AIM： To investigate electroacupunture（EA） at the acupoints of Stomach Meridian of Foot-Yangming（SMFY）, Gallbladder Meridian of Foot-Yangming（SMFY） on gastric mucosal intestinal trefoil factor （ITF） gene expre...AIM： To investigate electroacupunture（EA） at the acupoints of Stomach Meridian of Foot-Yangming（SMFY）, Gallbladder Meridian of Foot-Yangming（SMFY） on gastric mucosal intestinal trefoil factor （ITF） gene expression detection in stress-induced rats with gastric mucosal lesion, and to explore the regulatory mechanism and significance of EA-related gastric mucosal protective effect. METHODS： Forty rats were randomly divided into 4 groups： Blank group, Model group, Model group＋EA at acupoints of SMFY group（＂SMFY group＂）, and Model group＋EA at acupoints of GMFY group（GMFY group）. All rats （except blank group） were made model by water immersion and restraint stress （WRS）. Then the gastric mucosa tissue in each rat was taken off alter assessment of gastric mucosal lesion index（GUI）, and the expression of ITF mRNA of the tissues was detected by reverse transcdption-polymerase chain reaction（RT-PCR） method. RESULTS： Compared with Model group（S4.3± 1.34）, the GUI value in SMFY group （31±2.211 decreased significantly（P〈0.01）, so did that in GMFY group （39.8± 1.62, P〈0.05）, meanwhile GUI value in SMFY group was significantly lower than in GMFY group（P〈0.01）. Compared with Model group （0.65±0.01）, EA had a tendency to improve the expression of gastric mucosal ITFmRNA gene： such tendency existed in GMFY group （0.66±0.01） but with no signficant difference（P〉 0.05）, in SMFY group（0.76±0.01） with an extremely obvious difference （P〈0.01）, furthermore the expression in SMFY group was significantly higher than in GMFY group （P〈 0.01）.CONCLUSION： The gastric mucosal protective effect by EA at the acupoints of SMFY and GMFY was related to the expression variance of ITF, indicating certain meridian specificity exists, It could be one proof for the TCM theory ＂Relative pardcularity between SMFY and stomach＂.展开更多
In order to investigate the protective effects of intestinal trefoil factor (ITF) on colonic mucosa in experimental colitis of rats, ITF was detected by RT-PCR and immunohistochemistry at different time points. Three ...In order to investigate the protective effects of intestinal trefoil factor (ITF) on colonic mucosa in experimental colitis of rats, ITF was detected by RT-PCR and immunohistochemistry at different time points. Three days after colitis induction, rats were treated with either 0.9 % saline solution or rhITF. Pathological changes and the expression of iNOS mRNA, NO, MDA and SOD were measured respectively. It was found that ITF was mainly located in goblet cells, significantly higher in model group than in normal group (P<0.05). rhITF could increase the iNOS mRNA expression and NO contents, and there was statistically significant difference between rhITF group and model group (P<0.05). rhITF also caused an increase of MDA and a decrease of SOD, but there was no significant difference between two groups. These results indicated that ITF has apparent therapeutic effects in ulcerative colitis, which may be associated with iNOS and NO.展开更多
BACKGROUND Stress-related gastric mucosal damage or ulcer remains an unsolved issue for critically ill patients.Stress ulcer prophylaxis has been part of routine intensive care,but uncertainty and controversy still ex...BACKGROUND Stress-related gastric mucosal damage or ulcer remains an unsolved issue for critically ill patients.Stress ulcer prophylaxis has been part of routine intensive care,but uncertainty and controversy still exist.Co-secreted with mucins,intestinal trefoil factor(ITF)is reported to promote restitution and regeneration of intestinal mucosal epithelium,although the mechanism remains unknown.AIM To elucidate the protective effects of ITF on gastric mucosa and explore the possible mechanisms.METHODS We used a rat model of gastric mucosal damage induced by water immersion restraint stress and lipopolysaccharide-treated human gastric epithelial cell line to investigate the potential effects of ITF on damaged gastric