本文選題：氟　切入點(diǎn)：支持細(xì)胞　出處：《山西農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：[目的]通過染氟支持細(xì)胞和淋巴細(xì)胞體外共培養(yǎng)后Fas/FasL系統(tǒng)相關(guān)基因和蛋白表達(dá)的檢測(cè),來探討氟對(duì)睪丸支持細(xì)胞免疫豁免的毒性機(jī)理,從而為支持細(xì)胞協(xié)助器官移植方面提供理論依據(jù)。[方法]本實(shí)驗(yàn)設(shè)置了不同劑量氟影響下的支持細(xì)胞和淋巴細(xì)胞共培養(yǎng),模擬體內(nèi)睪丸曲細(xì)精管中二者的相互作用。通過MTT和TUNEL檢測(cè)了淋巴細(xì)胞的相對(duì)存活率和凋亡率；應(yīng)用QRT-PCR和western blot檢測(cè)了染氟支持細(xì)胞內(nèi)FasLmRNA和FasL蛋白的表達(dá)變化；同時(shí)也檢測(cè)了共培養(yǎng)后淋巴細(xì)胞內(nèi)Fas蛋白的表達(dá)和Fas、caspase8和caspase3 mRNA的表達(dá)變化。[結(jié)果](1)支持細(xì)胞鑒定結(jié)果：熒光顯微鏡的藍(lán)光激發(fā)下,可見支持細(xì)胞相互交織,都像膜一樣鋪成一層,胞體呈現(xiàn)綠色熒光。計(jì)數(shù)后可以發(fā)現(xiàn)支持細(xì)胞的純度在90%左右,活性達(dá)95%以上。可以用來建立細(xì)胞模型。(2)氟對(duì)淋巴細(xì)胞相對(duì)存活率影響MTT結(jié)果顯示：隨著氟化鈉濃度的上升,淋巴細(xì)胞的相對(duì)存活率呈現(xiàn)下降趨勢(shì),10-6、10-5、10-4mol/L組與對(duì)照組淋巴細(xì)胞相對(duì)存活率相比變化不明顯,10-3mol/L組的氟濃度對(duì)淋巴細(xì)胞相對(duì)存活率影響極大,與對(duì)照組相比差異極顯著。(3)共培養(yǎng)后淋巴細(xì)胞相對(duì)存活率MTT結(jié)果顯示：淋巴細(xì)胞的相對(duì)存活率隨著支持細(xì)胞攻氟濃度的增加也呈現(xiàn)上升趨勢(shì),10-6、10-5mol/L的染氟濃度下,淋巴細(xì)胞的相對(duì)存活率與對(duì)照組相比差異不顯著；10-4mol/L組與對(duì)照組相比差異顯著,10-3mol/L組支持細(xì)胞對(duì)淋巴細(xì)胞相對(duì)存活率影響最小,差異極顯著。(4)共培養(yǎng)后淋巴細(xì)胞凋亡率TUNEL結(jié)果：與不同染氟濃度的支持細(xì)胞共培養(yǎng)后,淋巴細(xì)胞的凋亡率都有所變化,趨勢(shì)是隨著氟化鈉濃度的升高而降低,與對(duì)照組相比,10-6mol/L組的細(xì)胞凋亡率差異不顯著(P0.05); 10-5mol/L組與對(duì)照組相比凋亡顯著(p0.01); 10-4mol/L組差異極顯著(p0.01); 10-3mol/L組差異極極顯著(5) western blot結(jié)果顯示：與對(duì)照組相比支持細(xì)胞各實(shí)驗(yàn)組FasL蛋白表達(dá)水平有所下降,低劑量(10-5)染氟時(shí),FasL達(dá)到顯著性差異,10-4和10-3 mol/L的染氟劑量,FasL的表達(dá)與對(duì)照組相比差異極顯著；共培養(yǎng)后淋巴細(xì)胞中Fas的表達(dá)呈下降趨勢(shì),低劑量染氟組淋巴細(xì)胞中Fas的表達(dá)差異不顯著,10-4和10-3mol/L的支持細(xì)胞染氟劑量,淋巴細(xì)胞中Fas的表達(dá)差異極顯著。(6) QRT-PCR結(jié)果顯示：與對(duì)照組比,支持細(xì)胞FasL mRNA的表達(dá)量隨著染氟濃度的升高呈現(xiàn)下降趨勢(shì)；其中10-5mol/L的氟濃度,FasL mRNA的表達(dá)無顯著性變化；染氟后與對(duì)照組組比,10-4、10-5mol/L的氟濃度,FasL mRNA的表達(dá)顯著升高；共培養(yǎng)后各組淋巴細(xì)胞中Fas、caspase3-caspase8三個(gè)基因mRNA的表達(dá)量與對(duì)照組相比,有所下降。其中,Fas、caspase3和caspase8的10-3mol/L氟暴露組與對(duì)照組相比,mRNA的表達(dá)量均達(dá)到顯著性差異； Fas和caspase3的10-4mol/L氟暴露組與對(duì)照組相比,差異顯著；Fas的10-5mol/L氟暴露組與對(duì)照組相比,差異顯著；[結(jié)論]氟通過降低支持細(xì)胞FasL基因和蛋白的表達(dá),影響了支持細(xì)胞殺傷淋巴細(xì)胞的功能,降低了支持細(xì)胞的免疫豁免功能。
[Abstract]:[Objective] by detecting expression of Fas/FasL related genes and proteins of fluoride in vitro support cells and lymphocytes after co culture, to explore the mechanism of toxicity of fluoride on Sertoli cell immune privilege, so as to provide the theory basis. The experimental setup method] support cells and lymphocytes of different doses of fluoride under the influence of co culture of Sertoli cells assist in organ transplantation, the interaction in vivo in seminiferous tubules. In two by MTT and TUNEL to detect the relative survival rate and apoptosis rate of lymphocytes; application of QRT-PCR and Western blot to detect the expression changes of fluoride in the supporting cells FasLmRNA and FasL protein; also detected the expression of Fas and Fas protein co culture after the lymphocytes within the expression of caspase8 and Caspase3. The results of mRNA (1)] support the identification of cells: fluorescence excited by blue light, visible support The cells are intertwined, like the film as a paved layer, the cell body showed green fluorescence. After counting Sertoli cells can be found in the purity of about 90%, the activity of more than 95%. Can be used to establish the cell model. (2) fluoride on the relative survival rate of lymphocyte MTT results showed that: with the increase of concentration of sodium