新生儿坏死性小肠结肠炎的临床分析
The Clinical Analysis of Necrotizing Enterocolitis
-
摘要: 目的 对2010年5月至2013年5月及2013年6月至2016年6月云南省第一人民医院儿科新生儿病区诊断为坏死性小肠结肠炎 (necrotizing enterocolitis, NEC) 的患儿进行临床分析及比较, 了解NEC的发生率、NEC分期与预后的关系, 并对前后3 a NEC的转归进行比较分析, 为以后的临床工作提供诊治经验.方法 对云南省第一人民医院诊断为NEC的患儿临床资料进行回顾性分析, 按照改良Bell分级诊断标准进行病情分期, 用SPSS统计软件包, 并对前后3 a NEC患儿的早期诊断率、转归进行比较差异有统计学意义 (P<0.05) .结果 前3 a云南省第一人民医院共诊断了40例NEC患儿, 1发病率早产儿与足月儿经比较差异有统计学意义 (P<0.05) .治疗方法中32例内科保守治, Ⅰ期与Ⅱ期内科治疗比较差异有统计学意义 (P<0.05) .10例手术治疗的患儿, 外科治疗效果比较差异无统计学意义 (P>0.05) , Ⅱ期和Ⅲ期病人治疗的有效率无统计学意义.病死率比较差异有统计学意义 (P<0.05) .后3 a该院共诊断为NEC患儿41例, 发病率早产儿与足月儿经比较差异有统计学意义 (P<0.05) .35例经内科保守治疗, Ⅰ期与Ⅱ期内科治疗比较, 差异有统计学意义 (P<0.05) .4例手术治疗患儿, Ⅱ期与Ⅲ期手术治愈率比较差异无统计学意义, 前后3 a病死率比较差异有统计学意义 (P<0.05) , 住前后3 a早期诊断率比有统计学意义 (P<0.05) .结论 早期诊断NEC是降低病死率的关键, 而肠型脂肪酸结合蛋白 (I-FABP) 是早期诊断NEC的血清学指标, 降低早产儿发生率是降低NEC发病率的重要因素.Abstract: Objective From May 2010 to May 2013 and from June 2013 to June 2016, we compared the clinical analysis of patients with necrotizing enterocolitis (NEC) in the Department of Pediatrics, studied incidence rate of NEC and the relationship between stages of NEC and prognosis, and compared the prognosis between the two three-year periods to provide the experience of diagnosis and treatment for clinical practice in the future. Me thods The clinical data of patients diagnosed with NEC were retrospectively analyzed according to the diagnostic criteria of modified Bell staging classification using SPSS statistical software package. Early diagnosis rate and prognosis were compared between the two three-year periods. P <0.05 showed that statistical significance was found. Re s ults From May 2010 to May 2013, 40 cases of NEC were diagnosed in our hospital, among which 8 was in the first stage (20%) , 24 in the second stage (60%) , and 8 in the third stage (20%) . The number of premature infants was 32, accounting for 80%, among which 25 cases were infants with very low birth weight accounting for 62.5% and 8 cases were full-term infants, accounting for 20% (P < 0.05) . In the study, 32 cases (80%) received traditional treatment, which was effective in stage I. During the second stage, two abandoned the treatment was confirmed death in the follow-up, two underwent surgery successfully due to the progression of the disease and four (25%) died in the hospital. Statistic significance of treatment of internal medicine was found when comparing stage I with stage II (P<0.05) . Ten children underwent surgery, among which one gave up treatment after the surgery because of financial problems and was found dead in the follow-up and four (60%) died in the hospital. The comparison of surgical treatment in patients in stage II and III showed no statistical significance (P>0.05) . Fatality rates were statistically significant (P<0.05) . From June 2013 to June 2016, 41 children were diagnosed with NEC in our hospital, including 24 cases in stage I (59%) , 14 in stage II (34%) , and 3 in stage III (7%) . The number of premature infants was 33, accounting for 80%, among which 26 cases were infants with very low birth weight accounting for 62.5% and 8 cases were full-term infants, accounting for 20% (P<0.05) . In the study, 35 cases (85.3%) received traditional treatment, which was effective in stage I. During the second stage, one underwent surgery successfully due to the progression of the disease. Statistic significance of treatment of internal medicine was found when comparing stage I with stage II (P<0.05) . Four children underwent surgery, among which one gave up treatment after the surgery because of financial problems and was found dead in the follow-up and the surgery was successful in other three. The comparison of surgical treatment in patients in stage II and III showed statistical significance (P <0.05) . Fatality rates were statistically significant comparing the two three-year periods (P<0.05) . The comparison of the rates of early diagnosis in the two three-year periods showed statistically significance (P <0.001) . Conclus ion Early diagnosis of NEC is the key to reduce mortality.Intestinal fatty acid binding protein (I-FABP) is a serological marker for early diagnosis of NEC. As an important factor, the reduction of the incidence of premature birth will result in the drop in the incidence of NEC.
