肠三叶因子对新生鼠NEC模型PI3K和caspase-3/9的影响

Objective To study the effects of ITF on PI3K (phosphatidylinositol 3-kinase) and caspases-3/9 (cysteinyl aspartate-specific protea-ses) in neonatal rat NEC model. Methods NEC model of neonatal rats was established. Asphyxia stress was accomplished by exposure to l00% nitrogen for 60s, followed by exposure to coldness (4 ℃ ) for 10 min twice daily. Neonatal rats were fed formula (200 kcal/kg/day) every 3 h via an gavage tube. The feeding volume began at 0. 1 cc every 3 h and was increased incrementally. This procedure is done once everyday and continued for 3 days. On the 4th day,all the subjects were sacrificed. Fifty neonatal rats were randomized into five groups: A) NEC + NS0. 2mi, (B) NEC + ITF 0. 2 mg, (C) NEC(Wortmannin (0. 1 mg/kg) (D) NEC + ITF 0. 2 mg + Wortmannin (0. 1 mg/kg), (E) control. The intestinal tissue located at the boundary of ileum and cecum was sampled for histology. The remaining intestinal tissue was homogenized. After the homogenate was centrifuged,supernatants were used to assay PI3K and Caspase-3 and caspase-9. Results The pathological lesions showed that intestinal necrosis was severe in group A、C and D, which was graded as 3points. They were significantly decreased in group B,which was graded 1 point. The level of PI3K(pg/ml)in Group A was higher than those in group E (P<0. 05), the latter had difference with those in group D(P>0. 05). The level of PI3K(pg/ml) were the strongest in group B and were lowest in group C. The activity of Caspase-3 and Caspase-9 were significantly increased in group A compared to those in group B and E (P<0. 01 ),and was significantly increased in Group C(P<0. 01 ). Caspase-3 and Caspase-9 levels of group A and D,group B and E showed no significant difference(P>0. 05). Conclusions Intestinal inflammation was ameliorated by intraperitoneal ITF. ITF may provide a new therapy for NEC; PI3K/Akt signal pathways might play important roles in signal transduction during NEC;ITF may protect the intestinal injury of neonatal Wistar rat by activation of PI3K/Akt signal transduction pathway, and down regulation of caspase-9.     

肠三叶因子对新生鼠NEC模型PI3K和caspase-3/9的影响

Objective To study the effects of ITF on PI3K (phosphatidylinositol 3-kinase) and caspases-3/9 (cysteinyl aspartate-specific protea-ses) in neonatal rat NEC model. Methods NEC model of neonatal rats was established. Asphyxia stress was accomplished by exposure to l00% nitrogen for 60s, followed by exposure to coldness (4 ℃ ) for 10 min twice daily. Neonatal rats were fed formula (200 kcal/kg/day) every 3 h via an gavage tube. The feeding volume began at 0. 1 cc every 3 h and was increased incrementally. This procedure is done once everyday and continued for 3 days. On the 4th day,all the subjects were sacrificed. Fifty neonatal rats were randomized into five groups: A) NEC + NS0. 2mi, (B) NEC + ITF 0. 2 mg, (C) NEC(Wortmannin (0. 1 mg/kg) (D) NEC + ITF 0. 2 mg + Wortmannin (0. 1 mg/kg), (E) control. The intestinal tissue located at the boundary of ileum and cecum was sampled for histology. The remaining intestinal tissue was homogenized. After the homogenate was centrifuged,supernatants were used to assay PI3K and Caspase-3 and caspase-9. Results The pathological lesions showed that intestinal necrosis was severe in group A、C and D, which was graded as 3points. They were significantly decreased in group B,which was graded 1 point. The level of PI3K(pg/ml)in Group A was higher than those in group E (P<0. 05), the latter had difference with those in group D(P>0. 05). The level of PI3K(pg/ml) were the strongest in group B and were lowest in group C. The activity of Caspase-3 and Caspase-9 were significantly increased in group A compared to those in group B and E (P<0. 01 ),and was significantly increased in Group C(P<0. 01 ). Caspase-3 and Caspase-9 levels of group A and D,group B and E showed no significant difference(P>0. 05). Conclusions Intestinal inflammation was ameliorated by intraperitoneal ITF. ITF may provide a new therapy for NEC; PI3K/Akt signal pathways might play important roles in signal transduction during NEC;ITF may protect the intestinal injury of neonatal Wistar rat by activation of PI3K/Akt signal transduction pathway, and down regulation of caspase-9.     