mucosa both in vivo and in vitro.RESULTS ITF promoted the proliferation and migration and inhibited necrosis of gastric mucosal epithelia in vitro.It also preserved the integrity of gastric mucosa by upregulating expressions of occludin and zonula occludens-1.In the rat model,pretreatment with ITF ameliorated the gastric mucosal epithelial damage and facilitated mucosal repair.The protective effects of ITF were confirmed to be exerted via activation of Akt signaling,and the specific inhibitor of Akt signaling LY249002 reversed the protective effects.CONCLUSION ITF might be a promising candidate for prevention and treatment of stressinduced gastric mucosal damage,and further studies should be undertaken to verify its clinical feasibility.展开更多
Objective：To construct a Pichia pastoris （P. pastoris） expression vector of human intestinal trefoil factor （hITF） and study its expression and purification procedures. Methods：hITF gene encoding mature peptide ...Objective：To construct a Pichia pastoris （P. pastoris） expression vector of human intestinal trefoil factor （hITF） and study its expression and purification procedures. Methods：hITF gene encoding mature peptide was modified with a polybistidine tag sequence at the N-terminal, and then inserted into the P. pastoris expression vector pGAPZaA at the downstream of the s-mating factor signal. After gene sequencing, the recombinant pGAPZaA-hITF was transformed into the P. pastoris strain X-33 with lithium chloride, rhITF was induced to constitutively express in shake flask, and then analyzed with Tricine SDS-PAGEand Western blotting. The obtained rhITF was isolated from the cultured supernatants by ammonium sulfate precipitation, Ni-NTA affinity chromatography, and ultrafiltration. Results：The correctness and integrity of rhITF were identified by restriction digestion and gene sequencing, rhITF was successfully expressed to 50 mg/L as a secretive protein. After purification, the purity was above 95%. Tricine SDS-PAGE and Western-blot analysis showed that rhITF presented as a single band with a molecular weight of 10 kDa, a little larger than 7 879 Da as assayed by mass spectrometry analysis. Conclusion： hITF P. pastoris expression vector is successfully constructed and rhITF is expressed in P. pastoris at commercially relevant level. This research lays foundation for the further functional studying of hITF.展开更多
目的构建hITF毕赤酵母分泌型表达载体,表达重组hITF,为功能研究奠定基础。方法通过PCR获得hITFcDNA片段,将目的基因插入酵母表达载体pGAPZαA分泌信号下游,得到重组载体pGAPZαA-hITF。BspHⅠ线性化后氯化锂转化进入X-33,Zeoc in筛选转...目的构建hITF毕赤酵母分泌型表达载体,表达重组hITF,为功能研究奠定基础。方法通过PCR获得hITFcDNA片段,将目的基因插入酵母表达载体pGAPZαA分泌信号下游,得到重组载体pGAPZαA-hITF。BspHⅠ线性化后氯化锂转化进入X-33,Zeoc in筛选转化酵母菌,PCR鉴定目的基因。阳性转化子经摇瓶表达,取上清TCA沉淀后做Tric ine SDS-PAGE分析及W estern b lot检测。结果经测序及PCR证实,hITFcDNA准确插入酵母表达载体pGAPZαA中,氯化锂转化后,重组载体通过同源重组整合进入酵母基因组中。Tric ine SDS-PAGE分析证明hITF的分子量约为10×103,W estern b lot分析表明,表达蛋白具有良好的抗原性和特异性。结论成功构建出酵母表达载体pGAPZαA-hITF,获得重组hITF,为深入研究hITF奠定了基础。展开更多
目的:研究肠三叶因子(intestinal trefoil factor,ITF)对自身启动子活性的影响及Janus激酶/信号转导和转录激活因子(Janus kinase/signal transducers and activators of transcription,JAK/STAT)信号通路对其的调控作用。方法:以人全...目的:研究肠三叶因子(intestinal trefoil factor,ITF)对自身启动子活性的影响及Janus激酶/信号转导和转录激活因子(Janus kinase/signal transducers and activators of transcription,JAK/STAT)信号通路对其的调控作用。方法:以人全血基因组DNA为模板,PCR获取ITF基因5'侧翼序列,插入p GL3-Basic载体,构建ITF启动子重组载体;以不同浓度的ITF刺激,检测双荧光素酶的活性;运用JAK-STAT3通路特异性抑制剂AG490阻断JAKSTAT3信号通路,观察AG490对ITF启动子活性的影响。结果:酶切和直接测序法证实包含ITF启动子的重组质粒构建成功;瞬时转染实验显示,ITF能显著提高ITF启动子的活性(P<0.05);加入AG490后ITF启动子活性显著下降(P<0.05)。结论:ITF通过JAK-STAT3途径上调自身启动子的活性。展开更多
基金Supported by the National Natural Science Foundation of China No. 90209023
