莱菔硫烷对染镉小鼠睾丸间质细胞毒性缓解作用的研究

发布时间：2018-02-13 11:06

  本文关键词： 氯化镉 莱菔硫烷 睾丸间质细胞 Nrf2-ARE通路 抗氧化　出处：《沈阳农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：[目的]本试验旨在研究莱菔硫烷(TM3)通过激活Nrf2通路,调控下游Ⅱ相解毒酶和抗氧化酶活性,进而对镉诱导的小鼠睾丸间质细胞(TM3)氧化损伤的保护作用。通过体外试验对其作用机制进一步研究,为镉生殖毒性的防治提供理论依据。[方法]以氯化镉(CdCl2,Cd)和莱菔硫烷(sulforaphane,SFN)联合培养TM3细胞,建立细胞试验模型,通过检测TM3细胞的相对存活率、睾酮分泌量、乳酸脱氢酶(LDH)活性、细胞抗氧化水平、活性氧(ROS)阳性率、细胞凋亡情况以及谷胱甘肽过氧化物酶(GSH-PX)、细胞核因子E2相关因子2(Nrf2)及其调控的下游靶基因血红素加氧酶1(HO-1)、醌氧化还原酶1(NQO1)、γ-谷氨酰半胱氨酸合成酶(y-GCS)等mRNA及蛋白的表达量,研究SFN对镉诱导睾丸TM3细胞毒性的影响及作用机制。[结果]1.采用MTT法检测发现,Cd对TM3细胞具有损伤作用,当Cd浓度为10μmol/L时,TM3细胞的相对存活率显著降低(p0.05),当Cd浓度大于20μmol/L时,TM3细胞的相对存活率降低程度更加显著(p0.01),同时测得Cd的IC50为51.4μmol/L;在一定浓度范围内,SFN对TM3细胞有保护作用,但当SFN浓度大于20μmol/L时,对TM3细胞具有抑制作用(p0.05),且呈量效关系;Cd与SFN联合作用于TM3细胞时,SFN可缓解Cd引起的细胞毒性作用。2.通过对LDH及抗氧化指标的检测发现,与对照组相比,Cd组TM3细胞的GSH含量、T-SOD活性及GSH-PX活力显著降低(p0.01),LDH活性、MDA含量显著升高(p0.05);SFN组LDH活性、MDA含量显著降低(p0.01),GSH含量、T-SOD活性及GSH-PX活力显著升高(p0.05);Cd+SFN组GSH含量、T-SOD活性及GSH-PX活力与Cd组相比呈增加趋势;LDH活性、MDA含量则呈下降趋势,表明SFN可有效的缓解镉诱导TM3细胞的氧化损伤。3.通过ELISA酶联免疫吸附法发现,与对照组相比,Cd组睾酮分泌量显著降低(p0.01),SFN组睾酮分泌量略有升高,呈上升趋势。与Cd组相比,Cd+SFN作用组睾酮分泌量有不同程度的增加。表明Cd可降低TM3细胞分泌孕酮的量,且Cd对TM3细胞分泌睾酮存在剂量-效应关系。相反,SFN对TM3细胞睾酮分泌有促进作用,并且可缓解Cd对TM3细胞睾酮分泌的损害作用。4.利用荧光探针DCFH-DA法检测TM3细胞中活性氧的释放量,发现空白对照组TM3细胞的ROS释放量少,与对照组相比,其余各组细胞ROS释放量均显著升高(p0.01),且单独加Cd组细胞ROS释放量极显著升高。但与Cd组相比,SFN组以及Cd+SFN组细胞ROS的释放量又显著低于单独加Cd组。结果表明,任何物质的刺激都会使TM3细胞活性氧在短时间内有所增加,但Cd对TM3细胞ROS的作用尤为突出,Cd对TM3细胞有损伤作用,可导致细胞ROS含量显著升高。5.通过AO/EB双染法和流式细胞仪检测TM3细胞凋亡情况,发现与对照组相比,Cd组细胞凋亡率显著提高(p0.01),SFN组凋亡率明显降低。与单独加Cd相比,加SFN组TM3细胞凋亡率显著降低(p0.01),Cd与SFN联合作用组细胞凋亡率也低于单独加Cd组。表明Cd对TM3细胞有损伤作用,而加入SFN可缓解损伤作用,减少TM3细胞凋亡。6.应用荧光定量和Western blot法检测TM3细胞mRNA及蛋白表达量发现,与对照组相比,镉组的Nrf2及其靶基因HO-1、γ-GCS、NQO1的mRNA和蛋白表达量略有升高,而GSH-PX的mRNA及蛋白表达量降低,SFN组与对照组相比显著或极显著升高(p0.01,p0.05),与镉组相比,Cd+SFN组的Nrf2、GSH-PX、HO-1、γ-GCS、NQO1 等靶基因mRNA和蛋白的表达量明显上升(p0.O1,p0.05)。[结论]莱菔硫烷对镉诱导的小鼠睾丸间质细胞氧化损伤具有拮抗作用,莱菔硫烷对镉诱导的小鼠睾丸间质细胞损伤的保护作用可能通过激活Nrf2-ARE通路途径实现。
[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.

【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S859.8
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本文编号：1508013