-
九十年代初期的研究表明，在动脉粥样硬化（atherosclerosis，AS）形成过程中，动脉内膜和外膜层聚集着肥大细胞（mast cells，MCs），首次提出MCs参与AS进程[1]。
血管的微环境一旦发生变化，炎性细胞便会从血管外膜的滋养血管中渗出，参与疾病的变化[2-3]。MCs激活时释放出来的趋化因子、细胞因子、组胺、白三烯等炎症因子[4]使血管的通透性增加，导致单核巨噬细胞和淋巴细胞聚集到斑块处[5]，释放出大量的中性蛋白酶，可以加速低密度脂蛋白（low-density lipoprotein，LDL）进入到血管内膜局部并聚集[6]，促进AS斑块形成。
在骨髓细胞向MCs分化及活化过程中白细胞介素3（interleukin-3，IL-3）和干细胞因子（stem cell factor，SCF）起到重要作用[7-8]。本研究旨在通过提取乳兔骨髓细胞，利用IL-3、SCF、β-巯基乙醇联合作用诱导培养骨髓来源肥大细胞并鉴定其纯度与活性，为进一步研究肥大细胞与AS的关系提供依据。
1. 材料与方法
1.1 材料
IL-3、SCF（美国Peprotech公司）；FBS、DMEM、DMEM/F12、RPMI-1640（美国Gibco公司）；实验动物：2周龄乳兔，昆明医科大学实验动物学部，环境条件符合国家实验动物环境及设施标准要求，室内保持安静、清洁、干燥和通风。自由饮水。实验动物使用许可证号：SCXK（滇）2015-0002。
1.2 方法
1.2.1 兔骨髓细胞的获取
麻醉：（经2周龄乳兔耳缘静脉注射3%戊巴比妥钠，1 mL/kg）→浸泡（75%C2H5OH，10 min）→剥离（后肢皮肤切开，逐层分离，剥离肌肉）→暴露股骨→取出→浸泡（75% C2H5OH的无菌培养皿中浸泡3～5 min）→股骨两端剪去→吹打骨髓（吸取DMEM高糖培养基后吹打骨髓，股骨变白）→获取细胞（1000 r/min，5 min。如果在获取的细胞中有较多红细胞，则应用红细胞裂解液进行处理）→培养体系（含84 mL DMEM高糖培养基、15 mL FBS、1 mL双抗）。
1.2.2 细胞的培养
接种到T25瓶中（含DMEM高糖培养基）→隔天换液→细胞铺满→消化细胞→新培养瓶→贴壁→改用F12诱导培养基（含84 mL DMEM/F12、15 mL FBS、1 mL双抗、10 ng/mL SCF、10 ng/mL IL-3及0、10×10-5 mol/L的β-巯基乙醇）→放置37 ℃、5% CO2 饱和湿度环境→隔天换液→镜下观察细胞有变圆→更换为1640促成熟培养基（含84 mL RPMI 1640、15 mFBS、1 mL双抗、10 ng/mL SCF、10 ng/mL IL-3及0、10×10-5 mol/L的β-巯基乙醇）→7 d换液→直至细胞诱导成熟。
1.2.3 肥大细胞功能学鉴定
诱导成熟的重悬细胞液→载玻片→干燥→甲苯胺蓝染色→95% C2H5OH脱色→清洗→镜下观察。
1.2.4 肥大细胞形态学鉴定
通过光镜下观察不同培养时间的细胞形态，直至诱导成熟。
消化细胞→洗2次（PBS）→细胞密度1×10 7个/mL→每管100 μL→单染管各加入5.0 μL的 CD117-PE抗体及FcεR1α-FITC抗体→双染管加两种抗体→空白对照管（无抗体）→避光孵育30 min→每管加1 mL PBS→离心10 min（400 g）→去上清→上法洗涤1次→去上清→每管加100 μL PBS→检测。
2. 结果
2.1 光镜下观察肥大细胞生长形态
2 d后开始出现以形态为长梭形的贴壁细胞；7 d时：细胞基本将培养瓶铺满；2周时：细胞逐渐变形；4周时：细胞基本变为圆形，悬浮细胞变多；6周时：细胞基本变为类圆形，且形态均匀具有折光性（图1）。