肠三叶因子对新生鼠坏死性小肠结肠炎Bim和Bcl-xl基因表达的影响及意义

目的 分析肠三叶因子(ITF)对坏死性小肠结肠炎(NEC)新生鼠肠黏膜组织Bim基因与Bcl-xl基因表达水平的影响,探讨ITF对NEC的保护作用机制.方法 采用简单随机法将30只新生鼠分为对照组、NEC组及ITF组,每组10只,对照组不作处理,NEC组建立NEC模型后予以腹腔注射生理盐水0.2ml;ITF组为NEC模型后予以腹腔注射ITF 0.2 mg.对切片行HE染色并作组织病理学检查及免疫组织化学法检测Bim基因与Bcl-xl基因表达,并进行图像分析.结果 病理切片显示NEC组肠壁损伤轻重不一,可见全肠黏膜绒毛坏死,病理组织学改变中位积分为3分;ITF组肠上皮细胞少量脱落,顶端绒毛坏死,病理组织学改变中位积分为1分;图像分析结果显示NEC组Bim基因表达(7.87±0.14)高于对照组(2.15±0.28)及ITF组(3.27±0.34),三组比较差异均有统计学意义(P<0.05);ITF组Bcl-xl基因表达(11.23±0.22)高于对照组(1.89±0.28)及NEC组(2.51±0.13),三组比较差异均有统计学意义(P<0.05).结论 通过腹腔注射ITF可减轻NEC大鼠的肠道损伤,而ITF可能通过改变Bim基因与Bcl-xl基因表达水平保护NEC大鼠肠道损伤.     

肠三叶因子在新生鼠坏死性小肠结肠炎模型中对Bax和Bcl-2及Caspase-3表达的影响及意义

目的 通过研究肠三叶因子(ITF)对新生大鼠坏死性小肠结肠炎(NEC)模型肠组织病理学改变及肠道组织中蛋白酶Caspase-3、蛋白Bax和Bcl-2的含量变化,探讨ITF对NEC保护作用的可能机制.方法 30只新生1日龄Wistar大鼠随机分为3组,正常对照组、实验组、干预组,每组10只.实验组为NEC模型后加生理盐水0.2 ml腹腔注射;干预组为NEC模型后予以腹腔注射TTF 0.2 mg(0.2 ml);正常对照组未予处理.第4天处死所有大鼠,取肠组织待检,取近回盲部1～2 cm肠道组织,采用分光光度法检查Caspase-3的表达、采用免疫组化法检测肠道组织中Bax及Bcl-2的含量变化并做病理学检查.结果 实验组Caspase-3表达高于正常对照组和干预组(P均＜0.05),干预组与正常对照组比较差异无统计学意义(P＞0.05);实验组Bax表达高于正常对照组和干预组(P均＜0.05),干预组与正常对照组相近(P＞0.05);干预组Bcl-2表达高于正常对照组和干预组(P均＜0.05),实验组高于正常对照组(P＜0.05).正常对照组的肠组织病理学未见异常,病理评分为0分;实验组HE染色切片见肠壁损伤轻重不一,可见全肠黏膜绒毛坏死,病理评分的中位积分为3分;干预组肠上皮细胞少量脱落,顶端绒毛坏死,病理评分的中位积分为1分.与实验组比较,ITF治疗后NEC导致的组织病理学改变明显减轻.结论 注射ITF可通过减少Caspase-3、Bax表达和增加Bcl-2表达减轻NEC肠道损伤.     

