胰高血糖素样多肽-2的重组表达及其对肠道保护作用机制的实验研究
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摘要
目的意义:严重烧伤后造成肠粘膜血流量下降,肠粘膜组织损伤及通透性增加,使肠道屏障功能障碍,造成肠道细菌移位,引发多器官功能障碍,是导致伤后高代谢的重要原因。因此促进严重烧伤后肠道修复,能减轻脏器炎症反应,防止肠源性感染,改善代谢障碍,加快创面愈合,是临床救治重要措施之一。肠道作为人体最大的内分泌器官,其自身分泌许多肠道激素对肠道结构功能起着重要的保护作用,随着近年来对胰高血糖素样多肽-2(Glucagons like peptide 2,GLP-2)认识的不断深入,发现其对肠道有特异性保护作用。且有广泛临床应用前景。
本研究利用基因重组技术设计构建了GLP-2原核表达载体并诱导表达,初步观察其对烧伤后大鼠肠道及肠上皮细胞的保护作用,利用体外细胞模型,探讨GLP-2促细胞增殖的作用机制及调控因素,为今后临床应用提供理论依据。
材料与方法:
1.利用pET31b(+)表达载体,对GLP-2序列进行重组设计,构建表达质粒进行原核表达,优化条件后表达并进行亲和层析纯化、脱盐。溴化氰裂解后行western blot(WB)鉴定,并低温干燥保存。
2.采用30%TBSAⅢ度烧伤大鼠模型作为体内实验模型,随机分为正常对照组(N);烧伤对照组(C);重组GLP-2治疗组(Gr)及化学合成GLP-2治疗组(G)。检测大鼠伤后第7天肠粘膜通透性,肠粘膜湿重与肠段及躯壳重比,肠粘膜蛋白及肠道病理形态学的变化。RT-PCR及WB检测GLP-2R的表达,利用脂质体基因转染构建稳定表达GLP-2R的Caco-2/GLP-2R(+)细胞作为体外研究模型,随机分为正常对照组(N);缺氧对照组(C);缺氧后重组GLP-2处理组(Gr)及缺氧后化学合成GLP-2处理组(G)。观察不同GLP-2对缺氧后细胞增殖、乳酸脱氢酶及蔗糖酶活性的影响,并检测细胞CDK4及ZO1蛋白表达的变化。
3.构建caveolin-1/pEGFPN2质粒及体外合成caveolin-1 siRNA后瞬时转染Caco-2/GLP-2R(+)细胞,将细胞分为正常对照组(AN)、缺氧对照组(AC)、缺氧后GLP-2处理组(AG)、caveolin-1上调细胞缺氧后GLP-2处理组(CG)、caveolin-1下调细胞缺氧后GLP-2处理组(EG),检测细胞cAMP浓度,GRK2(ser280)磷酸化蛋白变化及细胞增殖,同时应用信号通路抑制剂观察GLP-2信号转导,WB检测ERK信号转导通路中相关蛋白及Akt(ser473)蛋白磷酸化表达的变化,免疫共沉淀检测caveolin-1与GLP-2R的作用,。观察GLP—2对缺氧后细胞增殖信号转导途径的影响以及caveolin-1对GLP-2R跨膜信号的调控
结果:
1.成功设计构建GLP-2/pET31b(+)质粒质粒,测序正确;在BLR(DE3)感受态大肠杆菌种诱导表达最佳条件为1mMIPTG诱导表达4h,通过亲和层析纯化及溴化氰裂解法获得单体GLP-2约100mg,重组得率5mg/100ml。
2.给予重组GLP-2及合成GLP-2后,烧伤后第7天,与烧伤对照组相比,大鼠的肠粘膜通透性明显降低,肠粘膜蛋白含量、肠粘膜与肠段重及与躯壳重之比均明显升高(P＜0.05),而两种GLP-2之间差异无显著性意义(P＞0.05)。形态学观察两种GLP-2均能减轻烧伤后肠粘膜病理损伤。体外实验结果显示,细胞严重缺氧8h立即加入1000nM的GLP-2,24、72h后能够明显增加Caco-2/GLP-2R(+)细胞的细胞增殖(P＜0.05),重组GLP-2与合成GLP-2效果无明显差别(P＞0.05)。而GLP-2处理72小时后,细胞乳酸脱氢酶及蔗糖酶活性无明显变化(P＞0.05)。CDK4及ZO1蛋白表达较缺氧对照组增强。
缺氧后立即给予1000nM的GLP-2处理,24h后,缺氧对照组cAMP含量明显降低(P＜0.01),而GLP-2处理组的cAMP却维持在一个较高水平。CG组细胞的cAMP明显增加,而EG组cAMP含量下调。GLP-2处理24h后,能够明显增加缺氧细胞的增殖(P＜0.05),在CG组细胞增殖更加明显(P＜0.05),EG组增殖作用下降。GLP-2处理后30min,磷酸化GRK2蛋白表达较缺氧对照组增强,无论上调或下调caveolin-1后,磷酸化GRK-2表达下降。给予不同信号通路抑制剂后发现,MEK1/2特异性抑制剂U0126及PI3特异抑制剂LY294002能够明显抑制GLP-2的促增殖作用(P＜0.05),而P38特异性抑制剂SB203580却不能抑制GLP-2促增殖作用。。缺氧后给予GLP-2能增强ERK信号途径中c-Raf、MEK1/2、ERK1/2及核内p90RSK磷酸化蛋白的表达,也能增强PI3信号途径中Akt磷酸化蛋白的表达。caveolin-1上调后ERK信号通路中相关磷酸化蛋白的表达增强增强,caveolin-1下调后相关磷酸化蛋白的表达减弱。
结论:
1.构建GLP-2原核表达载体。诱导表达纯化,为GLP-2获得提供了一种新的方法。
