【作者】 贾刚；

【导师】 王康宁；

【作者基本信息】 四川农业大学 ， 动物营养与饲料科学， 2009， 博士

【摘要】 胰高血糖素样肽-2（Glucagon-like Peptide-2,GLP-2）通过促进小鼠、大鼠、胎猪、新生仔猪等动物的肠上皮细胞增殖、抑制细胞凋亡而影响肠黏膜发育、调节肠道营养、免疫功能已得到众多营养学家的认同,但能否影响断奶仔猪的肠道适应,对具有快速更新特点的断奶仔猪肠上皮细胞的作用效果尚不得而知。为回答这个问题,本研究采用以下几个实验来探讨GLP-2与断奶仔猪肠道发育的关系及对肠上皮细胞增殖、代谢、凋亡的作用特点,为进一步直接采用动物试验研究GLP-2对仔猪断奶应激综合征的调节及作用机理提供理论和试验依据。试验1不同基因型仔猪断奶前后GLP-2的分泌规律及与肠道发育的关系本试验通过考察太湖猪、大×太二杂猪和长白仔猪断奶前后GLP-2的分泌规律、肠道发育状况的差异,初步探讨具有不同生长速度仔猪的GLP-2分泌水平的高低及与肠道发育的关系。试验选择1日龄健康正常的太湖猪、大×太二杂猪和长白猪各20头,将每个不同基因型猪作为一个处理,每个处理5个重复,每个重复4头猪,仔猪饲养于环控实验室,25日龄断奶,分别于仔猪24、26和30日龄时每个重复选取一头接近平均体重的仔猪前腔静脉采血测定GLP-2浓度,并于30日龄时屠宰仔猪测定肠道发育参数,并分析30日龄小肠绒毛高度与24日龄、26日龄、30日龄血清GLP-2浓度的关系。试验结果如下:仔猪30日龄时,大×太猪小肠长、小肠相对长、小肠相对重都显著高于长白猪（P＜0.05）,太湖猪小肠相对重显著高于长白猪（P＜0.05）,而小肠相对长极显著高于长白猪（P＜0.01）;30同龄时,大×太猪小肠绒毛高度高于长白猪和太湖猪（P＞0.05）,且血清GLP-2含量极显著高于长白猪和太湖猪（P＜0.01）;24日龄和26日龄时,大×太猪血清GLP-2含量极显著高于太湖猪（P＜0.01）;26日龄时,大×太猪血清GLP-2含量显著高于长白猪（P＜0.05）;30日龄小肠绒毛高度与各猪种血清GLP-2含量达极显著相关水平（P＜0.01）。研究结果表明,不同基因型仔猪断奶后GLP-2的活性逐渐升高,大×太猪GLP-2变化幅度最大,30日龄时其活性最高,肠道发育状况优于太湖猪和长白猪;三种猪30日龄血清GLP-2含量与30日龄小肠绒毛高度极显著相关。研究结果提示我们,选择大×太猪或其肠上皮细胞作为研究GLP-2调节仔猪断奶前后的肠道适应及可能的作用机理是可行的。在证实不同基因型仔猪断奶前后GLP-2的分泌水平及活性逐渐升高,而仔猪肠绒毛高度与循环中GLP-2浓度高度相关的基础上,课题组进一步研究了GLP-2对大×太断奶仔猪肠黏膜上皮细胞的营养效应（试验2）,研究发现GLP-2可以剂量依赖性地促进体外培养的28d断奶仔猪小肠黏膜上皮细胞增殖,抑制细胞凋亡,维持细胞形态和功能的完整性,课题组随即进行了下列研究。试验3 GLP-2和DPP-Ⅳ抑制剂联合使用对对断奶仔猪肠上皮细胞的影响本试验旨在研究不同浓度的胰高血糖素样肽-2（GLP-2）和二肽基肽酶Ⅳ（DPP-Ⅳ）抑制剂（KR62436）联合使用对体外原代培养的28日龄断奶仔猪肠上皮细胞增殖、代谢及相关酶活力的影响。试验以体外培养的28日龄断奶仔猪回肠上皮细胞作为模型,首先采用单因子试验设计探讨不同浓度的KR62436对细胞增殖及代谢的影响;然后采用2×3因子设计,考察添加不同浓度hGLP-2(1×10^-10、1×10^-9、1×10^-8 mol/L)和KR62436(0和1×10^-11mol/L)对细胞增殖、代谢及凋亡的影响,细胞培养时间共计144h。结果如下:单独添加KR62436,在倒置显微镜下观察发现细胞无明显变化,细胞数量虽有上升的趋势,但细胞MTT OD值、细胞沉积蛋白量、细胞总蛋白量呈现先增加后下降趋势（P＞0.05）,胞外LDH、CK活力在KR62436低剂量时呈降低趋势（P＞0.05）,但当其浓度较高时,细胞MTT OD值显著低于对照组（P＜0.05）,其胞外LDH、CK活力显著高于对照组（P＜0.05）,Na⁺,K⁺-ATP酶活力变化不明显;在培养液中同时添加KR62436和hGLP-2,随着hGLP-2浓度的增加,细胞数量极显著上升（P＜0.01）,MTT OD值极显著增加（P＜0.01）,细胞蛋白沉积量极显著增加（P＜0.01）,细胞总蛋白含量极显著增加（P＜0.01）,胞外LDH、CK活力显著降低（P＜0.05）,Na⁺,K⁺-ATP酶活力极显著增加（P＜0.01）。试验结果提示我们:低浓度DPP-Ⅳ抑制剂对细胞生长的影响不显著,高浓度抑制剂虽能促进仔猪肠上皮细胞的增殖,但对细胞的代谢和完整性有负面效应;在培养液中添加DPP-Ⅳ抑制剂能够增强外源GLP-2对细胞增殖、代谢和细胞完整性的影响,两者具有明显的协同效应。试验结果提示我们应用KR62436来提高GLP-2的作用效果要慎重。试验4 GLP-2对β-伴球蛋白损伤的28日龄断奶仔猪肠上皮细胞的保护、修复作用本试验主要考察GLP-2对28日龄断奶的大×太仔猪的肠上皮细胞被β-伴球蛋白损伤后存活、增殖、代谢、凋（死）亡的影响及可能的作用机理。试验首先采用单因子设计,分别用1.2mg/ml和2.4mg/ml的β-伴球蛋白对原代培养的断奶仔猪肠上皮细胞进行攻毒,通过考察对细胞存活、增殖、代谢及凋亡的影响而建立细胞损伤模型;然后再采用2×3因子设计,考察添加不同浓度的hGLP-2(1×10^-9、1×10^-8、1×10^-7mol/l)对致敏的培养细胞的影响。试验结果如下:使用β-伴球蛋白提取物攻毒,细胞MTTOD值显著降低（P＜0.05）,细胞蛋白质沉积量和细胞消耗蛋白量极显著降低（P＜0.01）,胞外LDH活力极显著增加（P＜0.01）,Na⁺,K⁺-ATP酶活力显著（P＜0.05）或者极显著（P＜0.01）降低,Caspase-3酶活力极显著（P＜0.01）升高;使用β-伴球蛋白攻毒的同时添加不同浓度的hGLP-2,细胞MTT OD值、细胞蛋白质沉积量、细胞消耗蛋白量和Na⁺,K⁺-ATP酶活力均显著或极显著（P＜0.05或0.01）升高,且随着hGLP-2浓度的增加而升高,而胞外LDH活力则随着hGLP-2浓度的增加而逐渐下降（P＜0.05或0.01）,Caspase-3酶活力极显著（P＜0.01）降低。研究结果表明β-伴球蛋白对断奶仔猪小肠上皮细胞的增殖和细胞完整性有不利影响,而GLP-2能够减轻或者避免β-伴球蛋白对断奶仔猪小肠上皮细胞的增殖和细胞完整性的不利影响,这种效应可能是通过调节细胞Caspase-3的活力而实现的。综上所述,GLP-2可影响断奶仔猪肠粘膜的发育,仔猪肠绒毛高度与GLP-2的分泌变化高度相关;GLP-2在体外可促进断奶仔猪肠上皮细胞增殖,抑制细胞凋（死）亡,这种效应具有明显的剂量依赖关系;使用DPP-Ⅳ抑制剂（KR62436）可以进一步提高GLP-2的作用效果,两者具有明显的协同效应,但单独使用KR62436可能会造成细胞完整性损伤;β-伴球蛋白对断奶仔猪小肠上皮细胞的增殖和细胞完整性有不利影响,而GLP-2能够通过细胞保护作用减轻或者避免β-伴球蛋白对断奶仔猪小肠上皮细胞的增殖和细胞凋亡的不利影响。研究结果为我们进一步探讨GLP-2对断奶仔猪的肠道适应的直接影响及相应的作用机理提高了有力支持,为使用GLP-2调节仔猪断奶应激提供了一个新思路。更多还原