文摘AIM： To investigate electroacupunture（EA） at the acupoints of Stomach Meridian of Foot-Yangming（SMFY）, Gallbladder Meridian of Foot-Yangming（SMFY） on gastric mucosal intestinal trefoil factor （ITF） gene expression detection in stress-induced rats with gastric mucosal lesion, and to explore the regulatory mechanism and significance of EA-related gastric mucosal protective effect. METHODS： Forty rats were randomly divided into 4 groups： Blank group, Model group, Model group＋EA at acupoints of SMFY group（＂SMFY group＂）, and Model group＋EA at acupoints of GMFY group（GMFY group）. All rats （except blank group） were made model by water immersion and restraint stress （WRS）. Then the gastric mucosa tissue in each rat was taken off alter assessment of gastric mucosal lesion index（GUI）, and the expression of ITF mRNA of the tissues was detected by reverse transcdption-polymerase chain reaction（RT-PCR） method. RESULTS： Compared with Model group（S4.3± 1.34）, the GUI value in SMFY group （31±2.211 decreased significantly（P〈0.01）, so did that in GMFY group （39.8± 1.62, P〈0.05）, meanwhile GUI value in SMFY group was significantly lower than in GMFY group（P〈0.01）. Compared with Model group （0.65±0.01）, EA had a tendency to improve the expression of gastric mucosal ITFmRNA gene： such tendency existed in GMFY group （0.66±0.01） but with no signficant difference（P〉 0.05）, in SMFY group（0.76±0.01） with an extremely obvious difference （P〈0.01）, furthermore the expression in SMFY group was significantly higher than in GMFY group （P〈 0.01）.CONCLUSION： The gastric mucosal protective effect by EA at the acupoints of SMFY and GMFY was related to the expression variance of ITF, indicating certain meridian specificity exists, It could be one proof for the TCM theory ＂Relative pardcularity between SMFY and stomach＂.
文摘In order to investigate the protective effects of intestinal trefoil factor (ITF) on colonic mucosa in experimental colitis of rats, ITF was detected by RT-PCR and immunohistochemistry at different time points. Three days after colitis induction, rats were treated with either 0.9 % saline solution or rhITF. Pathological changes and the expression of iNOS mRNA, NO, MDA and SOD were measured respectively. It was found that ITF was mainly located in goblet cells, significantly higher in model group than in normal group (P<0.05). rhITF could increase the iNOS mRNA expression and NO contents, and there was statistically significant difference between rhITF group and model group (P<0.05). rhITF also caused an increase of MDA and a decrease of SOD, but there was no significant difference between two groups. These results indicated that ITF has apparent therapeutic effects in ulcerative colitis, which may be associated with iNOS and NO.
基金Social Development Projects of Jiangsu Province,No.BE2017720the National Natural Science Foundation of China,No.81701894+4 种基金Jiangsu Provincial Medical Youth Talent,No.QNRC2016909 and No.QNRC2016908Natural Science Foundation of Jiangsu Province,No.BK20190247Science Foundation of Jiangsu Health Commission,No.H2018039China Postdoctoral Science Foundation,No.2018M643890Jiangsu Postdoctoral Science Foundation,No.2018K048A and No.2020Z193。
文摘BACKGROUND Stress-related gastric mucosal damage or ulcer remains an unsolved issue for critically ill patients.Stress ulcer prophylaxis has been part of routine intensive care,but uncertainty and controversy still exist.Co-secreted with mucins,intestinal trefoil factor(ITF)is reported to promote restitution and regeneration of intestinal mucosal epithelium,although the mechanism remains unknown.AIM To elucidate the protective effects of ITF on gastric mucosa and explore the possible mechanisms.METHODS We used a rat model of gastric mucosal damage induced by water immersion restraint stress and lipopolysaccharide-treated human gastric epithelial cell line to investigate the potential effects of ITF on damaged gastric mucosa both in vivo and in vitro.RESULTS ITF promoted the proliferation and migration and inhibited necrosis of gastric mucosal epithelia in vitro.It also preserved the integrity of gastric mucosa by upregulating expressions of occludin and zonula occludens-1.In the rat model,pretreatment with ITF ameliorated the gastric mucosal epithelial damage and facilitated mucosal repair.The protective effects of ITF were confirmed to be exerted via activation of Akt signaling,and the specific inhibitor of Akt signaling LY249002 reversed the protective effects.CONCLUSION ITF might be a promising candidate for prevention and treatment of stressinduced gastric mucosal damage,and further studies should be undertaken to verify its clinical feasibility.
