镉致体外培养鸡支持—生精细胞凋亡机理的研究
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摘要
镉是一种工业上应用广泛的具有潜在毒性的重金属,已逐渐成为环境中重要的污染物之一。大量研究表明,镉能够引起雄性动物和人生殖机能减退,诱导生殖细胞凋亡、坏死等不可逆变化。但大多数试验集中于对啮齿类动物和人的研究,对于禽类的研究较少。本试验以体外培养鸡原代支持-生精细胞为研究对象,在培养液中加入不同浓度CdCl_2,通过对鸡支持-生精细胞活性、贴壁性能、细胞凋亡、抗氧化功能、DNA损伤、细胞内游离钙离子浓度及细胞内bcl-2、Fas、FasL、CaM、caspase-3mRNA表达水平等的检测,探讨了镉致鸡支持-生精细胞凋亡的机理。研究结果表明:
1.应用台盼蓝拒染试验、MTT比色法、二硝基苯肼比色法检测了支持-生精细胞的活性和培养上清液中LDH活性,并观察了细胞贴壁性能,结果表明镉能够抑制体外培养鸡支持-生精细胞的活性,影响支持细胞的贴壁性能及支持细胞与生精细胞之间的黏附力,同时测定了染镉24h对支持-生精细胞的半数抑制浓度为28.41±0.42μmol/L,表明镉对鸡支持-生精细胞具有毒性作用。
2.应用AO/EB双荧光染色法测定了染镉后支持-生精细胞的凋亡率和坏死率,结果表明,低浓度镉主要引起细胞凋亡,而高浓度镉使细胞凋亡和细胞坏死同时发生。
3.应用碱性单细胞凝胶电泳测定了镉对支持-生精细胞DNA的损伤效应,结果表明镉能够诱导鸡生殖细胞DNA损伤,这可能是其致禽类生殖细胞损伤的机制之一。
4.通过对细胞内抗氧化功能的检测,结果表明镉能够导致支持-生精细胞内SOD、GSH-Px活性下降,MDA含量增加,细胞抗氧化功能降低,发生氧化应激,这可能是镉诱导禽类生殖细胞凋亡的机制之一。
5.应用半定量RT-PCR法和荧光探针法检测细胞内CaMmRNA表达水平和游离钙离子浓度,结果表明镉能够引起细胞内CaMmRNA表达水平下降,游离钙离子浓度升高,干扰细胞钙离子的转运,导致细胞钙稳态失衡,这可能是镉诱导禽类生殖细胞凋亡的机制之一。
6.应有半定量RT-PCR法检测了鸡支持-生精细胞内bcl-2、Fas、FasL、caspase-3mRNA表达水平,结果表明镉能够降低了支持-生精细胞内bcl-2mRNA的表达水平,增加Fas/FasL和caspase-3mRNA的表达水平,进而诱导生殖细胞的凋亡。
本试验从细胞、分子水平上探讨镉致禽类生殖细胞毒性作用,揭示了镉能够降低生殖细胞的活性、诱发氧化应激、损伤细胞DNA、干扰细胞钙稳态、影响细胞内基因表达、诱导细胞凋亡,阐明了镉致禽类生殖细胞毒性机制,为畜禽镉中毒的防治提供了理论依据,丰富了毒理学和比较医学的资料,对保护畜牧业的健康发展,保护野生禽类,维持生态平衡具有重要意义。
Cadmium, a heavy metal of potential toxicity is extensively used in the industry and become one of the most important pollutants in the enviroment. A large amount of researches have indicated that Cadmium can lead to the functional decrease of reproduction in male animals and human and induce the nonreversible change such as apoptosis and necrosis of germ cells. Most of the studies focus on the rodent and human being, while only a few focus on the avian. This study took cock primary sertoli-germ cell as a model and added different concentration of CdCl2 into the culture medium. Mechanisms in cadmium-induced apoptosis of cock sertoli-germ cell were revealed through detecting the activation of the sertoli-germ cell, the capability of adherence, apoptosis, anti-oxidative function, DNA dammages, [Ca~(2+)]i changes and gene expression. The results showed as follows:
1. Determine the activation of cock sertoli-germ cell and the level of LDH by the tapan blue method ;MTT assay and dinitro-phenylhydrazine(DPNH) chromometry. The capability of adherence was observed. The result revealed that the activation of cock sertoli-germ cell cultured in vitro can be inhibited by cadmium and the capability of adherence as well as the sticky interaction between the spermatogenic cells and the support cells was influenced by Cadmium. IC50 of cadmium was 28.41±0.42μmol/L in 24h. Our result showed that cadmium had toxic effect to chock sertoli-germ cells.
2.The apoptosis and necrosis percentage of the sertoli-germ cells were detected by AO/EB double fluorescence staining method. The results proved that low concentration cadmium can predominantly induce apoptosis, while high dose cadmium increased apoptosis and necrosis simultaneously.
3.The effects of cadmium on DNA damage were detected by SCGE. The results showed that cadmium caused DNA damage, which exhibited dose-dependent effect. It revealed that the effects of cadmium on DNA damage of germ cells may be one of the mechanisms of damage of germ cells induced by cadmium.
