非酒精性脂肪性肝病患者CD44定量检测及其临床价值

Quantitative detection and clinical value of CD44 in patients with nonalcoholic fatty liver disease

目的定量检测非酒精性脂肪性肝病(NAFLD)患者CD44表达并比较分析。方法于2018年5月至10月间收集南通大学附属医院住院慢性肝病伴/不伴NAFLD患者,包括慢性乙型肝炎、慢性乙型肝炎后肝硬化及肝细胞肝癌,以健康献血员作为正常对照;以流式细胞术分析CD44阳性细胞比例;用酶联免疫吸附法定量检测CD44水平,常规分析血清天冬氨酸转氨酶、丙氨酸转氨酶活性,总胆固醇和甘油三酯等生物化学指标;并按自身配对法收集肝癌伴/不伴NAFLD患者术后癌及癌旁组织,以蛋白质印迹法和组织化学法分析并以CD44积分光密度进行比较。用Image pro plus 6.0、Image J、Graphpad prism 5.0和Photoshop分析统计和绘制图片,Microsoft Excel和IBM SPSS Statistics 23软件分析及绘制表格,用独立样本t检验行组间差异比较。结果NAFLD患者伴肝细胞损伤、血脂异常;NAFLD患者、慢性肝病患者血清CD44水平均显著高于正常对照组(P<0.001);NAFLD患者CD44阳性淋巴细胞比率为(78.19%±16.33%),慢性乙型肝炎组为(68.47%±20.91%),均高于对照组(46.51%±20.52%);慢性乙型肝炎伴脂肪变组CD44浓度(181.42±49.36)ng/ml显著高于慢性乙型肝炎组CD44浓度(142.52±53.87)ng/ml和正常对照组(99.47±15.23)ng/ml;肝癌组CD44/GAPDH比值(1.306±0.614)显著高于癌旁组(0.477±0.291),两组比较,t=3.451,P=0.004,差异有统计学意义。NAFLD相关肝癌组CD44的积分吸光度值为25.721±5.881,癌旁组积分吸光度值为14.155±4.001,组间差异有统计学意义(t=14.544,P<0.001);肝细胞肝癌患者血CD44高表达的病理特征表现与HBV感染、肿瘤大小、单/多中心、淋巴结转移、分化程度、TNM分级、Child-Pugh分级、门静脉癌栓和肝外转移等显著相关;肝细胞肝癌伴NAFLD患者血CD44(234.62±69.40)ng/ml,显著高于不伴NAFLD患者(186.49±58.89)ng/ml(t=-3.191,P=0.002),但肝细胞肝癌患者伴NAFLD的血CD44高/低组间的临床病理特征差异均无统计学意义。结论结果提示CD44异常活化,其机制可能在NAFLD进展中发挥重要作用。

Objective To quantitatively detect CD44 expression in patients with nonalcoholic fatty liver disease(NAFLD)for comparative analysis.Methods Patients with chronic liver diseases accompanied with or without NAFLD,including chronic hepatitis B,cirrhosis and hepatocellular carcinoma after chronic hepatitis B,and healthy blood donors as normal controls who admitted to the Affiliated Hospital of Nantong University from May to October 2018 were selected.The proportion of CD44 positive cells was analyzed by flow cytometry.CD44 level was quantified by an enzyme-linked immunosorbent assay,and the biochemical indicators such as serum aspartate aminotransferase,alanine aminotransferase activity,total cholesterol and triglyceride were routinely analyzed.The cancerous and adjacent cancerous tissues of patients accompanied with or without NAFLD were collected by self-matching method and analyzed by immunoblotting and histochemistry and compared by CD44 integrated optical density.Image-Pro Plus version 6.0,Image J,GraphPad Prism 5.0,Photoshop,Microsoft Excel and IBM SPSS statistics 23 were used to analyze and draw pictures.An independent sample t-test was used to compare the differences between groups.Results Patients accompanied with NAFLD had hepatocyte injury and dyslipidemia.NAFLD and chronic liver disease patients had significantly elevated serum CD44 levels than normal control group(P<0.01).CD44 positive lymphocyte ratio was 78.19%±16.33%in NAFLD patients and 68.47%±20.91%in chronic hepatitis B group,which was higher than the control group(46.51%±20.52%).Chronic hepatitis B group with steatosis had significantly higher CD44 concentration(181.42±49.36)ng/ml than chronic hepatitis B group(142.52±53.87)ng/ml and normal control group(99.47±15.23)ng/ml.CD44/GAPDH ratio in the liver cancer group(1.306±0.614)was significantly higher than paracancerous group(0.477±0.291)and the difference between the two groups was statistically significant(t=3.451,P=0.004).The integrated optical density of CD44 in the NAFLD-related liver cancer and paracancerous group were 25.721±5.881 and 14.155±4.001 and the difference between the groups was statistically significant(t=14.544,P<0.001).The pathological features of high expression of CD44 in patients with hepatocellular carcinoma were significantly correlated with HBV infection,tumor size,single/multi-center,and lymph node metastasis,degree of differentiation,TNM grade,Child-Pugh score,portal vein tumor thrombus and extrahepatic metastasis.HCC patients with NAFLD had significantly higher serum CD44(234.62±69.40)ng/ml than patients without NAFLD(186.49±58.89)ng/ml(t=-3.191,P=0.002),but there was no statistically significant difference in the clinicopathological characteristics between the high/low CD44 groups of HCC patients with NAFLD.Conclusion The results suggest that CD44 is abnormally activated and its mechanism may play an important role in the progression of NAFLD.