图 1 肥大细胞变形过程（100×）A：7 d时长梭形贴壁细胞；B：2周时细胞逐渐变形；C：4周时细胞逐渐变圆；D：6周时变为类圆形细胞。Figure 1. Changes in mast cells（100×）2.2 肥大细胞的甲苯胺蓝染色
培养6周细胞成熟后利用甲苯胺蓝染色：可以观察到细胞质为紫红色，细胞核为蓝色。异染颗粒的细胞加入β-巯基乙醇比不加入β-巯基乙醇的能得到更多（图2）。
图 2 甲苯胺蓝染色（100×）A：β-巯基乙醇浓度0 mol/L；B：β-巯基乙醇浓度10×10-5 mol/L。Figure 2. Toluene blue staining（100×）2.3 流式细胞术检测肥大细胞
培养6周收集细胞，利用流式细胞术检测细胞表面CD117及FcεR1α的表达情况，加入β-巯基乙醇双阳性细胞的比例达96.2%，大于不加入β-巯基乙醇的（图3），且与不加β-巯基乙醇相比有统计学意义（P < 0.05），见表1。
图 3 流式细胞仪检测细胞的双阳性率A：β-巯基乙醇为0 mol/L；B：β-巯基乙醇为10×10-5 mol/L。Figure 3. Double positive rate of cells detected by flow cytometry表 1 不同浓度β-巯基乙醇诱导后CD117+ FcεRIα+肥大细胞的比例Table 1. The proportion of CD117+ FcεRIα+ mast cells induced by different concentrations of β-mercaptoethanolβ-巯基乙醇
（mol/L）实验次数 CD117+ FcεRIα+
肥大细胞的比例（%）P 0 5 48.97 ± 3.12 0.7069 10×10−5 5 95.64 ± 4.66# 与β-巯基乙醇浓度为0 mol/L比较，#P < 0.05。 3. 讨论
在正常人的心脏、主动脉及脂肪组织中有少量的MCs存在，当人类发生疾病时MCs的数量就会增多，比如AS。近年来，研究表明MCs在AS的发生、发展及斑块稳定性中有着重要作用[9]。
当下有研究认为MCs是利用其表面的趋化因子受体-3与AS斑块中表达的嗜酸细胞活化CC趋化因子亚族中趋化因子配体-11结合而向病变部位聚集[10]。MCs起源于骨髓造血干细胞[11]，促进MCs成熟的分子主要有SCF和神经生长因子（nerve growthfactor，NGF）[17]，SCF可能在MCs向受累区域聚集发挥作用，而MCs侵入受累区域可能会进一步导致更多的炎性细胞浸润，促进AS斑块形成。
IL-3又被叫做肥大细胞生长因子，在MCs的生长、分化、迁移和效应中具有重要作用。活化T细胞、天然杀伤（NK）细胞和MCs都可产生IL-3，且对于SCF在MCs前体的发生、发展、扩增具有促进意义[12-13]。SCF的配体CD117（c-kit）除在其表面外，各种造血祖细胞中均可存在。MCs发挥重要表面标志的是FcεRIα，但FcεRIα还表达于嗜酸性粒细胞等细胞表面，只有MCs同时表达CD117和FcεRIα[14]。甲苯胺蓝染色是一种常用于识别及判断MCs功能状态的方法，同时也是MCs的特异性染色，可染细胞核为蓝色，胞质内为异染性紫红色颗粒，说明其具有吞噬功能[15]。通过甲苯胺蓝染色和流式细胞仪检测结果对诱导获得的MCs的纯度进行鉴定，就目前来说，利用兔的骨髓间质干细胞来诱导培养MCs的报道是极少的。
本研究选用2周龄的乳兔来获取骨髓间质干细胞，因为当兔龄大于或者小于2周龄时，笔者发现细胞生长速度变得缓慢，可能是2周龄的骨髓间质干细胞的活性更好，更有利于细胞的培养。当IL-3浓度10 ng/mL、SCF浓度为10 ng/mL，加入β-巯基乙醇时：甲苯胺蓝染色及流式检测均较不加入β-巯基乙醇时可以获取更多、双阳性率更高的MSc，原因可能为β-巯基乙醇作为一种还原剂，在降低氧对细胞产生氧化损伤的同时促进干细胞生长。所以该培养体系是一种良好的体外诱导培养体系，其操作简单，且能获得更加成熟、更具有典型特征的MCs，为下一步对兔行进炎性损伤研究提供了优势。综上所述，本研究利用形态学、功能学2个方面对诱导的兔骨髓来源MCs进行鉴定，培养出的细胞不仅具有成熟MCs的生物学特性，还具有其功能，这便为后续的基础研究及临床研究奠定基础。
