miR-200及其靶基因ZEB1/2在急性肺损伤后早期肺纤维化中上皮-间质转化(EMT)过程
The Effect of miR-200 and Their Targets ZEB1/2 on Epithelial-mesenchymal Transition(EMT) in Early Pulmonary Fibrosis Caused by Acute Lung Injury
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摘要: 目的 观察脂多糖(lipopolysaccharide,LPS)诱发小鼠急性肺损伤后早期肺纤维化过程中miR-200b/c及其靶基因ZEB1/2的表达变化.方法 应用LPS 3次打击的方法构建小鼠急性肺损伤后早期肺纤维化模型,分别于造模后第3、7、14、21天处死小鼠,留取肺组织备用.各组小鼠肺组织切片行HE和Masson染色并在光学显微镜下观察病理改变;Real-time PCR检测肺组织miR-200b、miR-200c、ZEB1 m RNA、ZEB2 m RNA表达;Western blot检测肺组织ZEB1、ZEB2、E-cadherin、Vimentin、α-SMA蛋白表达.结果 (1)病理结果:与对照组相比,LPS处理后第3天肺组织胶原纤维开始沉积,随着时间延长,肺纤维化程度逐渐加重;(2)Real time-PCR结果:随着肺纤维化程度加重,miR-200b、miR-200c水平均呈下降趋势,第7、14、21天时均显著低于对照组(P<0.01);ZEB1 m RNA、ZEB2 m RNA水平呈上升趋势,且ZEB2 m RNA较ZEB1 m RNA表达增加更显著;(3)Western blot结果:随着急性肺损伤后肺纤维化的进展,ZEB1、ZEB2蛋白表达亦升高,与其m RNA表达变化相一致;上皮标志物E-cadherin蛋白表达逐渐减少,间质标志物Vimentin、α-SMA蛋白表达逐渐增多.结论 在LPS诱发急性肺损伤后早期肺纤维化过程中miR-200b/c表达降低,并通过负性调控其靶控基因转录抑制因子ZEB1/2的表达促进上皮向间质转化.
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关键词:
- 脂多糖 /
- 急性肺损伤 /
- 早期肺纤维化 /
- miR-200b/c /
- ZEB1/2
Abstract: Objective To observe the expression profiles of miR-200b/c and their targets ZEB1/2 in early pulmonary fibrosis caused by acute lung injury induced by lipopolysaccharide in mice.Methods Early pulmonary fibrosis caused by acute lung injury is built via a lipopolysaccharide three-hit regimen.Mice were sacrificed at post-injective day 3, 7, 14, 21 respectively and the lung tissue specimens were collected.The lung tissue sections were stained with HE and Masson staining and pathological changes were observed by optical microscope.The expression profiles of miR-200 b, miR-200 c, ZEB1 m RNA and ZEB2 m RNA were detected by real-time PCR.Western blot was utilized to detect the levels of ZEB1,ZEB2,E-cadherin,Vimentin,α-SMA proteins.Results(1) pathological findings:compared with the control group,the collagen fibers deposited on on the third day after LPS treatment and pulmonary fibrosis gradually worsened;(2) Real time-PCR results:With theaggravation of pulmonary fibrosis,miR-200 b and miR-200 c levels were declined and the levels of miR-200b/c at post-injective day 7,14,21 were significantly lower than that of control group(P<0.01).ZEB1 m RNA and ZEB2 m RNA levels were gradually increased in the process of pulmonary fibrosis and the increased magnitude of ZEB2 m RNA was more significant than that of ZEB1 m RNA;(3) Western blot results:ZEB1 