fluoride, lymphocyte the relative survival rate decreased, the relative survival rate of 10-6,10-5,10-4mol/L group and the control group did not change significantly compared to lymphocytes, fluoride concentration in 10-3mol/L group the relative survival rate of lymphocyte of great impact, compared with the control group the difference was significant. (3) the relative survival rate of MTT lymphocytes after co culture results showed: with the increase of the relative survival rate Sertoli cells attack fluoride concentration increased lymphocytes, fluoride concentration of 10-6,10-5mol/L lymphocytes, the relative survival rate compared with the control group, the difference was not significant 10-4mol/L; compared with the control group, 10-3mol/L group support cell survival rate with minimal impact on the lymphocyte, the difference was very significant. (4) after the lymphocyte apoptosis rate TUNEL co culture results: co cultured with different concentrations of fluoride in Sertoli cells, lymphocyte apoptosis rate change, the trend is decreased with increasing fluoride the concentration of sodium, compared with the control group, the apoptosis rate of 10-6mol/L group had no significant difference (P0.05); 10-5mol/L group compared with the control group (P0.01); 10-4mol/L apoptosis was significant difference (P0.01); 10-3mol/L group was extremely significant (5) Western blot results showed that: compared with the control cells the experimental group, the expression level of FasL protein decreased, low dose of fluoride (10-5), FasL had significant differences, the fluoride dose 10-4 and 10-3 mol/L, the expression of FasL compared with the control group significantly; The expression of Fas in lymphocytes decreased expression of co cultured, low dose fluoride group Fas in lymphocytes was not significant, and the support of the 10-3mol/L 10-4 cell dose of fluoride, differential expression of Fas in lymphocytes significantly. (6) QRT-PCR results showed: compared with control group, the expression of FasL mRNA cells with the increase of fluoride concentration decreased; fluoride concentration of the 10-5mol/L, no significant changes in the expression of FasL mRNA; fluoride group with the control group, the fluoride concentration of 10-4,10-5mol/L, the expression of FasL mRNA increased significantly after co cultured groups; lymphocyte Fas, caspase3-caspase8 three gene expression of mRNA in with the control group decreased. Among them, Fas, 10-3mol/L Caspase3 and caspase8 fluoride exposed group compared with the control group, the expression of mRNA had significant difference; 10-4mol /L and Caspase3 Fas fluoride exposure Compared with the control group were significantly different; 10-5mol/L Fas fluoride exposed group compared with the control group had significant difference; [Conclusion] fluoride support cells by reducing the expression of FasL gene and protein, the effect of Sertoli cell killer lymphocyte function, decreased immune function from Sertoli cells.

【學(xué)位授予單位】：山西農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R114

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 Seong-Jae Kim;Hwajin Kim;Jeongsook Park;Inyoung Chung;Hyug-Moo Kwon;Wan-Sung Choi;Ji-Myong Yoo;;Tonicity response element binding protein associated with neuronal cell death in the experimental diabetic retinopathy[J];International Journal of Ophthalmology;2014年06期

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本文編號(hào)：1619124