【作者】 姚晓宾；

【导师】 张忠英；

【作者基本信息】 福建医科大学 ， 临床检验诊断学， 2009， 硕士

【摘要】 背景与目的:原发性肝癌（hepatocellular carcinoma,HCC）是世界上常见的恶性肿瘤之一,其恶性程度高,发展迅速,若治疗不及时或治疗方案选择不当,平均生存时间为半年。中国是HCC高发区,约占全球患者的55%,而厦门市同安区是中国两大肝细胞癌高发区之一,年发病率在十万分之三十以上。由于肝细胞癌早期不易发现,患者大多错失最佳手术时机。目前的常规化疗药物疗效不佳,致使肝细胞癌的治愈率较低。因此,研究HCC的发生发展机制,探明重要的相关基因,将为HCC的诊治提供新的思路,对攻克肝细胞癌意义重大。本课题以人肝癌细胞株BEL-7402为研究对象,通过检测全反式维甲酸对肝癌细胞增殖、凋亡以及Cks1、Cks2基因蛋白表达的影响,分析瘤细胞增殖、凋亡与Cks1、Cks2表达的相关性,初步探讨可能存在的作用机制,为全反式维甲酸治疗肝癌提供理论和实验依据,为Cks1、Cks2基因及蛋白进一步的实验研究积累经验。方法:1、MTT法测定ATRA对BEL-7402细胞增殖的影响,实验组加入相应量的ATRA至终浓度为（0.1,1,5,10,20）μmol/L,对照组只加相应量的RPMI 1640,分别于加药后继续培养24 h、48 h、72 h、96 h检测各组细胞和对照组及相邻实验组间增殖抑制率的差异。2、流式细胞仪Annexin V/PI双染法检测ATRA对BEL-7402细胞周期及凋亡的影响,实验组加入相应量的ATRA至终浓度为10μmol/L,对照组加入1‰的无水乙醇,于加药后继续培养24 h、48 h、72 h检测各组细胞周期分布和凋亡的差异。3、RT-PCR法检测ATRA对BEL-7402细胞Cksl、Cks2基因mRNA表达的影响,实验组加入相应量的ATRA至终浓度为10μmol/L,对照组加入1‰的无水乙醇,于加药后继续培养24 h、48 h、72 h检测各组细胞之间Cks1、Cks2基因mRNA表达的差异。4、免疫细胞化学ABC法检测ATRA对BEL-7402细胞Cks1、Cks2、P27蛋白表达的影响,实验组加相应量的ATRA至终浓度为10μmol/L,对照组加入1‰的无水乙醇,于加药后继续培养24 h、48 h、72 h检测Cks1、Cks2、P27蛋白表达的变化,采用HSCORE积分法分析ATRA对Cks1、Cks2、P27蛋白表达的影响。结果:1、ATRA对BEL-7402细胞增殖具有明显的抑制作用,抑制效应呈现明显的时间、剂量依赖性。10μmol/L组ATRA作用24小时抑制率为17.45±1.93,96小时抑制率达到50.23±2.44。各组之间的吸光度A值和抑制率IR相比差异有显著统计学意义（P＜0.01）。2、BEL-7402细胞经10μmol/L ATRA作用后,细胞周期各期细胞比例发生明显变化,随培养时间延长,G₀-G₁期细胞比例不断增高,细胞大量阻滞在G₀-G₁期,对照组G₀-G₁期细胞平均为59.87±1.07,ATRA作用72小时后升高到72.78±1.13,S期细胞比例相应下降,G₂/M期细胞比例各组之间差异无统计学意义（P＞0.05）。3、10μmol/LATRA作用24小时引起部分BEL-7402细胞发生凋亡6.48±0.45,并随作用时间延长,细胞凋亡率不断增加,72小时凋亡率达到17.31±1.00,实验组和对照组及各时间组间比较差异有显著统计学意义（P＜0.01）。4、ATRA对Cks1、Cks2基因mRNA表达无明显影响（P＞0.05）。5、BEL-7402细胞随ATRA作用时间延长,Cks1、Cks2蛋白表达量呈减低趋势,Cks1组化染色HSCORE积分从1.88±0.06降低到1.47±0.06,Cks2组化染色HSCORE积分从1.58±0.09降低到1.39±0.03,而P27蛋白表达量不断增加,组化染色HSCORE积分从2.32±0.08升高到2.97±0.11,各组之间的HSCORE积分相比差异均有显著统计学意义（P＜0.01或P＜0.05）。结论:1、全反式维甲酸对BEL-7402绌胞具有明显的增殖抑制作用,并呈现一定的时间、剂量依赖性。2、全反式维甲酸具有诱导BEL-7402细胞凋亡的作用。3、全反式维甲酸不影响BEL-7402细胞Cks基因mRNA的表达,却能够在蛋白水平减低Cks蛋白的表达,增强P27蛋白的表达。4、全反式维甲酸抑制BEL-7402细胞增殖,使大量细胞阻滞在G₀-G₁期,可能通过下调Cks1蛋白的表达,从而抑制P27蛋白的泛素化降解,使得P27蛋白在细胞内积聚,对细胞周期起负调控作用。5、全反式维甲酸诱导BEL-7402细胞凋亡的分子机制可能与Cks2蛋白表达下调有关。更多还原