肠三叶因子对坏死性小肠结肠炎新生鼠白细胞介素-6的影响

目的探讨肠三叶因子（ITF）对坏死性小肠结肠炎（NEC）新生大鼠肠黏膜组织IL-6水平的影响，分析ITF对NEC的保护作用机制。方法新生鼠32只随机分为4组，每组各8只，正常对照组（A组）；NEC模型组（B组）；NEC模型后予以皮下注射9g/L盐水0．2mL（C组）；NEC模型后予以皮下注射ITF0.2mg（D组）。取近回盲部1-2cm肠道组织固定包埋、切片、HE染色做病理学检查，其他肠道组织制备组织匀浆取上清液检测IL-6水平。结果B、C组组织匀浆IL-6的水平较A、D组显著增高（Pa〈0.05，0．01），A与D组无明显差异（P〉0．05）。病理切片显示C、D组HE染色切片见肠壁损伤轻重不一，可见全肠黏膜绒毛坏死，病理评分的中位积分为3分；D组肠上皮细胞少量脱落，顶端绒毛坏死，病理评分的中位积分为1分。结论通过皮下注射ITF可减轻NEC后的肠道炎性反应。ITF有可能为治疗NEC提供新的方法。     

肠三叶因子在新生鼠坏死性小肠结肠炎模型中对Bax和Bcl-2及Caspase-3表达的影响及意义

目的通过研究肠三叶因子(ITF)对新生大鼠坏死性小肠结肠炎(NEC)模型肠组织病理学改变及肠道组织中蛋白酶Caspase-3、蛋白Bax和Bcl-2的含量变化,探讨ITF对NEC保护作用的可能机制。方法30只新生1日龄Wistar大鼠随机分为3组,正常对照组、实验组、干预组,每组10只。实验组为NEC模型后加生理盐水0.2ml腹腔注射;干预组为NEC模型后予以腹腔注射ITF 0.2 mg(0.2 ml);正常对照组未予处理。第4天处死所有大鼠,取肠组织待检,取近回盲部1～2 cm肠道组织,采用分光光度法检查Caspase-3的表达、采用免疫组化法检测肠道组织中Bax及Bcl-2的含量变化并做病理学检查。结果实验组Caspase-3表达高于正常对照组和干预组(P均<0.05),干预组与正常对照组比较差异无统计学意义(P>0.05);实验组Bax表达高于正常对照组和干预组(P均<0.05),干预组与正常对照组相近(P>0.05);干预组Bcl-2表达高于正常对照组和干预组(P均<0.05),实验组高于正常对照组(P<0.05)。正常对照组的肠组织病理学未见异常,病理评分为0分;实验组HE染色切片见肠壁损伤轻重不一...     

新生鼠坏死性小肠结肠炎动物模型建立及评价

目的 利用新生SD大鼠建立缺氧、冷刺激、鼠乳代用品人工喂养等多因素造成的新生鼠坏死性小肠结肠炎动物模型,并对其进行评价.方法 按析因设计,32只新生SD大鼠出生48 h开始随机分成4组,每组动物各8只.A组采用鼠乳代用品人工喂养,并给予100%氮气缺氧90 s,4 ℃冷刺激10 min,每天2次,连续3 d;B组为单纯采用鼠乳代用品人工喂养,未进行缺氧冷刺激;C组采用鼠乳喂养,给予同样的缺氧冷刺激;D组为正常对照组,采用鼠乳喂养,未进行缺氧冷刺激.HE染色后光镜下观察回盲部近端肠组织形态学改变,采用肠损伤病理评分进行评价,组织学评分≥2分确定为坏死性小肠结肠炎(NEC).结果 A、B、C组新生SD大鼠相继出现腹泻、腹胀、萎靡、活动减少,生长减慢,A组程度最为严重.A、B、C、D 4组肠损伤病理评分(±s)分别为(3.25±0.89)分、(2.63±0.92)分、(1.13±1.36)分、(0.25±0.46)分,各组间差异均有统计学意义(H=19.30,P＜0.01).NEC发病率分别为100%、87.5%、37.5%和0.喂养方式和缺氧冷刺激为肠组织损伤病理评分值影响因素,两因素间无明显交互作用.结论 新生鼠在剔除鼠乳喂养,改用鼠乳代用品人工喂养的前提下,经过连续多次的缺氧冷刺激后,诱导的新生鼠坏死性小肠结肠炎模型与人类新生儿NEC临床特征、病理改变相一致,是一种理想的新生儿坏死性小肠结肠炎动物模型.     

