【作者】 王娜；

【导师】 姚树坤；

【作者基本信息】 河北医科大学 ， 内科学， 2005， 硕士

【摘要】 目的:原发性肝癌(Primary liver carcinoma,PLC)是我国常见的恶性肿瘤之一,死亡率占第二位。我们应用自研中成药肝癥口服液治疗PLC,临床疗效比较理想。含中药血清对肝癌细胞SMMC-7721 增殖、细胞凋亡、细胞周期及细胞因子的影响等方面尚未进行深入研究。本实验探讨肝癥口服液对PLC 的作用机制。方法:参照2001 年中国肝癌协会肝癌委员会修订的原发性肝癌的诊断标准及临床分期标准,选择Ⅱb 期或Ⅲ期(无远处转移,肝功能Child-Pugh B 级),不能手术的并且尚未接受任何治疗的PLC 患者10 名。再选10 例健康志愿者同样剂量和方式给予肝癥口服液作为对照。服药前均清晨空腹抽血10ml,分离血清。服药对象于首次采血后,开始服用肝癥口服液40ml/日,持续8 周,于末次服药2h 后抽血10ml,分离血清。服药前后血清均56℃、30min 灭活,无菌条件下分装入EP 管,-80℃冻存。1 应用血清药理学方法通过四唑盐比色法(简称MTT)筛选TGF-β1 对人肝癌细胞SMMC-7721 增殖抑制的最佳时间及浓度;比较PLC 患者及健康志愿者服用肝癥口服液前后,不同浓度下的含药血清及与TGF-β1 联合使用的含药血清对人肝癌细胞SMMC-7721 增殖的抑制作用; 2 采用流式细胞术检测含中药血清对肝癌细胞凋亡、更多还原

【Abstract】 Objective:Primary liver carcinoma (PLC) is one of the commonest malignant diseases in our country. It’s mortality rate is the second of all diseases’. We treat PLC with Ganzheng Peroral Solution(GZPS) which produced by ourselves, and have acquired sound effect. But it has not been studied that the effects of GZPS serum to human hepatocellular carcinoma SMMC-7721 cell proliferation, apoptosis, cellular cycling and cytokines. This research project is to observe the mechanism of GZPS treating PLC. Methods: According to a prognostic and clinical pathological criterion which mended by Society of Liver Cancer(CACA) in the 2001 Chinese liver cancer conference, 10 pathological stageⅡb or Ⅲ(no faraway organ metastasis, liver function rank Child-Pugh B) PLC patients which had lost an operation chance and not been treated by any methods were chosen to be administered GZPS.And 10 control healthy volunteers were treated by GZPS in the same way.10ml blood was taken from all of the fasting subjects in the morning. The twenty subjects began to take GZPS 40ml per-day for 8 week’s after the first blood was drawn. 10ml blood was taken again after two hours of the last medicated. All blood was centrifuged to extract serum. Serum was pretreated at 56℃for 30 minute and put in Ep capsule at sterile condition, and was stored at -80℃until being used. 1 The inhibitory effect of TGF-β1 on SMMC-7721 cell was investigated by MTT assay to determine an optimal time and concentration. MTT assay was used as well to compare the different effects between patients’and volunteers’serum to SMMC-7721 cell proliferation before and after GZPS was administrated, and also to investigate the synergetic effect of TGF-β1 and serum. 2 After treated by serum,cellular cycling and apoptosis associated gene-bax and bcl-2 of SMMC-7721 were detected by flow cytometry assay; 3 To observe the mechanism of GZPS, the serum levels of IL-2,IL-12,TNF-a,TGF-β1 and IFN-γwere measured by ELISA after administering GZPS to PLC patients and healthy volunteers. Resluts : 1 The inhibitory effects of GZPS serum and TGF-β1 on SMMC-7721 cell proliferation: MTT assay of SMMC-7721 cell showed that in lower concentration, TGF-β1 showed an inhibitory effect but in some higher concentrations, such as 8ng/ml and 10ng/ml(P<0.05,P<0.01); TGF-β1 showed promoted effect on SMMC-7721 cell proliferation.TGF-β1 had the highest inhibitory effect in 48h and concentration of 2ng/ml.The OD value and inhibitory rate was 17.95% (P<0.01). At the time point of 48h, the cultured SMMC-7721 cells were stronger inhibited by patient’s serum in concentration of 20% than in concentration of 10% (P<0.01) . There was a kind of dose-dependent effect. And 2ng/ml TGF-β1 had cooperated the inhibitory effect of patient’s serum (P<0.01). But the healthy volunteers’serum before and after administrated GZPS showed no significant inhibitory effect on SMMC-7721 proliferation. 2 The effect of GZPS serum and TGF-β1 on SMMC-7721 cell apoptosis: The flow cytometry results showed that the apoptosis rate of SMMC-7721 cell increased in both PLC patient serum-treated group(P<0.01), and there was a cooperation effect by combined using TGF-β1(P<0.01). The expression of bax gene increased, and bcl-2 decreased. Cell cycle was restrained from G0/1 to S phase. The effect of healthy volunteer’serum was of inhibitory effect as PLC patients’, but it was not supported by statistic test. The GZPS serum could affect cell cycle and the expression of apoptosis-related gene such as bax and bcl-2, thus could result in SMMC-7721 apoptosis. 3 The effect of GZPS on cytokine of PLC patients’and healthy volunteers’serum: The ELISA assay showed that serum levels of IL-2 and IFN-γin PLC patients were lower than that in healthy volunteers (P<0.05). And they increased after treatment of GZPS(P<0.05). It was found that the IL-12,TNF-aand TGF-β1 serum levels of PLC patients were much higher than those of healthy volunteers(P<0.05 or P<0.01). But theydecreased obviously after treated by GZPS (P<0.05).Those data showed an increasing level of Th1 cell cytokine and a reduced level of Th2 cell cytokine by GZPS treatment in PLC. But the same change in healthy volunteers had not been found. Conclusions: The serum of patients taken GZPS could inhibit SMMC-7721 cell’s proliferation, induce apoptosis of SMMC-7721 cell and have synergism effect with TGF-β1. It also could retrain cell cycle at G0/1-phase so that the cells number at S-phase reduced. And it could up-regulate the expression of bax gene and down-regulate the expression of bcl-2 gene. So treatment of GZPS was effective to cell cycle, apoptosis, bax and bcl-2 gene expression. The Th1/Th2 ratio was down-regulated in the serum of PLC patients, whose Th1 cytokine levels such as IL-2 and IFN-γwere lower than healthy people. On the contrary, patients’Th2 cytokine levels such as IL-12 and TNF-awere higher than healthy people. And the serum level of TGF-β1 was down-regulated as well.That dominant Th2 cytokine resulted in an action of immunosuppression in PLC. GZPS can partly correct the immunological misbalance above-mentioned. GZPS could up-regulate the Th1 cytokines and down-regulate the Th2 cytokines. Therefore, it is necessary to break out the immunosuppression and balance the Th1/Th2 ratio in the course of treating primary hepatic cancer.GZPS can hold the superiority of Th1 cytokines, resume and preserve the balance of cell and body fluid immunity.GZPS can also relieve immunosuppression更多还原