【作者】 周艳；

【导师】 揭盛华；

【作者基本信息】 华中科技大学 ， 内科学， 2011， 硕士

【摘要】 目的:研究Leukotriene D4(LTD4)在慢性乙型肝炎(CHB)和原发性肝癌(HCC)患者血清中的表达水平,并分析其与血清ALT、AST、γ-GT、HBV-DNA和AFP间的相关性,进而探讨LTD4在CHB和HCC发生发展中的作用。方法:选取CHB患者21例(男16例,女5例,平均年龄37.6±12.7岁),其中慢性肝炎14例、慢性重型肝炎7例;HCC患者71例(男64例,女7例,平均年龄48.9±11.1岁),其中合并HBV者62例;及健康体检者(对照组)20例为研究对象。应用酶联免疫法(ELISA)检测血清中LTD4水平,全自动生化仪检测ALT、AST、γ-GT水平,化学发光仪和基因扩增仪分别检测AFP和HBV-DNA。结果:CHB和HCC患者血清LTD4水平均明显高于对照组(P<0.01),且其水平在CHB组、HCC合并HBV组、单独HCC组及对照组中逐渐下降。在CHB中,慢性肝炎与重型肝炎比较,LTD4水平差异无统计学意义(P>0.05);且LTD4水平与ALT、AST、γ-GT及HBV-DNA含量间无相关性(P>0.05)。在HCC中,合并HBV组与单独HCC组相比,LTD4水平有显著性差异(P<0.01);分别比较转移组与无转移组、治疗组与未治疗组,结果差异均无统计学意义(P>0.05);LTD4水平仅与ALT呈正相关(P<0.05),而与AST、γ-GT、AFP无相关性(P>0.05)。结论:LTD4作为重要的炎症介质,可能参与了CHB的发生发展,并在促进CHB向HCC进展的过程中起着一定的作用,而且对肝癌细胞自身的肿瘤生物学产生影响。目的:研究LTD4对肝癌细胞的增殖、周期、凋亡及迁移的影响,探讨其在HCC发生发展机制方面的作用。方法:培养人肝癌细胞系SMMC-7721细胞进行实验,不同浓度LTD4处理细胞24h、48h、72h后,采用CCK-8法测定SMMC-7721细胞的增殖活性及LTD4拮抗剂对细胞增殖的抑制作用;流式细胞术检测细胞周期变化;Annexin V/PI双标法检测细胞凋亡变化;8μm Transwell法检测肝癌细胞的迁移作用。结果:在1nmol/L处LTD4作用48h时促进SMMC-7721细胞的增殖作用最强(P < 0.01),可达59%;抑制剂MK571浓度在10-8-10-6mol/L时可部分拮抗LTD4,而在10-5mol/L处能明显抑制细胞生长(P < 0.05);LTD4在有效浓度内作用SMMC-7721细胞后,M1/M2期细胞的比例明显下降(P < 0.05),促进细胞周期从M1期向M2期分化,呈剂量依赖性;其次,它可明显抑制细胞凋亡(P<0.05),以早期凋亡为主,呈剂量依赖性;此外,LTD4还诱导癌细胞的迁移(P<0.05),其促进作用亦呈剂量依赖性。结论:LTD4作为一种重要的炎症介质在肝癌的发生发展中发挥了一定的作用,有可能作为一个新的指标来辅助肝癌的诊断及预后的判断,其拮抗剂也可能成为化学预防慢性肝炎向肝癌发展的潜在的新药物。更多还原

【Abstract】 Objective: To detect the expression of serum leukotriene D4 in paitents with chronic hepatitis B(CHB) and hepatocellular carcinoma(HCC) in order to investigate the relationship between LTD4 levels and the development of CHB and HCC,and to probe its roles on the progression of HCC.Methods: This study included 21 CHB paitents, 71 HCC patients and 20 controls.Therein, the average age of CHB paitents was 37.6±12.7 years with 16 males and 5 females including light~heavy chronic hepatitis 7 cases and severe hepatitis 14 cases; 71 HCC patients with an average age of 48.9±11.1 years included male 64 cases and female 7 cases, for 62 cases among them accompanying with HBV. In addition, LTD4 levels was detected by Enzyme-linked immunosorbent assay (ELISA), the levels of serum ALT, AST, gamma-GT were examined by Automatic biochemical analyzer, and serum AFP and HBVDNA were measured using Chemiluminescence instrument and Gene amplification instrument,respectively.Results: The serum LTD4 levels of paitients with CHB and HCC were obvously higher than controls (P < 0.01),and its levels gradually declined followed by patients with CHB, HCC with HBV, HCC alone and control group. In CHB group, there was no significant difference between light~heavy hepatitis and severe hepatitis (P > 0.05). Moreover, LTD4 levels were not associated with levels of ALT, AST, gamma-GT and HBV-DNA content. In HCC group, there was significant difference (P < 0.05) between HCC mergered with HBVand HCC alone. Moveover, in metastasis and nonmetastasis group as well as treatment and non-treatment group, there were no statistically difference (P > 0.05). Additonally, there was no correlation between LTD4 and AST,γ-GT or AFP(P > 0.05), but a positive correlation between LTD4 and ALT (P < 0.05). Conclusion: LTD4, as an important proinflammatory mediator, is not only involved in the pathogensis of CHB and could play a role in promoting the progression process from CHB to HCC, but also have some directly effects on HCC cells. Objective: Chronic inflammation plays an important role in carcinogenesis. A pro-inflammatory mediator leukotriene D4(LTD4) not only has been implicated in the pathophysiology of asthma and inflammations, but also in several cancers. However, little is known about the effects of the LTD4 on human hepatocellular carcinoma (HCC). The aim of this study is to investigate the roles of LTD4 on the SMMC-7721 cell proliferation, cell cycle, apoptosis and migration.Methods: By culture human hepatocellular carcinoma cell line SMMC-7721, treated with LTD4 at different concentration and different time and cysteinyl leukotrienes receptor 1 (CysLT1 R) antagonist MK571, cell proliferation were examined by CCK-8 assay in vitro. Then, effects of LTD4 on cell cycle and apoptosis were assessed by flow cytometry. In addition, 8μm Transwell plate was used to detect LTD4 role on the cell migration.Results: The proliferation rate of SMMC-7721 cell was obviously enhanced by LTD4 (P < 0.05), reaching a maximum about 59 percent at a concentration of 1 nmol/L for 48 hour. Furthermore, antagonist MK571 with 10-8-10-6mol/L concentrations prevented partially the roles of LTD4 on cell proliferation by competitive inhibition, while over 10-5mol/L concentration it could significantly inhibit cell growth (P < 0.05). Additonally, SMMC-7721 cell treated by LTD4 within the effective concentrations, the proportion of M1/M2 phase cell declined significantly (P < 0.05) with a dose-dependent manner, promoting cell cycle from M1 from M2; Moreover, LTD4 could apparently inhibit cell apoptosis (P < 0.05) in a dose-dependent, mainly in early-stage apoptosis.Besides, LTD4 also induced cancer cell migration (P < 0.05) and the promotion roles also emerged in the dose-dependent.Conclusion: As an important proinflammatory mediator, LTD4 play roles in the pathogenesis and progression of HCC, indeed. Hence, LTD4 would be as a new potential index to assist the diagnosis of HCC and judgement the prognosis, and its antagonists may also become to new potential drugs chemopreventing the development of chronic hepatitis to HCC in the future.更多还原