延迟断脐对新生儿生理意义的研究现状

20世纪后，新生儿出生时立即结扎脐带为产科临床上的常规操作。但是，国内外大量研究结果表明，延迟断脐(DCC)可改善新生儿营养状况与健康结局，而且该项技术受到包括世界卫生组织(WHO)在内的多个国际专业机构及组织推荐。目前研究结果表明，DCC有益于新生儿，具有可以增加新生儿血容量、降低其贫血发生率及减少缺血缺氧性脑损伤等并发症发生率等优势。但是，对于DCC的最佳时机及其潜在益处、风险等问题，目前则尚未达成共识。关于DCC对新生儿生理意义的研究，不但可明确DCC的生理机制，还可更新医护人员对断脐时间的传统固有观念，对DCC的临床应用及推广具有积极作用。笔者拟就DCC时间选择、DCC与胎盘输血、DCC与新生儿呼吸功能及DCC对新生儿影响的最新研究现状进行阐述。     

Development of a standardized definition for Hirschsprung's-associated enterocolitis: a Delphi analysis

Purpose

The reported incidence of Hirschsprung's-associated enterocolitis (HAEC) is extremely variable. A standardized definition would permit comparison of different studies and provide an interpretable outcome measure for future prospective studies in patients with Hirschsprung's disease.

Methods

The Delphi method is a technique for achieving consensus among a panel of experts. A list of 38 potential criteria from the history, physical examination, radiologic studies, and pathologic specimens was made available to pediatric surgeons and gastroenterologists who have contributed to the literature on Hirschsprung's disease. Each expert ranked the diagnostic importance of each item using a Likert scale. In subsequent surveys, the same process was used, but the means and SDs from previous rounds were included as a way of influencing the experts toward consensus. Cronbach's α was used after each round to measure variability among the experts. Once consensus was reached, an overall “HAEC score” was developed by assigning a value of 1 or 2 to each item that was considered important by the expert panel. The score was then validated by circulating 10 clinical cases to the panel and asking if each represented HAEC or not.

Results

Twenty-seven experts completed the survey. Cronbach's α increased from 0.93 after the first round to 0.97 after the second. Criteria receiving the highest scores were diarrhea, explosive stools, abdominal distension, and radiologic evidence of bowel obstruction or mucosal edema. Eighteen items were included in the score. During the validation process, the score agreed with the experts in 9 of the 10 case scenarios.

Conclusion

The most important clinical diagnostic criteria for HAEC were identified from a larger pool of potential diagnostic items through a consensus approach using the Delphi method. A score was developed and validated and can now be used as a standardized and reproducible outcome measure for future studies in children with Hirschsprung's disease.     

The ITGB2 immunomodulatory gene (CD18), enterocolitis, and Hirschsprung's disease

Hirschsprung's disease (HSCR)–associated enterocolitis (HAEC) remains a major contributor to morbidity and mortality associated with HSCR, being sometimes difficult to diagnose in its subclinical form. Its pathogenesis appears to include impaired local defense mechanisms as well as dysfunctional immune response and leukocyte function. In this context, the ITGB2 (CD18) immunomodulation-related gene is a possible candidate in HAEC pathogenesis as it codes for the β-subunit of leukocyte adhesion molecule lymphocyte function-associated antigen 1, which has an established role in T-cell development and function. ITGB2/CD18 has also been linked to chronic colitis in both human and animal models involving defense mechanisms within colonic mucosa. There is therefore a fairly compelling case for the potential involvement of the ITGB2 (CD18) in HAEC pathogenesis.

Aim

The aim of this study was to investigate the ITGB2 immunomodulatory gene (CD18) in a cohort of patients with HSCR and explore its correlation with enterocolitis.

Patients and methods

Screening for mutations of the ITGB2 (CD18) gene was performed on DNA extracted from colonic tissue samples and whole blood of 33 HSCR patients controlled by analysis of 60 unaffected individuals from the diverse South African population. Polymerase chain reaction amplification was performed, followed by heteroduplex single-strand conformation polymorphism analysis and bidirectional semiautomated DNA sequencing analysis.

Results

Heteroduplex single-strand conformation polymorphism banding patterns of the ITGB2 gene showed variations in 22 HSCR patients (66%), 13 of whom had severe episodes of HAEC, and 6 others had milder symptoms. Of the 13, 6 (46%) had Down's syndrome–associated HSCR. Genetic variations included 1 mutation (D77N), 2 known (V367, V441), and 4 novel polymorphisms (−111T/C, 24G/T, 295G/A, 892A/G). Significant associations were identified in the exon 5′ untranslated promotor region (P < .0001), exon 10 (P < .0007), and the 3′ untranslated promotor region at 122G/A (P < .0001) and 370 G/T positions (P = .04). Those regions of the gene most frequently associated with HAEC and severe symptoms were those with more than 1 variant identified in the gene.

