镉诱导仔猪睾丸支持细胞凋亡及机理研究
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摘要
镉作为机体生长发育非必需元素,少量进入体内即可通过生物放大和生物蓄积对肝、肾、肺、睾丸、脑、骨骼以及血液系统产生一系列损伤,镉进入身体后,分布于全身各个器官,睾丸是重要的代谢器官。镉的生殖毒性的研究大都是对生精细胞.但镉对睾丸支持细胞,特别是猪睾丸支持细胞毒性研究国内外罕见报道。本研究以体外培养的仔猪睾丸支持细胞为模型,采用形态学、生化及分子生物学方法,研究氯化镉诱导支持细胞氧化损伤、细胞凋亡及其机理,以期为镉的雄性生殖毒性的研究提供参考性资料。
1.支持细胞的分离、培养、鉴定、传代、冻存和复苏
采用二步酶解法分离后的支持细胞并用含15%FBS的DMEM/F12培养基培养,等到纯度较高的支持细胞。通过油红0和免疫细胞化学法鉴定。进行传代培养,再进行冻存复苏培养。为后续试验提供了试验材料。
2.氯化镉对支持细胞抑制率的影响
O、10、20、40及80μmol／L氯化镉分别作用支持细胞24 h后,采用MTT法观察氯化镉对细胞生长的影响。结果显示：随着给氯化镉剂量的增加,对细胞抑制作用增强,同对照组比较差异具有显著性,存在着剂量-效应关系。
2.氯化镉对支持细胞的氧化损伤作用
0、10、20、40及80μmol／L氯化镉分别作用支持细胞24 h后,利用生化方法检测其对细胞氧化损伤的情况。结果显示：随着氯化镉剂量的升高,SOD的活性降低,同对照组比较差异具有显著性；MDA含量逐渐增加,同对照组比较差异具有显著性；GSH-px的活性下降,各实验组与对照组比较差异有显著性。
3.氯化镉对支持细胞的DNA损伤作用
0、10、20、40及80 p mol/L氯化镉分别作用支持细胞24 h后,利用彗星试验检测其对细胞DNA损伤的情况。结果显示：随着氯化镉剂量的升高,支持细胞DNA损伤加重,存在着剂量-效应关系。
4.氯化镉对支持细胞超微结构的影响
0、10、20、40及80μmol／L氯化镉分别作用支持细胞24 h后,支持细胞超微结构发生病变,随着氯化镉剂量的升高有较重趋势。
5.氯化镉对支持细胞凋亡的影响
0、10、20、40及80μmol／L氯化镉分别作用支持细胞24 h后, AO／EB双荧光标记,荧光显微镜观察细胞。不同剂量组均有凋亡细胞出现。正常细胞的细胞核DNA呈绿色荧光,核饱满；出现凋亡的细胞可见细胞体积缩小,细胞呈黄绿色,形状不规则,细胞膜粗糙,部分细胞呈鲜亮的橙色斑点,随着药物浓度的增加,细胞数量明显减少,并出现坏死细胞,细胞体积增大,胞质内黄绿色或桔黄色荧光减弱甚至消失,可见红染细胞。随着剂量的增加,各实验组细胞凋亡率升高。采用FITC-AnnexinV/PI双荧光标记,流式细胞术检测细胞凋亡的变化。结果显示：各实验组均观察到明显的细胞凋亡,同对照组比较差异具有显著性,并随着剂量的升高凋亡率增加。
6氯化镉对支持细胞线粒体电位膜的影响
0、10、20、40及80μmol／L氯化镉分别作用支持细胞24 h后,罗丹明123染色,荧光显微镜和荧光分光光度计检测：氯化镉能促使线粒体膜电位降低,随着氯化镉作用浓度的增加,弱荧光部分细胞含量逐渐增高。
6. RT-PCR法检测Bax, Bcl-2, CytC, AIF, Caspase-9和Caspase-3 mRNA水平表达变化
采用RT-PCR方法检测氯化镉作用支持细胞24 h后细胞Bax,Bcl-2, CytC, AIF, Caspase-9和Caspase-3 mRNA水平的变化。结果显示：随着给氯化镉剂量的增加,bcl-2的基因表达呈现降低的趋势,其余各基因表达呈增加趋势。说明bcl-2基因有抑制凋亡的作用,其余基因有促进凋亡的作用。
7. Western blot法检测Bax,Bcl-2, CytC, AIF, Caspase-9和Caspase-3的蛋白水平的变化
采用Western blot检测检测氯化镉作用支持细胞24 h后细胞Bax,Bcl-2,CytC, AIF, Caspase-9和Caspase-3蛋白水平的变化。结果显示：随着给氯化镉剂量的增加,bcl-2的蛋白表达呈现降低的趋势,其余各基因蛋白表达呈增加趋势。
Cd, as a kind of non-essential element to growth and development of organism, only a little in the human body, can induce a series of damage, including to liver, kidney, testicle, brain, bones and hematological system by bioamplification or biological accumulation. Cd goes in the human body, then distributes to all parts of the whole body and testicle is an important metabolic organ. Nowadays, mostly research about cadmium toxicity on male reproductive is directed at spermatogenic cells and the reprots on Cd to texicity of tescticle sertoli cells, especially to piglet sertoli cells are rare both at home and abroad. In this study, sertoli cells of piglet testicle as a model, were used to detect oxdiase damage, cell apoptosis and mechanism induced by cadmium chloride in vitro by the methods of morphology, biochemisty and molecular biology. The goal was in order to provide theoretical basis to study cadmium toxicity on male reproductive.