and ZEB2 protein levels were also gradually increased, consistent with their m RNA levels and the expression of E-cadherin protein was decreased while Vimentin and α-SMA protein levels increased with the evolution of pulmonary fibrosis caused by acute lung injury.Conclusion miR-200 b and miR-200 c promote epithelial-mesenchymal transition by inhibiting their targets ZEB1/2 in early pulmonary fibrosis caused by acute lung injury induced by LPS.-
Key words:
- LPS /
- Acute lung injury /
- Early pulmonary fibrosis /
- miR-200b/c /
- ZEB1/2
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急性阑尾炎是普外科最多见的急腹症之一，发病率可达8%[1]，过去临床上急性阑尾炎的诊断一直是以症状为主，依靠患者的病史、临床症状、体征和实验室检查，但30%患者早期临床症状并不典型[2]，且临床表现多样，需与多种腹痛疾病相鉴别，据报道，急性阑尾炎临床误诊率为4%～5%[3]。超声是诊断急性阑尾炎的一种简便、快捷的检查方法，本研究选取2019年1月至2020年6月所收治的423例临床急性阑尾炎患者的超声检查结果，术后所有患者均病理确诊为急性阑尾炎，分析探讨超声在急性阑尾炎诊断中的诊断价值。
1. 资料与方法
1.1 一般资料
回顾性分析2019年1月至2020年6月昆明医科大学第二附属医院所收治的423例右下腹痛患者资料，所有患者术前均行超声检查，术后均病理确诊为急性阑尾炎，将其按照不同病理类型分成急性单纯性阑尾炎、急性化脓性阑尾炎以及急性坏疽性阑尾炎三个组，临床表现为右下腹痛、脐周痛，麦氏点固定压痛，或伴发热、恶心、呕吐、乏力、稀便等症状，此423例患者均按以下标准纳入研究范围：（1）临床资料完整；（2）术前均行超声检查，术后均病理确诊为急性阑尾炎；（3）自愿参与，配合检查；（4）不伴有精神方面疾病，意识清楚；（5）无哺乳期及妊娠期妇女。本研究共纳入男性患者280例，女性患者143例，年龄7～60岁，平均年龄（29.5±2.8）岁。本研究经医学伦理会批准，患者签署知情同意书。
1.2 检查方法
全部患者均采用超声检查，患者采用平卧位，然后以麦氏点为中心进行多方位、多切面连续性探查[4]，观察阑尾的形态、管腔直径、管壁厚度、管壁层次及连续性，以及右下腹回盲部管壁增厚，局部积液，局部强回声，淋巴结肿大情况，加压探头观察是否变形，肠道是否扩张，同时测量其直径长度与管壁厚度，并记录下各指标数据。
1.3 统计学处理
采用IBM SPSS Statistics V21.0统计学软件对数据进行分析，计数资料用百分率（%）表示，计量资料用均数±标准差（
$ \bar x \pm s $ ）表示，组间差距采用χ2和q检验。P < 0.05为差异有统计学意义。2. 结果
2.1 诊断准确率比较
超声共检出374例，诊断率88.42%（374/423）。120例急性单纯性阑尾炎，准确率83.92%（120/143）；162例急性化脓性阑尾炎，准确率90.00%（162/180）；92例急性坏疽性阑尾炎，准确率92.00%（92/100）。超声诊断与病理检查结果对比有差异（P < 0.05），见表1。
表 1 不同病理类型急性阑尾炎超声诊断与术后病理诊断结果的对比（n）Table 1. Comparison of ultrasonographic diagnosis and postoperative pathological diagnosis of acute appendicitis in all patients（n）组别 病理诊断 超声诊断 符合率（%） 急性单纯性阑尾炎 143 120 83.9 急性化脓性阑尾炎 180 162 90.0 急性坏疽性阑尾炎 100 92 92.0 χ2 22.74 19.91 19.91 P 0.000 0.000 0.000 2.2 不同病理类型急性阑尾炎的影像学像特征
急性单纯性阑尾炎超声图像显示阑尾轻度肿大，管腔直径在5.80～8.40 mm之间，管壁厚度在2.30～4.10 mm之间，阑尾管壁层次清晰，连续性完整，横切面呈“同心圆”或“靶环征”样，受压后未发生变形，四周未显示明显液性暗区；彩超显示管壁的血流信号较稀疏，见图1A、1B，表2，表3。