Conclusions

This study shows that impaired CD18 leukocyte and T regulatory cell regulation can probably be linked to a genetic (ITGB2) predisposition to HAEC. It furthermore provides a possible genetic link to HAEC patient selection, identifying a potential molecular target.     

生长抑素在先天性巨结肠术后小肠结肠炎治疗中的应用效果观察

目的 探讨生长抑素在先天性巨结肠（HD）术后小肠结肠炎治疗中的应用效果.方法 将2000-01~2011-01期间收治的HD术后出现小肠结肠炎27例患儿,随机分为生长抑素加常规治疗组（治疗组,12例）和常规治疗组（对照组,15例）,观察两组病例腹胀消失时间、呕吐消失时间、治愈时间等各项指标,对结果分别进行对比.结果 治疗组在腹胀消失时间、呕吐消失时间和治愈时间均较对照组提前.结论 生长抑素可全面抑制消化液的分泌,减压肠管扩张和缺血性改变;可明显改善HD术后小肠结肠炎的临床症状,提高保守治疗的成功率.     

肠三叶因子对新生鼠NEC模型PI3K和caspase-3/9的影响

目的 利用PI3K的特异性抑制剂Wortmannin阻断PI3K/Akt细胞信号转导通路,观察ITF对新生鼠NEC模型caspase-3及caspase-9水平的影响,探讨ITF对NEC的保护作用机制.方法 建立NEC模型,对新生一日龄Wistar大鼠予100%氮气,1 min后放4℃冷环境中持续10min,每日2次,随后放回自制的保温箱内人工喂养,第四天处死,取肠组织待检.新生鼠50只随机分为5组,每组10只,A组为NEC模型后予以腹腔注射生理盐水0.2 ml;B组NEC模型后予以腹腔注射ITF0.2 mg;C组为NEC模型后腹腔内注射wortmannin(0.1 mg/kg);D组为NEC模型后腹腔内注射ITF+wortmannin;E组为正常对照组.取近回盲部1～2 cm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应用ELISA试剂盒检测PI3K的含量及用分光光度法检测caspase-3/9的活性.结果 病理切片显示A组HE染色见肠壁损伤轻重不一,可见全肠黏膜绒毛坏死,病理评分中位积分3分;B组病变明显减轻,肠上皮细胞少量脱落,顶端绒毛坏死,病理评分中位积分1分;C、D组病理评分中位积分3分.A组组织匀浆中PI3K的含量(pg/ml)较E组略升高(P<0.05),与D组差异无统计学意义(P>0.05);B组PI3K表达水平较其他组显著升高(P<0.01);C组与E组比较显著降低,差异有统计学意义(P<0.01).Caspase-3及caspase-9活性在A组较B、E组明显升高,差异有统计学意义(P<0.01),C组更加明显升高(P<0.01),A、D组及B、E组之间,差异无统计学意义(P>0.05).结论 通过腹腔注射ITF可以减轻NEC后的肠道炎症反应,ITF有可能为治疗NEC提供新的方法 ;PI3K/Akt细胞信号转导通路参与了NEC发病过程,并起着信号转导作用.而ITF可能是通过激活PI3K/Akt信号转导途径来下调caspase-3/9的水平,从而保护新生Wistar大鼠NEC模型肠道损伤.     

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.     

新生鼠坏死性小肠结肠炎肠组织一氧化氮和一氧化氮合酶变化

目的动态观察新生大鼠坏死性小肠结肠炎(NEC)发病过程中肠组织一氧化氮(NO)含量、一氧化氮合酶(NOS)活性变化及其与肠损伤关系,为进一步阐明NEC发病机制、寻找新的治疗方法提供实验依据。方法40只新生SD大鼠按简单随机法分成模型组(M)32只,对照组(C)8只。模型组大鼠出生48h开始鼠配方奶人工喂养,并予以3次缺氧和冷刺激建立NEC模型,缺氧冷刺激开始后24h(M24)、48h(M48)、72h(造模结束,M72)及最后一次缺氧和冷刺激后24h(M96)分别空腹断头处死8只;实验结束时处死对照组大鼠,分别留取肠管进行肠组织损伤评分、肠组织中NO含量和NOS活性检测。结果建模后,模型组出现腹泻、腹胀、萎靡、活动减少。M24、M48、M72、M96及对照组肠组织损伤评分分别为(1.25±0.56)、(1.46±0.31)、(2.79±0.40)、(3.33±0.59)和(0.08±0.15)分,肠组织NO含量分别为(2.07±0.38)、(2.88±0.32)、(3.09±0.40)、(3.98±1.15)和(0.94±0.44)μmol/gprot,总NOS活性分别为(2.21±0.42)、(2.77±0...     