1. Isolation, culture, identification, subculture, cryopreservation and resuscitation of sertoli cells.
Sertoli cells were isolated from piglet testes using a two-step enzyme digestion and cultured by DMEM/F culture-medium including 15% FBS. High purify sertoli cells were isolated by the two-step enzyme digestion and were identificated by oil red 0 staining and immunocytochemical method. Sertoli cells were used to subculture and then were cryopreserved and resuscitated. All studies were in order to provide materials for the rear test.
2. Effects of cadmium chloride on the inhibitory rate of sertoli cells.
Piglet sertoli cells were exposed to cadmium chloride at 10,20,40 and 80μmol/L, and MTT assay was applied to observe the inhibitory effects of CdCl2 after 24h. The results showed that the inhibitory rate of sertoli cells increased significantly with the increasing dosage of CdCl2 compared with control group. The exists an obvious dose-effect relationship.
3. Effects of cadmium chloride on oxidase damage of sertoli cells.
Biochemical kits were applied to study activity of SOD, GSH-px and content of MDA in sertoli cells,24h after 10,20,40 and 80μmol/L of cadmium chloride treatment. The results showed that the activity of SOD was decreased, and it had a significant changes between CdCl2 administration groups and control group; The content of MDA was increased significantly at different exposed goups compared with control goup; The activity of GSH-px in all exposed groups were decreased significantly compared with control group.
4. Effects of cadmium chloride on DNA damage of sertoli cells.
Sertoli cells were treated with 10,20,40 and 80μmol/L cadmium chloride for 24h and then comet assay was used to detected effects of cadmium chloride on DNA damage of sertoli cells. The results showed that with the increasing of CdCl2 dosage, DNA damage of sertoli cells was worsened. The exists an obvious dose-effect relationship.
5. Effects of cadmium chloride on ultrastructure of sertoli cells.
Ultrastructure of sertoli cells treated with 10,20,40 and 80μmol/L cadmium chloride for 24h emerged lesions and the increasing dosage could result in worsening of ultrastructure lesions.
6. Effects of cadmium chloride on apoptosis of sertoli cells.
Sertoli cells were treated with 10,20,40 and 80μmol/L cadmium chloride for 24h. AO/EB double fluorescent staining assay was used to observe cell apoptosis.The results showed the apoptotic cells appeared in all the CdCl2 groups. Normal cell had a regular nuclear with green fluorimetric stain, while apoptotic cell had unregular nuclear with yellowish green stain, and the capacity of cell became smaller. The amount of cell was decrease with increasing dosage. And necrosis cell appeared with red stained.
Apoptosis was measured by AnnexinⅤ-FITC/PI double staining using flow cytometry. The results showed that the percentage of apoptotic cells was increased significantly with increasing dosage, and there were significantly diff rences among the exposure groups.
7. Effects of cadmium chloride on mitochondria△Ψm of sertoli cells.
Sertoli cells were treated with 10,20,40 and 80μmol/L cadmium chloride for 24h and stained with Regand 123, then detected by fluorescence microscopy and fluorospectrophotometer. Results showed cadmium chloride could cause to the decrease of mitochondria△Ψm of sertoli cells, but the content of the characteristic light fluorescence was increased with increasing dosage.
8. Changes in mRNA level of Bax, Bcl-2, Cyt C, AIF, Capase-9 and Capase-3 of sertoli cells.
RT-PCR assay was used to measure the mRNA level of Bax, Bci-2, Cyt C, AIF, Capase-9 and Capase-3 of sertoli cells. The results showed that Bcl-2 mRNA level was decreased with the increasing dosage of cadmium chloride. mRNA level of the other genes were increased with the increasing dosage. Results indicated Bcl-2 gene could inhibit apoptosis of sertoli cells, but the other genes could accelate apoptosis of sertoli cells.
