本文關(guān)鍵詞： 氯化鎘 萊菔硫烷 睪丸間質(zhì)細(xì)胞 Nrf2-ARE通路 抗氧化　出處：《沈陽農(nóng)業(yè)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：[目的]本試驗旨在研究萊菔硫烷(TM3)通過激活Nrf2通路,調(diào)控下游Ⅱ相解毒酶和抗氧化酶活性,進(jìn)而對鎘誘導(dǎo)的小鼠睪丸間質(zhì)細(xì)胞(TM3)氧化損傷的保護(hù)作用。通過體外試驗對其作用機(jī)制進(jìn)一步研究,為鎘生殖毒性的防治提供理論依據(jù)。[方法]以氯化鎘(CdCl2,Cd)和萊菔硫烷(sulforaphane,SFN)聯(lián)合培養(yǎng)TM3細(xì)胞,建立細(xì)胞試驗?zāi)Ｐ?通過檢測TM3細(xì)胞的相對存活率、睪酮分泌量、乳酸脫氫酶(LDH)活性、細(xì)胞抗氧化水平、活性氧(ROS)陽性率、細(xì)胞凋亡情況以及谷胱甘肽過氧化物酶(GSH-PX)、細(xì)胞核因子E2相關(guān)因子2(Nrf2)及其調(diào)控的下游靶基因血紅素加氧酶1(HO-1)、醌氧化還原酶1(NQO1)、γ-谷氨酰半胱氨酸合成酶(y-GCS)等mRNA及蛋白的表達(dá)量,研究SFN對鎘誘導(dǎo)睪丸TM3細(xì)胞毒性的影響及作用機(jī)制。[結(jié)果]1.采用MTT法檢測發(fā)現(xiàn),Cd對TM3細(xì)胞具有損傷作用,當(dāng)Cd濃度為10μmol/L時,TM3細(xì)胞的相對存活率顯著降低(p0.05),當(dāng)Cd濃度大于20μmol/L時,TM3細(xì)胞的相對存活率降低程度更加顯著(p0.01),同時測得Cd的IC50為51.4μmol/L;在一定濃度范圍內(nèi),SFN對TM3細(xì)胞有保護(hù)作用,但當(dāng)SFN濃度大于20μmol/L時,對TM3細(xì)胞具有抑制作用(p0.05),且呈量效關(guān)系;Cd與SFN聯(lián)合作用于TM3細(xì)胞時,SFN可緩解Cd引起的細(xì)胞毒性作用。2.通過對LDH及抗氧化指標(biāo)的檢測發(fā)現(xiàn),與對照組相比,Cd組TM3細(xì)胞的GSH含量、T-SOD活性及GSH-PX活力顯著降低(p0.01),LDH活性、MDA含量顯著升高(p0.05);SFN組LDH活性、MDA含量顯著降低(p0.01),GSH含量、T-SOD活性及GSH-PX活力顯著升高(p0.05);Cd+SFN組GSH含量、T-SOD活性及GSH-PX活力與Cd組相比呈增加趨勢;LDH活性、MDA含量則呈下降趨勢,表明SFN可有效的緩解鎘誘導(dǎo)TM3細(xì)胞的氧化損傷。3.通過ELISA酶聯(lián)免疫吸附法發(fā)現(xiàn),與對照組相比,Cd組睪酮分泌量顯著降低(p0.01),SFN組睪酮分泌量略有升高,呈上升趨勢。與Cd組相比,Cd+SFN作用組睪酮分泌量有不同程度的增加。表明Cd可降低TM3細(xì)胞分泌孕酮的量,且Cd對TM3細(xì)胞分泌睪酮存在劑量-效應(yīng)關(guān)系。相反,SFN對TM3細(xì)胞睪酮分泌有促進(jìn)作用,并且可緩解Cd對TM3細(xì)胞睪酮分泌的損害作用。4.利用熒光探針DCFH-DA法檢測TM3細(xì)胞中活性氧的釋放量,發(fā)現(xiàn)空白對照組TM3細(xì)胞的ROS釋放量少,與對照組相比,其余各組細(xì)胞ROS釋放量均顯著升高(p0.01),且單獨加Cd組細(xì)胞ROS釋放量極顯著升高。但與Cd組相比,SFN組以及Cd+SFN組細(xì)胞ROS的釋放量又顯著低于單獨加Cd組。結(jié)果表明,任何物質(zhì)的刺激都會使TM3細(xì)胞活性氧在短時間內(nèi)有所增加,但Cd對TM3細(xì)胞ROS的作用尤為突出,Cd對TM3細(xì)胞有損傷作用,可導(dǎo)致細(xì)胞ROS含量顯著升高。5.通過AO/EB雙染法和流式細(xì)胞儀檢測TM3細(xì)胞凋亡情況,發(fā)現(xiàn)與對照組相比,Cd組細(xì)胞凋亡率顯著提高(p0.01),SFN組凋亡率明顯降低。與單獨加Cd相比,加SFN組TM3細(xì)胞凋亡率顯著降低(p0.01),Cd與SFN聯(lián)合作用組細(xì)胞凋亡率也低于單獨加Cd組。表明Cd對TM3細(xì)胞有損傷作用,而加入SFN可緩解損傷作用,減少TM3細(xì)胞凋亡。6.應(yīng)用熒光定量和Western blot法檢測TM3細(xì)胞mRNA及蛋白表達(dá)量發(fā)現(xiàn),與對照組相比,鎘組的Nrf2及其靶基因HO-1、γ-GCS、NQO1的mRNA和蛋白表達(dá)量略有升高,而GSH-PX的mRNA及蛋白表達(dá)量降低,SFN組與對照組相比顯著或極顯著升高(p0.01,p0.05),與鎘組相比,Cd+SFN組的Nrf2、GSH-PX、HO-1、γ-GCS、NQO1 等靶基因mRNA和蛋白的表達(dá)量明顯上升(p0.O1,p0.05)。[結(jié)論]萊菔硫烷對鎘誘導(dǎo)的小鼠睪丸間質(zhì)細(xì)胞氧化損傷具有拮抗作用,萊菔硫烷對鎘誘導(dǎo)的小鼠睪丸間質(zhì)細(xì)胞損傷的保護(hù)作用可能通過激活Nrf2-ARE通路途徑實現(xiàn)。
[Abstract]:[Objective] this experiment was conducted to study the sulforaphane (TM3) through the activation of the Nrf2 pathway, regulating downstream phase II detoxification enzymes and antioxidant enzyme activity, and the mouse testis interstitial cells induced by cadmium (TM3) oxidative injury by in vitro test. Further research on its mechanism, provide a theoretical basis for the method. For the prevention and treatment of reproductive toxicity of cadmium in cadmium chloride (CdCl2, Cd) and sulforaphane (sulforaphane, SFN) co cultured TM3 cells, a cell model test, the relative survival rate of TM3 cells was detected, testosterone secretion, lactate dehydrogenase (LDH) activity, cellular antioxidant levels, reactive oxygen species (ROS). The rate of cell apoptosis, and glutathione peroxidase (GSH-PX), nuclear factor E2 related factor 2 (Nrf2) of downstream target genes and regulation of heme oxygenase 1 (HO-1), quinone reductase 1 (NQO1), gamma glutamylcysteine synthetase (y-GC S) expression of mRNA and protein, the effects of SFN on cadmium induced testicular TM3 cell toxicity and mechanism. The results of]1. detected by MTT found that Cd on TM3 cells with injury, when the Cd concentration is 10 mol/L, the relative survival rate of TM3 cells decreased significantly (P0.05), when Cd the concentration is greater than 20 mol/L, the relative survival rate of TM3 cells decreased more significantly (P0.01), while the measured Cd IC50 51.4 mol/L; in a certain concentration