壬基酚对中华绒螯蟹（Eriocheir sinensis）的免疫毒性及雌激素效应研究
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摘要
中华绒螯蟹(Eriocheir sinensis)又名河蟹,是我国极为重要的水产经济动物。随养殖规模的扩大,中华绒螯蟹病害也随之加剧,给养殖业造成了极大经济损失。自身的免疫能力下降、病原体增加以及其生存水环境污染是导致河蟹疾病的三大主要原因。其中环境因素起着重要作用,不仅影响甲壳动物机体抗病能力同时也导致病原生物的大量繁殖。
对甲壳动物来说,水体污染物引起的环境压力是其免疫力下降的一个重要原因。甲壳动物机体变化或患病率的上升可以作为环境压力的标志。目前对中华绒螯蟹的环境胁迫研究主要集中在氨氮,温度,pH值,重金属离子等环境因子方面,有关壬基酚(NP)等环境内分泌干扰化学物质对河蟹的毒性作用的研究未见报道。NP是一种常见的、化学性质稳定的,并且容易在生物体内积累的污染物。随着工业的发展,污染加剧,我国长江流域水体和沉积物中发现了NP的存在。目前已有大量研究证实NP可对生物体产生的不良作用：包括对内分泌、生殖和发育、致癌、免疫等方面。中华绒螯蟹的生存环境也受到NP的威胁,因此,有必要探索NP对水产养殖虾蟹类的胁迫效应,为建立相应的评价体系提供理论指导。
本研究借助免疫检测技术和分子生物学手段,通过对免疫相关酶活性的测定,对血细胞密度(THC)、酚氧化酶激活系统(proPO、PPAF)、抗菌肽(crustin)、抗氧化酶系统(SOD、CAT、GPX)等免疫相关基因表达量以及卵黄蛋白原(VTG)的变化的检测,分别从细胞和分子水平检测腹腔注射NP对中华绒螯蟹的免疫毒性及雌激素效应,为NP对中华绒螯蟹作用的研究丰富资料。
主要研究结果如下：
1、NP暴露10d后,中华绒螯蟹总血淋巴细胞THC密度下降,且与NP的剂量(10mk／kg～1000mg／kg)呈反比线性关系。在本实验的中剂量300mg／kgNP进行的时间效应实验中,中华绒螯蟹THC密度在腹腔注射暴露后的3d内呈下降；暴露6d后,血细胞数回升,此后血细胞数呈现明显的回升,并在第14d接近对照组。
2、较低剂量(≤100mg／kg)NP可提高中华绒螯蟹血细胞ProPO和PPAF基因的表达量,但是高于此浓度时,诱导效应反而下降。NP对中华绒螯蟹ProPO和PPAF基因表达的诱导具有时间效应,注射后6d内表达量一路上调,其后回落。
3、低剂量的NP同样可以使中华绒螯蟹血淋巴细胞抗菌肽甲壳素crustin mRNA表达的上调。经低剂量NP预暴露1d的中华绒螯蟹,感染嗜水汽单胞菌时血淋巴细胞中crustin的表达量远高于未预暴露组,但是NP暴露14d后的个体,受细菌感染时血淋巴细胞中crustin的表达量低于未感染组,显示出短时间免疫应激效应。
4、中华绒螯蟹肝胰腺超氧化物歧化酶SOD、过氧化氢酶CAT和过氧化物酶GPX对NP暴露的感应灵敏。最低剂量(10mg／kg)的NP即可诱导酶活性的升高,并且有明显的剂量效应关系。NP对中华绒螯蟹抗氧化酶类的作用是双向的,即较低剂量的NP暴露可以显著提高酶的活性,并诱导抗氧化酶基因的表达,而较高剂量组暴露反而使得酶活性显著低于对照组,表现为抑制效应。此外,NP对中华绒螯蟹的毒性作用有时间变化过程,即呈现先缓慢上升,后下降的过程。
5、NP对中华绒螯蟹也有雌激素效应。不仅可提高雌蟹肝胰腺中卵黄蛋白原基因的表达水平,也可诱导雄性中华绒螯蟹肝胰腺VTG基因的表达。10mg／kg剂量组NP暴露就检测到了VTG基因的表达量的升高,500mg／kg表达量达到最大值。
综合分析各项实验结果可知,低剂量的NP可刺激中华绒螯蟹产生免疫应激反应,这种应激反应随NP在体内的代谢消除而减弱,并会对中华绒螯蟹的免疫能力产生一定的负效应。高浓度的NP,通过减少中华绒螯蟹血淋巴细胞影响免疫功能。与高等脊椎动物的反应相似,NP对中华绒螯蟹也有雌激素效应。
Chinese mitten crab (Eriocheir sinensis) is a very important economic aquatic animal in our country. With the expansion of the scale in recent years, aquaculture, environment deterioration is serious, leading to increase the crab diseases, a crab breeding caused tremendous economic losses. There are three reasons for crab's diseases:crab's own immunity, pathogens and the quality of water where crabs are living in. Environmental factors play an important role, not only affects the crab's resistance to disease but also affect bloom of the pathogens.
