胰高血糖素样肽-2对28日龄断奶仔猪肠上皮细胞完整性及其增殖的影响
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摘要
本试验以原代培养的28日龄断奶仔猪小肠上皮细胞为研究模型,研究胰高血糖素样肽-2(GLP-2)对断奶仔猪肠上皮细胞增殖及其功能的影响。研究共包括两个试验:试验一、28日龄断奶仔猪肠道上皮细胞的原代培养及生长规律;试验二、胰高血糖素样肽-2对28日龄断奶仔猪肠道上皮细胞增殖和功能的影响。
试验一共设6个处理(1-1、1-2、1-3、1-4、1-5、1-6),每个处理设8个重复,每个重复1孔,在37℃,5%CO_2条件下培养144小时,在培养的第48h、72h、96h、120h、144h和168h分别测定细胞的MTT OD值,同时用倒置显微镜观察细胞数量和形态,并摄影记录。结果表明:28日龄断奶仔猪小肠上皮细胞体外接种48h贴壁生长,在细胞接种的48h至72h,细胞大量增殖,在72h至96h,细胞增殖减缓,到96h时达到最大值,在接种120-168h,细胞增殖减少和凋亡增加,细胞数量逐渐减少。
试验结果表明:要确定某一物质对断奶仔猪IEC的作用和其它定量测试最佳时间应该在细胞接种的48-96h。
试验二共设6个处理(2-1、2-2、2-3、2-4、2-5、2-6),每个处理设16个重复,每个重复1孔,在37℃,5%CO_2条件下培养48h后分别换为含GLP-2浓度为0、10~(-11)、10~(-10)、10~(-9)、10~(-8)和10~(-7)mol/l的培养液,继续培养48h,收集培养液上清和细胞裂解液进行指标测定,并分别在接种后48、72、和95h利用倒置显微镜观察细胞的形态和生长情况。
结果如下:在细胞培养液中加入GLP-2处理48h,各处理组细胞数量显著(P＜0.05)或极显著(P＜0.01)多于对照组,MTT OD值均极显著高于对照组(P＜0.01)。各处理组培养液乳酸含量、总蛋白含量、蛋白沉积量和Na~+,K~+-ATP酶活力都显著(P＜0.05)或极显著(P＜0.01)高于对照组。此外,各处理组胞外碱性磷酸酶、乳酸脱氢酶和肌酸激酶活力均极显著低于对照组(P＜0.01)。
综上试验结果表明,GLP-2可以促进断奶仔猪小肠上皮细胞的增殖,增加断奶仔猪小肠上皮细胞蛋白沉积,减轻断奶仔猪小肠上皮细胞受损伤的程度,维持细胞膜结构和功能的完整性,其作用效果与GLP-2浓度存在剂量依赖效应,这种剂量依赖效应具有明显的阶段性,GLP-2作用于断奶仔猪小肠上皮细胞的最佳浓度范围可能在1×10~(-9)与1×10~(-7)mol/l之间。
28-day-old weaned piglet intestinal epithelial cells in primary culture were selected to evaluate the effects of Glucagon-like peptide-2 on the proliferation and function of weaned piglet intestinal-epithelial cells.Firstly,the growth characteriztion of weaned piglet intestinal epithelial cells was investigated.The quantity and morphous of cells was observed by invert microscope.in the 48h、72h、96h、120h、144h and 168h,at the same time, the cell MTT absorption at 490nm wavelength was observed.
Secondly,the alkaling phosphatase activities,creatine phoshhokinase activites,lactic acid dehydrogenase activites,lactic acid content,protein retention and total protein content were investigation when the weaned piglet intestinal epithelial cells was growing 96h in medium of containing 0,1×10~(-11),1×10-~(10),1×10~(-9),1×10~(-8) and 1×10~(-7)mol/L Glucagon-like peptide-2 respectively.
The results shows:1.the quantity of cell increased from 48h to 96h after cell inoculation and followed decreased;2.After addition of GLP-2 96h,the quantity of cell is significant or extremely significant more than the control group(P＜0.05),the MTT OD value is extremely significant more than the control group.Compare with the control group, the Lactic Acid concentration,total protein content,protein retention and Na~+,K~+-ATP enzyme activity significant or extremely significant increased in each GLP-2 groups(P＜0.05). the alkaling phosphatase activity,creatine phoshhokinase activity and lactic acid dehydrogenase activites in culture media extremely significant decreased(P＜0.01)
The results indicated GLP-2 can encourage small intestine enterocyte of 28-day-old weaned pig cell proliferation,increase small intestine enterocyte of 28-day-old weaned pig cell protein retention,degrade the impaired degree of 28-day-old weaned pig cell,keep cell appearance and function Integrity.These effects is dose-dependent effects,and it has evident stage characteristic,the best density of GLP-2 on the proliferation and function of weaned piglet Intestinal-epithelial cells maybe between 1×10~(-9) and 1×10~(-7)mol/L.