Are the long-term results of the transanal pull-through equal to those of the transabdominal pull-through? A comparison of the 2 approaches for Hirschsprung disease

Purpose

The transanal endorectal pull-through (TERPT) is becoming the most popular procedure in the treatment of Hirschsprung disease (HD), but overstretching of the anal sphincters remains a critical issue that may impact the continence. This study examined the long-term outcome of TERPT versus conventional transabdominal (ABD) pull-through for HD.

Methods

Records of 41 patients more than 3 years old who underwent a pull-through for HD (TERPT, n = 20; ABD, n = 21) were reviewed, and their families were thoroughly interviewed and scored via a 15-item post-pull-through long-term outcome questionnaire. Patients were operated on between the years 1995 and 2003. During this time, our group transitioned from the ABD to the TERPT technique. Total scoring ranged from 0 to 40: 0 to 10, excellent; 11 to 20 good; 21 to 30 fair; 31 to 40 poor. A 2-tailed Student t test, analysis of covariance, as well as logistic and linear regression were used to analyze the collected data with confidence interval higher than 95%.

Results

Overall scores were similar. However, continence score was significantly better in the ABD group, and the stool pattern score was better in the TERPT group. A significant difference in age at interview between the 2 groups was noted; we therefore reanalyzed the data controlling for age, and this showed that age did not significantly affect the long-term scoring outcome between groups.

Conclusion

Our long-term study showed significantly better (2-fold) results regarding the continence score for the abdominal approach compared with the transanal pull-through. The stool pattern and enterocolitis scores were somewhat better for the TERPT group. These findings raise an important issue about the current surgical management of HD; however, more cases will need to be studied before a definitive conclusion can be drawn.     

Chronic Colitis in IL-10-/- Mice: Insufficient Counter Regulation of a Th1 Response

IL-10-deficient (IL-10^-/-) mice, generated by a gene-targeted mutation, develop abnormal immune responses as a result of uncontrolled interactions between antigen presenting cells and lymphocytes. The studies reviewed herein have focused on the enterocolitis that spontaneously develops in IL-10^-/- mice. Not unexpectedly, heightened production of proinflammatory mediators accompanied pathologic changes in the gastrointestinal tract of young mutants. In a series of studies, the proinflammatory mediators responsible for initiating the pathogenic response were distinguished from those that were elicited as a consequence of persistent inflammation. We have also investigated the possibility that different mediators are involved in the inductive versus the maintenance phase of disease. The findings of these mechanistic studies as they relate to our understanding of progressive inflammatory disease and the role of IL-10 in controlling the acute and chronic stages are discussed.     

丁酸钠对大鼠小肠上皮细胞株-6基质金属蛋白酶-3表达的影响

目的 探讨丁酸钠对大鼠小肠上皮细胞株-6(intestinal epithelial cell line No.6,IEC-6)细胞基质金属蛋白酶-3(matrix metalloproteinase-3,MMP-3)和组织金属蛋白酶抑制物-1(tissue inhibitor of metalloproteinase-1,TIMP-1)表达的影响,为预防新生儿NEC提供理论依据.方法将对数生长期IEC-6细胞分3组进行实验,低浓度丁酸钠组:培养液中加入终浓度为2 mmol/L的丁酸钠;高浓度丁酸钠组:丁酸钠终浓度为14 mmol/L;另设不含丁酸钠的对照组.共培养24 h后获取细胞,采用逆转录聚合酶链反应技术检测MMP-3和TIMP-1 mRNA的水平.培养48 h后,采用Western印迹法检测MMP-3和TIMP-1蛋白的表达水平.组间差异比较采用单因素方差分析及Dunnett-t和Dunnett's T3进行两两检验.结果 高浓度丁酸钠组MMP-3 mRNA水平为0.566±0.140,MMP-3酶原水平为0.756±0.128,MMP-3活性形式蛋白水平为0.255±0.057,均较对照组(分别为0.166±0.041、0.312±0.057和0.042±0.038)明显升高,差异均有统计学意义(P均＜0.01).低浓度丁酸钠组MMP-3 mRNA水平(0.213±0.039)、酶原水平(0.407±0.088)和活性形式蛋白水平(0.103±0.050)与对照组差异均无统计学意义(P＞0.05).高、低浓度丁酸钠组和对照组TIMP-1 mRNA为0.427±0.042,0.383±0.043和0.355±0.048;蛋白水平分别为0.576±0.140,0.524±0.123和0.355±0.062,组间差异均无统计学意义(F=1.97和3.10,P均＞0.05).结论高浓度丁酸钠能选择性诱导IEC-6细胞MMP-3的表达,而低浓度丁酸钠对其没有明显影响.