For crustaceans, water pollution which causes the environmental stress is the important reason for the decline of the immunity. The changes of the body or the increase of the prevalence can be markers of environmental stress. The research on crustaceans's environmental stress focuses on nitrogen, temperature, pH value, heavy metals and other environmental factors,
Toxic effects of NP on Eriocheir sinensis have rarely been studied. NP is common in the water, having good chemical stability, and easy to accumulate in the organisms. With the development of the industry, the pollution is increasingly serious. High dose of NP have found in Plenty of the rivers of our country, the living environment of Eriocheir sinensis is threatened. Many studies have shown that NP can be toxic on organisms in many aspects, like the endocrine, reproductive and developmental, cancer, immune and so on. Therefore, it's neccessery to explore the NP stress effect on shrimps and crabs, for providing theoretical guidance to the establishment of appropriate evaluation system.
1. NP exposure to can lead to a significant decline of THC in Chinese mitten crab, and relationship between NP dose and THC was nagative. In the time-effect experiment of 300mg/kg NP, THC were exposed down at the first 3d; while it recovery after 6d, after this day, it arised significantly, and close to the control group on 14d.
2. Low dose (≤100mg/kg) of NP can increase ProPO and PPAF gene expression in hemocyte of Eriocheir sinensis, but above this concentration, the induction effect decreased. The inductive effect on ProPO and PPAF of NP with time-effect relationship. The expression conatant rise after injection 6d, then become decrease.
3. Low dose of NP can increase the expression of crustin mRNA of Eriocheir sinensis. The expression crustin crab which pre-exposure to low doses of NP for 1d, infection with Aeromonas hydrophila, much higher than non-exposure group, but in the group which exposure to NP for 14d the expression of crustin gene is lower the the non-infected group. Show short time-effects of immune stress.
4:The three antioxidant enzymes (SOD, CAT and GPX) are sensitive to NP. low doses can increase their activity. low dosage of NP can increase the expression of antioxidant enzymes, while the higher dosage inhibit it. In addition, we also observed that, the toxicity of NP changes by the exposed time. that is slowly increased at first, then decreased in the process.
5. NP have estrogenic effect in Eriocheir sinensis. Not only to enhance the VTG gene expression level in female carb, but also can induce VTG gene expression in the male crab. VTG gene expression increased can been detected in 10mg/kg dose NP, and in the group of 500mg/kg dosage NP, the expression reach the highest level.
Comprehensive analysis the experimental results we can see that low doses of NP may stimulate the immune stress response in Chinese mitten crab, this stress weakened with the metabolic elimination of NP in the body, and it will have a certain negative effects for the immunity of Eriocheir sinensis. High concentrations of NP, by reducing hemocyte to effect. the immune function of Eriocheir sinensis. Similar to the vertebrates, NP has estrogenic effects on Eriocheir sinensis.
对甲壳动物来说,水体污染物引起的环境压力是其免疫力下降的一个重要原因。甲壳动物机体变化或患病率的上升可以作为环境压力的标志。目前对中华绒螯蟹的环境胁迫研究主要集中在氨氮,温度,pH值,重金属离子等环境因子方面,有关壬基酚(NP)等环境内分泌干扰化学物质对河蟹的毒性作用的研究未见报道。NP是一种常见的、化学性质稳定的,并且容易在生物体内积累的污染物。随着工业的发展,污染加剧,我国长江流域水体和沉积物中发现了NP的存在。目前已有大量研究证实NP可对生物体产生的不良作用：包括对内分泌、生殖和发育、致癌、免疫等方面。中华绒螯蟹的生存环境也受到NP的威胁,因此,有必要探索NP对水产养殖虾蟹类的胁迫效应,为建立相应的评价体系提供理论指导。
本研究借助免疫检测技术和分子生物学手段,通过对免疫相关酶活性的测定,对血细胞密度(THC)、酚氧化酶激活系统(proPO、PPAF)、抗菌肽(crustin)、抗氧化酶系统(SOD、CAT、GPX)等免疫相关基因表达量以及卵黄蛋白原(VTG)的变化的检测,分别从细胞和分子水平检测腹腔注射NP对中华绒螯蟹的免疫毒性及雌激素效应,为NP对中华绒螯蟹作用的研究丰富资料。
主要研究结果如下：
1、NP暴露10d后,中华绒螯蟹总血淋巴细胞THC密度下降,且与NP的剂量(10mk／kg～1000mg／kg)呈反比线性关系。在本实验的中剂量300mg／kgNP进行的时间效应实验中,中华绒螯蟹THC密度在腹腔注射暴露后的3d内呈下降；暴露6d后,血细胞数回升,此后血细胞数呈现明显的回升,并在第14d接近对照组。
2、较低剂量(≤100mg／kg)NP可提高中华绒螯蟹血细胞ProPO和PPAF基因的表达量,但是高于此浓度时,诱导效应反而下降。NP对中华绒螯蟹ProPO和PPAF基因表达的诱导具有时间效应,注射后6d内表达量一路上调,其后回落。
3、低剂量的NP同样可以使中华绒螯蟹血淋巴细胞抗菌肽甲壳素crustin mRNA表达的上调。经低剂量NP预暴露1d的中华绒螯蟹,感染嗜水汽单胞菌时血淋巴细胞中crustin的表达量远高于未预暴露组,但是NP暴露14d后的个体,受细菌感染时血淋巴细胞中crustin的表达量低于未感染组,显示出短时间免疫应激效应。