9. Changes in protein expression of Bax, Bcl-2, CytC, AIF, Capase-9 and Capase-3 of of sertoli cells.
The western blot assay was used to detect the Bax, Bcl-2, CytC, AIF, Capase-9 and Capase-3 expression of total protein in sertoli cells treated with CdCl2 for 24h. Resutls showed that the Bcl-2 expression decreased with the increasing of CdCl2 dosage but the Bax, CytC, AIF, Capase-9 and Capase-3 expression all increased with the increasing dosage. Keywords:cadmium chloride; sertoli cell; oxidase damage; ultrastructure; mitochondria△Ψm; apoptosis
1.支持细胞的分离、培养、鉴定、传代、冻存和复苏
采用二步酶解法分离后的支持细胞并用含15%FBS的DMEM/F12培养基培养,等到纯度较高的支持细胞。通过油红0和免疫细胞化学法鉴定。进行传代培养,再进行冻存复苏培养。为后续试验提供了试验材料。
2.氯化镉对支持细胞抑制率的影响
O、10、20、40及80μmol／L氯化镉分别作用支持细胞24 h后,采用MTT法观察氯化镉对细胞生长的影响。结果显示：随着给氯化镉剂量的增加,对细胞抑制作用增强,同对照组比较差异具有显著性,存在着剂量-效应关系。
2.氯化镉对支持细胞的氧化损伤作用
0、10、20、40及80μmol／L氯化镉分别作用支持细胞24 h后,利用生化方法检测其对细胞氧化损伤的情况。结果显示：随着氯化镉剂量的升高,SOD的活性降低,同对照组比较差异具有显著性；MDA含量逐渐增加,同对照组比较差异具有显著性；GSH-px的活性下降,各实验组与对照组比较差异有显著性。
3.氯化镉对支持细胞的DNA损伤作用
0、10、20、40及80 p mol/L氯化镉分别作用支持细胞24 h后,利用彗星试验检测其对细胞DNA损伤的情况。结果显示：随着氯化镉剂量的升高,支持细胞DNA损伤加重,存在着剂量-效应关系。
4.氯化镉对支持细胞超微结构的影响
0、10、20、40及80μmol／L氯化镉分别作用支持细胞24 h后,支持细胞超微结构发生病变,随着氯化镉剂量的升高有较重趋势。
5.氯化镉对支持细胞凋亡的影响
0、10、20、40及80μmol／L氯化镉分别作用支持细胞24 h后, AO／EB双荧光标记,荧光显微镜观察细胞。不同剂量组均有凋亡细胞出现。正常细胞的细胞核DNA呈绿色荧光,核饱满；出现凋亡的细胞可见细胞体积缩小,细胞呈黄绿色,形状不规则,细胞膜粗糙,部分细胞呈鲜亮的橙色斑点,随着药物浓度的增加,细胞数量明显减少,并出现坏死细胞,细胞体积增大,胞质内黄绿色或桔黄色荧光减弱甚至消失,可见红染细胞。随着剂量的增加,各实验组细胞凋亡率升高。采用FITC-AnnexinV/PI双荧光标记,流式细胞术检测细胞凋亡的变化。结果显示：各实验组均观察到明显的细胞凋亡,同对照组比较差异具有显著性,并随着剂量的升高凋亡率增加。
6氯化镉对支持细胞线粒体电位膜的影响
0、10、20、40及80μmol／L氯化镉分别作用支持细胞24 h后,罗丹明123染色,荧光显微镜和荧光分光光度计检测：氯化镉能促使线粒体膜电位降低,随着氯化镉作用浓度的增加,弱荧光部分细胞含量逐渐增高。
6. RT-PCR法检测Bax, Bcl-2, CytC, AIF, Caspase-9和Caspase-3 mRNA水平表达变化
采用RT-PCR方法检测氯化镉作用支持细胞24 h后细胞Bax,Bcl-2, CytC, AIF, Caspase-9和Caspase-3 mRNA水平的变化。结果显示：随着给氯化镉剂量的增加,bcl-2的基因表达呈现降低的趋势,其余各基因表达呈增加趋势。说明bcl-2基因有抑制凋亡的作用,其余基因有促进凋亡的作用。
7. Western blot法检测Bax,Bcl-2, CytC, AIF, Caspase-9和Caspase-3的蛋白水平的变化
采用Western blot检测检测氯化镉作用支持细胞24 h后细胞Bax,Bcl-2,CytC, AIF, Caspase-9和Caspase-3蛋白水平的变化。结果显示：随着给氯化镉剂量的增加,bcl-2的蛋白表达呈现降低的趋势,其余各基因蛋白表达呈增加趋势。
Cd, as a kind of non-essential element to growth and development of organism, only a little in the human body, can induce a series of damage, including to liver, kidney, testicle, brain, bones and hematological system by bioamplification or biological accumulation. Cd goes in the human body, then distributes to all parts of the whole body and testicle is an important metabolic organ. Nowadays, mostly research about cadmium toxicity on male reproductive is directed at spermatogenic cells and the reprots on Cd to texicity of tescticle sertoli cells, especially to piglet sertoli cells are rare both at home and abroad. In this study, sertoli cells of piglet testicle as a model, were used to detect oxdiase damage, cell apoptosis and mechanism induced by cadmium chloride in vitro by the methods of morphology, biochemisty and molecular biology. The goal was in order to provide theoretical basis to study cadmium toxicity on male reproductive.
