發(fā)布時間：2018-06-24 15:21

  本文選題：HaCaT細胞 + 硝酸銀　； 參考：《遵義醫(yī)學院》2013年碩士論文

【摘要】：目的：探討低濃度硝酸銀對體外培養(yǎng)角朊細胞增殖的影響。 方法：建立HaCaT(人KC細胞株)角朊細胞的體外培養(yǎng)模型,分別于接種細胞貼壁后,隨機分為(1)對照組,不添加藥物試劑；(2) AgN03一組,培養(yǎng)基中添加終濃度為1×10-5mol/l AgNO3;(3) AgNO3二組,培養(yǎng)基中添加終濃度為1×10-6mol/l AgNO3;(4) CFTRinh-172抑制劑組,培養(yǎng)液中添加終濃度為10umol/ml CFTRinh-172溶液。每24h更換相應藥物濃度的培養(yǎng)液。并在細胞貼壁后即刻、24h、36h、48h、72h、84h、96h進行每組0D測量。24h及48h進行MQAE染色,利用酶聯(lián)免疫檢測儀檢測細胞內(nèi)氯離子熒光含量,DAPI染色后,免疫熒光顯微鏡檢測。 結(jié)果：HaCaT細胞接種2小時后,細胞沉降至培養(yǎng)板內(nèi)底部,各實驗組間的基礎0D均值(P0.05),隨后添加硝酸銀組較其他組0D值高(P0.05),生長曲線提示,添加硝酸銀實驗組HaCaT細胞呈指數(shù)生長,約在72H后達生長平臺期,較其他兩組有明顯促進作用,添加終濃度為10umol/1的CFTRinh-172溶液實驗組細胞生長較其他三組慢。添加不同濃度的AgNO3對細胞生長有明顯的促進作用,添加終濃度為10μmol/m1的CFTRinh-172溶液實驗組細胞生長較其他三組慢(P0.05)。添加硝酸銀組中熒光強度明顯強于對照組及抑制劑組,說明細胞內(nèi)部Cl-含量明顯下降,48h后各組細胞內(nèi)熒光強度進一步增強,提示細胞內(nèi)氯離子外流增加。在無鈣改良RPMI1640培養(yǎng)液中添加不同濃度的AgNO3離體培養(yǎng)HaCaT細胞,其HaCaT細胞外流氯離子濃度明顯高于CFTRinh-172抑制劑組(P0.05)和對照組(P0.05)；本實驗中兩種濃度的AgNO3實驗組沒有顯著性差異(P0.05)；CFTRinh-172抑制劑組HaCaT細胞內(nèi)氯離子濃度明顯高于對照組(P0.05)。HaCaT細胞經(jīng)MQAE孵育染色及DAPI染色下,細胞漿呈淡綠色熒光反應,圍繞細胞核,分布均勻；細胞核呈藍色熒光,高亮。AgNO3溶液實驗組較對照組細胞胞漿綠色熒光分布增多,CFTRinh-172抑制劑組較對照組細胞胞漿綠色熒光較少。 結(jié)論：低濃度硝酸銀對離體培養(yǎng)HaCaT細胞增殖有促進作用,HaCaT細胞內(nèi)含有相關(guān)氯離子通道蛋白,硝酸銀影響HaCaT細胞的氯離子通道促進細胞增殖作用。
[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.
【學位授予單位】：遵義醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R644

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