【作者】 丁磊；

【导师】 陈孝平；

【作者基本信息】 华中科技大学 ， 普外， 2006， 博士

【摘要】 肝癌（Hepatocellular carcinoma，HCC）是人体最常见的恶性肿瘤之一。以手术为主的个体化综合治疗已成为目前肝癌治疗的规范化治疗方案。化疗作为肝癌一项重要的辅助治疗手段在临床仍占据重要的地位，但多药耐药现象的产生却是制约着化疗效果的重要因素之一。近年来，寻找更加有效的基因治疗手段和高效、低毒的药物治疗肝癌的耐药问题仍是学者们研究的热点课题之一。因为任何单一的手段逆转肝癌的多药耐药现象面对复杂的耐药机制均不能达到理想的治疗效果，联合两种有效的方法更有可能达到显著增强化疗药物毒效的目的。研究发现利用基因工程技术将细胞因子基因转染肿瘤细胞，可克服其全身应用的副反应，且细胞因子释放到肿瘤周围可增加其刺激机体免疫机能的抗肿瘤作用，并且可改变肿瘤细胞对其它治疗方法如化疗的敏感性。人肿瘤坏死因子（hTNF-α）是迄今为止所发现的抗肿瘤活性最强的细胞因子之一。另有学者发现溴隐亭有抑制ATP酶的活性从而抑制P-gp的药物外泵功能。溴隐亭是一种D₂多巴胺受体拮抗剂，临床上主要用于治疗巴金森氏综合征和肢端肥大症，由于其毒副作用少而易于被患者接受。本实验即是联合人肿瘤坏死因子（hTNF-α）和溴隐亭对肝癌耐药性的治疗进行了系统的体内外实验研究，共分以下四部分。第一部分真核表达载体pBK-hTNFα的构建及其在人肝癌耐药细胞株HepG₂／ADM中的稳定表达目的：构建可在真核细胞内表达人肿瘤坏死因子alpha（hTNF-α）基因的真核表达载体。经脂质体转染人肝癌耐药细胞株HepG₂／ADM，建立稳定表达hTNF-α基因的细胞模型，为基因治疗奠定基础。方法：应用RT—PCR的方法从分离的正常人外周血单核细胞（经脂多糖，LPS刺激）中，扩增出hTNF-αcDNA，连接至载体pMD18-T vector中，经酶切鉴定与序列测定证实；以亚克隆法构建于真核表达载体pBK—CMV的相应酶切位点，转化至大肠杆菌DH5α，最后将表达的pBK-hTNFα重组蛋白行SDS-PAGE电泳，并作考马斯亮蓝染色分析，Westernblot鉴定；经脂质体Lipofectamine2000转染HepG₂／ADM细胞后，经G418筛选，通过ELISA、RT-PCR方法检测其在体外转染肝癌耐药细胞HepG₂／ADM后hTNFα基因和蛋白的表达。结果：自正常人外周血单核细胞中克隆的hTNF-αcDNA成功连接到pMD18-T vector，用BamHⅠ和HindⅢ双酶切、经序列测定证实，与Genbank的序列完全一致；pBK-hTNFα的克隆表达重组蛋白经Westernblot证实此蛋白为hTNFα。克隆基因正确插入载体pBK-CMV中。pBK-hTNFα经脂质体介导转染肝癌耐药细胞后，ELISA、RT-PCR方法检测到其在转染的细胞中稳定表达。结论：通过DNA重组技术成功地构建了可在体外稳定表达hTNF-αcDNA全长的真核表达载体pBK-hTNFα。第二部分人肿瘤坏死因子α联合溴隐亭对肝癌耐药细胞株化疗敏感性的研究目的：研究溴隐亭（Bromocriptine，BCT）联合人TNF-α基因（human tumor necrosis factor-α，hTNFα）处理人肝癌耐药细胞株HepG₂／ADM对化疗敏感性的影响。方法：首先脂质体法将hTNFα基因转染耐药细胞HepG₂／ADM，ELISA法检测转染细胞后上清液hTNFα的含量及RT-PCR方法检测转染细胞hTNFα基因表达情况；实验共分4组：空白对照组HepG₂（A）、耐药组HepG₂／ADM（B）、转染hTNFα基因组（C）及联合BCT和hTNFα组（D）；分别用流式细胞仪检测各组罗丹明123的细胞内潴留率变化、MTT法检测C、D两组细胞耐药指数的变化以及用免疫组化、Westernblot和PT-PCR方法测定各组PKC-α、P-gp蛋白、MDR1mRNA的表达变化，流式细胞仪检测Bcl-2蛋白表达，同时流式细胞仪检测各组肿瘤细胞化疗后凋亡的变化。结果：检测转染hTNFα基因耐药细胞组上清液hTNFα有明显表达，且扩增出hTNFα基因条带，说明转染成功。MTT测定C、D两组之间耐药逆转率有显著统计学意义（P＜0.01），罗丹明123潴留率实验发现C、D两组均有明显增强其潴留的作用且两组之间差异显著（P＜0.01）；P-gp蛋白和MDR1基因表达显示C、D两组之间MDR1基因和P-gp蛋白的表达无统计学差异（P＞0.05），但C与B组之间比较有显著统计学意义（P＜0.01）；PKC-α蛋白表达在D组明显下调（P＜0.01）；B组细胞Bcl-2蛋白表达明显增强，C、D组细胞Bcl-2蛋白表达明显下调，与A组比较均有统计学意义（P＜0.01），但C、D组细胞Bcl-2蛋白表达无统计学意义（P＞0.05）。流式测定细胞凋亡率C组与D组比较有显著统计学意义（P＜0.01），D组与A组比较差异无显著性（P＞0.05）。结论：溴隐亭和hTNFα通过不同的机制逆转肿瘤的多药耐药性，两者联合明显加强抗肿瘤药物对细胞的毒力。第三部分B超引导下裸鼠原位肝细胞癌多药耐药模型的建立目的：B超引导下建立裸鼠原位肝癌多药耐药模型。方法：含10％胎牛血清的1640培养基培养人肝癌细胞系HepG₂和多药耐药细胞系HepG₂/ADM（盐酸多柔比星，Adriamycin），分别在B超引导下将HepG₂和HepG₂/ADM两种细胞经皮种植于裸鼠的肝包膜下建立原位肝癌模型（分别为对照组15只、耐药组15只）。用B超、剖腹探查检测两组肝脏肿瘤生长情况。利用长链PCR技术对耐药细胞系HepG₂/ADM和相应种植瘤组织MDRi基因全长进行扩增并测序，再分别用RT-PCR、免疫组化、Westernblot方法检测两组肿瘤耐药基因MDR1mRNA和P-gp蛋白的表达。结果：原位肝脏种植肿瘤成瘤率均为100％（30/30），耐药诱导成功率为100％（15/15）；用长链PCR技术对耐药细胞系HepG₂/ADM和相应种植瘤组织进行MDR1基因扩增，均能扩增出全长为3.8Kbp基因条带，双向DNA测序结果均与Genebank报道一致。耐药组MDR1mRNA和P-gp蛋白（Westernblot）的表达均明显高于对照组（t₁=3.72，t₂=2.98，P＜0.01）。免疫组化结果示耐药组P-gp蛋白表达为（42.6±1.7）％远高于对照组（2.6±0.1）％，两者有极显著差异（t=6.43，P＜0.01）。同时对耐药细胞系HepG₂/ADM种植瘤第2周及第8周时肿瘤组织应用蛋白印迹技术进行P-gp蛋白测定显示其表达无统计学意义（t=1.27，P＞0.05），说明在短期内进行多药耐药研究的可靠性。结论：成功在B超引导下建立肝癌多药耐药动物模型为研究肝癌多药耐药逆转提供动物平台。第四部分人肿瘤坏死因子-α联合溴隐亭对人肝癌裸鼠耐药模型耐药性逆转的研究目的：研究人肿瘤坏死因子α（hTNFα）基因（Human tumor necrosiS factor-α，hTNFα）联合溴隐亭（Bromocriptine，BCT）对人肝癌裸鼠耐药模型耐药性逆转的作用。