图 1 不同病理类型急性阑尾炎的超声表现A、B：急性单纯性阑尾炎；C：急性化脓性阑尾炎；D：急性坏疽性阑尾炎。Figure 1. Ultrasonographic features of acute appendicitis with different pathological types表 2 超声在不同病理类型急性阑尾炎中的直接征象对比（$\bar x \pm s$ ）Table 2. Comparison of direct signs of ultrasonography in acute appendicitis of different pathological types（$\bar x \pm s$ ）组别 n 管腔直径（mm） 管壁厚度（mm） 管壁层次 管壁连续性 急性单纯性阑尾炎 120 7.1 ± 1.3 3.2 ± 0.9 清晰 完整 急性化脓性阑尾炎 162 10.0 ± 1.9 4.3 ± 1.1 欠清晰 欠完整 急性坏疽性阑尾炎 92 12.4 ± 2.2 5.3 ± 1.3 不清晰 连续性中断 q 16.52 13.43 P 0.001 0.010 表 3 超声在不同病理类型急性阑尾炎中的间接征象对比[n （%）]Table 3. Comparison of indirect signs of ultrasonography in acute appendicitis of different pathological types [n （%）]组别 n 回盲部管壁增厚 右下腹局部积液 右下腹局部强回声 右下腹淋巴结肿大 急性单纯性阑尾炎 120 62（51.7） 24（20.0） 8（6.7） 39（32.5） 急性化脓性阑尾炎 162 130（80.2） 105（64.8） 97（59.9） 81（50.0） 急性坏疽性阑尾炎 92 83（90.2） 86（93.5） 67（72.8） 61（66.3） χ2 26.45 50.07 71.52 14.63 P 0.000 0.000 0.000 0.001 急性化脓性阑尾炎超声图像显示阑尾肿胀明显，管腔直径在8.10～11.90 mm之间，阑尾管壁厚度在3.20～5.40 mm之间，阑尾管壁层次欠清晰，连续性欠完整，纵切面呈“腊肠样”结构，阑尾周围存在少量液性暗区，彩超显示管壁的血流信号较丰富，见图1C，表2，表3。
急性坏疽性阑尾炎超声图像显示阑尾肿胀，形态呈不规则性，管腔直径在10.20～14.60 mm之间，管壁厚度在4.00～6.60 mm之间，阑尾管壁层次模糊，回声杂乱，纵切面呈断续管样，阑尾周围、肠间隙以及盆腔可发现不规则的液性暗区，彩超显示阑尾管壁未有血流信号，见图1D，表2，表3。
3. 讨论
近年超声检查在临床上急性阑尾炎患者的应用中逐渐增多，与CT相比，超声具有无创伤性、可重复性高、分辨率高等优点，能够清楚显示阑尾管腔直径，管壁厚度，管壁层次和连续性，周围渗出情况，并依据超声图像特征快速判断患者的病情[5-6]，Whang等[7]研究表明超声检查是急性阑尾炎的有效首要诊断工具，对超声检查结果不确定的患者再行CT检查。本研究中超声诊断的准确率为88.42%（374/423），与Trout等[8]报道的准确率85%～91%相符，各研究报道差异较大，可能与阑尾炎病理类型、超声直接及间接征象的选择有关。
急性阑尾炎主要是由于淋巴滤泡增生，阑尾管腔粪石阻塞，细菌入侵损伤粘膜上皮而发生急性炎性病变，不同的发展过程呈现出不同的病理类型，随着阑尾管腔直径不断增大，管壁厚度不断增粗，阑尾管腔可在早期表现为充血水肿，进而腔内大量积脓，压力升高，致使阑尾壁血液循环障碍，层次消失，局部坏死穿孔[9]。Augustin等[10]研究发现，55岁以上的阑尾炎患者，症状持续时间 < 36 h的穿孔率为29%，36～48 h的穿孔率为67%，穿孔率随临床症状持续的时间延长呈指数增加，临床上可通过超声动态观察管腔直径和管壁厚度，管壁层次及连续性的变化，判断阑尾是否穿孔，并尽早选择手术治疗。有学者研究发现超声间接征象能确切反映阑尾周围组织炎症，其阳性率与炎症程度有关[11]，本研究中，急性坏疽性阑尾炎和急性化脓性阑尾炎合并回盲部管壁增厚，右下腹局部积液，局部强回声，淋巴结肿大的比例均高于急性单纯性阑尾炎，说明间接征象指标阳性率越高，阑尾周围组织的炎性浸润越重，预后也就越差。
本研究中急性化脓性阑尾炎和急性坏疽性阑尾炎超声诊断准确率较高，可能与其炎性病变渗出液较多，累及范围较广、层次较深有关，急性单纯性阑尾炎超声诊断准确率较低，可能是炎症早期，阑尾肿胀不明显，超声图像缺乏相应特异性。分析本组资料中的漏诊与误诊，主要是由于阑尾位于盲肠后位，受肠气干扰，显示不清，阑尾穿孔后管径回缩，炎性渗出，周围大网膜包裹，显示不清，除此之外，体型肥胖，有下腹部手术史，腹腔出现粘连等也可造成漏诊[12-13]。针对这种情况，临床上可采用高低频探头联合应用[14-15]、改变体位、探头加压推开肠管减少肠气的干扰[16]，同时将患者的病史、临床症状、体征及实验室检查，结合超声图像特征综合分析，扩大探查范围，排外其他外科急腹症（如胃十二指肠溃疡穿孔、右侧输尿管结石、妇产科疾病、急性肠系膜淋巴结炎、急性胃肠炎、胆道系统感染性疾病、回盲部肿瘤、Meckel憩室等）[17-18]，以此提高急性阑尾炎的诊断准确率，避免漏诊误诊的发生。有学者报道胃癌阑尾转移并发急性化脓性阑尾炎发作并穿孔1例[19]，患者以发热为首发症状，无典型转移性右下腹痛症状，有可能误诊为急性阑尾炎及阑尾原发肿瘤，患者发病隐匿，需紧密结合患者详细病史、体征以及相关影像学检查，综合分析患者病情，以免误诊及漏诊。
综上所述，急性化脓性阑尾炎及急性坏疽性阑尾炎的超声诊断准确率较高，阑尾肿胀明显，管腔直径、管壁厚度均高于急性单纯性阑尾炎，并且阑尾周围组织炎症的严重程度也明显高于急性单纯性阑尾炎，超声能够准确测量管腔直径，管壁厚度，清晰地观察阑尾位置、肿胀情况以及各壁层结构的改变，腔内回声特征，右下腹回盲部是否增厚，周围有无积液积脓，从而更深层次详细地了解阑尾炎症进展，故临床上可将急性阑尾炎患者的超声图像特征作为连续动态观察患者病情变化的手段，准确地诊断阑尾炎的病理分型及病变严重程度，并为治疗及预后提供相应的指导。
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