【作者】 张鑫；

【导师】 陈元仲；

【作者基本信息】 福建医科大学 ， 内科学， 2008， 硕士

【摘要】 一、背景:原发性肝癌是我国常见的恶性肿瘤之一,高度恶性、预后差,如不经治疗,一般于出现症状后4～6个月死亡。手术切除、化疗、放疗、介入治疗是本病的常用治疗方法,其中手术切除为首选,但是大多数患者确诊时已发展到中晚期,此时多数病人的肝功能和体质状况较差,能做肝切除手术者不到20%。常用的化疗药物目前有阿霉素、氟尿嘧啶、顺铂等,但效果不够理想,因此人们目前仍在寻求高效低毒的化疗药物。雷公藤内酯醇(Triptolide,以下简称TPL)是从卫矛科雷公藤属植物雷公藤中分离到的二萜内酯化合物,分子式C20H24O6,分子量为360.4,临床上已广泛应用于自身免疫性疾病,如类风湿关节炎、肾炎、红斑狼疮等的治疗。近年来的研究发现TPL具有广泛且高效的抗肿瘤作用。通过30多年的研究发现TPL对人白血病、淋巴瘤、乳腺癌、骨肉瘤、胃癌、胰腺癌、肝癌、宫颈癌、卵巢癌、肺癌、结肠癌和黑色素瘤等60多种肿瘤细胞均有很好的抑制作用。TPL对肝癌细胞同样具有很好的抑制作用,近年来的研究发现TPL能抑制SMMC-7721、Hepg2等肝癌细胞增殖及诱导其凋亡,但未见有关TPL对肝癌Bel-7402细胞体内外作用的报道。二、目的:本课题以肝癌细胞株Bel-7402、Hepg2及Bel-7402细胞裸鼠移植瘤为对象,研究TPL体内外抗肝癌作用,并对其机制进行初步研究,为临床应用提供理论基础。三、方法:1.采用MTT比色法、集落形成实验检测不同浓度TPL(5～80ng/ml)对Bel-7402细胞及Hepg2细胞增殖的抑制作用。2.采用AO-EB荧光染色、TdT酶介导的缺口末端标记(TUNEL)法、DNA片段化检测及电镜观察细胞超微结构等方法检测不同浓度TPL(5～80ng/ml)对肝癌Bel-7402及Hepg2细胞凋亡的影响。3.采用RT-PCR技术检测经TPL (20、40、80ng/ml)作用后Bel-7402细胞bcl-2、bax、c-myc基因mRNA表达水平的改变。4.采用肿瘤生长曲线及抑瘤率评估TPL对Bel-7402细胞裸鼠异种移植瘤的作用:于裸鼠皮下接种对数生长期的Bel-7402细胞,建立肝癌Bel-7402细胞裸鼠异种移植瘤模型。待肿瘤直径达0.5cm时随机分组,每组6只,分为5组,分别为:(1)阴性对照组:给予含2%丙二醇的生理盐水;(2)阳性对照组:给予30mg/kg 5-氟尿嘧啶;(3)TPL组:分别给予100μg/kg、200μg/kg、400μg/kg TPL。给药方式为尾静脉注射,隔天给药,共7次。给药期间定期测量肿瘤的长径(a)、短径(b),按公式V=ab2/2计算肿瘤体积,绘制肿瘤生长曲线。并定期观察裸鼠体重的变化及进食量等。末次给药后24小时,处死裸鼠。完整剥离瘤块并称取离体瘤块重量、计算抑瘤率;透射电镜观察TPL对Bel-7402细胞裸鼠移植瘤组织细胞超微结构的影响;同时摘除眼球取血检测血常规及肝肾功能,并取心、肝、脾、肺、肾等组织作病理检查以检测TPL对裸鼠的毒性作用。四、结果:1.TPL可抑制肝癌Bel-7402细胞及Hepg2细胞增殖,且这种作用具有时间及浓度依赖性。TPL对Bel-7402细胞及Hepg2细胞作用48h的IC50分别为42.82ng/ml、7.32ng/ml。2.TPL可诱导肝癌Bel-7402细胞及Hepg2细胞凋亡。AO-EB双染法结果证实TPL可诱导肝癌Bel-7402及Hepg2细胞凋亡,透射电镜也显示TPL可诱导Bel-7402细胞凋亡。TUNEL法检测5、10、20、40、80ng/ml TPL处理48h的Bel-7402细胞及Hepg2细胞,显示两种细胞均有不同程度的凋亡细胞出现,Bel-7402细胞的凋亡率依次为9.2%、19.8%、30.2%、52.0%、64.6%,Hepg2细胞的凋亡率依次为15.0%、31.6%、49.2%、58.7%、69.1%。DNA片段化检测可见TPL处理组细胞呈现典型的梯形条带。3. TPL可调控Bel-7402细胞凋亡相关基因的表达。RT-PCR显示,分别以20、40、80ng/ml的TPL处理Bel-7402细胞48小时后,c-myc、bcl-2基因的mRNA表达下调,bax基因的mRNA表达上调,且呈浓度依赖性。4. TPL可抑制Bel-7402细胞裸鼠异种移植瘤的生长。尾静脉注射TPL 100μg/kg、200μg/kg和400μg/kg后,TPL对Bel-7402细胞裸鼠异种移植瘤的抑瘤率分别为27.2%(p>0.05)、50.0%(p<0.01)和69.5%(p<0.01),且400μg/kg组较200μg/kg组明显(p<0.05),说明TPL对Bel-7402细胞裸鼠移植瘤的作用呈浓度依赖性,且400μg/kg组效果明显优于5-FU组(p<0.01);透射电镜发现TPL处理组瘤组织中可见较多凋亡细胞;TPL治疗组与对照组比较荷瘤裸鼠的体重无明显减轻(p>0.05),血常规及肝肾功能亦未见明显异常,心、肝、脾、肺、肾等脏器未发现明显病理改变。五、结论:1. TPL可抑制体外培养的人肝癌Bel-7402及Hepg2细胞的增殖,并呈时间及浓度依赖性;2. TPL可诱导体外培养的人肝癌Bel-7402及Hepg2细胞的凋亡,并呈浓度依赖性;3. TPL诱导人肝癌Bel-7402细胞的凋亡作用,可能与上调bax基因,下调bcl-2、c-myc基因表达有关;4. TPL可抑制Bel-7402细胞裸鼠异种移植瘤的生长,呈剂量依赖性,且效果优于5-氟尿嘧啶。更多还原

【Abstract】 Introduction:Hepatocarcinoma is one of the most commonly seen tumors in China. It is highly malignant. These patients of hepatocarcinoma have poor prognosis. These patients often died within 4~6 months without treatment. Resection、radiotherapy、chemotherapy and intervention are common therapic methods of hepatocarcinoma. At present, resection offers the best chance for cure of these patients. Unfortunately, it is always metaphase or afternoon when the hepatocarcinoma is diagnosed. These