方法：将人肝癌细胞系HepG₂、耐药细胞系HepG₂/ADM和耐药细胞转染hTNFα基因后再分别原位种植裸鼠肝脏[分别称对照组（HepG₂组，A）、耐药组（ADM组，B）、转染组]，建立原位人肝癌耐药模型。其中转染组再分两组[一组单纯转hTNFα基因组称（hTNF组，C），另一组加口服溴隐亭称联合治疗组（BCT组，D）]；每组同时设对照组（注射生理盐水）和化疗组（联合小剂量持续化疗），B超观察种植瘤的大小变化，病理观察组织学结构及裸鼠生长状况和化疗药物的敏感性。采用免疫组化和逆转录—聚合酶链反应（RT-PCR）检测各种植瘤的相关耐药基因MDR1和LRP在mRNA水平、蛋白水平的变化，原位凋亡检测法（TUNEL法）检测化疗后肿瘤组织凋亡指数情况。SPECT检测种植瘤裸鼠在服用溴隐事前后各组分别注射^99mTc-methoxyisobutyl isonitrile（MIBI）在肝脏肿瘤部位的潴留情况。结果：原位种植成功率100％，种植瘤组织学特点符合人肝癌特征；转染组种植组肿瘤生长速度最慢，与其它两组比较有显著差异（P＜0.05）。采用相同的化疗方案治疗14天后发现BCT组质量抑瘤率（67％）最明显，大于HepG₂组质量抑瘤率（54％），与其他两组均有显著差异（P＜0.01）。RT-PCR结果只有单纯转染hTNF组和联合治疗组（BCT组）扩增出hTNFα基因条带（725bp），四组均有MDR1和LRPmRNA表达，组间差异显著（P＜0.05）；免疫组化显示hTNFα和BCT组肿瘤组织MDR1蛋白表达与ADM组比较有显著差异（P＜0.01），凋亡指数（TUNEL法）比ADM组均明显增高（P＜0.05），且hTNFα和BCT两组之间亦有显著性差异（P＜0.05），但与HepG₂组之间比较均无差异（P＞0.05）。SPECT检测发现转染hTNFα基因组和联合口服溴隐亭后肝肿瘤组织对^99mTc-MIBI的潴留率较耐药组明显提高（P＜0.01）。结论：TNFα基因能下调MDR1和LRPmRNA及蛋白表达，联合溴隐亭能加强对化疗药物的敏感性，降低耐药肝癌细胞的致瘤性。更多还原

【Abstract】 Human hepatocellular carcinoma （HCC） is one of the most common malignancy tumors. General given by individual treatment but giving priority to operation had been become the standard therapy project for HCC. Chemotherapy, as a critical adjuvant treatment method, still hold the important status in clinical therapy, but the MDR phenomena was one of the major factors which are restrict the chemotherapy effect.In recent years, to find an effective gene therapy method and high effective but low virulence drugs for HCC treatment was still the hot topic for the scholars. Any single method for reversing HCC multidrug resistance couldnot obtain the ideal treatment effect because of the complicated mechanism to MDR, and combining two effective means were more likely to win enhancing the susceptibility of HCC cells to cytotoxic drugs. However, cytokines administered by the conventional intravenous method can cause severe side effects. Transduction of cytokine genes into tumor cells constitutes an alternative approach for production and release of the cytokine proteins in the local tumor microenvironment, which may reduce problems of toxicity associated with systemic administration but enhance the susceptibility of the tumor cells to the cytotoxic drugs. Human tumor necrosis factor-a was one of the most powerful anti-cancer cytokines up to now. Some scholars also found that BCT, described as a dopaminergic receptor agonist, could inhibit the P-gp ATPase activity and modulates P-glycoprotein function. BCT is used in clinics to treat hyperprolactinemia and Parkinson’s disease and accepted by patients because of its little side-effect. Therefore systemic study on reversing HCC multidrug resistance combining with human tumor necrosis factor-a and bromocriptine would be carried through in this experiment which concludes four parts: Part 1 Construction of a recombinant eukaryotic vectorexpressing human tumor necrosis factor alpha andexpressing stably in cell line HepG₂/ADM[Objective] To construct a eukaryotic expression vector expressing human tumor necrosis factor alpha（hTNF-α） and expressing stably in cell line HepG₂/ADM.