本文選題：γ-GT　切入點(diǎn)：肝癌　出處：《北京中醫(yī)藥大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：臨床研究目的報(bào)道顯示γ-谷胺酰轉(zhuǎn)肽酶(γ-glutamyltransferase,γ-GT)水平與肝細(xì)胞癌(hepatocellular carcinoma,HCC)的預(yù)后相關(guān)。將確診時(shí)血清γ-GT基線水平作為預(yù)測(cè)HCC患者不良預(yù)后的預(yù)測(cè)指標(biāo)進(jìn)行評(píng)估,并將其預(yù)測(cè)價(jià)值與其他預(yù)后因子進(jìn)行比較。方法回顧性地分析了 2008年1月至2013年3月初次診斷為HCC的858例患者基線數(shù)據(jù)和臨床結(jié)局�；�數(shù)據(jù)包括確診時(shí)記錄的γ-GT、Child-Pugh分級(jí)和MELD評(píng)分等。我們運(yùn)用Cox單因素、多因素和ROC曲線分析研究變量與預(yù)后的關(guān)系。進(jìn)行Kaplan-Meier生存分析,以評(píng)估不同巴塞羅那分期(Barcelona Clinic Liver Cancer,BCLC)或腫瘤淋巴結(jié)轉(zhuǎn)移(Tumor Node Metastasis,TNM)分期及不同血清甲胎蛋白(alpha fetoprotein,AFP)水平下,γ-GT作為HCC預(yù)后因子的價(jià)值。結(jié)果研究顯示患者存活率與γ-GT以及血清生物標(biāo)志物,包括中性粒細(xì)胞計(jì)數(shù)(absolute neutrophil count,ANC),淋巴細(xì)胞計(jì)數(shù)(absolute lymphocyte count,ALC),血清AFP顯著相關(guān)。血清γ-GT≥75U/L強(qiáng)烈提示HCC患者預(yù)后不良。在BCLC和TNM分期為進(jìn)展期及任何血清AFP水平,γ-GT≥75U/L患者的生存時(shí)間都明顯縮短。結(jié)論基線γ-GT是HCC患者臨床結(jié)局的有價(jià)值的預(yù)測(cè)指標(biāo)。γ-GT的預(yù)測(cè)價(jià)值優(yōu)于腫瘤數(shù)量、腫瘤大小、淋巴結(jié)侵犯、血清ANC、ALC、AFP和Child-Pugh分級(jí),并且不受酒精攝入量的影響。實(shí)驗(yàn)研究目的扶正解毒消積方是首都醫(yī)科大學(xué)附屬北京地壇醫(yī)院中西醫(yī)結(jié)合中心治療原發(fā)性肝癌的經(jīng)驗(yàn)方。既往研究表明,該方能提高患者實(shí)體瘤的緩解率和穩(wěn)定率、控制瘤體的生長(zhǎng)速度、延長(zhǎng)患者的生存時(shí)間,但其作用機(jī)制尚不明確。擬通過體內(nèi)和體外實(shí)驗(yàn)評(píng)價(jià)扶正解毒消積方的治療效果,并初步探討其抑制肝癌進(jìn)展的作用機(jī)制。方法1.體內(nèi)實(shí)驗(yàn):SPF級(jí)雄性SD大鼠66只,隨機(jī)分成正常組(n=6)、模型組(n=30)、中藥組(n=30)。模型組和中藥組給予DEN腹腔注射制備HCC模型。12周成瘤后,中藥組每天給予28g/kg體重中藥灌胃,正常組及模型組大鼠每天以等體積生理鹽水灌胃,各組灌胃體積均為10ml/kg體重。灌胃第3周末次給藥后,正常組6只大鼠,模型組、中藥組各隨機(jī)選取10只大鼠,禁食12小時(shí),麻醉大鼠腹主動(dòng)脈取血,處死大鼠取肝臟、脾臟并稱重。模型組、中藥組其余大鼠繼續(xù)灌胃,至其自然死亡。檢測(cè)指標(biāo)包括:各組大鼠成瘤率、存活率、肝臟濕重、脾臟濕重、肝臟指數(shù)、脾臟指數(shù);血清 ALT、AST、TBil、γ-GT、ALB;酶聯(lián)免疫吸附測(cè)定(enzyme linked immunosorbent assay,Elisa)血清 AFP 水平;Western Blot 法檢測(cè)肝腫瘤組織中 Rab27B、P53、MMP-2蛋白的表達(dá);明膠酶譜法檢測(cè)MMP-2活性。2.體外實(shí)驗(yàn):扶正解毒消積方含藥血清干預(yù)Bel7402細(xì)胞,通過MTT、平板克隆、Transwell實(shí)驗(yàn)觀察其在體外的增殖、侵襲能力;Rab27B、P53、MMP-2水平通過Western Blot實(shí)驗(yàn)檢測(cè)。結(jié)果1.體內(nèi)實(shí)驗(yàn):與正常組比較,模型組和中藥組大鼠體重(P0.01)顯著減輕,肝指數(shù)(P0.01)、脾指數(shù)(p0.01)顯著升高;模型組大鼠血清ALT(P0.01)、AST(P0.01)、TBil(P0.05)、γ-GT(P0.01)、AFP(P0.05)水平明顯升高,ALB 無明顯差異(P0.05);中藥組大鼠血清 ALT(P0.01)、AST(P0.05)、TBil(P0.01)、γ-GT(P0.01)、AFP(P0.05)、ALB(P0.01)明顯升高。肝癌組織中,模型組大鼠Rab27B(P0.01)、MMP-2(P0.01)蛋白表達(dá)及活性顯著升高,P53蛋白表達(dá)顯著下降(P0.01);中藥組大鼠Rab27B(P0.05)、MMP-2(P0.01)蛋白表達(dá)及活性升高(P0.05),P53蛋白表達(dá)明顯下降(P0.05)。與模型組相比,中藥組大鼠存活率(P0.05)、體重(P0.01)明顯升高,肝指數(shù)(P0.05)、脾指數(shù)(P0.05)明顯降低;血清ALT、AST水平與模型組無統(tǒng)計(jì)學(xué)差異(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);肝癌組織中Rab27B、MMP-2蛋白表達(dá)水平顯著降低(P0.01),P53蛋白表達(dá)水平顯著升高(P0.01),MMP-2活性降低(P0.05)。2.體外實(shí)驗(yàn):與正常血清組相比,含藥血清組Bel7402細(xì)胞增殖能力下降(P0.05);平板克隆形成能力(P0.01)、遷移(P0.01)及侵襲能力(P0.01)減弱;Rab27B、MMP-2蛋白表達(dá)降低,P53蛋白表達(dá)升高。結(jié)論以上實(shí)驗(yàn)結(jié)果證實(shí):在體內(nèi)及體外實(shí)驗(yàn)中,扶正解毒消積方可以通過抑制Rab27B的表達(dá)水平,增強(qiáng)P53表達(dá),抑制MMP-2的表達(dá)及活性,抑制腫瘤的進(jìn)展及轉(zhuǎn)移,延長(zhǎng)肝癌大鼠生存期,該結(jié)論與臨床前期研究結(jié)果一致。扶正解毒消積方抑制肝癌的作用可能是通過Rab27B/P53/MMP-2通路實(shí)現(xiàn)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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