4.The result of detection on anti-oxidative function dedicated that cadmium lead to the decease of SOD activity and GSH-Px activity, the incease of MDA content and the anti-oxidative function, which lead to the oxidative stress. Possibly, this was one of the mechanisms of apoptosis induced by cadmium.
5.CaMmRNA expression and [Ca~(2+)]i were separatly detected by semiquantitative PCR and
1.应用台盼蓝拒染试验、MTT比色法、二硝基苯肼比色法检测了支持-生精细胞的活性和培养上清液中LDH活性,并观察了细胞贴壁性能,结果表明镉能够抑制体外培养鸡支持-生精细胞的活性,影响支持细胞的贴壁性能及支持细胞与生精细胞之间的黏附力,同时测定了染镉24h对支持-生精细胞的半数抑制浓度为28.41±0.42μmol/L,表明镉对鸡支持-生精细胞具有毒性作用。
2.应用AO/EB双荧光染色法测定了染镉后支持-生精细胞的凋亡率和坏死率,结果表明,低浓度镉主要引起细胞凋亡,而高浓度镉使细胞凋亡和细胞坏死同时发生。
3.应用碱性单细胞凝胶电泳测定了镉对支持-生精细胞DNA的损伤效应,结果表明镉能够诱导鸡生殖细胞DNA损伤,这可能是其致禽类生殖细胞损伤的机制之一。
4.通过对细胞内抗氧化功能的检测,结果表明镉能够导致支持-生精细胞内SOD、GSH-Px活性下降,MDA含量增加,细胞抗氧化功能降低,发生氧化应激,这可能是镉诱导禽类生殖细胞凋亡的机制之一。
5.应用半定量RT-PCR法和荧光探针法检测细胞内CaMmRNA表达水平和游离钙离子浓度,结果表明镉能够引起细胞内CaMmRNA表达水平下降,游离钙离子浓度升高,干扰细胞钙离子的转运,导致细胞钙稳态失衡,这可能是镉诱导禽类生殖细胞凋亡的机制之一。
6.应有半定量RT-PCR法检测了鸡支持-生精细胞内bcl-2、Fas、FasL、caspase-3mRNA表达水平,结果表明镉能够降低了支持-生精细胞内bcl-2mRNA的表达水平,增加Fas/FasL和caspase-3mRNA的表达水平,进而诱导生殖细胞的凋亡。
本试验从细胞、分子水平上探讨镉致禽类生殖细胞毒性作用,揭示了镉能够降低生殖细胞的活性、诱发氧化应激、损伤细胞DNA、干扰细胞钙稳态、影响细胞内基因表达、诱导细胞凋亡,阐明了镉致禽类生殖细胞毒性机制,为畜禽镉中毒的防治提供了理论依据,丰富了毒理学和比较医学的资料,对保护畜牧业的健康发展,保护野生禽类,维持生态平衡具有重要意义。
Cadmium, a heavy metal of potential toxicity is extensively used in the industry and become one of the most important pollutants in the enviroment. A large amount of researches have indicated that Cadmium can lead to the functional decrease of reproduction in male animals and human and induce the nonreversible change such as apoptosis and necrosis of germ cells. Most of the studies focus on the rodent and human being, while only a few focus on the avian. This study took cock primary sertoli-germ cell as a model and added different concentration of CdCl2 into the culture medium. Mechanisms in cadmium-induced apoptosis of cock sertoli-germ cell were revealed through detecting the activation of the sertoli-germ cell, the capability of adherence, apoptosis, anti-oxidative function, DNA dammages, [Ca~(2+)]i changes and gene expression. The results showed as follows:
1. Determine the activation of cock sertoli-germ cell and the level of LDH by the tapan blue method ;MTT assay and dinitro-phenylhydrazine(DPNH) chromometry. The capability of adherence was observed. The result revealed that the activation of cock sertoli-germ cell cultured in vitro can be inhibited by cadmium and the capability of adherence as well as the sticky interaction between the spermatogenic cells and the support cells was influenced by Cadmium. IC50 of cadmium was 28.41±0.42μmol/L in 24h. Our result showed that cadmium had toxic effect to chock sertoli-germ cells.
2.The apoptosis and necrosis percentage of the sertoli-germ cells were detected by AO/EB double fluorescence staining method. The results proved that low concentration cadmium can predominantly induce apoptosis, while high dose cadmium increased apoptosis and necrosis simultaneously.
3.The effects of cadmium on DNA damage were detected by SCGE. The results showed that cadmium caused DNA damage, which exhibited dose-dependent effect. It revealed that the effects of cadmium on DNA damage of germ cells may be one of the mechanisms of damage of germ cells induced by cadmium.
4.The result of detection on anti-oxidative function dedicated that cadmium lead to the decease of SOD activity and GSH-Px activity, the incease of MDA content and the anti-oxidative function, which lead to the oxidative stress. Possibly, this was one of the mechanisms of apoptosis induced by cadmium.
5.CaMmRNA expression and [Ca~(2+)]i were separatly detected by semiquantitative PCR and
引文
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