patients had resectable tumors is less than 20%. Drugs most in use are adriamycin、fluracil、PDD and so on. But the effects of these drugs are not satisfactory. So, lowly-toxicity and highly-effect drugs have been explored.Triptolide(Molecular formula,C20H24O6 ; molecular weight,360.4)is a diterpenoid triepoxide derived from the Chinese herb Tripterygium wilfordii Hook f. It has been demonstrated to be effective in Patients with a variety of inflammatory and autoimmune disease , such as rheumatoid arthritis、nephritis and lupus erythematosus. Recent studies showed that TPL possessed extensive and efficient anti-tumors properties. Through 30 years research , it was found that TPL can inhibit the growth of more than 60 tumors ,such as leukemia、lymphoma、breast cancer、osteosarcoma、stomach carcinoma、pancreatic cancer、hepatocellular carcinoma、cervix cancer、ovarian cancer、lung cancer、colon cancer、melanocarcinoma. TPL can inhibit the growth of hepatocellular carcinoma cell. Refering to the records of past years, TPL can inhibit the growth of SMMC-7721、Hepg2 and induce the apoptosis. But there were no study in TPL to hepatocellular carcinoma cells Bel-7402 in vitro and in vivo.Objectives: We chose Bel-7402、Hepg2 cells and xenografts of Bel-7402 in nude mice as objective and tried to reveal the hepatocarcinoma effects in vitro and in vivo and investigate the probable mechanism, provided theory for clinic.Methods:1.MTT and colony forming assay were used to detect the effect of TPL(5～80ng/ml) on the cell proliferation of Bel-7402 and Hepg2.2.AO-EB (acridine orange-thidium bromide ) staining、TUNEL assay、DNA fragmentation and transmission electron microscope (TEM) were used to detect the effect of TPL(5～80ng/ml) on the cell apoptotic of Bel-7402 and Hepg2.3. Reverse transcriptase-polymerase chain reaction analysis (RT-PCR) were used to examine the level of bcl-2、c-myc、bax mRNA. The mRNA was abstracted from Bel-7402 cells which treated by TPL(20、40、80ng/ml).4. The graph of tumor growth and the inhibitory rate of tumor weight were used to evaluate the effect of TPL(100、200、400μg/kg) on the xenograft. The xenograft models were established by injecting the Bel-7402 cells into nude mice. When the diameters of tumors were 0.5 centimeters, these mice bearing growing tumors were divided into six groups. There were 2% propylene glycolsaline (NS) negative control group,5-FU (30mg/kg) positive control and three treatment groups in which the dose of TPL in every animal was 400μg/kg,200μg/kg、100μg/kg. Reagents were administered via injection into the caudal veins every two days for seven times. We observed the length (a) and width (b) regularly, account volume of tumors with the formula V=ab2/2 and paint graph of tumor growth. We also observed the weight and appetite of nude mice regularly. These mice were executed after 24 hours of the last treatment. Tumors