【作者】 赵娜；

【导师】 李艳佳；

【作者基本信息】 河北医科大学 ， 皮肤病与性病学， 2008， 硕士

【摘要】 目的:Ca²⁺是人体内最普遍使用的信号转导因子,在细胞生命活动中起着重要的作用,所有类型细胞的各种功能均不同程度的被Ca²⁺调控。阳光中的紫外线辐射是一种非常显著的环境诱变因子,对角质形成细胞的损伤作用已经明确,而维生素C的抗氧化作用也有很多研究,其重要的抗氧化作用越来越受到重视。但目前在细胞水平上,关于维生素C对角质形成细胞光损伤的干预作用机制尚不完全清楚,是否与其钙调控有关尚未见报道。本实验首先用窄谱中波紫外线（NB-UVB）对角质形成细胞进行辐射,然后给予不同浓度的维生素C,着重通过观察细胞形态、细胞活性及细胞内Ca²⁺浓度变化来探讨维生素C的抗氧化作用与细胞内Ca²⁺浓度变化的相关性。方法:角质形成细胞的分离与培养:收集我院泌尿外科青少年包皮环切术后的包皮组织,利用中性分离酶和胰蛋白酶联合消化法分离细胞,加入DMEM培养基制成细胞混悬液,转入25cm2培养瓶中,于37℃、5%CO2培养箱中培养,隔天换液。原代角质形成细胞培养至15天左右细胞接近融合状态时胰蛋白酶消化,按1:3的比例传代,37℃、5%CO2培养箱中培养,次日更换培养基。以后视细胞生长情况隔天更换培养基。确定最佳辐射剂量:选择第四代对数生长期的细胞,消化后制成细胞混悬液,按1×105/ml接种于24孔培养板中,37℃、5%CO2孵箱中孵育24h,待细胞70%融合时辐射紫外线,分为8组:分别为空白对照组、30s组、1min组、3min组、4min组、5min组、6min组、8min组,每组设立三个复孔。照射后继续培养24h,倒置显微镜下观察细胞形态,并用台盼兰活细胞计数法检测细胞存活率,以细胞轻度受损,并在照射后可以很快恢复,细胞存活率在80%左右为最佳照射时间,以此确定最佳辐射剂量。分组处理:将对数生长期的细胞分别接种于6孔、24孔、96孔培养板中,待细胞接近融合时进行分组处理,实验分为以下6组:空白对照组、单纯NB-UVB损伤组、NB-UVB+0.10mmol/L维生素C组、NB-UVB+0.25mmol/L维生素C组、NB-UVB+ 0.50mmol/L维生素C组、NB-UVB+1.0mmol/L维生素C组,每组均设立3个复孔,各实验组均置于NB-UVB光源下辐射5min,然后给予不同浓度的维生素C,继续培养24h。倒置显微镜下观察细胞形态:将处理后的细胞置于倒置显微镜下观察空白对照组、单纯NB-UVB组及不同浓度维生素C组角质形成细胞的形态学变化。台盼兰染色检测细胞存活率:细胞按照上述处理方法处理,消化后制成细胞混悬液,用移液管将细胞悬液移入小试管中,加0.3%台盼兰染液浸染4min后,在倒置显微镜高倍镜下随机观察100个细胞,计数其中存活细胞数（蓝染的细胞为死亡细胞）,计算细胞存活率。MTT法检测细胞活性:孵育及照射方法同上,照射24h后各孔加入MTT,孵育4h后加入150μl DMSO,常温振荡,酶标仪492nm波长下测定各孔吸光光度值（OD值）。激光共聚焦扫描显微镜检测活细胞内游离Ca²⁺浓度:细胞按照上述方法处理后,加入浓度为5μmol/L的钙离子荧光探针rhod-2/AM,避光孵育30min,然后在激光共聚焦显微镜下扫描观察各组细胞的荧光强度。结果:（1）.最佳辐射剂量:以10.2mW/cm2的NB-UVB强度照射各组角质形成细胞30s、1min、3min、4min、5min、6min、8min后观察细胞形态,5min组细胞受到中等程度的损伤,但损伤也逐渐恢复,细胞存活率轻度下降,各组差异有统计学意义（P<0.05）。因此确定光照时间为5min,照射剂量为3060mJ/cm2。（2）.细胞形态学观察:空白对照组角质形成细胞呈扁平的三角形或多角形,细胞之间排列紧密,互相衔接成片,如铺路石样,各实验组细胞均有不同程度损伤,表现为细胞肿胀,严重者细胞碎片明显增多,部分漂浮于培养液中。（3）.角质形成细胞存活率:各实验组细胞存活率分别为74.6%、75.1%、81.4%、85.1%、65.6%,与空白对照组均存在显著差异,差异有统计学意义（P<0.05）;表明3060mJ/cm2的NB-UVB照射可使角质形成细胞受损,生长受抑制,不同浓度的维生素C作用后虽可改变细胞的活性,但仍然低于对照组;0.25mmol/L维生素C组、0.50mmol/L维生素C组细胞存活率较单纯NB-UVB组均有升高,差异有统计学意义（P<0.05）;而1.0mmol/L维生素C组的细胞存活率较单纯NB-UVB照射组显著降低,差异有统计学意义（P<0.05）。（4）.OD值:各实验组OD值分别为0.81、0.75、0.76、0.98、0.40,与空白对照组（1.31）比较均降低,差异有统计学意义（P<0.05）;0.50mmol/L维生素C组细胞OD值较单纯NB-UVB组有显著升高,差异有统计学意义（P<0.05）;1.0mmol/L维生素C组的细胞OD值较单纯NB-UVB照射组明显降低,差异有统计学意义（P<0.05）。（5）.各组细胞Ca²⁺荧光强度:各实验组荧光强度分别为54.5、55.1、54.6、40.0、7.6,与对照组（27.0）比较均有改变,差异有统计学意义（P<0.05）;NB-UVB+0.1mmol/L维生素C组、NB-UVB+0.25mmol/L维生素C组与单纯NB-UVB组比较差异无统计学意义（ P>0.05 ） ;NB-UVB+0.50mmol/L维生素C组比单纯NB-UVB组荧光强度小,差别有统计学意义（P<0.05）;NB-UVB+1.0mmol/L维生素C组的细胞荧光强度为7.6,远小于其他各组,差别均有统计学意义（P<0.05）。结论:（1）窄谱中波紫外线对细胞的损伤作用较小,与普通中波紫外线相比,在相同累计剂量下副作用较小,但超大剂量的窄谱中波紫外线辐射也会对细胞造成不可逆转的损伤。（2）维生素C对光损伤后的角质形成细胞具有保护作用,且这种保护作用在一定浓度下呈剂量相关性,超过一定浓度后反可加重细胞损伤。（3）细胞内游离Ca²⁺浓度与细胞损伤程度呈正相关,结果提示维生素C对角质形成细胞光损伤的作用机制可能与细胞内的Ca²⁺调控有关更多还原

