miR-182、miR-183-5p和miR-96-5p与非酒精性脂肪肝的相关性研究 (1).docx

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1、中文摘要miR-182、miR-183-5p和miR-96-5p与非酒精性脂肪肝的相关性研究摘要目的:非酒精性脂肪性肝病(NAFLD)可加重代谢紊乱，增加相关并发症的发病风险。因此，早期发现NAFLD是避免并发症不良影响的关键。本研究旨在识别miRNAs，以早期诊断及治疗NAFLD。方法：1.动物实验：采用高脂饮食建立NAFLD大鼠模型。把实验大鼠的肝脏取出，作为本次实验的标本，检测肝组织甘油三酯含量，然后切取大鼠肝组织并使用HE染色和油红O染色观察其组织学改变。采用qRT-PCR方法检测大鼠肝脏组织中miR-182、miR-183-5p和miR-96-5p的表达水平。2.临床实验：在研究对象

2、的选择上，选取生活习惯正常的健康人群和NAFLD患者进行对比，从河北省人民医院体检中心收集研究对象血清，用qRT-PCR方法检测研究对象血清中miR-182、miR-183-5p和miR-96-5p的表达水平并进行比较。结果:动物实验1.各组实验鼠的重量、鼠的肝脏湿重、实验鼠在未进食情况下的血糖数值、TG含量的比较HFD组大鼠体重、肝湿重高于ND组（P0.05）。TG含量在HFD组明显升高，且P值小于0.01。但两组实验组中的实验鼠未进食情况下的血糖值无明显差异，P值大于0.05。2肝脏组织病理学检测结果比较 HE染色：ND组肝组织健康情况良好，没有出现细胞发生炎症情况，也没有出现肝组织内脂肪

3、发生病变。与之相比，HFD组肝脏情况就发生病变，病变肝细胞气球样变肿大且间质组织中，出现均匀一致、无结构、半透明状的蛋白质蓄积，肝小叶功能混乱，小叶内也有大量细胞出现炎症。油红O染色：ND组未见明显脂滴积累，HFD组可见大量脂滴积累。3.各组大鼠肝脏miRNA的表达与ND组比，HFD组肝脏中miR-182、miR-183-5P、miR-96-5P表达明显减少，差异有统计学意义（P0.01）；临床实验1.基本数据的比较获得了100名符合入选标准的参与者(50名男性，50名女性)的数据。收集以下指标进行比较：身体体脂指数(Body Mass Index, BMI)、血压（Blood Pressur

4、e，BP）、空腹血糖（Fasting Blood -Glucose，FBG）、尿酸（Uric Acid，UA）、白蛋白（Albumin，ALB）、免疫球蛋白(Globulin，GLB)、总蛋白(Total Protein，TP)、总胆固醇（Total Cholesterol，TCHO）、(RCOO)3C3H5(Triglyceride，TG)、a1脂蛋白(High-Density Lipoprotein Cholesterol，HDL-C）、低密度脂蛋白（Low Density Lipoprotein Cholesterol，LDL-C）。与健康对照组相比，NAFLD患者的BMI（P0.05）

5、、DBP（P0.05）较高，NAFLD患者的血清中AST（P0.05）、ALT（P0.01）、TG（P0.01）、LDL（P0.05）、UA（P0.01）显著升高，HDL（P0.01）显著降低。2.血清microRNA测定结果与正常组（Normal)相比，mir -182在非酒精性脂肪肝患者(NAFLD)的表达明显下降和减少,取得的差异值具有重要意义（P5%）。与Normal组相比，miR-183-5P在NAFLD组表达明显减少，取得的差异值具有重要意义（P5%）。与Normal组相比，miR-96-5P在NAFLD组表达明显减少，取得的差异值具有重要意义（P5%）。结论:1.高脂饮食可以诱导

6、大鼠肝脏脂肪变性。2.miR-182、miR-183-5P、miR-96-5p在脂肪肝组的大鼠肝脏中表达显著降低。3.miR-182、miR-183-5P、miR-96-5p在NAFLD患者的血清中表达显著降低，血清中miR-182、miR-183-5p、miR-96-5p可能成为筛查NAFLD新的标志物及潜在的治疗靶点。关键词：非酒精性脂肪肝；miR-182；miR-183-5p；miR-96-5p2英 文摘要ASSOCIATION OF MICRORNAS (MIR-182, MIR-183-5P AND MIR-96-5P) WITH NON-ALCOHOLIC FATTY LIVER

7、DISEASEABSTRACTObjective: To investigate the relationship between miR-182, miR-183-5p and miR-96-5p and non-alcoholic fatty liver disease (NAFLD), and to provide a strong basis for the early diagnosis and treatment of NAFLD.Methods:1. Animal experiment: NAFLD rat model was established by high fat di

8、et. The liver of the experimental rats was taken out as the specimen of this experiment, It is necessary to determine the content of triglyceride in liver tissue. Histological changes were observed after he staining and oil red O staining.The expression levels of mir-182, mir-183-5p and mir-96-5p we

9、re detected by QRT PCR.2. Clinical experiment: in the selection of research objects, healthy people with normal living habits and NAFLD patients were selected for comparison. The serum of research objects was collected from the physical examination center of Hebei Provincial Peoples Hospital, and th

10、e expression levels of mir-182, mir-183-5p and mir-96-5p in the serum of research objects were detected by QRT PCR.Results:Animal experiment 1. Comparison of body weight, wet liver weight, fasting blood glucose and TG contents in each groupThe liver weight and wet weight of HFD group were higher tha