【Abstract】 Although most nutritionists acknowledge that Glucagon-like Peptide-2 affects intestinal mucosa development,regulates small intestinal adaptation and immune function by promoting proliferation and inhibiting apoptosis of intestinal enterocyte cells such as mice,rats,fetal pigs and neonatal piglets,it is not clear yet whether GLP-2 can regulate the intestinal adaptation of weaned piglets or not and what are the operative properties of GLP-2 function on the intestinal enterocyte cells of weaned piglets with the features of rapid renewal.To anwer this question,the study used 4 series experiments to explore the effects of GLP-2 on the intestinal growth of weaned piglets and the operative properties of GLP-2 function on proliferation,metabolism and apoptosis of weaned piglets’ intestinal enterocyte cells,which will provide theoretical and experimental basis for further studying regulation and mechanism of GLP-2 on weanling stress by using weaned piglets in situ.Exp.1 The relationship between GLP-2’s secretion and intestinal growth of different genotypes pigletsThe experiment preliminarily studied the relationship between GLP-2’s secretion and intestinal growth of piglets with different growth rate by studying GLP-2 secretion rule and intestinal growth parameters of piglets with different genotypes during weaning.1-d old healthy piglets（20 MeiShan Pigs,20 Yorkshire×MeiShan Pigs and 20 Landraee）, were assigned to three treatments with five replicates and four piglets per replicate according to Single-factor design principle.The piglets were fed in environment control laboratory and weaned on the 25^th day.Blood samples from 24-d,26-d and 30-d old piglets close to average weight in each replicate were collected through precaval vein for the determination of GLP-2 concentration.30-d old piglets were slaughtered for measuring intestinal growth parameters and investigating the relationship between the height of intestinal villis of 30-d piglets and serum GLP-2 concentrations of 24-d,26-d and 30-d old piglets.The results were as follows:at 30-d old,the length and the intestines length index of the Yorkshire×MeiShan Pig were longer than that of Landrace（P＜0.05） and the intestines weight index also outweighed that of Landraee（P＜0.05）;the intestines weight index of the MeiShan Pig outweighed that of Landrace（P＜0.05） and the intestines length index was also longer than that of Landrace（P＜0.01）.In addition,at 30-d old,the height of intestinal villis of Yorkshire×MeiShan Pigs was highter than that of Landrace and MeiShan Pig（P＞0.05） and the serum GLP-2 content was higher than Landrace and MeiShan Pig（P＜0.01）;at 24-d old and at 26-d old,the serum GLP-2 content of Yorkshire×MeiShan Pigs was higher than MeiShan Pig（P＜0.01）;at 26-d old,the serum GLP-2 content of Yorkshire×MeiShan Pigs was higher than Landrace（P＜0.05）. Furthermore,at 30-d old,there was a significant correlation between the height of intestinal villis and serum GLP-2 content of different genotypes（P＜0.01）.The study showed that GLP-2 activity of piglets with different genotypes increased gradually after weaning.Yorkshire×MeiShan Pigs had the largest change range and reached the highest activity at 30-d old,with intestinal growth superior to MeiShan Pigs and Landrace.Serum GLP-2 content of the three 30-d old genotypes was