[Methods) The full length gene of hTNFα cDNA was identified and cloned（cDNA） from human monocyte stimulated by LPS using reverse transcription polymerase chain reaction（RT-PCR）. The cDNA was incorporated into the pMD18-T plasmid and then inserted into a dual expression vector pBK-CMV and called pBK-hTNF-α. Furthermore, pBK-hTNF-α vector was used to infect E.coli DH5α. The expression of the recombinant hTNFα protein（rhTNFα） by E.coli DH5α was analyzed using SDS-PAGE and westernblot test. After gene sequence of hTNFα was confirmed by cleavage of restriction enzymes and DNA sequence analyzed, expression of hTNFα mRNA and protein of hTNF-α transfected into drug resistant cell line HepG₂/ADM were detected by RT-PCR and ELISA.[Results] The sequence of hTNFα cDNA cloned from human monocyte was completely correct,compared with the sequence in Genbank. Digestion with BamH I and Hind III confirmed that hTNFα cDNA was inserted correctly into pBK-CMV eukaryotic expression vector. The genetically engineered E.coli DH5α did express hTNF-α confirmed by westernblot and the drug resistant cell line of HepG₂/ADM was transfected with an eukaryotic expression plasmid containing hTNFα gene by Lipofectamine2000, and then expressed hTNFα cDNA steadily. [ Conclusion ] The pBK-hTNFα, a eukaryotic expression plasmid for the full length gene of hTNFα cDNA was constructed successfully by DNA recombinant technique and expressed steadily in vitro . Part 2 Tumor necrosis factor-α and bromocription induceapoptosis and sensitize resistant hepatic cancercells to chemotherapy[Objective] To investigate the effect of bromocriptine（BCT） combining with human tumor necrosis factor a（hTNFα） inducing apoptosis in resistant hepatic cancer cells.[Methods] Firstly, to isolate and identification of hTNFα expressing cells, and to use liposome carrying hTNFα gene to transfect HepG₂ /ADM cell line and establish a cell model expressing the hTNFα protein stably. The hTNFα secreting cell clone HepG₂ /ADM/ TNF-α was obtained by G418 selection, and the integrating and secreting of hTNFα were analyzed by RT-PCR and ELISA method. All experiments were divided into four groups and named blank control group（A）、 drug resistant group HepG₂/adriamycin（ADM）（B） 、 hepatocarcinoma cell line transfected with hTNFα gene HepG₂/ADM/TNF （C ） and group BCT（D） respectively. Among these groups, group BCT was that group C treated with bromocriptine simultaneously. MTT assay was tested to detect the sensitivity to ADM for every groups’ cells, and Rhodaminel23（Rhl23） applied to test the function of P-gp by Flow Cytometric Analysis（FCM）. MDR associated genes and proteins（MDRmRNA, P-gp） and PKC-α protein were detected by immunohistochemistry （IHC）、 Westernblot and reverse transcriptase polymerase chain reaction （RT-PCR） methods respectively, and Bcl-2 protein expression and apoptosis rate of hepatocarcinoma cells was detected by FCM.