半胱胺对瘘管手术后山羊外周血T淋巴细胞增殖的影响及其机理研究
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摘要
为了研究半胱胺对山羊瘘管手术后T淋巴细胞增殖的影响及其机理研究,本文进行了如下试验：
试验一：口服半胱胺盐酸盐对瘘管手术后山羊外周血T淋巴细胞增殖及血清中几项指标的影响
8只本地山羊分为试验组和对照组,分圈饲养,每只羊分别先后施行瘤胃、十二指肠瘘管手术。术前每只山羊每天饲喂精料300g,自由采食花生杆和饮水,瘤胃瘘管安装手术前第3天试验组在精料中添加半胱胺盐酸盐微胶囊45mg/kg·BW·d(CT2000,含半胱胺30%),并持续添加直至试验结束；瘤胃术手后恢复14天,安装十二指肠瘘管,恢复期与手术后饲喂方案同瘤胃手术。分别于瘤胃手术前第3天、术后1、2、3、4、8天,十二指肠手术前、术后1、3、8天颈静脉无菌采血,检测外周血T淋巴细胞转化率。另外采集部分血液,分离血清,检测血清中IL-2和GSH含量。结果显示,对照组瘤胃手术后1天T淋巴细胞转化率下降了60.52%(P<0.05),此后逐渐恢复,但术后8天仍低于术前水平；十二指肠手术后1天T细胞转化率也显著下降(31.73%,P<0.05),于第8天恢复,表明手术应激抑制了外周血T淋巴细胞的增殖功能。在口服半胱胺盐酸盐山羊观察到手术对外周血T淋巴细胞增殖的影响较小,与未施加手术相比,两次手术后的T淋巴细胞转化率均无显著改变,且有逐渐上升的趋势。组间相比,试验组T淋巴细胞转化率在瘤胃手术后1、2、3、4、8天分别比对照组高151.66%、52.07%、30.17%、96.96%和120.04%(P<0.05)。十二指肠手术前和术后1、8天也显著高于对照组148.28%、58.92%、55.36%(P<0.05)。表明半胱胺提高手术后T淋巴细胞的增殖能力,缓解手术造成的细胞免疫抑制。
瘤胃手术后第1天,对照组血清中IL-2含量有下降的趋势(31.54%,P=0.121),在十二指肠手术后也有所下降。而试验组在两次手术后IL-2含量均无明显变化(P=0.984,P=0.776)。组间相比,血清中的IL-2含量在瘤胃手术后第1天和十二指肠手术后第1天,试验组显著高于对照组(P<0.05),在十二指肠手术后第3天,也趋向于高于对照组(P=0.142)。提示手术创伤可引起血清中IL-2含量的下降,口服半胱胺盐酸盐可以阻止手术创伤引起的血清中IL-2含量的下降。
瘤胃手术后第1天,对照组血清中的GSH含量显著下降(35.90%,P<0.05),在十二指肠手术后也有所下降。试验组在瘤胃手术后第1天无明显变化(P=0.439),在十二指肠手术后显著下降(45.35%,P<0.05),与其在术前水平较高有一定的关系,但恢复较快,在术后三天已高于术前水平。组间相比,十二指肠手术后第3天,试验组显著高于对照组14.67%(P<0.05)；在瘤胃手术后第1天、十二指肠手术后第8天有高于对照组的趋势(0.05 试验二：半胱胺影响外周血T淋巴细胞增殖的体外研究
无菌采集山羊新鲜肝素抗凝血,分离得到淋巴细胞。细胞分为对照组(培养液)和半胱胺组(半胱胺40μg/m1)培养后检测其转化率和淋巴细胞内GSH含量。此外,分离的淋巴细胞分为对照组(培养液)和IGF-Ⅰ组(IGF-Ⅰ25ng/ml),培养后检测其转化率。结果显示,半胱胺组T淋巴细胞转化率高于对照组105.24%(P<0.05),同时,半胱胺显著地增加了T淋巴细胞内的GSH含量(225.94%, P<0.05); IGF-Ⅰ组T淋巴细胞转化率增加39.27%(P<0.05)。结果表明半胱胺可以直接刺激T淋巴细胞增殖,还可以通过增加T淋巴细胞内的GSH、或通过IGF-Ⅰ系统而间接促进T淋巴细胞增殖。
综上所述,半胱胺促进手术后山羊外周血T淋巴细胞增殖、增强免疫功能的作用可经多条途径实现,除了直接刺激作用外,半胱胺也可通过GSH、IL-2或IGF-Ⅰ介导而增强细胞免疫功能。
In order to investigate the effect of cysteamine on T lymphocyte proliferation of peripheral blood in pre- and post- fistula operation and its mechanism, the following experiments were carried out:
1 Effecr of cysteamin on T lymphocyte proliferation of peripheral blood and some index in serum in post- fistula operation goats
8 local goats fed with peanut stems, were randomly divided into 2 groups:control and experiment group. Every goat underwent operation of fixing permanent fistula on rumen and duodenum respectively. Concentrate (300g/goat-d) was added to the diet of all goats. Cysteamine (CT2000,45mg/kg-BW-d) added only to the diet of experiment group 3 days before operation on rumen and lasted until the end of the whole experiment. The fresh blood were collected on the third day before operation, on the first, second, third,4th,8th day after operation on rumen, and also on the day before duodenal operation, on the first, third,8th day after operation on duodenum. And the serum was separated to detect the concentration of IL-2 and GSH. The fresh blood samples were used to test the transformation of peripheral blood lymphocyte (TLC)in 30 minutes. The results was that on the first day after the two operations,TLC decreased 60.52% and 31.73% in the control (p<0.05), showed the depression of surgery on proliferation of T lymphocyte of peripheral blood. while there was no obvious change in experiment group yet had the tendency to increase. Between the groups, TLC was significantly higher in experiment group than in control on the first(151.66%), second (52.07%), third (30.17%),4th (96.96%),8th (120.04%)day after rumen operation(p<0.05), and also higher before duodenum operation(148.28%), on the first (58.92%),8th(55.36%) day after duodenum operation(p<0.05), which demonstrated that cysteamine improved TLC after operation and alleviated the cell immune suppression.