significantly correlated with the height of intestinal villis at 30-d old.The experimental results suggest that it is a feasible approach to select the Yorkshire×MeiShan Pig or its intestinal enterocyte cells as model to study GLP-2’s possible mechanism and its regulation on the intestinal adaptation of piglets during weaning.After the confirmation that the secretion level and activity of GLP-2 in different genotypes piglets during weaning increased gradually,and that the height of intestinal villis was significantly correlated with serum GLP-2 concentration in circulation,the studying team further studied nutritional effects of GLP-2 on intestinal enterocyte cells of Yorkshire×MeiShan Pigs（Exp.2）.The results indicated that GLP-2 in vitro was capable of promoting the proliferation of intestinal enterocyte cells of 28-d old weaned piglets, inhibiting cell apoptosis and maintaining the integrity of cell morphology and function.On this ground,the studying team made the following studies.Exp.3 Effects of the combined use of Glucagon-like peptide-2 and dipeptidyl peptidase-ⅣInhibitors on Intestinal Enterocyte Cells of Weaned Piglets in VitroThis study was conducted to investigate in vitro the effects of the combined use of Glucagon-like peptide-2（GLP-2） and dipeptidyl peptidase-Ⅳ（DPP-Ⅳ） inhibitors with different concentrations on cell proliferation,metabolism and enzyme activity of 28-d old weaned piglets’ intestinal enterocyte cells.The experiment,taking in vitro 28-d old weaned piglets’ intestinal enterocyte cells as model,studied effects of DPP-Ⅳ（KR62436） inhibitors with different concentrations on the cell proliferation and metabolism according to the single-factor design principle.The method of 2×3 factorial design was adopted to study effects of the combined use of different GLP-2 concentrations(1×10^-10,1×10^-9 and 1×10^-8 mol/L) and different KR62436 concentrations(0 and 1×10^-11 mol/L) on cell proliferation,metabolism and apoptosis,with cell culture time 144h.The results were as follows:with the single use of DPP-Ⅳinhibitors,there was no significant change in cell under the observation of inverted microscope;although the cell quantity presented an increasing trend,MTT OD,protein retention and protein consumption firstly increased and then decreased（P＞0.05）.LDH activity and CK activity presented a decreasing trend with low dosage of KR62436（P＞0.05）.With higher concentration of KR62436,MTT OD value was signigicantly lower than control groups（P＜0.05）,LDH activity and CK activity were signigicantly higher（P＜0.05）,but Na⁺,K⁺-ATP enzyme activity changed insignificantly. When GLP-2 and KR62436 were both added into the cultura media,with the increasing of hGLP-2 concentrations,cell quantity increased signigicantly（P＜0.01）,MTT OD value increased signigicantly（P＜0.01）,total protein content and protein retention increased signigicantly（P＜0.01）,and Na⁺,K⁺-ATP enzyme activity increased signigicantly（P＜0.01） but LDH activity and CK activity decreased signigicantly（P＜0.01）.The results indicated that DPP-Ⅳinhibitors with a lower concentration had insignificant effects on cell growth. Although the DPP-Ⅳinhibitor with a higher concentration promoted the proliferation of intestinal enterocyte cells,it