【Abstract】 Background and Objective:Hepatocellular carcinoma,HCC is one of the word’s common malignant tumor with high malignancy,rapid development,average survival time only for six months if treatment is not timely or inappropriate.China is one of the regions of high incidence of liver cancer,accounting for about 55%of worldwide patients,and Tongan District, Xiamen City,is one of Chinese two major areas,which annual incidence rate is about 30/100000.Hepatocellular carcinoma is easy to miss the best timing of surgery because of difficulty to early detect.At present,the poor efficacy of conventional chemotherapy drugs resulted in lower cure rates.Therefore,studing the occurrence and development mechanism of HCC,verifying the important related genes,that will provide the new mentality to HCC diagnosis,means a great deal to conquer the HCC. This topic raked the person liver cancer BEL-7402 cell as the object,provided experimental and theoretical basis for clincal application with all-trans retinoic acid, piled up experience and data to further study of Cks1 and Cks2,through detecting effect of all-trans retinoic acid on liver cancer cell proliferation,apoptosis and Cks1, Cks2 expression,analysed the correlation between cell proliferation,apoptosis and Cks1,Cks2 expression,elementary studied the possible mechanisms that may exist.Methods:1.The method of MTT was used to examine the cell proliferation suppression activity of ATRA to cell BEL-7402.The experimental groups was cultured with ATRA in （0.1,1,5,10,20）μmol/L different concentration and control groups only adds the corresponding quantity RPMI 1640.After a further culture 24 h,48 h,72 h,96 h.The effect of different group cells’ inhibition rate was evaluated with MTT method.2.Cell cycle and apoptosis rate was detected by flow cytometry（FCM） with PI and Annexin V/PI double stainings.The experimental groups was cultured with ATRA in 20μmol/L concentration and control groups with 1‰anhydrous ethanol.The Cell cycle and apoptosis rate was detected by FCM after a further culture 24 h,48 h,72 h.3.Cks1,Cks2 mRNA were detected by RT-PCR.The experimental groups was cultured with ATRA in 20μmol/L concentration and control groups with 1‰anhydrous ethanol.The differential expression of Cks1,Cks2 mRNA was detected by RT-PCR after a further culture 24 h,48 h,72 h.4.The change of protein Cks1,Cks2,P27 expression was examined by immunocytochemistry.The experimental groups was cultured with ATRA in 20μmol/L concentration and control groups with 1%0 anhydrous ethanol.The results of protein Cks1,Cks2,P27 expression was analysed by HSCORE.Results:1.Cell BEL-7402 proliferation was significantly inhibited in time-dependent and dose-dependent manners after induced by ATRA.Inhibition rate of proliferation was 17.45±1.93 after 24 h culture and 50.23±2.44 after 96 h culture.The difference of absorbance A and inhibition rate were statistically significant among different groups （P＜0.01）.2.After induced by ATRA,G₀-G₁ phase ratio of BEL-7402 cells increased,and plenty of cells were blocked in G₀-G₁ phase,while S phase ratio decreased.G₀-G₁ phase ratio was 59.87±1.07 after 24 h culture and 72.78±1.13 after 72 h culture.There was no difference among experimental groups and control groups of G₂/M phase ratio（P＞0.05）.3.The apoptotic rates were increased gradually along with time of culture.The ratio of apoptosis was 6.48±0.45 after 24 h and 17.31±1.00 after 72 h.there was significantly difference between experimental groups,the control groups or different time goups（P＜0.01）.4.ATRA has no significant effect on the expression of Cks1,Cks2 mRNA（P＞0.05）.5.Expression of protein Cks1,Cks2 decreased after induced by ATRA.HSCORE of protein Cks1 decreased from 1.88±0.06 to 1.47±0.06 and protein Cks2 from 1.58± 0.09 to 1.39±0.03,while protein P27 increased from 2.32±0.08 to 2.9±0.11. There was significant difference among groups（P＜0.01 or P＜0.05）.Conclusions:1.ATRA inhibits the proliferation of human hepatoma cell line BEL-7402 significantly in time-dependent and dose-dependent manners;2.The apoptosis in BEL-7402 cells could be induced by ATRA;3.ATRA has no significant effect on the expression of Cks1,Cks2 mRNA,but down-regulation of their protein and up-regulation protein P27.4.ATRA caused the massive ceils to hinder in the G₀-G₁ phase,probably through the mechanism of down regulation of protein Cks1 and inhibition ubiquitination of the protein P27 degradation.The protein P27 accumulated in cells and played a negative regulation effect during the cell mitosis.5.The molecular mechanism of inducing apoptosis of cell BEL-7402 may be related to down-regulation of protein Cks2.更多还原