1. Isolation, culture, identification, subculture, cryopreservation and resuscitation of sertoli cells.
Sertoli cells were isolated from piglet testes using a two-step enzyme digestion and cultured by DMEM/F culture-medium including 15% FBS. High purify sertoli cells were isolated by the two-step enzyme digestion and were identificated by oil red 0 staining and immunocytochemical method. Sertoli cells were used to subculture and then were cryopreserved and resuscitated. All studies were in order to provide materials for the rear test.
2. Effects of cadmium chloride on the inhibitory rate of sertoli cells.
Piglet sertoli cells were exposed to cadmium chloride at 10,20,40 and 80μmol/L, and MTT assay was applied to observe the inhibitory effects of CdCl2 after 24h. The results showed that the inhibitory rate of sertoli cells increased significantly with the increasing dosage of CdCl2 compared with control group. The exists an obvious dose-effect relationship.
3. Effects of cadmium chloride on oxidase damage of sertoli cells.
Biochemical kits were applied to study activity of SOD, GSH-px and content of MDA in sertoli cells,24h after 10,20,40 and 80μmol/L of cadmium chloride treatment. The results showed that the activity of SOD was decreased, and it had a significant changes between CdCl2 administration groups and control group; The content of MDA was increased significantly at different exposed goups compared with control goup; The activity of GSH-px in all exposed groups were decreased significantly compared with control group.
4. Effects of cadmium chloride on DNA damage of sertoli cells.
Sertoli cells were treated with 10,20,40 and 80μmol/L cadmium chloride for 24h and then comet assay was used to detected effects of cadmium chloride on DNA damage of sertoli cells. The results showed that with the increasing of CdCl2 dosage, DNA damage of sertoli cells was worsened. The exists an obvious dose-effect relationship.
5. Effects of cadmium chloride on ultrastructure of sertoli cells.
Ultrastructure of sertoli cells treated with 10,20,40 and 80μmol/L cadmium chloride for 24h emerged lesions and the increasing dosage could result in worsening of ultrastructure lesions.
6. Effects of cadmium chloride on apoptosis of sertoli cells.
Sertoli cells were treated with 10,20,40 and 80μmol/L cadmium chloride for 24h. AO/EB double fluorescent staining assay was used to observe cell apoptosis.The results showed the apoptotic cells appeared in all the CdCl2 groups. Normal cell had a regular nuclear with green fluorimetric stain, while apoptotic cell had unregular nuclear with yellowish green stain, and the capacity of cell became smaller. The amount of cell was decrease with increasing dosage. And necrosis cell appeared with red stained.
Apoptosis was measured by AnnexinⅤ-FITC/PI double staining using flow cytometry. The results showed that the percentage of apoptotic cells was increased significantly with increasing dosage, and there were significantly diff rences among the exposure groups.
7. Effects of cadmium chloride on mitochondria△Ψm of sertoli cells.
Sertoli cells were treated with 10,20,40 and 80μmol/L cadmium chloride for 24h and stained with Regand 123, then detected by fluorescence microscopy and fluorospectrophotometer. Results showed cadmium chloride could cause to the decrease of mitochondria△Ψm of sertoli cells, but the content of the characteristic light fluorescence was increased with increasing dosage.
8. Changes in mRNA level of Bax, Bcl-2, Cyt C, AIF, Capase-9 and Capase-3 of sertoli cells.
RT-PCR assay was used to measure the mRNA level of Bax, Bci-2, Cyt C, AIF, Capase-9 and Capase-3 of sertoli cells. The results showed that Bcl-2 mRNA level was decreased with the increasing dosage of cadmium chloride. mRNA level of the other genes were increased with the increasing dosage. Results indicated Bcl-2 gene could inhibit apoptosis of sertoli cells, but the other genes could accelate apoptosis of sertoli cells.
9. Changes in protein expression of Bax, Bcl-2, CytC, AIF, Capase-9 and Capase-3 of of sertoli cells.
The western blot assay was used to detect the Bax, Bcl-2, CytC, AIF, Capase-9 and Capase-3 expression of total protein in sertoli cells treated with CdCl2 for 24h. Resutls showed that the Bcl-2 expression decreased with the increasing of CdCl2 dosage but the Bax, CytC, AIF, Capase-9 and Capase-3 expression all increased with the increasing dosage. Keywords:cadmium chloride; sertoli cell; oxidase damage; ultrastructure; mitochondria△Ψm; apoptosis
引文
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