硝酸银对角朊细胞增殖的作用

发布时间：2018-06-24 15:21

本文选题：HaCaT细胞 + 硝酸银　；参考：《遵义医学院》2013年硕士论文

【摘要】：目的：探讨低浓度硝酸银对体外培养角朊细胞增殖的影响。方法：建立HaCaT(人KC细胞株)角朊细胞的体外培养模型,分别于接种细胞贴壁后,随机分为(1)对照组,不添加药物试剂；(2) AgN03一组,培养基中添加终浓度为1×10-5mol/l AgNO3;(3) AgNO3二组,培养基中添加终浓度为1×10-6mol/l AgNO3;(4) CFTRinh-172抑制剂组,培养液中添加终浓度为10umol/ml CFTRinh-172溶液。每24h更换相应药物浓度的培养液。并在细胞贴壁后即刻、24h、36h、48h、72h、84h、96h进行每组0D测量。24h及48h进行MQAE染色,利用酶联免疫检测仪检测细胞内氯离子荧光含量,DAPI染色后,免疫荧光显微镜检测。结果：HaCaT细胞接种2小时后,细胞沉降至培养板内底部,各实验组间的基础0D均值(P0.05),随后添加硝酸银组较其他组0D值高(P0.05),生长曲线提示,添加硝酸银实验组HaCaT细胞呈指数生长,约在72H后达生长平台期,较其他两组有明显促进作用,添加终浓度为10umol/1的CFTRinh-172溶液实验组细胞生长较其他三组慢。添加不同浓度的AgNO3对细胞生长有明显的促进作用,添加终浓度为10μmol/m1的CFTRinh-172溶液实验组细胞生长较其他三组慢(P0.05)。添加硝酸银组中荧光强度明显强于对照组及抑制剂组,说明细胞内部Cl-含量明显下降,48h后各组细胞内荧光强度进一步增强,提示细胞内氯离子外流增加。在无钙改良RPMI1640培养液中添加不同浓度的AgNO3离体培养HaCaT细胞,其HaCaT细胞外流氯离子浓度明显高于CFTRinh-172抑制剂组(P0.05)和对照组(P0.05)；本实验中两种浓度的AgNO3实验组没有显著性差异(P0.05)；CFTRinh-172抑制剂组HaCaT细胞内氯离子浓度明显高于对照组(P0.05)。HaCaT细胞经MQAE孵育染色及DAPI染色下,细胞浆呈淡绿色荧光反应,围绕细胞核,分布均匀；细胞核呈蓝色荧光,高亮。AgNO3溶液实验组较对照组细胞胞浆绿色荧光分布增多,CFTRinh-172抑制剂组较对照组细胞胞浆绿色荧光较少。结论：低浓度硝酸银对离体培养HaCaT细胞增殖有促进作用,HaCaT细胞内含有相关氯离子通道蛋白,硝酸银影响HaCaT细胞的氯离子通道促进细胞增殖作用。
[Abstract]:Objective: to investigate the effect of low concentration of silver nitrate on the proliferation of keratinocytes in vitro. Methods: the culture model of keratinocytes of HaCaT (human KC cell line) was established in vitro. After inoculation, the keratinocytes were randomly divided into (1) control group, (2) Agn03 group, 1 脳 10-5mol/l Agno _ 3, (3) Agno _ 3 group, Agno _ 3 group and Agno _ 3 group. (4) in the CFTRinh-172 inhibitor group, the final concentration in the culture medium was 10umol/ml CFTRinh-172 solution. The culture medium of the corresponding drug concentration was replaced every 24 hours. The MQAE staining was performed at 24 h, 36 h, 48 h, 72 h, 84 h and 96 h, respectively. The fluorescence content of chloride in the cells was detected by enzyme linked immunoassay (Elisa) and then detected by immunofluorescence microscope. Results two hours after inoculation, the cells were deposited to the bottom of the culture plate, and the basal D value was higher in the silver nitrate group than in the other groups (P0.05). The growth curve indicated that the HaCaT cells in the silver nitrate experimental group grew exponentially. After 72H, the cells reached the stage of growth plateau, which was obviously promoted compared with the other two groups. The growth of the cells in the experimental group was slower than that in the other three groups in the CFTRinh-172 solution with the final concentration of 10umol/1. Addition of different concentrations of Agno _ 3 significantly promoted cell growth, and the growth of the experimental group was slower than that of the other three groups (P0.05) when the final concentration of 10 渭 mol/m1 was added to CFTRinh-172 solution. The fluorescence intensity in the silver nitrate group was significantly higher than that in the control group and the inhibitor group, which indicated that the intracellular Cl-content in the cells decreased significantly after 48 hours, and the fluorescence intensity in the cells increased further, suggesting the increase of the chloride ion outflow in the cells. The concentration of chloride in HaCaT cells was significantly higher than that in CFTRinh-172 inhibitor group (P0.05) and control group (P0.05), but there was no significant difference between the two groups (P0.05). The concentration of chloride in HaCaT cells in CFTRinh-172 inhibitor group was significantly higher than that in control group (P0.05). Under the conditions of MQAE incubation and DAPI staining, the cytoplasm of HaCaT cells showed a light green fluorescence reaction, distributed evenly around the nucleus, and showed blue fluorescence. The distribution of green fluorescence in the cytoplasm of the experimental group was higher than that of the control group, and the green fluorescence of the cytoplasm in the inhibitor group was less than that in the control group. Conclusion: low concentration of silver nitrate can promote the proliferation of HaCaT cells in vitro.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R644

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