2.重组GLP-2及化学合成GLP-2能够降低严重烧伤后大鼠肠粘膜通透性,增加粘膜重量及蛋白含量,减轻肠粘膜的损伤程度。对Caco2/GLP-2R(+)细胞具有明显的促增殖作用,但对细胞无明显毒性,对蔗糖酶活性没有明显的影响,能够增强缺氧后细胞CDK4及ZO1蛋白表达,可能参与了细胞分裂及细胞间紧密连接的调控机制。
3.GLP-2对细胞的促增殖作用伴有cAMP的增加,而磷酸化GRK2蛋白负反馈机制可能参与了对cAMP的调控。GLP-2对细胞增殖的作用依赖于ERK及PI3途径,其可以引起c-Raf,MEK1/2,ERK1/2及P90RSK磷酸化蛋白表达增强,也使Akt磷酸化蛋白表达增强。而可能不依赖于P38信号途径。Caveolin-1对GLP-2R信号转导可能有调控作用,本实验中,其可促进GLP-2介导的细胞增殖及激活ERK,PI3信号途径。
Introduction:It is well known that the dysfunction of intestinal mucosal barrier is the key to initiating bacterial translocation,MODS and enterogenous hypermetabolism after severe burn injury.Maintaining the structure and function of intestinal mucosa and promoting the recovery of injuried intestine can lower enterogenic infection,inflammatory response and enterogenous hypermetabolism.The hormones secreted from intestifial endocrine cells are the most important messengers that regulate intestinal function, including cytoprotection,nutrition and cell proliferation.Among these is the intestinotrophic peptide glucagon-like peptide-2(GLP-2),which plays a significant role in the adaptive regulation of bowel mass and mucosal integrity.However,the mechanism of GLP-2 has not yet been elucidated.
Objectives:This study was intended to design prokaryotic expression vector,induce GLP-2 expression in E.coli BLR(DE3),and investigate the protective effects of recombinant GLP-2 on intestinal mucosa of burned rats in vivo and on hypoxic intestinal epithelial cells in vitro.The intestinal epithelial cell line stably expressing GLP-2 receptor (GLP-2R) was constructed to explore the mechanisms of enteroprotection of GLP-2 and the regulation of transmembrane signal transduction of GLP-2R by caveolin-1.
Material and Methods:
1.AlwN I-digested and dephosphorylated pET-31b(+) DNA was used.The second amino acid,alanine,of GLP-2 coding sequences was replaced by glycine.The new sequences were cloned as tandem repeats interspersed with single methionine residues and placed downstream of a 125aa ketosteroid isomerase(KSI) gene and upstream of a His·Tag. The recombinant vector,GLP-2/pET31b(+),was transfected into the BLR(DE3) expression cells,and the target peptide production was induced by adding IPTG to the culture media. The purified GLP-2 with affinity chromatograph and CNBr cleavage was stored after cryodrying.