基金Supported by the National Natural Science Foundation of China (No. 30200294)
文摘Objective：To construct a Pichia pastoris （P. pastoris） expression vector of human intestinal trefoil factor （hITF） and study its expression and purification procedures. Methods：hITF gene encoding mature peptide was modified with a polybistidine tag sequence at the N-terminal, and then inserted into the P. pastoris expression vector pGAPZaA at the downstream of the s-mating factor signal. After gene sequencing, the recombinant pGAPZaA-hITF was transformed into the P. pastoris strain X-33 with lithium chloride, rhITF was induced to constitutively express in shake flask, and then analyzed with Tricine SDS-PAGEand Western blotting. The obtained rhITF was isolated from the cultured supernatants by ammonium sulfate precipitation, Ni-NTA affinity chromatography, and ultrafiltration. Results：The correctness and integrity of rhITF were identified by restriction digestion and gene sequencing, rhITF was successfully expressed to 50 mg/L as a secretive protein. After purification, the purity was above 95%. Tricine SDS-PAGE and Western-blot analysis showed that rhITF presented as a single band with a molecular weight of 10 kDa, a little larger than 7 879 Da as assayed by mass spectrometry analysis. Conclusion： hITF P. pastoris expression vector is successfully constructed and rhITF is expressed in P. pastoris at commercially relevant level. This research lays foundation for the further functional studying of hITF.
文摘目的构建hITF毕赤酵母分泌型表达载体,表达重组hITF,为功能研究奠定基础。方法通过PCR获得hITFcDNA片段,将目的基因插入酵母表达载体pGAPZαA分泌信号下游,得到重组载体pGAPZαA-hITF。BspHⅠ线性化后氯化锂转化进入X-33,Zeoc in筛选转化酵母菌,PCR鉴定目的基因。阳性转化子经摇瓶表达,取上清TCA沉淀后做Tric ine SDS-PAGE分析及W estern b lot检测。结果经测序及PCR证实,hITFcDNA准确插入酵母表达载体pGAPZαA中,氯化锂转化后,重组载体通过同源重组整合进入酵母基因组中。Tric ine SDS-PAGE分析证明hITF的分子量约为10×103,W estern b lot分析表明,表达蛋白具有良好的抗原性和特异性。结论成功构建出酵母表达载体pGAPZαA-hITF,获得重组hITF,为深入研究hITF奠定了基础。
文摘目的:研究肠三叶因子(intestinal trefoil factor,ITF)对自身启动子活性的影响及Janus激酶/信号转导和转录激活因子(Janus kinase/signal transducers and activators of transcription,JAK/STAT)信号通路对其的调控作用。方法:以人全血基因组DNA为模板,PCR获取ITF基因5'侧翼序列,插入p GL3-Basic载体,构建ITF启动子重组载体;以不同浓度的ITF刺激,检测双荧光素酶的活性;运用JAK-STAT3通路特异性抑制剂AG490阻断JAKSTAT3信号通路,观察AG490对ITF启动子活性的影响。结果:酶切和直接测序法证实包含ITF启动子的重组质粒构建成功;瞬时转染实验显示,ITF能显著提高ITF启动子的活性(P<0.05);加入AG490后ITF启动子活性显著下降(P<0.05)。结论:ITF通过JAK-STAT3途径上调自身启动子的活性。