【Abstract】 Objective: Calcium ion, the most widespread signal transduction element in corpore, plays an important role in vital movement of cells, and regulates the function of various kinds of cells. Ultraviolet radiation from sunlight is a highly significant factor of environmental mutagenesis. The significant damage to keratinocytes has been testified. Much research on antioxidation of vitamin C has been done. But now the mechanism of interventional effects on damaged keratinocytes of vitamin C is still unclear, with no reports of whether it is relevant with calcium ion. This study is to investigate the interventional effects of different levels of vitamin C on cytosolic calcium in cultured keratinocytes after NB- UVB irradiation and to explore the dependablity with intra-cellular calcium ion.Methods: Normal human keratinocytes derived from adolescent foreskins were isolated by Dispase and trypsin, and were kept in keratinocyte culture medium (Dulbecco’s modified Eagle’s medium) supplemented with 15% foetal calf serum. Cultures were incubated in a humidified 5% CO2 atmosphere at 37°C and medium was changed every 1~2 days. Primary cultures of keratinocytes were subcultured after about 15 days by treating keratinocytes with a trypsin-EDTA wash for 10~15 min and reseeding them at a 1:3 ratio. Primary keratinocytes were cultured. After three passages, the cells were treated with NB-UVB radiation for various time(0, 30s, 1min, 3min, 4min, 5min, 6min, 8min).After radiation,cells were incubated in a humidified 5% CO2 atmosphere at 37°C for 24h. Then the cells were transferred to microscope to observe the morphology,and then Trypan Blue was used to detect the cell livability. The best radiation dose is when the cells were injured slightly,and cell livability was about 80%. Primary cells were cultured. After three passages, cells were inoculated into culture plates with 6 holes, 24 holes and 96 holes. Then cells were divided into 6 groups: untreated controls, NB-UVB radiation alone, NB-UVB+0.10mmol/L vitamin C, NB-UVB+ 0.25mmol/L vitamin C, NB-UVB+0.50mmol/L vitamin C, NB-UVB +1.0mmol/L vitamin C. All the experimental groups were irradiated under the NB-UVB light for 5 minites, and then given to various concentrations of vitamin C. Cultures were incubated in a humidified 5% CO2 atmosphere at 37°C for another 24h. 4. Light microscope was used for morphological change,cell count, Trypan Blue was used to detect the cell livability and MTT method to detect proliferation of keratinocytes.5. Ca2+-imaging experiments and membrane staining Cells were stained with 5μmol/L rhod-2/AM (Molecular Probes) in medium at room temperature away from light for 30 minutes. The staining solution was removed by washing three times with PBS. Cells on cover glasses were transferred to custom-built observation chambers to be scanned. To compare sets of data, we used analysis of variance (ANOVA). Differences were considered to be statistically significant when P<0.05Results: (1). Compared with untreated controls, cells irradiated for 5 minutes were injured moderately, and a slightly decrease was observed in cell livability (P<0.05). Therefore 5 minutes was considered the best suitable time. (Irradiation dose was 3060mJ/cm2)(2). Morphological observation: Flat and triangle- or polygon-shaped keratinocytes, closed arranged, were seen in the control group. These cells adjoined to each other such like cobble-stones in the road. While the cells in experimental group were injured of different levels. Cellular swelling, even much cell debris floating in the culture fluid were seen in these groups.(3). Cell livability: Compared with untreated controls, a significant increase was observed in cell livability (74.6%, 75.1%, 81.4%, 85.1%, 65.6%) of all experimental groups (P<0.05). Compared with cells irradiated alone, a significant increase was observed in cell livability of NB-UVB+ 0.25mmol/L vitamin C group and NB-UVB+0.50mmol/L vitamin C group, and a significant decrease was observed in cell livability of NB-UVB+ 1.0mmol/L vitamin C group(P<0.05).(4). Optical absorption value: Cells in all the experimemtal groups showed a significant lower optical absorption value than those in untreated controls. The optical absorption value of cells in NB-UVB+ 0.50mmol/L vitamin C group was higher than in NB-UVB alone, and it showed a decrease in NB-UVB+ 0.50mmol/L vitamin C group. The results showed statistical significance.(5).Intracellular fluorescence intensity: The intracellular fluorescence intensity of every experimental group was, respectively, 54.5, 55.1, 54.6, 40.0, and 7.6, lower than that of untreated controls. The results showed a statistical significance (P<0.05). The intracellular fluorescence intensity of NB-UVB+ 0.10mmol/L vitamin C group and NB-UVB+ 0.25mmol/L vitamin C group showd nonsignificant difference from NB-UVB group(P>0.05). Intracellular fluorescence intensity of cells in NB-UVB+ 1.0mmol/L vitamin C group was 7.6, far lower than that of all the other groups. There showed a statistical significance(P<0.05).Conclusions: (1). Compared with general UVB, NB-UVB is less harmful to keratinocytes. But over-dosage of NB-UVB radiation can also be fatal to keratinocytes.(2). Vitamin C is protective to keratinocytes injured by UVB radiation, which shows a dose-dependent manner. But it can aggravate the injury over a certain concentration.(3). The concentration of intracellular calcium is positively correlated with injury level of keratinocytes. The results indicate that the mecanism of UV-radiation on apoptosis, and vitamin C’s protective function might be related with the increase of intracellular free calcium ion.更多还原