2.Rats of either sex infliced with 30%TBSAⅢ°burns were randomly divided into 4 groups:normal group(N),burn control group(C),recombinant GLP-2 group(Gr,treated with recombinant GLP-2,100nM.kg~(-1).d~(-1) subcutaneously for 7 days) and synthesized GLP-2 treated group(G,treatment with synthesized GLP-2,100nM.kg~(-1).d~(-1)subcutaneously for 7 days).The rats were killed on 7d postburn and the following indexes were measured, including mucosal pathological examination,mucosa permeability,the ratio of mucosa wet weight and bowel mass or carcase weight,and the content of intestinal mucosal protein. For in vitro study,the GLP-2R/PCDNA3.1(+) was transfected into Caco-2 with Lipofectamine2000,and the stably transfected cells were selected and maintained by growth in G418.Individual cell clones were obtained by limited dilution cloning,verified by determining the GLP-2R protein expression with Western blot method,expanded for further studies,and named Caco2/GLP-2R(+).The cells were divided into 4 groups: normal group(N),hypoxia control group(C),recombinant GLP-2 group(Gr.treatment with recombinant GLP-2 after hypoxia for 8h,1000nM for 30min,4,24,72h) and synthesized GLP-2 treated group(G,treatment with synthesized GLP-2 after hypoxia for 8h,1000nM for 30min,4,24,72h),The following indexes were measured including cell proliferation. activities of lactate dehydrogenase and sucrase,the protein expressions of CDK4 and ZO1.
3.The caveolin-1/pEGFPN2 plasmid was constructed.The small interference RNA of caveolin-1 obtained from an in vitro transcription reaction was transiently transfected into Caco2/GLP-2R(+).The cells were divided into 5 groups:normal group(AN),hypoxia control group(AC),GLP-2 group(AG,treatment with synthesis GLP-2 after hypoxia 8h, 1000nM for 30min,24h),caveolin-1 up-regulation group(CG,treatment with synthesized GLP-2 after hypoxia 8h,1000nM for 30min,24h);caveolin-1 down-regulation group(EG, treatment with synthesized GLP-2 after hypoxia for 8h,1000nM for 30min,24h).The kinase inhibitors including U0126,LY294002,SB203580 were uesed to explore the GLP-2 signal transduction pathway.The cAMP content and cell proliferation were detected.The phosphorylation of c-Raf,MEK1/2,ERK1/2,Akt and p90RSK were analysized to investigate the mechanism of enteroprotection of GLP-2 and regulation of transmembrane signal transduction of GLP-2 receptor by caveolin-1.
Results:
1.The sequence of GLP-2/pET31b(+) is confirmed by DNA sequencing,and the expressed GLP-2 peptide was verified by Western Blot.Approximate 100mg of the GLP-2 monomer were obtained in the current expression system.
2.Histologically,the structure of intestinal mucosa was damaged as evidenced by the thinning of mucosa,shortening of villus,and decreasing of villus surface area in burn group on 7d postburn,while both recombinant GLP-2 and synthesis GLP-2 could alleviate intestinal mucosa injury obviously.Compared with those in burn group,the mucosa permeability significantly decreased in both GLP-2 groups.The ratio of mucosa wet weight and bowel mass or carcase weight,the content of intestinal mucosal protein were more significantly increased in both GLP-2 groups than those in burn control group.The in vitro results showed that GLP-2 directly stimulated proliferation of the hypoxic Caco2/GLP-2R(+) cells at 24,72h after GLP-2 treatment.There was no significant difference between recombinant GLP-2 and synthesized GLP-2.The activities of lactate dehydrogenase and sucrase had no significant changes at 72h after GLP-2 treatment.The protein expressions of CDK4 and ZO1 were increased in hypoxic Caco2/GLP-2R(+) cells at 72h after GLP-2 treatment.
3.The data also showed that the cAMP content were increased and maintained at high level in cells treated with GLP-2.The phosphorylated GRK2 level increased after treated with 1000nM GLP-2.The effect of GLP-2 on stimulating cells proliferation was blocked by the kinase inhibitors U0126 and LY294002,but not SB203580.Western blot analysis of hypoxic Caco2/GLP-2R(+) cell treated with GLP-2 showed that ERK1/2 was activated,the expressions of phosphorylated c-Raf,MEK1/2,ERK1/2 and p90RSK of cellular nucleus were increased.The expression of phosphorylated Akt was enhanced too. Our results showed that caveolin-1 ehanced the phosphorylation of ERK and PI3 pathway and promoted cell proliferation.Meanwhile,the phosphorylation of cell signal transduction was weakened after knocking down the caveolin-1 by transfecting caveolin-1 siRNA into Caco2/GLP-2R(+) cell
Conlusions:
1.The prokaryotic expression is a new approach to product the GLP-2. 2.Both the recombinant GLP-2 and synthesized GLP-2 could alleviate intestinal mucosa injury in burn rats and promote proliferation of the hypoxic Caco2/GLP-2R(+) cells
3.The GLP-2 stimulate proliferation of the hypoxic Caco2/GLP-2R(+) cells by activating ERK/PI3- but not P38-dependent pathway.