were peeled off fully and quantified. The changes of tumor weights were used to evaluate the effects of TPL on xenografts growth of Bel-7402 cells. The apoptosis of tumor cell was tested by transmission electron microscope assay. The blood was gained to detect the cell numbers, liver and renal function. The important organs were detected by histology assay to evaluate the toxic action of TPL to nude mice.Results: 1. TPL evidently inhibit proliferation of Bel-7402 and Hepg2 cells in a time dependent and dose dependent manner. The IC50 values were 42.82ng/ml and 7.32ng/ml respectively after 48 hours treatment.2. TPL could induce apoptosis in Bel-7402 and Hepg2 cells: AO-EB assay showed that TPL can induce apoptosis of Bel-7402 and Hepg2 cells. Transmission electron microscope also revealed that TPL can induce apoptosis of Bel-7402 cells. TUNEL assay showed that TPL induced apoptosis of Bel-7402 and Hepg2 cells in a dose-dependent manner. The apoptotic percentage of Bel-7402 cells treated by TPL (5、10、20、40、80ng/ml) were 9.2%、19.8%、30.2%、52.0% and 64.6% respectively. The apoptotic percentage of Hepg2 cells treated by TPL (5、10、20、40、80ng/ml) were 15.0%、31.6%、49.2%、58.7% and 69.1% respectively. DNA LADDER displayed typical echelon stripe.3. TPL could regulate the expression of apoptosis-related genes: RT-PCR analysis showed that the expression of c-myc、bcl-2 were down-regulated in Bel-7402 cells treated with TPL after 48 hours, whereas the expression of bax were up-regulated. These changes were in a dose dependent manner.4. In vivo TPL could inhibit xenograft growth of Bel-7402 cells. The inhibition rate Were 27.5%(p﹥0.05)、50.0% (p﹤0.01)and 69.5%( p﹤0.01) in the group which TPL was injected to each mouse with a dose of 100μg/kg、200μg/kg and 400μg/kg every two days,the group of 400μg/kg TPL was superior to the group of 200μg/kg TPL(p﹤0.05). These showed that the inhibition was in a dose dependent manner. the group of 400μg/kg TPL was superior to the group of 5-FU(p﹤0.01).Transmission electron microscope showed there were more apoptotic cells in TPL groups. The mean weights of mice were uniform in TPL groups and negative control group(p﹥0.05). The blood cells count and liver and renal function of nude mice treated with TPL was not different to the nude mice of negative control group also. The pathologic examination of heart、liver、spleen、lung and kidney showed there were no pathological changes.Conclusion:1. TPL evidently inhibit proliferation of Bel-7402 and Hepg2 cells in a time dependent and dose dependent manner.2. TPL induce apoptosis of Bel-7402 and Hepg2 cells in a dose dependent manner.3. The mechanism that TPL induce Bel-7402 apoptosis was possibly related to down-regulated c-myc、bcl-2 and up-regulated bax.4. TPL could inhibit xenograft growth of Bel-7402 cells in vivo in a dose dependent manner and it is superior to 5-FU.更多还原