-
[1]许光, 李碧香, 周崇高等.新生儿坏死性小肠结肠炎的外科治疗[J].医学临床研究, 2008, 25 (11) :2028--2031. [2] [2]TRINLZI F, PARAZZINI C, RIGHINI A.Patterna of damage in the nature neonatal brain[J].Pediatr Radio J, 2006, 36:608-620 [3]邵肖梅, 叶鸿瑁, 岳小汕.实用新生儿学[M].第4版.北京:人民卫生出版社, 2011:477. [4] [4]KAFETZIS DA, SKEVAKI C, COSTALOS C.Neonatal nerotizing enterocolitis;an overview[J].Curropin Znfect Dis, 2003, 16:345-355. [5]唐瑟, 李炜如, 熊英.早产极低出生体质量儿坏死性小肠结肠炎29例[J].实用儿科临床杂志, 2004, 1 (8) :670-671. [6]杨蕾.张冰.刘朝阳.45例坏死性小肠结肠炎临床分析[J].中国实用医药, 2011, 25 (8) :27-28. [7] [7] OCKNER R K.Historic overview of studies on fatty acid binding proteins[J].Science, 1972, 177 (43) :56-58. [8] [8]PELSERS M M, HERMENS W T, GLATA J F C.Fatty acid-binding proteina as plasma markers of tissue injury[J].Clin Chim Acta, 2005, 352 (1-2) :15-35. [9] [9] STORCH J, THUMSER A E.Tissure-specific functions in the FABP (fatty acid-binding protein) family[J].J Biol Chem, 2010, 285 (43) :32679-32683. [10] [10]MOCHIZUKI K, SURUGA K, YAGI E, et al.The expression of PPAR-associated genes is modulated through postnatal development of PPAR subtypes in the small intestine[J].Biophys Acta, 2001, 1531 (1-2) :68-76. [11] [11]LEVY M M, MENARD D, DELVIN E, et al.Localization, function and regulation of the two intestinal fatty acid-binding protein types[J].Histochem Cell Biol, 2009, 132 (3) :351-367. [12]田云粉, 李利, 正常新生儿血、尿肠型脂肪酸结合蛋白浓度的探讨[J].昆明医科大学学报, 2015, 36 (5) :52-54. -
下载:
点击查看大图
计量
- 文章访问数: 2464
- HTML全文浏览量: 879
- PDF下载量: 79
- 被引次数: 0
下载:
