中华眼镜蛇毒对大鼠炎性细胞因子影响的研究
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摘要
目的：通过腹腔内注射中华眼镜蛇毒(CCV)建立眼镜蛇伤大鼠模型,以检测血清及心肌组织中肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)的表达,从而探讨炎性细胞因子在CCV所致大鼠眼镜蛇伤中的致炎机制以及心肌损伤的机制。
方法：60只健康清洁级Sprague-Dawley (SD)大鼠在广西医科大学动物实验中心饲养3天后开始实验。SD大鼠被随机(随机数字法)等分为四组,即：对照组、染毒1h组、染毒3h组及染毒6h组。将CCV冻干粉按lmg/m1与生理盐水配成蛇毒溶液,染毒各组大鼠予以腹腔内注射1.272mg/kg CCV溶液；对照组大鼠给予等剂量生理盐水溶液腹腔内注射。
(1)染毒后,连续观察各组大鼠中毒表现直至实验终止；
(2)染毒各组大鼠于染毒后相应时间点即染毒1h、3h、6h行腹主动脉采血2.0 ml,对照组大鼠于1h后同法采血,标本在室温下静置20分钟后离心(3000r/min,10min),收集血清,采用酶联免疫吸附法(ELISA法)测定血清TNF-a和IL-6抗原含量,以探讨炎性细胞因子TNF-αIL-6在CCV所致大鼠炎症反应中的致炎机制；
(3)同时,染毒各组大鼠于染毒后各相应时间点切取心肌组织,行HE染色；对照组大鼠于观察1h后同法切取心肌组织,通过光学显微镜观察心肌组织结构的变化；
(4)按上述方法,染毒各组和对照组分别切取心肌组织,采用免疫组织化学方法检测心肌组织中TNF-α及工L-6的表达,进行图像分析并计算其平均灰度值,研究染毒后大鼠心肌组织中炎性细胞因子TNF-α和IL-6的表达变化。
结果：
(1)腹腔内注射CCV溶液后,染毒各组大鼠逐渐出现中毒反应,而对照组大鼠在整个实验观察期间未出现明显的中毒症状；
(2)与对照组比较,大鼠血清TNF-a、IL-6抗原含量在染毒各组中表达增高：血清TNF-α含量在染毒1h组开始增加,染毒6h组明显增加；血清工L-6含量在染毒1h组增加不明显,染毒3h组开始增加,染毒6h组增加明显；
(3)光学显微镜下观察染毒6h组大鼠心肌结构出现明显的病理改变,主要表现为心肌纤维出现部分断裂、肿胀,间质水肿,出现炎性细胞的浸润；对照组大鼠心肌结构无明显改变。
(4)本实验结果显示,与对照组比较,染毒1、3、6h大鼠心肌细胞内可见明显TNF-α和工L-6阳性表达细胞,呈棕黄色颗粒状,分布于细胞浆内,且阳性细胞平均灰度值明显降低。结论：
(1)本研究采用腹腔内注射CCV溶液的方法建造眼镜蛇伤大鼠模型,在造模后染毒各组大鼠均出现相应的中毒表现以及心肌组织结构的变化,表明大鼠染毒模型建造成功。
(2)染毒各组大鼠血清TNF-α和工L-6抗原含量明显增加,表明炎性细胞因子在CCV所致大鼠眼镜蛇伤炎症反应发生机制中有促炎作用。
(3)本研究表明CCV能够使大鼠心肌组织中TNF-α和工L-6表达明显增加以及心肌组织结构形态发生改变,提示TNF-α、IL-6在大鼠眼镜蛇伤心肌损伤发生机制中可能起到重要作用。
Objective:This study was to establish rat model by intraperitoneal injection of Chinese cobra venom (CCV) and observe the mechanism of inflammation and acute myocardial injury by tumor necrosis factor alpha (TNF-a) and interleukin-6 (IL-6).
Methods:Sixty health Sprague-Dawley (SD) rats fed for three days in Guangxi medical university experiment room were randomly (random number) divided into four groups, including the control group, one hour group, three hours group and six hours group after administration by intraperitoneal injection of CCV. The lyophilized CCV paired with saline solution 1mg/ml, and then CCV was injected to every experimental group, by a dose of 1.272mg/kg (dissolved by normal sodium). According to the above methods, the control group controlled with the same doses of normal sodium accordingly.
(1) To investigate the performance of every group rats by continuous after injection.
(2) According to the mentioned above methods, two milliliters abdominal aorta blood were collected to measure serum TNF-a and IL-6 in every experiment group after administration by intraperitoneal injection of CCV. The control group was collected the same dose of sample after one hour. Levels of TNF-a and IL-6 were determined by enzyme-linked immune-absorbent assay (ELISA), to study the inflammatory mechanism of cytokine caused by CCV.
(3) After injection, every experiment group was sacrificed at every time point, the blocks of the myocardial tissue from the middle portion of the left ventricular free wall were collected and fixed in the 4% paraformaldehyde solution. Then the injection of the myocardial tissue was observed by light microscopy.
(4) According to the mentioned above way, the changes of TNF-a and IL-6 in rat myocardial cells were observed with immunohistochemistry assay and the images were analysed.
Results:(1) After administration by intraperitoneal injection of CCV, the symptoms of poisoning were markedly emerged in every expeiment group, but during the entire time, but there was no toxic manifestation in the control group.
(2) One hour after injection of CCV, the levels of serum TNF-a rapidly increased, the markedly levels appeared 6-hour later injection; Significantly higher levels of serum IL-6 in the experiment groups as compared with the control group were shown on 3 hours group and 6 hours group but not on 1 hour group.
(3) In every experiment group, the morphologic changes of myocardial tissue injection was obviously observed with light microscopy. These changes were significally shown on the 6 hours group but not on the 1 hour group,3 hours group and the control group.
(4) Compared with the control group, we observed a great quantity of TNF-a and IL-6 positive cell among the myocardial cells of the rats, the average gray was decreased significantly on injecting CCV groups, there was much brown positive cell in the cytoplasm.
Conclusion:(1) We successfully established rat model of snakebite by CCV and the symptoms of poisoning and morphologic changes of the myocardial tissue were observed after administration by intraperitoneal injection of CCV in rats.
(2) CCV could increase the serum contents of TNF-a and IL-6 in rat and play an important role in the inflammatory mechanism.
(3) TNF-a and IL-6 may play an important role in CCV induced acute myocardial injury in rats.
方法：60只健康清洁级Sprague-Dawley (SD)大鼠在广西医科大学动物实验中心饲养3天后开始实验。SD大鼠被随机(随机数字法)等分为四组,即：对照组、染毒1h组、染毒3h组及染毒6h组。将CCV冻干粉按lmg/m1与生理盐水配成蛇毒溶液,染毒各组大鼠予以腹腔内注射1.272mg/kg CCV溶液；对照组大鼠给予等剂量生理盐水溶液腹腔内注射。
(1)染毒后,连续观察各组大鼠中毒表现直至实验终止；
(2)染毒各组大鼠于染毒后相应时间点即染毒1h、3h、6h行腹主动脉采血2.0 ml,对照组大鼠于1h后同法采血,标本在室温下静置20分钟后离心(3000r/min,10min),收集血清,采用酶联免疫吸附法(ELISA法)测定血清TNF-a和IL-6抗原含量,以探讨炎性细胞因子TNF-αIL-6在CCV所致大鼠炎症反应中的致炎机制；
(3)同时,染毒各组大鼠于染毒后各相应时间点切取心肌组织,行HE染色；对照组大鼠于观察1h后同法切取心肌组织,通过光学显微镜观察心肌组织结构的变化；
(4)按上述方法,染毒各组和对照组分别切取心肌组织,采用免疫组织化学方法检测心肌组织中TNF-α及工L-6的表达,进行图像分析并计算其平均灰度值,研究染毒后大鼠心肌组织中炎性细胞因子TNF-α和IL-6的表达变化。
结果：
(1)腹腔内注射CCV溶液后,染毒各组大鼠逐渐出现中毒反应,而对照组大鼠在整个实验观察期间未出现明显的中毒症状；
(2)与对照组比较,大鼠血清TNF-a、IL-6抗原含量在染毒各组中表达增高：血清TNF-α含量在染毒1h组开始增加,染毒6h组明显增加；血清工L-6含量在染毒1h组增加不明显,染毒3h组开始增加,染毒6h组增加明显；
(3)光学显微镜下观察染毒6h组大鼠心肌结构出现明显的病理改变,主要表现为心肌纤维出现部分断裂、肿胀,间质水肿,出现炎性细胞的浸润；对照组大鼠心肌结构无明显改变。
(4)本实验结果显示,与对照组比较,染毒1、3、6h大鼠心肌细胞内可见明显TNF-α和工L-6阳性表达细胞,呈棕黄色颗粒状,分布于细胞浆内,且阳性细胞平均灰度值明显降低。结论：
(1)本研究采用腹腔内注射CCV溶液的方法建造眼镜蛇伤大鼠模型,在造模后染毒各组大鼠均出现相应的中毒表现以及心肌组织结构的变化,表明大鼠染毒模型建造成功。
(2)染毒各组大鼠血清TNF-α和工L-6抗原含量明显增加,表明炎性细胞因子在CCV所致大鼠眼镜蛇伤炎症反应发生机制中有促炎作用。
(3)本研究表明CCV能够使大鼠心肌组织中TNF-α和工L-6表达明显增加以及心肌组织结构形态发生改变,提示TNF-α、IL-6在大鼠眼镜蛇伤心肌损伤发生机制中可能起到重要作用。
Objective:This study was to establish rat model by intraperitoneal injection of Chinese cobra venom (CCV) and observe the mechanism of inflammation and acute myocardial injury by tumor necrosis factor alpha (TNF-a) and interleukin-6 (IL-6).
Methods:Sixty health Sprague-Dawley (SD) rats fed for three days in Guangxi medical university experiment room were randomly (random number) divided into four groups, including the control group, one hour group, three hours group and six hours group after administration by intraperitoneal injection of CCV. The lyophilized CCV paired with saline solution 1mg/ml, and then CCV was injected to every experimental group, by a dose of 1.272mg/kg (dissolved by normal sodium). According to the above methods, the control group controlled with the same doses of normal sodium accordingly.
(1) To investigate the performance of every group rats by continuous after injection.
(2) According to the mentioned above methods, two milliliters abdominal aorta blood were collected to measure serum TNF-a and IL-6 in every experiment group after administration by intraperitoneal injection of CCV. The control group was collected the same dose of sample after one hour. Levels of TNF-a and IL-6 were determined by enzyme-linked immune-absorbent assay (ELISA), to study the inflammatory mechanism of cytokine caused by CCV.
(3) After injection, every experiment group was sacrificed at every time point, the blocks of the myocardial tissue from the middle portion of the left ventricular free wall were collected and fixed in the 4% paraformaldehyde solution. Then the injection of the myocardial tissue was observed by light microscopy.
(4) According to the mentioned above way, the changes of TNF-a and IL-6 in rat myocardial cells were observed with immunohistochemistry assay and the images were analysed.
Results:(1) After administration by intraperitoneal injection of CCV, the symptoms of poisoning were markedly emerged in every expeiment group, but during the entire time, but there was no toxic manifestation in the control group.
(2) One hour after injection of CCV, the levels of serum TNF-a rapidly increased, the markedly levels appeared 6-hour later injection; Significantly higher levels of serum IL-6 in the experiment groups as compared with the control group were shown on 3 hours group and 6 hours group but not on 1 hour group.
(3) In every experiment group, the morphologic changes of myocardial tissue injection was obviously observed with light microscopy. These changes were significally shown on the 6 hours group but not on the 1 hour group,3 hours group and the control group.
(4) Compared with the control group, we observed a great quantity of TNF-a and IL-6 positive cell among the myocardial cells of the rats, the average gray was decreased significantly on injecting CCV groups, there was much brown positive cell in the cytoplasm.
Conclusion:(1) We successfully established rat model of snakebite by CCV and the symptoms of poisoning and morphologic changes of the myocardial tissue were observed after administration by intraperitoneal injection of CCV in rats.
(2) CCV could increase the serum contents of TNF-a and IL-6 in rat and play an important role in the inflammatory mechanism.
(3) TNF-a and IL-6 may play an important role in CCV induced acute myocardial injury in rats.
引文
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[6]龚非力.医学免疫学(第二版)[M].北京：科学出版社,2006,61-84.
[7]曾令铝,陈熹,杨威,等.中华眼镜蛇咬伤大鼠模型多项电生理指标的动态观察[J].蛇志,2007,19(2)：89-93.
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