【摘要】：目的觀察雙酚A (BPA)對(duì)體外大鼠睪丸支持細(xì)胞緊密連接occludin蛋白表達(dá)的影響,探討B(tài)PA對(duì)精子發(fā)生的損傷機(jī)制。 方法原代培養(yǎng)Wistar大鼠睪丸支持細(xì)胞(Sertoli細(xì)胞)4～5天,雙室培養(yǎng)模型的基礎(chǔ)上建立體外緊密連接(Tight Junction, TJ)滲透屏障。含有系列濃度(10-3,10-2,10-1,100,101,102,103μmol/L) BPA的無(wú)血清培養(yǎng)基(DMEM)孵育Sertoli細(xì)胞24h,采用CCK8法檢測(cè)BPA對(duì)Sertoli細(xì)胞增殖活性的影響。根據(jù)CCK8結(jié)果用SPSS17.0軟件計(jì)算BPA半數(shù)致死量(LD50)為150μmol/L,選定梯度濃度的100μmol/L,50μmol/L,25μmol/L BPA作為后續(xù)實(shí)驗(yàn)組。以0.1%二甲基亞砜(DMSO)為對(duì)照組(BPA濃度為0μmol/L),分別以0μmol/L,25μmol/L,50μmol/L,100μmol/L BPA處理Sertoli細(xì)胞24h,細(xì)胞免疫熒光染色觀察occludin陽(yáng)性率和表達(dá)強(qiáng)度；Western blot半定量檢測(cè)緊密連接蛋白o(hù)ccludin的表達(dá)變化。 結(jié)果成功分離并培養(yǎng)Wistar大鼠Sertoli細(xì)胞,建立了良好的體外TJ屏障模型。CCK8結(jié)果顯示,不同劑量的BPA染毒24h后,Sertoli細(xì)胞的吸光度(OD值)隨染毒劑量的增加呈下降趨勢(shì)。與對(duì)照組(0μmol/L BPA)相比,102μmol/L、103μmol/L組的OD值明顯降低(P0.05),10-2μmol/L、10-1μmol/L、100μmol/L和101μmol/L組的OD值確無(wú)明顯變化(P0.05)。細(xì)胞免疫熒光染色顯示,各染毒組均有occludin表達(dá)。隨著B(niǎo)PA染毒劑量的增加,Occludin熒光表達(dá)強(qiáng)度逐漸降低。25μmol/L及50μmol/L組occludin表達(dá)陽(yáng)性率和表達(dá)強(qiáng)度均明顯降低(P0.05),未見(jiàn)有強(qiáng)陽(yáng)性表達(dá)；100μmol/L組只有極小部分呈弱陽(yáng)性表達(dá)。Western blot結(jié)果顯示,Occludin在各劑量組中均有表達(dá),其表達(dá)隨BPA染毒劑量的增加呈下降趨勢(shì)。與對(duì)照組相比,-25μmol/L、50μmol/L及100μmol/L組的occludin表達(dá)明顯降低(P0.05)；與25μmol/L組相比,50μmol/L及100μmol/L組的occludin表達(dá)亦明顯降低(P0.05)。 結(jié)論雙酚A可削減Sertoli細(xì)胞occludin的正常表達(dá),由此可能損傷緊密連接的滲透性屏障,從而影響正常的精子發(fā)生過(guò)程。
[Abstract]:Objective to observe the effect of bisphenol A (BPA) on the expression of tight junction occludin protein in rat testicular Sertoli cells in vitro and to explore the mechanism of spermatogenesis induced by BPA. Methods Wistar rat testicular Sertoli cells (Sertoli cells) were cultured for 4 days for 5 days. The tight (Tight Junction, TJ) osmotic barrier was established on the basis of two-compartment culture model. Sertoli cells were incubated in serum-free medium (DMEM) containing a series of concentrations (10: 3, 10: 2, 10-1100101102103 渭 mol / L) BPA) for 24 h. The effect of BPA on the proliferation of Sertoli cells was detected by CCK8 assay. According to the results of CCK8, the BPA half lethal dose (LD50) was calculated to be 150 渭 mol / L by SPSS17.0 software, and the gradient concentration of 100 渭 mol / L, 50 渭 mol / L and 25 渭 mol / L BPA was selected as the follow-up experimental group. Sertoli cells were treated with 0.1% dimethyl sulfoxide (DMSO) as control group (the concentration of BPA was 0 渭 mol / L, 25 渭 mol / L, 50 渭 mol / L, 100 渭 mol / L BPA, respectively). The positive rate and expression intensity of occludin were observed by immunofluorescence staining. The expression of tight junction protein occludin was detected by Western blot semi-quantitatively. Results Wistar rat Sertoli cells were successfully isolated and cultured, and a good TJ barrier model in vitro was established. CCK8 results showed that the absorbance (OD value) of Sertoli cells decreased with the increase of exposure dose after exposure to different doses of BPA for 24 hours. Compared with the control group (0 渭 mol / L), the OD values of 102 渭 mol / L and 103 渭 mol / L groups were significantly lower than those of the control group (P 0.05). There was no significant change in the OD values of 10-1 渭 mol / L, 100 渭 mol / L and 101 渭 mol / L groups (P 0.05). Cellular immunofluorescence staining showed that occludin was expressed in all groups. With the increase of BPA exposure dose, the fluorescence expression intensity of Occludin decreased gradually. The positive rate and expression intensity of occludin expression decreased significantly in 25 渭 mol / L and 50 渭 mol / L groups (P 0.05), but no strong positive expression was found. In 100 渭 mol / L group, only a small number of them were weakly positive. Western blot results showed that Occludin was expressed in all dose groups, and its expression decreased with the increase of BPA dose. Compared with the control group, the expression of occludin in-25 渭 mol / L, 50 渭 mol / L and 100 渭 mol / L groups decreased significantly (P 0.05), and the expression of occludin in 50 渭 mol / L and 100 渭 mol / L groups was also significantly lower than that in 25 渭 mol / L group (P 0.05). Conclusion Bisphenol A can reduce the normal expression of occludin in Sertoli cells, which may damage the tightly connected permeability barrier and affect the normal spermatogenesis.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R114
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本文編號(hào)：2484703