肠细胞凋亡在新生鼠坏死性小肠结肠炎肠损伤中动态变化

目的 动态观察新生大鼠坏死性小肠结肠炎(NEC)发病过程中肠细胞凋亡率变化及其与肠损伤关系.方法 40只新生SD大鼠随机分成对照组(C)和模型组(M).对照组8只;模型组32只,在出生48 h开始给予鼠配方奶人工喂养,100%氮气缺氧90 s,4℃冷刺激10 min,每天2次,连续3 d,建立新生大鼠NEC模型;模型组开始造模后24 h(M24)、48 h(M48)、72 h(造模结束,M72)及造模结束后24 h(M96)分别处死8只,留取肠管进行肠组织损伤评分和肠细胞凋亡率检测(流式细胞仪).组织学评分≥2确定为NEC.各组随机选取1份回盲部近端小肠标本进行肠黏膜透射电镜检查.采用SPSS 11.0统计学软件进行统计分析,α =0.05为显著性检验标准.结果 透射电镜显示模型组大鼠肠黏膜出现大量凋亡细胞,形成凋亡小体.对照组、M24、M48、M72和M96肠组织损伤评分分别为(0.08±0.15)、(1.38±0.42)、(1.46±0.69)、(1.58±0.30)分和(3.33±0.59)分,肠细胞凋亡率分别为4.8%±2.9%、12.8%±6.3%、14.9%±5.5%、17.7%±5.5%和27.6%±9.9%.肠损伤程度与肠细胞凋亡率呈显著正相关(r＜凋亡率=0.853,P＜0.01).结论 新生鼠肠细胞凋亡增加是NEC肠组织损伤起始事件;随时间延长,肠细胞凋亡增加程度进一步加重;肠细胞凋亡增加是造成新生鼠NEC肠道进行性损伤的病理基础.     

肠三叶因子对坏死性小肠结肠炎新生大鼠白细胞介素-1β的影响

目的 探讨肠三叶因子(ITF)对坏死性小肠结肠炎(NEC)新生大鼠肠黏膜组织IL-1β水平的影响,分析ITF对NEC的保护作用.方法 50只新生鼠随机分为5组,每组10只:A组为正常对照组,未进行缺氧、低温处理和注射药物;B组为正常ITF组,予皮下注射ITF0.2 mg(0.2 mL);C组为NEC模型组,建立NEC模型后未予药物注射;D组为NEC加9 g/L盐水(NS)组,建立NEC模型后予皮下注射NS 0.2 mL;E组为NEC加ITF组,建立NEC模型后予皮下注射ITF0.2 mg(0.2 mL).第4天断头处死动物后取其回盲部肠组织1～2 cm固定、包埋,HE染色行病理学检查,其他肠道组织制备组织匀浆取上清液应用ELISA试剂盒榆测IL-1β水平.结果 病理切片示C、D组HE染色切片见肠壁损伤轻重不一,可见全肠黏膜绒毛坏死,病理组织学积分为2～4分,E组肠上皮细胞有少量脱落,顶端绒毛坏死,病理组织学积分为0～2分.C、D组组织匀浆IL-1β水平均较E组显著增高(Pa＜0.01);E组与A、B组比较均无显著性差异(Pa ＞0.05).结论 ITF下可通过降低IL-1β的水平减轻NEC模型的肠道炎性反应,ITF有可能为NEC的治疗提供新方法.     

短链脂肪酸对新生鼠肠黏膜屏障的作用研究

目的 通过在体外检测生理浓度短链脂肪酸(SCFAs)作用下的新生鼠肠黏膜,探索SCFAs对于维持正常肠黏膜屏障的作用.方法 取SD大鼠近端结肠组织,分断奶前组(n=8,日龄13～17 d),断奶后组(n=6,日龄24～31 d).在尤斯灌流系统内37℃水浴(95％ O2,5％ CO2),黏膜侧水槽分别加入含乙酸Kreb液(乙酸浓度分别为0、20、80 mmol/L,pH 6.0)或者是含丁酸Kreb液(丁酸浓度分别为0、5、20 mmol/L,pH 6.0).实时记录跨上皮电阻(TER),并检测不同时间段(0、30、60、90、120 mins)黏膜对侧FITC-dextran浓度.结果 在未加入任何SCFAs情况下,90 min后断奶后组与断奶前组相比,结肠黏膜透过率明显降低,而TER明显升高(P＜0.01).从120 min的实时记录来看,在黏膜侧加入80 mmol/L乙酸后,两组的TER均出现显著上升(P＜0.05),但在丁酸实验中未发现如此明显效应(P＞0.05).结论 断奶前的新生鼠肠黏膜屏障尚未完全成熟;在新生鼠结肠离体模型中,生理浓度乙酸在维持正常肠黏膜屏障功能比丁酸更为有效.     