range, SFN has a protective effect on TM3 cells, but when the SFN concentration is higher than 20 mol/L, has the inhibiting effect on TM3 cells (P0.05), and the dose effect relationship; Cd and SFN in TM3 cells, SFN cells can alleviate the toxicity of.2. Cd induced by detection of LDH and antioxidant indexes showed that compared with the control group, the content of GSH TM3 cells in Cd group, T-SOD activity and GSH-PX activity significantly decreased (P0.01). LDH activity, MDA content Increased significantly (P0.05); group SFN, LDH activity, MDA content decreased significantly (P0.01), GSH content, T-SOD activity and GSH-PX activity increased significantly (P0.05); group Cd+SFN, GSH content, T-SOD activity and GSH-PX activity compared with the Cd group showed an increasing trend; LDH activity, MDA content decreased, indicated that SFN effectively alleviate the oxidative damage of.3. cells induced by cadmium TM3 by ELISA enzyme linked immunosorbent assay showed that compared with the control group, Cd group, testosterone secretion decreased significantly (P0.01), SFN group, the testosterone secretion increased slightly increased. Compared with Cd group, Cd+SFN group the testosterone secretion increased in different degrees that Cd can reduce the amount of progesterone secretion of TM3 cells, and Cd on TM3 cells secreting testosterone dose-response relationship. On the contrary, SFN has a promoting effect on TM3 cells and the secretion of testosterone, can alleviate the Cd damage of TM3 cell secretion of testosterone.4. by fluorescence probe DCFH-DA The release amount of active oxygen was detected in TM3 cells, TM3 cells were found in blank control group, the release amount of ROS, compared with the control group, the release amount of ROS cells in other groups were significantly increased (P0.01), and with the release of ROS cells in Cd group increased significantly. Compared with the Cd group, the release amount of SFN group and ROS cells in Cd+SFN group were significantly lower than Cd group separately. The results show that any physical stimulus will cause TM3 cells reactive oxygen species increased in a short period of time, but the effect of Cd on TM3 cell ROS damage is particularly prominent. The effect of Cd on TM3 cells, ROS cells can lead to increased the content of.5. by AO/EB double staining and TM3 cell apoptosis was detected by flow cytometry, found that compared with the control group, the apoptosis rate of Cd group increased significantly (P0.01), the apoptosis rate of SFN group decreased significantly. Compared with Cd group, the apoptosis rate of TM3 cells with SFN significantly decreased (P0.01), Cd and SFN Co With the apoptosis rate of group is lower than that of Cd plus Cd group alone. The injury of TM3 cells, and SFN can alleviate the injury, reduce the apoptosis of TM3 cells using fluorescent quantitative.6. and Western blot method to detect the mRNA and protein expression of TM3 cells, compared with control group, Nrf2 and its target gene HO-1, cadmium group y -GCS, mRNA and protein expression of NQO1 was increased slightly, while the mRNA and protein expression of GSH-PX was decreased, SFN group compared with the control group increased significantly (P0.01, P0.05), compared with the CD group, Cd+SFN group Nrf2, GSH-PX, HO-1, R -GCS, the expression of target gene and mRNA NQO1 protein was significantly increased (p0.O1, P0.05). Conclusion: sulforaphane has an antagonistic effect on mouse testicular interstitial cells of cadmium induced oxidative damage, protective effect of sulforaphane on mouse testis induced by cadmium interstitial cell injury may activate the Nrf2-ARE pathway.

【學(xué)位授予單位】：沈陽農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S859.8
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本文編號：1508013