[Results] The cell line transfected with hTNFα gene were established successfully by RT-PCR. The levels of hTNFα secreted by HepG₂ /ADM/ TNF-α and HepG₂ /ADM cell line were （789.68±34.43） pg/ml、 （34.48±13.62） pg/ml respectively, there was significant difference between them. At the same time, there were significant difference between group C and group D in aspect of the rate of reversing resistance and the intracellular Rho 123 accumulation （P<0.01） . MDR1mRNA and P-gp protein expression in group C and D were low similar to that in group A, but no difference could be found among them（P<0.05）. As we found that PKC-a protein expression was downregulated in group D but Bcl-2 protein expression was downregulated in group C, and there were significant difference compared to other groups. The apoptosis rate of hepatocarcinoma cells was much higher in group D than that in group C（P<0.05） with FCM, but similar to that in group A（P>0.05）.[Conclusion] Synergistic effect of between bromocription and tumor necrosis factor-α on reversing hepatocellular carcinoma multidrug resistance could be obtained and enhancing the susceptibility of HepG₂/ADM cells to cytotoxic drugs Part 3 Establishing multidrug resistant model in nude micevia orthotopic implantation of human multidrug resistanthepatocellular carcinoma cells directed by B ultrosound[Objective] To establish multidrug resistant model in nude mice via orthotopic implantation of human multidrug resistant hepatocellular carcinoma cells directed by B ultrosound.[Methods] Human hepatocellular carcinoma cells HepG₂ and multidrug resistant human hepatocellular carcinoma cells HepG₂/ADM were maintained in 1640 containing 10% heat-inactivated fetal bovine serum （FBS） at 37 °C in a humid atmosphere of 5% CO2 and an orthotopic mdr1 hepatoma was obtained by injecting the cell lines HepG₂ and HepG₂/ADM subserosally into the mice liver directed by B ultrasound（control group 15 cases, multidrug resistant groups 15 cases）. Ultrasonography and laparotomy were used to detect the tumor growth, and integrity mdrl gene from multidrug resistant human hepatocellular carcinoma cells HepG₂/ADM and corresponding implantated tumor tissues were amplificated by long PCR technique and sequenced. Furthermore, the MDR1mRNA and P-gp protein expression were evaluated by means of reverse transcription and the polymerase chain reaction（RT-PCR）、 Westernblot 、 Immunohistochemistry （IHC） methods respectively.[Results] The successful rate of tumor implantation and multidrug resistant induced is 100%（30/30）、 100% （15/15）respectively. MDR1 integrity gene 3.8kbp band can be detected from both HepG₂/ADM cells line and corresponding implantated tumor tissues by long RT-PCR technique, and the MDR1gene sequence is in coincidence to that reported in genebank. The expression of MDRlmRNA and P-gp protein in multidrug resistant groups were significantly higher than that in control groups by RT-PCR and Westernblot methods respectively. There were notable difference between multidrug resistant groups and controls which P-gp protein expression were（42.6±1.7）%and（2.6±0.1）%respectively by immunohistochemistry. Furthermore, P-gp protein expression from implanted tumors after HepG₂/ADM cells implanted 2 and 8 weeks were detected respectively and no difference were found.