新生鼠坏死性小肠结肠炎动物模型建立及评价

目的利用新生SD大鼠建立缺氧、冷刺激、鼠乳代用品人工喂养等多因素造成的新生鼠坏死性小肠结肠炎动物模型,并对其进行评价。方法按析因设计,32只新生SD大鼠出生48h开始随机分成4组,每组动物各8只。A组采用鼠乳代用品人工喂养,并给予100%氮气缺氧90s,4℃冷刺激10min,每天2次,连续3d;B组为单纯采用鼠乳代用品人工喂养,未进行缺氧冷刺激;C组采用鼠乳喂养,给予同样的缺氧冷刺激;D组为正常对照组,采用鼠乳喂养,未进行缺氧冷刺激。HE染色后光镜下观察回盲部近端肠组织形态学改变,采用肠损伤病理评分进行评价,组织学评分≥2分确定为坏死性小肠结肠炎(NEC)。结果A、B、C组新生SD大鼠相继出现腹泻、腹胀、萎靡、活动减少,生长减慢,A组程度最为严重。A、B、C、D4组肠损伤病理评分(x-±s)分别为(3.25±0.89)分、(2.63±0.92)分、(1.13±1.36)分、(0.25±0.46)分,各组间差异均有统计学意义(H=19.30,P<0.01)。NEC发病率分别为100%、87.5%、37.5%和0。喂养方式和缺氧冷刺激为肠组织损伤病理评分值影响因素,两因素间无明显交互作用。结论新生鼠在剔除鼠乳喂养,改用鼠乳代用品人工喂养的前提下,经过连续多次的缺氧冷刺激后,诱导的新生鼠坏死性小肠结肠炎模型与人类新生儿NEC临床特征、病理改变相一致,是一种理想的新生儿坏死性小肠结肠炎动物模型。     

干细胞治疗坏死性小肠结肠炎的研究进展

坏死性小肠结肠炎(necrotizing enterocolitis,NEC)是新生儿尤其是早产儿、低出生体重儿的常见肠道疾病.随着现代医疗技术的进步,国内早产儿、极低出生体重儿的救治成功率增加,而NEC发病率有所上升,病死率仍较高.目前临床上对NEC的主要管理措施是保守治疗和手术治疗.干细胞治疗NEC虽仍处于动物实验阶段,其治疗机制尚未完全清楚,但干细胞逐渐成为NEC治疗的研究热点,可能成为新突破.干细胞治疗NEC用于临床实践之前,还需要更多的实验研究支持.     

肠细胞凋亡在新生鼠坏死性小肠结肠炎肠损伤中动态变化

目的动态观察新生大鼠坏死性小肠结肠炎(NEC)发病过程中肠细胞凋亡率变化及其与肠损伤关系。方法40只新生SD大鼠随机分成对照组(C)和模型组(M)。对照组8只;模型组32只,在出生48h开始给予鼠配方奶人工喂养,100%氮气缺氧90s,4℃冷刺激10min,每天2次,连续3d,建立新生大鼠NEC模型;模型组开始造模后24h(M24)、48h(M48)、72h(造模结束,M72)及造模结束后24h(M96)分别处死8只,留取肠管进行肠组织损伤评分和肠细胞凋亡率检测(流式细胞仪)。组织学评分≥2确定为NEC。各组随机选取1份回盲部近端小肠标本进行肠黏膜透射电镜检查。采用SPSS11.0统计学软件进行统计分析,α=0.05为显著性检验标准。结果透射电镜显示模型组大鼠肠黏膜出现大量凋亡细胞,形成凋亡小体。对照组、M24、M48、M72和M96肠组织损伤评分分别为(0.08±0.15)、(1.38±0.42)、(1.46±0.69)、(1.58±0.30)分和(3.33±0.59)分,肠细胞凋亡率分别为4.8%±2.9%、12.8%±6.3%、14.9%±5.5%、17.7%±5.5%和27.6%±9.9%。肠损伤程度与肠细胞凋亡率呈显著正相关(r凋亡率=0.853,P<0.01)。结论新生鼠肠细胞凋亡增加是NEC肠组织损伤起始事件;随时间延长,肠细胞凋亡增加程度进一步加重;肠细胞凋亡增加是造成新生鼠NEC肠道进行性损伤的病理基础。     