11、n those of ND group (P5%).TG content in HFD group was significantly increased (P1%).There was no obvious differences in blood glucose levels between the two groups when they did not eat (P 0 0.05).2. Comparison of liver histopathological examination resultsHE staining: In ND group, the liver tissue

12、of mice was intact, and the distribution of hepatocytes and liver lobules was regular and the cytoplasm was uniform. No obvious inflammation or fatty degeneration was found. HFD group: the structure of hepatic lobules was irregular, and there were many fat droplets in the hepatocytes, and the hepato

13、cytes became swollen and balloon shaped, with a large number of inflammatory cells infiltration in the portal area and lobules.Oil red O staining: There were no obvious lipid droplets in the ND group, and a large number of lipid droplets in the HFD group.3. Expression of miRNA in liver of rat in eac

14、h groupCompared with ND group, the expressions of miR-182, miR-183-5p and miR-96-5p in liver of HFD group were significantly decreased, with statistical significance (P0.01).Clinical experiment 1. Comparison of basic dataData were obtained from 100 participants (50 men and 50 women) who met the incl

15、usion criteria. We compared the following indicators:Body Mass Index（BMI)、Blood Pressure（BP）、Fasting Blood -Glucose（FBG）、Uric Acid（UA）、Albumin（ALB）、Globulin（GLB)、Total Protein（TP)、Total Cholesterol（TCHO）、Triglyceride（TG)、High-Density Lipoprotein Cholesterol（HDL-C）、Low Density Lipoprotein Cholesterol

16、（LDL-C） 1-抗胰蛋白酶对型糖尿病的药效学研究 。Compared with healthy control group, BMI (P 5%) and DBP (P 5%) of NAFLD patients were higher, AST (P 5%), ALT (P 1%), TG (P 1%), LDL (P 5%), UA (P 1%) of NAFLD patients were significantly higher, and HDL (P 0.01) was significantly lower.2. Serum microRNA assay resultsThe

17、expression of mir-182 in NAFLD group was significantly lower than that in normal group, which was statistically significant (P 5%); the expression of mir-183-5p in NAFLD group was also significantly lower than that in normal group, which was statistically significant (P 70g/周； 妊娠； 肾功能不全； 严重的炎症性疾病。2.

18、2标本采集采集各研究对象空腹静脉血5mL,3000r/min离心10min，吸取部分血清置于无RNase/DNase Ep管中，-80保存。剩余血清用于各项生化指标的检测。2.3血液生化取所有患者空腹血清用生化分析检测仪检测检测丙氨酸氨基转移酶（ALT)、空腹血糖(FPG)、三酰基甘油(TG)、总胆固醇(TC)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)、和白蛋白(ALB)、尿酸（UA)。2.4血清中miR-182、miR-183-5p、miR-96-5p表达的测定采用实时荧光定 PCR(RT-qPCR)测定miRNA的表达。2.4.1血清miRNA 的提取参考天根生化

19、科技有限公司的miRcute miRNA提取分离试剂盒，操作步骤如下：1.在200l全血、血清或血浆中加入等体积的MZ溶液裂解复合物，振荡器振荡30sec。2.室温条件下静止5 min，使得复合物完全分离呈核酸、蛋白质独立存在。3.室温条件下高速离心机12,000转离心10 min，提取上清液，转入一个新的离心管中。4.加入200l四氯甲烷，避光保存，剧烈振荡15sec，室温放置5min 。5.室温条件下高速离心机12,000 转离心15min，样品会分成三层：有机层，中间层和水层，有机层呈黄色、水层呈无色。RNA主要存在于水相中，把水相转移到新管中，进行后续操作。6.量取转移液的体积缓慢加入

20、转移液体积1/3体积的无水乙醇混匀（此时可能会出现沉淀）将得到的溶液和沉淀一起转入吸附柱miRspin室温放置2 min室温 12,000 rpm离心30 sec离心后弃掉吸附柱miRspin，保留流出液。7.量取流出液的体积，缓慢加入流出液体积2/3体积的无水乙醇混匀（此时可能会出现沉淀）将得到的溶液和沉淀一起转入吸附柱 miRelute，室温放置2 min室温 12,000 rpm离心30 sec离心后弃掉流出液，保留吸附柱miRelute。8向吸附柱miRelute中加入500l去蛋白液MRD（请先检查是否已加入乙醇）室温静置2 min室温 12,000 rpm (13,400g)离心3

21、0 sec弃废液。9.向吸附柱miRelute中加入500 l漂洗液RW（请先检查是否已加入乙醇）室温静置2 min室温 12,000 rpm (13,400g)离心30 sec弃废液。10. 重复操作步骤9。11.将吸附柱miRelute放入2 ml收集管中室温12,000rpm (13,400g)离心1 min去除残余液体。12.将吸附柱miRelute转入一个新的RNase-Free 1.5 ml离心管中加15-30 l RNase-Free ddH2O室温放置2 min室温 12,000 rpm (13,400g)离心2 min。 产品内容产品组成 2.4.2血清miRNA 的逆转录反应参考北京天根生化科技有限公司 miRcute 增强型 miRNA cDNA 第一链合成试剂盒操作，反应体系如下：Total RNA2L2miRNA RT Reaction Buffer10LmiRNA RT Enzyme Mix2LRNase-Free ddH2O6uL总体积20L反应条件：42，60min，95，3 min，20冰箱保存。2.4.3 m