The experiments showed that IL-2 concentration in serum has the tendency to decrease (31.54%, P=0.121) on the first day after rumen operation in control. There were no evident change after rumen and duodenum operation (P=0.984, P=0.776) in experiment group. Between the groups, IL-2 concentration in serum were significant higher in experiment group than in control on the first day after rumen and duodenum operation (p<0.05), and had the tendency to increase on the third day after duodenum operation (P=0.142). The results indicated that trauma resulted in reduction of IL-2 concentration in serum and cysteamine could improve it.
The GSH concentration in serum decreased significantly in control (P<0.05) and did not vary apparently in experiment group on the first day after rumen operation. There was evident decrease in experiment group on the first day after duodenum operation (P<0.05), which was related with its higher level before duodenum operation, and returned quickly. Between the groups, it was significant higher in experiment group than in control on the third day after duodenal operation (P<0.05). and had a tendency to increase on the first day after rumen operation and on the 8th day after duodenum operation. The results testified surgery lead to the decrease of antioxidation and cysteamine could improve antioxidation induced by increased GSH concentration in serum.
2 Effect of cysteamine on T lymphocyte proliferation of peripheral blood in vitro
Lymphocytes were separated from peripheral blood of goats and cultured. There were three groups:control, cysteamine (40μg/ml)and IGF-Ⅰ(25ng/ml). Compared with the control group, cysteamine and IGF-Ⅰsignificantly inceased the TLC(105.24%,39.27%, P<0.05), and cysteaming can also significantly increase the GSH concentration in lymphocyte, which indicated that cysteamine can stimulate the TLC directly and indirectly by IGF-Ⅰand improved GSH in lymphocyte.
试验一：口服半胱胺盐酸盐对瘘管手术后山羊外周血T淋巴细胞增殖及血清中几项指标的影响
8只本地山羊分为试验组和对照组,分圈饲养,每只羊分别先后施行瘤胃、十二指肠瘘管手术。术前每只山羊每天饲喂精料300g,自由采食花生杆和饮水,瘤胃瘘管安装手术前第3天试验组在精料中添加半胱胺盐酸盐微胶囊45mg/kg·BW·d(CT2000,含半胱胺30%),并持续添加直至试验结束；瘤胃术手后恢复14天,安装十二指肠瘘管,恢复期与手术后饲喂方案同瘤胃手术。分别于瘤胃手术前第3天、术后1、2、3、4、8天,十二指肠手术前、术后1、3、8天颈静脉无菌采血,检测外周血T淋巴细胞转化率。另外采集部分血液,分离血清,检测血清中IL-2和GSH含量。结果显示,对照组瘤胃手术后1天T淋巴细胞转化率下降了60.52%(P<0.05),此后逐渐恢复,但术后8天仍低于术前水平；十二指肠手术后1天T细胞转化率也显著下降(31.73%,P<0.05),于第8天恢复,表明手术应激抑制了外周血T淋巴细胞的增殖功能。在口服半胱胺盐酸盐山羊观察到手术对外周血T淋巴细胞增殖的影响较小,与未施加手术相比,两次手术后的T淋巴细胞转化率均无显著改变,且有逐渐上升的趋势。组间相比,试验组T淋巴细胞转化率在瘤胃手术后1、2、3、4、8天分别比对照组高151.66%、52.07%、30.17%、96.96%和120.04%(P<0.05)。十二指肠手术前和术后1、8天也显著高于对照组148.28%、58.92%、55.36%(P<0.05)。表明半胱胺提高手术后T淋巴细胞的增殖能力,缓解手术造成的细胞免疫抑制。
瘤胃手术后第1天,对照组血清中IL-2含量有下降的趋势(31.54%,P=0.121),在十二指肠手术后也有所下降。而试验组在两次手术后IL-2含量均无明显变化(P=0.984,P=0.776)。组间相比,血清中的IL-2含量在瘤胃手术后第1天和十二指肠手术后第1天,试验组显著高于对照组(P<0.05),在十二指肠手术后第3天,也趋向于高于对照组(P=0.142)。提示手术创伤可引起血清中IL-2含量的下降,口服半胱胺盐酸盐可以阻止手术创伤引起的血清中IL-2含量的下降。
瘤胃手术后第1天,对照组血清中的GSH含量显著下降(35.90%,P<0.05),在十二指肠手术后也有所下降。试验组在瘤胃手术后第1天无明显变化(P=0.439),在十二指肠手术后显著下降(45.35%,P<0.05),与其在术前水平较高有一定的关系,但恢复较快,在术后三天已高于术前水平。组间相比,十二指肠手术后第3天,试验组显著高于对照组14.67%(P<0.05)；在瘤胃手术后第1天、十二指肠手术后第8天有高于对照组的趋势(0.05
无菌采集山羊新鲜肝素抗凝血,分离得到淋巴细胞。细胞分为对照组(培养液)和半胱胺组(半胱胺40μg/m1)培养后检测其转化率和淋巴细胞内GSH含量。此外,分离的淋巴细胞分为对照组(培养液)和IGF-Ⅰ组(IGF-Ⅰ25ng/ml),培养后检测其转化率。结果显示,半胱胺组T淋巴细胞转化率高于对照组105.24%(P<0.05),同时,半胱胺显著地增加了T淋巴细胞内的GSH含量(225.94%, P<0.05); IGF-Ⅰ组T淋巴细胞转化率增加39.27%(P<0.05)。结果表明半胱胺可以直接刺激T淋巴细胞增殖,还可以通过增加T淋巴细胞内的GSH、或通过IGF-Ⅰ系统而间接促进T淋巴细胞增殖。
综上所述,半胱胺促进手术后山羊外周血T淋巴细胞增殖、增强免疫功能的作用可经多条途径实现,除了直接刺激作用外,半胱胺也可通过GSH、IL-2或IGF-Ⅰ介导而增强细胞免疫功能。
In order to investigate the effect of cysteamine on T lymphocyte proliferation of peripheral blood in pre- and post- fistula operation and its mechanism, the following experiments were carried out:
1 Effecr of cysteamin on T lymphocyte proliferation of peripheral blood and some index in serum in post- fistula operation goats
8 local goats fed with peanut stems, were randomly divided into 2 groups:control and experiment group. Every goat underwent operation of fixing permanent fistula on rumen and duodenum respectively. Concentrate (300g/goat-d) was added to the diet of all goats. Cysteamine (CT2000,45mg/kg-BW-d) added only to the diet of experiment group 3 days before operation on rumen and lasted until the end of the whole experiment. The fresh blood were collected on the third day before operation, on the first, second, third,4th,8th day after operation on rumen, and also on the day before duodenal operation, on the first, third,8th day after operation on duodenum. And the serum was separated to detect the concentration of IL-2 and GSH. The fresh blood samples were used to test the transformation of peripheral blood lymphocyte (TLC)in 30 minutes. The results was that on the first day after the two operations,TLC decreased 60.52% and 31.73% in the control (p<0.05), showed the depression of surgery on proliferation of T lymphocyte of peripheral blood. while there was no obvious change in experiment group yet had the tendency to increase. Between the groups, TLC was significantly higher in experiment group than in control on the first(151.66%), second (52.07%), third (30.17%),4th (96.96%),8th (120.04%)day after rumen operation(p<0.05), and also higher before duodenum operation(148.28%), on the first (58.92%),8th(55.36%) day after duodenum operation(p<0.05), which demonstrated that cysteamine improved TLC after operation and alleviated the cell immune suppression.
The experiments showed that IL-2 concentration in serum has the tendency to decrease (31.54%, P=0.121) on the first day after rumen operation in control. There were no evident change after rumen and duodenum operation (P=0.984, P=0.776) in experiment group. Between the groups, IL-2 concentration in serum were significant higher in experiment group than in control on the first day after rumen and duodenum operation (p<0.05), and had the tendency to increase on the third day after duodenum operation (P=0.142). The results indicated that trauma resulted in reduction of IL-2 concentration in serum and cysteamine could improve it.
The GSH concentration in serum decreased significantly in control (P<0.05) and did not vary apparently in experiment group on the first day after rumen operation. There was evident decrease in experiment group on the first day after duodenum operation (P<0.05), which was related with its higher level before duodenum operation, and returned quickly. Between the groups, it was significant higher in experiment group than in control on the third day after duodenal operation (P<0.05). and had a tendency to increase on the first day after rumen operation and on the 8th day after duodenum operation. The results testified surgery lead to the decrease of antioxidation and cysteamine could improve antioxidation induced by increased GSH concentration in serum.
2 Effect of cysteamine on T lymphocyte proliferation of peripheral blood in vitro
Lymphocytes were separated from peripheral blood of goats and cultured. There were three groups:control, cysteamine (40μg/ml)and IGF-Ⅰ(25ng/ml). Compared with the control group, cysteamine and IGF-Ⅰsignificantly inceased the TLC(105.24%,39.27%, P<0.05), and cysteaming can also significantly increase the GSH concentration in lymphocyte, which indicated that cysteamine can stimulate the TLC directly and indirectly by IGF-Ⅰand improved GSH in lymphocyte.
引文
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