had negative effects on cell metabolism and cell integrality. The addition of DPP-Ⅳinhibitors in the culture media enhanced the effects of in vitro GLP-2 on cell proliferation,cell metabolism and cell integrality.GLP-2 and DPP-Ⅳinhibitors had significant synergistic effect.The experimental results suggested that KR62436 should be cautiously used to promote GLP-2 effects.Exp.4 Cytoprotection and repairing effects of GLP-2 on Intestinal Enterocyte Cells of 28-d Weaned Piglets following Injury byβ-conglycininThe experiment was conducted to study the effects of hGLP-2 on survival, proliferation,metabolism and apoptosis of intestinal enterocyte cell in 28-day-old weaned piglets after injury byβ-conglycinin and GLP-2’s possible mechanism.According to Single-factor design principle,differentβ-conglycinin concentrations of 1.2mg/ml and 2.4mg/ml were used for conteracting toxic substances to primary culture enterocyte cells of weaned piglets.The cell damage model was established on the investigation of the cell survival,proliferation,metabolism and apoptosis.The experiment of 2×3 factorial design was adopted to study the impacts of the sensitized cultured cell with different hGLP-2 concentrations of 1×10^-9,1×10^-8 and 1×10^-7mol/l.The results were as follows:when β-conglycinin were used for counteracting toxic substances,the MTT OD significantly decreased（P＜0.05）,especially protein retention and protein consumption extremely significantly decreased（P＜0.01）,Na⁺,K⁺-ATP enzyme activity significantly decreased （P＜0.05）,LDH activity and Caspase-3 significantly increased（P＜0.01）.Whenβ-conglycinin were used for conteracting toxic substances after addition of hGLP-2,the MTT OD,protein retention and protein consumption and Na⁺,K⁺-ATP enzyme activity significantly（P＜0.05）.LDH activity gradually decreased（P＜0.05） and Caspase-3 extremely significantly decreased（P＜0.01） along with the increase of hGLP-2 concentration.The results indicated thatβ-conglycinin had adverse effects on intestinal enterocyte cell proliferation and cell integrality in vitro.GLP-2 could relieve or avoid the adverse effects on intestinal enterocyte cell proliferation and cell integrality by regulating the Caspase-3 activity.To summarize,GLP-2 has effects on intestinal development of weaned piglets,and the height of intestinal villis is highly correlated with GLP-2 secretion levels.GLP-2 in vitro is capable of promoting the proliferation of the intestinal enterocyte cells and inhibiting their apoptosis in a dose-dependent manner.Although the single use of KR62436 may impair cell integrity,the addition of DPP-IV inhibitors enhances GLP-2 effects,which indicates that the combination of DPP-Ⅳinhibitors and GLP-2 has good synergistic effect,β-conglycinin extracts had adverse effects on proliferation and integrality of intestinal enterocyte cells.GLP-2 could relieve or avoid the adverse effects on intestinal enterocyte cell proliferation and cell apoptosis by protecting cells.The experimental results effectively support the further research and related work on in situ effects of GLP-2 on intestinal adaptation and acting mechanism of weaned piglets,and provide a fresh thinking on how to use GLP-2 to regulate weanling stress as well.更多还原