试验一共设6个处理(1-1、1-2、1-3、1-4、1-5、1-6),每个处理设8个重复,每个重复1孔,在37℃,5%CO_2条件下培养144小时,在培养的第48h、72h、96h、120h、144h和168h分别测定细胞的MTT OD值,同时用倒置显微镜观察细胞数量和形态,并摄影记录。结果表明:28日龄断奶仔猪小肠上皮细胞体外接种48h贴壁生长,在细胞接种的48h至72h,细胞大量增殖,在72h至96h,细胞增殖减缓,到96h时达到最大值,在接种120-168h,细胞增殖减少和凋亡增加,细胞数量逐渐减少。
试验结果表明:要确定某一物质对断奶仔猪IEC的作用和其它定量测试最佳时间应该在细胞接种的48-96h。
试验二共设6个处理(2-1、2-2、2-3、2-4、2-5、2-6),每个处理设16个重复,每个重复1孔,在37℃,5%CO_2条件下培养48h后分别换为含GLP-2浓度为0、10~(-11)、10~(-10)、10~(-9)、10~(-8)和10~(-7)mol/l的培养液,继续培养48h,收集培养液上清和细胞裂解液进行指标测定,并分别在接种后48、72、和95h利用倒置显微镜观察细胞的形态和生长情况。
结果如下:在细胞培养液中加入GLP-2处理48h,各处理组细胞数量显著(P＜0.05)或极显著(P＜0.01)多于对照组,MTT OD值均极显著高于对照组(P＜0.01)。各处理组培养液乳酸含量、总蛋白含量、蛋白沉积量和Na~+,K~+-ATP酶活力都显著(P＜0.05)或极显著(P＜0.01)高于对照组。此外,各处理组胞外碱性磷酸酶、乳酸脱氢酶和肌酸激酶活力均极显著低于对照组(P＜0.01)。
综上试验结果表明,GLP-2可以促进断奶仔猪小肠上皮细胞的增殖,增加断奶仔猪小肠上皮细胞蛋白沉积,减轻断奶仔猪小肠上皮细胞受损伤的程度,维持细胞膜结构和功能的完整性,其作用效果与GLP-2浓度存在剂量依赖效应,这种剂量依赖效应具有明显的阶段性,GLP-2作用于断奶仔猪小肠上皮细胞的最佳浓度范围可能在1×10~(-9)与1×10~(-7)mol/l之间。
28-day-old weaned piglet intestinal epithelial cells in primary culture were selected to evaluate the effects of Glucagon-like peptide-2 on the proliferation and function of weaned piglet intestinal-epithelial cells.Firstly,the growth characteriztion of weaned piglet intestinal epithelial cells was investigated.The quantity and morphous of cells was observed by invert microscope.in the 48h、72h、96h、120h、144h and 168h,at the same time, the cell MTT absorption at 490nm wavelength was observed.
Secondly,the alkaling phosphatase activities,creatine phoshhokinase activites,lactic acid dehydrogenase activites,lactic acid content,protein retention and total protein content were investigation when the weaned piglet intestinal epithelial cells was growing 96h in medium of containing 0,1×10~(-11),1×10-~(10),1×10~(-9),1×10~(-8) and 1×10~(-7)mol/L Glucagon-like peptide-2 respectively.
The results shows:1.the quantity of cell increased from 48h to 96h after cell inoculation and followed decreased;2.After addition of GLP-2 96h,the quantity of cell is significant or extremely significant more than the control group(P＜0.05),the MTT OD value is extremely significant more than the control group.Compare with the control group, the Lactic Acid concentration,total protein content,protein retention and Na~+,K~+-ATP enzyme activity significant or extremely significant increased in each GLP-2 groups(P＜0.05). the alkaling phosphatase activity,creatine phoshhokinase activity and lactic acid dehydrogenase activites in culture media extremely significant decreased(P＜0.01)
The results indicated GLP-2 can encourage small intestine enterocyte of 28-day-old weaned pig cell proliferation,increase small intestine enterocyte of 28-day-old weaned pig cell protein retention,degrade the impaired degree of 28-day-old weaned pig cell,keep cell appearance and function Integrity.These effects is dose-dependent effects,and it has evident stage characteristic,the best density of GLP-2 on the proliferation and function of weaned piglet Intestinal-epithelial cells maybe between 1×10~(-9) and 1×10~(-7)mol/L.
引文
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