4、中华绒螯蟹肝胰腺超氧化物歧化酶SOD、过氧化氢酶CAT和过氧化物酶GPX对NP暴露的感应灵敏。最低剂量(10mg／kg)的NP即可诱导酶活性的升高,并且有明显的剂量效应关系。NP对中华绒螯蟹抗氧化酶类的作用是双向的,即较低剂量的NP暴露可以显著提高酶的活性,并诱导抗氧化酶基因的表达,而较高剂量组暴露反而使得酶活性显著低于对照组,表现为抑制效应。此外,NP对中华绒螯蟹的毒性作用有时间变化过程,即呈现先缓慢上升,后下降的过程。
5、NP对中华绒螯蟹也有雌激素效应。不仅可提高雌蟹肝胰腺中卵黄蛋白原基因的表达水平,也可诱导雄性中华绒螯蟹肝胰腺VTG基因的表达。10mg／kg剂量组NP暴露就检测到了VTG基因的表达量的升高,500mg／kg表达量达到最大值。
综合分析各项实验结果可知,低剂量的NP可刺激中华绒螯蟹产生免疫应激反应,这种应激反应随NP在体内的代谢消除而减弱,并会对中华绒螯蟹的免疫能力产生一定的负效应。高浓度的NP,通过减少中华绒螯蟹血淋巴细胞影响免疫功能。与高等脊椎动物的反应相似,NP对中华绒螯蟹也有雌激素效应。
Chinese mitten crab (Eriocheir sinensis) is a very important economic aquatic animal in our country. With the expansion of the scale in recent years, aquaculture, environment deterioration is serious, leading to increase the crab diseases, a crab breeding caused tremendous economic losses. There are three reasons for crab's diseases:crab's own immunity, pathogens and the quality of water where crabs are living in. Environmental factors play an important role, not only affects the crab's resistance to disease but also affect bloom of the pathogens.
For crustaceans, water pollution which causes the environmental stress is the important reason for the decline of the immunity. The changes of the body or the increase of the prevalence can be markers of environmental stress. The research on crustaceans's environmental stress focuses on nitrogen, temperature, pH value, heavy metals and other environmental factors,
Toxic effects of NP on Eriocheir sinensis have rarely been studied. NP is common in the water, having good chemical stability, and easy to accumulate in the organisms. With the development of the industry, the pollution is increasingly serious. High dose of NP have found in Plenty of the rivers of our country, the living environment of Eriocheir sinensis is threatened. Many studies have shown that NP can be toxic on organisms in many aspects, like the endocrine, reproductive and developmental, cancer, immune and so on. Therefore, it's neccessery to explore the NP stress effect on shrimps and crabs, for providing theoretical guidance to the establishment of appropriate evaluation system.
1. NP exposure to can lead to a significant decline of THC in Chinese mitten crab, and relationship between NP dose and THC was nagative. In the time-effect experiment of 300mg/kg NP, THC were exposed down at the first 3d; while it recovery after 6d, after this day, it arised significantly, and close to the control group on 14d.
2. Low dose (≤100mg/kg) of NP can increase ProPO and PPAF gene expression in hemocyte of Eriocheir sinensis, but above this concentration, the induction effect decreased. The inductive effect on ProPO and PPAF of NP with time-effect relationship. The expression conatant rise after injection 6d, then become decrease.
3. Low dose of NP can increase the expression of crustin mRNA of Eriocheir sinensis. The expression crustin crab which pre-exposure to low doses of NP for 1d, infection with Aeromonas hydrophila, much higher than non-exposure group, but in the group which exposure to NP for 14d the expression of crustin gene is lower the the non-infected group. Show short time-effects of immune stress.
4:The three antioxidant enzymes (SOD, CAT and GPX) are sensitive to NP. low doses can increase their activity. low dosage of NP can increase the expression of antioxidant enzymes, while the higher dosage inhibit it. In addition, we also observed that, the toxicity of NP changes by the exposed time. that is slowly increased at first, then decreased in the process.
5. NP have estrogenic effect in Eriocheir sinensis. Not only to enhance the VTG gene expression level in female carb, but also can induce VTG gene expression in the male crab. VTG gene expression increased can been detected in 10mg/kg dose NP, and in the group of 500mg/kg dosage NP, the expression reach the highest level.
Comprehensive analysis the experimental results we can see that low doses of NP may stimulate the immune stress response in Chinese mitten crab, this stress weakened with the metabolic elimination of NP in the body, and it will have a certain negative effects for the immunity of Eriocheir sinensis. High concentrations of NP, by reducing hemocyte to effect. the immune function of Eriocheir sinensis. Similar to the vertebrates, NP has estrogenic effects on Eriocheir sinensis.
引文
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