[Conclusion] Multidrug resistant model in nude mice via orthotopic implantation of multidrug resistant human hepatocellular carcinoma cells could be successfully established directed by B ultrasound, and provided good plateau for studying the reversal strategy of PHC multidrug resistant phenomenon. Part 4 Study on reversing HCC multidrug resistance by combination therapy with tumor necrosis factor a and bromocriptme in nude mice mdr model of liver neoplasm.[Objective] To investigate the reverse effect on HCC multidrug resistance（MDR） by combination with tumor necrosis factor a and bromocriptine in nude mice mdr model of liver neoplasm.[Methods] Human hepatocarcinoma cell line HepG₂、 drug resistant hepatocarcinoma cell HepG₂/ADM and hepatocarcinoma transfected into human tumor necrosis factor a gene cell HepG₂/ADM/TNF were injeceted into the liver of nude mice via orthotopic implantation respectively and MDR model of liver neoplasm in vivo was established[named group HepG₂（A）、 ADM（B）、 TNF（C）、 BCT（D） respectively]. BCT group was group TNF which treated together with bromocriptine using gastric canal.Each group was divided into control and chemotherapy group and size 、 weight of the tumors were measured ,tumor histological characters by HE and growth of the nude mice were observed and its chemosensitivity were tested.MDR associated genes and proteins（MRP、 LRP） of implantated tumors were detected by immunohistochemistry （IHC） and RT-PCR technique, and the apoptosis rates of hepatocarcinoma cell were measured with TUNEL assay.[Results] The nude mice model of every cell line were all inoculated successfully. The tumor growth rates and weights were different significantly among every groups（P<0.05）, with the lowest group by hTNF α transfected cell line compared to the other two groups. After the same chemotherapy though abdominal cavity tumor growth inhibitory rate was highest in D group （67%） compared with B and C group respectively （P<0.01）,and similar to HepG₂ group （54%） .MDR1 and LRPmRNA could all be detected in all groups, but TNFmRNA be detected only in C and D groups. Furthermore, MDR1 protein expression of tumors in C and D groups were lower than that in group B and were similar to group A, but difference could be detected between the group C and D. Additionally the apoptosis rates of hepatocarcinoma cell were much highest in group D than that of other groups（P<0.05） with TUNEL assay.[Conclusion] TNF-α gene can down-regulate the MDR associated genes andproteins expression for example MDR1 、 LRP, and lower its tumorgenesis. Moreover, enhancing the susceptibility of HepG₂/ADM cells to cytotoxic drugs combination therapy with bromocriptine,更多还原