2 (10s) group, 2.453J/cm2 (300s) group, 9.81 J/cm2 (1200s) group. The mitochondria in 6 groups were stained with red mitochondrial tracer Mito-Tracker, and the reactive oxygen species probes were incubated in each group. The relationship between the origin of ROS and mitochondria was observed under confocal microscope. Results: after incubation with DCFH-DA for 1 hour, 0 h, 0.5 h, 1 h, 2 h, the fluorescence values were measured. The energy density of the 4.91J/cm2 group was higher than that of the control group. The fluorescence values of the 2.453 J/cm2 group were higher than those of the control group (P < 0.05), and the fluorescence values of the two groups were higher than those of the control group. Compared with the control group, the fluorescence value of the experimental group with energy density above 4.91J/cm2 showed a decreasing trend, compared with the control group, the fluorescence value of the experimental group was significantly higher than that of the control group, and the fluorescence value of the control group was significantly higher than that of the control group. There was no statistical significance. After 4 hours of incubation with CCK-8, the fluorescence value of the experimental group below 4.91J/cm2 increased significantly, among which the 2.453 J/cm2 energy density group was the most obvious, and the fluorescence value of the experimental group below CCK-8 increased significantly. Compared with the control group, the fluorescent value of the experimental group with energy density above 4.91J/cm2 was lower than that of the control group, and there was no significant difference between the experimental group and the control group. Confocal microscopy showed that the production of ROS was consistent with the fluorescent value, and all the groups were coincident with mitochondria. Conclusion: low energy red light irradiation of 633nm can promote the proliferation of human keratinocytes, which is closely related to the increase of ROS produced by mitochondria of human keratinocytes stimulated by red light irradiation.
【學(xué)位授予單位】：遵義醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R622

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