γ-GT对肝癌患者结局的预测价值及扶正解毒消积方的作用机制研究

发布时间：2018-03-02 11:18

  本文选题：γ-GT　切入点：肝癌　出处：《北京中医药大学》2017年博士论文　论文类型：学位论文

【摘要】：临床研究目的报道显示γ-谷胺酰转肽酶(γ-glutamyltransferase,γ-GT)水平与肝细胞癌(hepatocellular carcinoma,HCC)的预后相关。将确诊时血清γ-GT基线水平作为预测HCC患者不良预后的预测指标进行评估,并将其预测价值与其他预后因子进行比较。方法回顾性地分析了 2008年1月至2013年3月初次诊断为HCC的858例患者基线数据和临床结局。基线数据包括确诊时记录的γ-GT、Child-Pugh分级和MELD评分等。我们运用Cox单因素、多因素和ROC曲线分析研究变量与预后的关系。进行Kaplan-Meier生存分析,以评估不同巴塞罗那分期(Barcelona Clinic Liver Cancer,BCLC)或肿瘤淋巴结转移(Tumor Node Metastasis,TNM)分期及不同血清甲胎蛋白(alpha fetoprotein,AFP)水平下,γ-GT作为HCC预后因子的价值。结果研究显示患者存活率与γ-GT以及血清生物标志物,包括中性粒细胞计数(absolute neutrophil count,ANC),淋巴细胞计数(absolute lymphocyte count,ALC),血清AFP显著相关。血清γ-GT≥75U/L强烈提示HCC患者预后不良。在BCLC和TNM分期为进展期及任何血清AFP水平,γ-GT≥75U/L患者的生存时间都明显缩短。结论基线γ-GT是HCC患者临床结局的有价值的预测指标。γ-GT的预测价值优于肿瘤数量、肿瘤大小、淋巴结侵犯、血清ANC、ALC、AFP和Child-Pugh分级,并且不受酒精摄入量的影响。实验研究目的扶正解毒消积方是首都医科大学附属北京地坛医院中西医结合中心治疗原发性肝癌的经验方。既往研究表明,该方能提高患者实体瘤的缓解率和稳定率、控制瘤体的生长速度、延长患者的生存时间,但其作用机制尚不明确。拟通过体内和体外实验评价扶正解毒消积方的治疗效果,并初步探讨其抑制肝癌进展的作用机制。方法1.体内实验:SPF级雄性SD大鼠66只,随机分成正常组(n=6)、模型组(n=30)、中药组(n=30)。模型组和中药组给予DEN腹腔注射制备HCC模型。12周成瘤后,中药组每天给予28g/kg体重中药灌胃,正常组及模型组大鼠每天以等体积生理盐水灌胃,各组灌胃体积均为10ml/kg体重。灌胃第3周末次给药后,正常组6只大鼠,模型组、中药组各随机选取10只大鼠,禁食12小时,麻醉大鼠腹主动脉取血,处死大鼠取肝脏、脾脏并称重。模型组、中药组其余大鼠继续灌胃,至其自然死亡。检测指标包括:各组大鼠成瘤率、存活率、肝脏湿重、脾脏湿重、肝脏指数、脾脏指数;血清 ALT、AST、TBil、γ-GT、ALB;酶联免疫吸附测定(enzyme linked immunosorbent assay,Elisa)血清 AFP 水平;Western Blot 法检测肝肿瘤组织中 Rab27B、P53、MMP-2蛋白的表达;明胶酶谱法检测MMP-2活性。2.体外实验:扶正解毒消积方含药血清干预Bel7402细胞,通过MTT、平板克隆、Transwell实验观察其在体外的增殖、侵袭能力;Rab27B、P53、MMP-2水平通过Western Blot实验检测。结果1.体内实验:与正常组比较,模型组和中药组大鼠体重(P0.01)显著减轻,肝指数(P0.01)、脾指数(p0.01)显著升高;模型组大鼠血清ALT(P0.01)、AST(P0.01)、TBil(P0.05)、γ-GT(P0.01)、AFP(P0.05)水平明显升高,ALB 无明显差异(P0.05);中药组大鼠血清 ALT(P0.01)、AST(P0.05)、TBil(P0.01)、γ-GT(P0.01)、AFP(P0.05)、ALB(P0.01)明显升高。肝癌组织中,模型组大鼠Rab27B(P0.01)、MMP-2(P0.01)蛋白表达及活性显著升高,P53蛋白表达显著下降(P0.01);中药组大鼠Rab27B(P0.05)、MMP-2(P0.01)蛋白表达及活性升高(P0.05),P53蛋白表达明显下降(P0.05)。与模型组相比,中药组大鼠存活率(P0.05)、体重(P0.01)明显升高,肝指数(P0.05)、脾指数(P0.05)明显降低;血清ALT、AST水平与模型组无统计学差异(P0.05),TBil(P0.05)和 γ-GT(P0.05)水平降低,ALB 水平升高(P0.05);AFP水平降低(P0.05);肝癌组织中Rab27B、MMP-2蛋白表达水平显著降低(P0.01),P53蛋白表达水平显著升高(P0.01),MMP-2活性降低(P0.05)。2.体外实验:与正常血清组相比,含药血清组Bel7402细胞增殖能力下降(P0.05);平板克隆形成能力(P0.01)、迁移(P0.01)及侵袭能力(P0.01)减弱;Rab27B、MMP-2蛋白表达降低,P53蛋白表达升高。结论以上实验结果证实:在体内及体外实验中,扶正解毒消积方可以通过抑制Rab27B的表达水平,增强P53表达,抑制MMP-2的表达及活性,抑制肿瘤的进展及转移,延长肝癌大鼠生存期,该结论与临床前期研究结果一致。扶正解毒消积方抑制肝癌的作用可能是通过Rab27B/P53/MMP-2通路实现的。
[Abstract]:Objective to report the clinical research shows that gamma glutamyl transpeptidase (-glutamyltransferase gamma, gamma -GT) and hepatocellular carcinoma (hepatocellular, carcinoma, HCC) and the prognosis. The diagnosis of serum gamma -GT baseline levels as a predictor to predict poor prognosis of patients with HCC were evaluated, and its predictive value with other prognostic factors were compared. Methods Retrospective analysis from January 2008 to March 2013 the first 858 patients who were diagnosed as HCC baseline data and clinical outcome. The baseline data including gamma -GT records the time of diagnosis, Child-Pugh grading and MELD. We use the Cox single factor, multi factor analysis and ROC curve relationship between variables and prognosis. The Kaplan-Meier survival analysis to assess the different stages of Barcelona (Barcelona Clinic Liver Cancer, BCLC) or lymph node metastasis (Tumor Node, Metastasis, TNM) in different stages and serum alpha fetoprotein Protein (alpha fetoprotein, AFP) level, -GT HCC as a prognostic factor of gamma value. Results showed that the patients survival rate and gamma -GT and serum biomarkers, including neutrophil count (absolute neutrophil, count, ANC), lymphocyte count (absolute lymphocyte, count, ALC) were significantly related to serum AFP serum gamma. -GT = 75U/L is strongly suggestive of poor prognosis of patients with HCC. BCLC and TNM at any stage and the level of serum AFP, gamma -GT = 75U/L survival time were significantly shortened. Conclusion -GT is a predictor of gamma baseline clinical outcomes in patients with HCC value. The number, the predictive value is better than that of tumor gamma -GT tumor size. Lymph node involvement, serum ANC, ALC, AFP and Child-Pugh grade, and is not affected by alcohol intake. Experimental study on Fuzheng Jiedu Xiaoji decoction is combination of traditional Chinese and Western medicine in Beijing Ditan Hospital affiliated to Capital Medical University The experience of the heart in the treatment of primary liver cancer. Previous studies showed that the tumor patients can improve the remission rate and stable growth rate, the speed control of tumor, prolong the survival time of patients, but the mechanism is still not clear. The consumer product through the evaluation in vivo and in vitro experiments of Fuzhengjiedu therapy, and to investigate the inhibition mechanism of HCC progression. Methods 1. in vivo experiment: SPF level 66 male SD rats were randomly divided into normal group (n=6), model group (n=30), Chinese medicine group (n=30). The model group and the medicine group were given intraperitoneal injection of DEN HCC model was prepared by.12 weeks after tumor formation the Chinese medicine group, Chinese medicine 28g/kg weight given daily gavage, the normal group and model group every day the rats in saline gavage group, intragastric volume was 10ml/kg weight. Intragastric administration after third weeks, normal group 6 rats, model group, Chinese medicine group, 10 rats each were randomly selected no. Eat 12 hours of anesthesia abdominal aortic blood in rats, the rats were sacrificed, liver, spleen and weighing. The model group, Chinese medicine group the rats to gavage, to its natural death. The indexes included: each rat tumor formation rate, survival rate, liver weight, liver index, spleen wet weight. Spleen index; serum ALT, AST, TBil, -GT gamma, ALB; enzyme linked immunosorbent assay (enzyme linked immunosorbent assay, Elisa) level of serum AFP; Rab27B detection of liver tumor Western Blot method in P53, the expression of MMP-2 protein; zymography to detect the activity of MMP-2.2. in vitro: Fuzheng Jiedu Xiaoji Decoction the serum Bel7402 cells by MTT, clone, observe the invasion ability in vitro proliferation of Transwell, Rab27B, P53, MMP-2; Western level by Blot assay. Results of the 1. experiment in vivo: comparing with normal group, the body weight of model group and Chinese medicine group rats (P0.01). 钁楀噺杞，

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