4.The transduction of transmembrane signal of GLP-2 receptor is regulated by caveolin-1
本研究利用基因重组技术设计构建了GLP-2原核表达载体并诱导表达,初步观察其对烧伤后大鼠肠道及肠上皮细胞的保护作用,利用体外细胞模型,探讨GLP-2促细胞增殖的作用机制及调控因素,为今后临床应用提供理论依据。
材料与方法:
1.利用pET31b(+)表达载体,对GLP-2序列进行重组设计,构建表达质粒进行原核表达,优化条件后表达并进行亲和层析纯化、脱盐。溴化氰裂解后行western blot(WB)鉴定,并低温干燥保存。
2.采用30%TBSAⅢ度烧伤大鼠模型作为体内实验模型,随机分为正常对照组(N);烧伤对照组(C);重组GLP-2治疗组(Gr)及化学合成GLP-2治疗组(G)。检测大鼠伤后第7天肠粘膜通透性,肠粘膜湿重与肠段及躯壳重比,肠粘膜蛋白及肠道病理形态学的变化。RT-PCR及WB检测GLP-2R的表达,利用脂质体基因转染构建稳定表达GLP-2R的Caco-2/GLP-2R(+)细胞作为体外研究模型,随机分为正常对照组(N);缺氧对照组(C);缺氧后重组GLP-2处理组(Gr)及缺氧后化学合成GLP-2处理组(G)。观察不同GLP-2对缺氧后细胞增殖、乳酸脱氢酶及蔗糖酶活性的影响,并检测细胞CDK4及ZO1蛋白表达的变化。
3.构建caveolin-1/pEGFPN2质粒及体外合成caveolin-1 siRNA后瞬时转染Caco-2/GLP-2R(+)细胞,将细胞分为正常对照组(AN)、缺氧对照组(AC)、缺氧后GLP-2处理组(AG)、caveolin-1上调细胞缺氧后GLP-2处理组(CG)、caveolin-1下调细胞缺氧后GLP-2处理组(EG),检测细胞cAMP浓度,GRK2(ser280)磷酸化蛋白变化及细胞增殖,同时应用信号通路抑制剂观察GLP-2信号转导,WB检测ERK信号转导通路中相关蛋白及Akt(ser473)蛋白磷酸化表达的变化,免疫共沉淀检测caveolin-1与GLP-2R的作用,。观察GLP—2对缺氧后细胞增殖信号转导途径的影响以及caveolin-1对GLP-2R跨膜信号的调控
结果:
1.成功设计构建GLP-2/pET31b(+)质粒质粒,测序正确;在BLR(DE3)感受态大肠杆菌种诱导表达最佳条件为1mMIPTG诱导表达4h,通过亲和层析纯化及溴化氰裂解法获得单体GLP-2约100mg,重组得率5mg/100ml。
2.给予重组GLP-2及合成GLP-2后,烧伤后第7天,与烧伤对照组相比,大鼠的肠粘膜通透性明显降低,肠粘膜蛋白含量、肠粘膜与肠段重及与躯壳重之比均明显升高(P＜0.05),而两种GLP-2之间差异无显著性意义(P＞0.05)。形态学观察两种GLP-2均能减轻烧伤后肠粘膜病理损伤。体外实验结果显示,细胞严重缺氧8h立即加入1000nM的GLP-2,24、72h后能够明显增加Caco-2/GLP-2R(+)细胞的细胞增殖(P＜0.05),重组GLP-2与合成GLP-2效果无明显差别(P＞0.05)。而GLP-2处理72小时后,细胞乳酸脱氢酶及蔗糖酶活性无明显变化(P＞0.05)。CDK4及ZO1蛋白表达较缺氧对照组增强。
缺氧后立即给予1000nM的GLP-2处理,24h后,缺氧对照组cAMP含量明显降低(P＜0.01),而GLP-2处理组的cAMP却维持在一个较高水平。CG组细胞的cAMP明显增加,而EG组cAMP含量下调。GLP-2处理24h后,能够明显增加缺氧细胞的增殖(P＜0.05),在CG组细胞增殖更加明显(P＜0.05),EG组增殖作用下降。GLP-2处理后30min,磷酸化GRK2蛋白表达较缺氧对照组增强,无论上调或下调caveolin-1后,磷酸化GRK-2表达下降。给予不同信号通路抑制剂后发现,MEK1/2特异性抑制剂U0126及PI3特异抑制剂LY294002能够明显抑制GLP-2的促增殖作用(P＜0.05),而P38特异性抑制剂SB203580却不能抑制GLP-2促增殖作用。。缺氧后给予GLP-2能增强ERK信号途径中c-Raf、MEK1/2、ERK1/2及核内p90RSK磷酸化蛋白的表达,也能增强PI3信号途径中Akt磷酸化蛋白的表达。caveolin-1上调后ERK信号通路中相关磷酸化蛋白的表达增强增强,caveolin-1下调后相关磷酸化蛋白的表达减弱。
结论:
1.构建GLP-2原核表达载体。诱导表达纯化,为GLP-2获得提供了一种新的方法。
2.重组GLP-2及化学合成GLP-2能够降低严重烧伤后大鼠肠粘膜通透性,增加粘膜重量及蛋白含量,减轻肠粘膜的损伤程度。对Caco2/GLP-2R(+)细胞具有明显的促增殖作用,但对细胞无明显毒性,对蔗糖酶活性没有明显的影响,能够增强缺氧后细胞CDK4及ZO1蛋白表达,可能参与了细胞分裂及细胞间紧密连接的调控机制。