益生菌防治新生儿坏死性小肠结肠炎机制研究进展

新生儿坏死性小肠结肠炎(necrotizing enterocolitis, NEC)是导致新生儿尤其是早产儿和低出生体重儿死亡的病因之一。NEC的发生与新生儿肠道菌群定植延迟和结构紊乱密切相关。近些年大量研究认为,益生菌可通过调节肠道菌群结构,增强肠道屏障功能,减少病原菌定植和迁移,同时促进新生儿肠道免疫细胞发育和功...     

Differentiation of food protein-induced enterocolitis syndrome and necrotizing enterocolitis in neonates by abdominal sonography

ObjectivesTo summarize and differentiate abdominal ultrasound findings of necrotizing enterocolitis and food protein-induced enterocolitis syndrome.MethodsFrom January 2017 to December 2018, the abdominal ultrasound results of 304 cases diagnosed necrotizing enterocolitis or food protein-induced enterocolitis syndrome were retrospectively analyzed. The presence of pneumatosis intestinalis, portal venous gas, bowel wall thickening, intestinal motility, focal fluid collections and hypoechoic change of gallbladder wall were calculated, and the results were compared and analyzed.ResultsPneumatosis intestinalis, portal venous gas, bowel wall thickening, intestinal motility weakened/absent, focal fluid collections and hypoechoic change of gallbladder wall can be found in both necrotizing enterocolitis and food protein-induced enterocolitis syndrome infants. However, in infants with necrotizing enterocolitis, intestinal motility was weakened/absent in whole abdomen, and in food protein-induced enterocolitis syndrome, it only involved isolated segment of bowel. The positive rates of above signs in necrotizing enterocolitis infants were significantly higher than those in food protein-induced enterocolitis syndrome (p < 0.01). Moreover, it was observed that the rate of weakened intestinal motility besides the lesion segment of bowel in necrotizing enterocolitis infants was 100%, and in food protein-induced enterocolitis syndrome infants, it was 0%, which is supposed to be a main sign for identification.ConclusionIn the early stage, abdominal ultrasound can be used to differentiate necrotizing enterocolitis and food protein-induced enterocolitis syndrome.     

肠三叶因子对新生大鼠坏死性小肠结肠炎的保护作用(英文)

目的　肠三叶因子(ITF)对消化道粘膜上皮细胞的损伤修复具有重要作用。本实验通过研究ITF 对新生大鼠坏死性小肠结肠炎(NEC)模型肠组织病理学改变,环氧合酶 2(COX 2)表达及前列腺素E2(PGE2)、血 栓素B2(TXB2)生成的影响,以探讨ITF对NEC是否有治疗作用。方法　40只新生1日龄Wistar大鼠随机分为5 组,每组8只。A组为正常对照组。B和C组大鼠为NEC模型鼠,分别予以0.5mL生理盐水腹腔或0.2mL皮下注 射。D和E组大鼠亦为NEC模型鼠,分别予以0.5mLITF(0.5mg)腹腔或0.2mL(0.2mg)皮下注射。连续3天 新生大鼠予以缺氧-复氧处理制成NEC模型。第4天处死所有大鼠,取肠组织检查组织病理学改变,COX 2表达 和PGE2与TXB2的生成。结果　A组的肠组织病理学未见异常,病理评分为0分。与相应的NEC组(B和C组) 比较,ITF治疗后(D和E组)NEC导致的组织病理学改变明显减轻(P<0.01)。B和C组的病理学评分为1～4 分,而D和E组评分为0～2分。与A组比较,B和C组PGE2与TXB2浓度显著增高,但ITF治疗后(D和E组)显 著下降,与A组无明显差异。免疫组化结果显示B和C组的COX 2表达显著高于A,D,E组(P<0.05)。D和E 组弱表达COX 2,其强度高于A组,但显著低于B和C组。结论　ITF通过抑制COX 2的表达,减少了PGE2和 TXB2含量,减轻肠组织炎