3.GLP-2对细胞的促增殖作用伴有cAMP的增加,而磷酸化GRK2蛋白负反馈机制可能参与了对cAMP的调控。GLP-2对细胞增殖的作用依赖于ERK及PI3途径,其可以引起c-Raf,MEK1/2,ERK1/2及P90RSK磷酸化蛋白表达增强,也使Akt磷酸化蛋白表达增强。而可能不依赖于P38信号途径。Caveolin-1对GLP-2R信号转导可能有调控作用,本实验中,其可促进GLP-2介导的细胞增殖及激活ERK,PI3信号途径。
Introduction:It is well known that the dysfunction of intestinal mucosal barrier is the key to initiating bacterial translocation,MODS and enterogenous hypermetabolism after severe burn injury.Maintaining the structure and function of intestinal mucosa and promoting the recovery of injuried intestine can lower enterogenic infection,inflammatory response and enterogenous hypermetabolism.The hormones secreted from intestifial endocrine cells are the most important messengers that regulate intestinal function, including cytoprotection,nutrition and cell proliferation.Among these is the intestinotrophic peptide glucagon-like peptide-2(GLP-2),which plays a significant role in the adaptive regulation of bowel mass and mucosal integrity.However,the mechanism of GLP-2 has not yet been elucidated.
Objectives:This study was intended to design prokaryotic expression vector,induce GLP-2 expression in E.coli BLR(DE3),and investigate the protective effects of recombinant GLP-2 on intestinal mucosa of burned rats in vivo and on hypoxic intestinal epithelial cells in vitro.The intestinal epithelial cell line stably expressing GLP-2 receptor (GLP-2R) was constructed to explore the mechanisms of enteroprotection of GLP-2 and the regulation of transmembrane signal transduction of GLP-2R by caveolin-1.
Material and Methods:
1.AlwN I-digested and dephosphorylated pET-31b(+) DNA was used.The second amino acid,alanine,of GLP-2 coding sequences was replaced by glycine.The new sequences were cloned as tandem repeats interspersed with single methionine residues and placed downstream of a 125aa ketosteroid isomerase(KSI) gene and upstream of a His·Tag. The recombinant vector,GLP-2/pET31b(+),was transfected into the BLR(DE3) expression cells,and the target peptide production was induced by adding IPTG to the culture media. The purified GLP-2 with affinity chromatograph and CNBr cleavage was stored after cryodrying.
2.Rats of either sex infliced with 30%TBSAⅢ°burns were randomly divided into 4 groups:normal group(N),burn control group(C),recombinant GLP-2 group(Gr,treated with recombinant GLP-2,100nM.kg~(-1).d~(-1) subcutaneously for 7 days) and synthesized GLP-2 treated group(G,treatment with synthesized GLP-2,100nM.kg~(-1).d~(-1)subcutaneously for 7 days).The rats were killed on 7d postburn and the following indexes were measured, including mucosal pathological examination,mucosa permeability,the ratio of mucosa wet weight and bowel mass or carcase weight,and the content of intestinal mucosal protein. For in vitro study,the GLP-2R/PCDNA3.1(+) was transfected into Caco-2 with Lipofectamine2000,and the stably transfected cells were selected and maintained by growth in G418.Individual cell clones were obtained by limited dilution cloning,verified by determining the GLP-2R protein expression with Western blot method,expanded for further studies,and named Caco2/GLP-2R(+).The cells were divided into 4 groups: normal group(N),hypoxia control group(C),recombinant GLP-2 group(Gr.treatment with recombinant GLP-2 after hypoxia for 8h,1000nM for 30min,4,24,72h) and synthesized GLP-2 treated group(G,treatment with synthesized GLP-2 after hypoxia for 8h,1000nM for 30min,4,24,72h),The following indexes were measured including cell proliferation. activities of lactate dehydrogenase and sucrase,the protein expressions of CDK4 and ZO1.
3.The caveolin-1/pEGFPN2 plasmid was constructed.The small interference RNA of caveolin-1 obtained from an in vitro transcription reaction was transiently transfected into Caco2/GLP-2R(+).The cells were divided into 5 groups:normal group(AN),hypoxia control group(AC),GLP-2 group(AG,treatment with synthesis GLP-2 after hypoxia 8h, 1000nM for 30min,24h),caveolin-1 up-regulation group(CG,treatment with synthesized GLP-2 after hypoxia 8h,1000nM for 30min,24h);caveolin-1 down-regulation group(EG, treatment with synthesized GLP-2 after hypoxia for 8h,1000nM for 30min,24h).The kinase inhibitors including U0126,LY294002,SB203580 were uesed to explore the GLP-2 signal transduction pathway.The cAMP content and cell proliferation were detected.The phosphorylation of c-Raf,MEK1/2,ERK1/2,Akt and p90RSK were analysized to investigate the mechanism of enteroprotection of GLP-2 and regulation of transmembrane signal transduction of GLP-2 receptor by caveolin-1.
Results:
1.The sequence of GLP-2/pET31b(+) is confirmed by DNA sequencing,and the expressed GLP-2 peptide was verified by Western Blot.Approximate 100mg of the GLP-2 monomer were obtained in the current expression system.
2.Histologically,the structure of intestinal mucosa was damaged as evidenced by the thinning of mucosa,shortening of villus,and decreasing of villus surface area in burn group on 7d postburn,while both recombinant GLP-2 and synthesis GLP-2 could alleviate intestinal mucosa injury obviously.Compared with those in burn group,the mucosa permeability significantly decreased in both GLP-2 groups.The ratio of mucosa wet weight and bowel mass or carcase weight,the content of intestinal mucosal protein were more significantly increased in both GLP-2 groups than those in burn control group.The in vitro results showed that GLP-2 directly stimulated proliferation of the hypoxic Caco2/GLP-2R(+) cells at 24,72h after GLP-2 treatment.There was no significant difference between recombinant GLP-2 and synthesized GLP-2.The activities of lactate dehydrogenase and sucrase had no significant changes at 72h after GLP-2 treatment.The protein expressions of CDK4 and ZO1 were increased in hypoxic Caco2/GLP-2R(+) cells at 72h after GLP-2 treatment.
3.The data also showed that the cAMP content were increased and maintained at high level in cells treated with GLP-2.The phosphorylated GRK2 level increased after treated with 1000nM GLP-2.The effect of GLP-2 on stimulating cells proliferation was blocked by the kinase inhibitors U0126 and LY294002,but not SB203580.Western blot analysis of hypoxic Caco2/GLP-2R(+) cell treated with GLP-2 showed that ERK1/2 was activated,the expressions of phosphorylated c-Raf,MEK1/2,ERK1/2 and p90RSK of cellular nucleus were increased.The expression of phosphorylated Akt was enhanced too. Our results showed that caveolin-1 ehanced the phosphorylation of ERK and PI3 pathway and promoted cell proliferation.Meanwhile,the phosphorylation of cell signal transduction was weakened after knocking down the caveolin-1 by transfecting caveolin-1 siRNA into Caco2/GLP-2R(+) cell
Conlusions:
1.The prokaryotic expression is a new approach to product the GLP-2. 2.Both the recombinant GLP-2 and synthesized GLP-2 could alleviate intestinal mucosa injury in burn rats and promote proliferation of the hypoxic Caco2/GLP-2R(+) cells
3.The GLP-2 stimulate proliferation of the hypoxic Caco2/GLP-2R(+) cells by activating ERK/PI3- but not P38-dependent pathway.
4.The transduction of transmembrane signal of GLP-2 receptor is regulated by caveolin-1
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