【作者】 郑核；

【导师】 钟德玝；

【作者基本信息】 中南大学 ， 外科学， 2007， 博士

【摘要】 研究背景：原发性肝癌是（以下简称肝癌），是我国常见的恶性肿瘤之一，以手术为主联合化疗、放疗是目前治疗肝癌的主要模式。由于肝癌对化疗不太敏感，因此，如何降低化疗药物的毒性并增强化疗效果，值得进一步深入地研究。而随着分子生物学技术的发展，利用基因研究的进展对肝癌进行基因治疗，为肝癌的治疗提供了新的策略。Plk1（Polo-like kinase 1）基因属于有丝分裂丝氨酸／苏氨酸激酶家族Plks（Polo-like kinases）中的一员，是G₂／M期DNA损伤检测点重要激酶，对中心体的成熟，双极纺锤体的形成，及胞浆分裂等起重要作用。Plk1作为参与细胞分裂启动和正性推进者，在细胞增殖过程中起重要作用。在多种肿瘤中，Plk1常表现为过表达并与不良预后密切相关。肝癌往往发生于慢性肝病，如肝硬化和慢性肝炎，这类疾病由于肝脏损伤后伴有持续的肝细胞增殖，细胞增殖加速使肝细胞增殖周期中的调控基因更容易发生随机改变，同时细胞增殖加速也容易使慢性肝病过程中致病因子所导致的DNA突变得以保留并迅速克隆性扩张，最后导致肝癌的发生。因此，我们选定Plk1作为潜在的肿瘤基因治疗靶点，调控G₂／M期的DNA损伤检测点功能，以期达到抑制肿瘤生长、增殖的目的。RNA干扰（RNA interference，RNAi）技术是近年来出现的一种高效地封闭特异性基因的新技术，近年来无论在功能基因组研究还是肿瘤的基因治疗等方面得到广泛的应用。RNAi是指内源性或外源性双链RNA（dsRNA）介导细胞内的mRNA发生特异性降解，导致靶基因的表达沉默，产生相应的功能表型缺失。因此，运用RNA干扰技术抑制肝癌细胞内异常表达的Plk1基因从而影响其生物学行为并增强化疗药物敏感性可能为肝癌的治疗带来新的思路。第一章Plk1在肝癌组织中表达及临床意义目的：探讨Plk1在原发性肝细胞性肝癌中的表达及其意义方法：用半定量RT-PCR、Western blot的方法检测21例原发性肝细胞性肝癌中Plk1的表达情况，并分析其与肝癌临床病理因素的关系。同时取肝硬化及正常肝组织各7例做对照。结果：肝癌组织中Plk1mRNA表达比值（Plk1／β—actin）为0.572±0.144，明显高于肝硬化组织中的0.250±0.057和正常肝组织的0.218±0.073（P＜0.01）。Plk1 mRNA表达水平在肿瘤直径大于5cm组中为0.641±0.110，而在肿瘤直径小于5cm组中为0.458±0.120，两组之间比较有统计学差异（P＜0.01）。其在有门静脉癌栓患者中的表达为0.652±0.107，明显高于无门静脉癌栓者中的表达0.522±0.144。（P＜0.05）。而与有无包膜，肿瘤病理分级和AFP阳性与否无明显相关。肝癌组织Plk1蛋白表达明显高于肝硬化及正常肝组织中的Plk1蛋白表达量。结论：1．原发性肝癌组织中Plk1mRNA及蛋白表达均明显增加；2．Plk1的表达高低与原发性肝癌的肿瘤直径大小及脉管侵犯有明显相关。第二章靶向Plk1的shRNA真核表达载体构建和鉴定目的：构建靶向Plk1的短发夹状RNA（shRNA）真核表达载体。方法：设计合成两条针对Plk1的短发夹状RNA及一条阴性对照无意义短发夹状RNA，将其导入pGenesi1-2质粒中。用菌液PCR、质粒双酶切及重组质粒测序的方法，检验构建载体的正确性。结果：分别合成针对Plk1基因251—269及1396—1417位点的siRNA序列及阴性序列，成功将其导入真核表达载体，经菌液PCR、质粒双酶切及重组质粒测序，均显示siRNA序列已成功装入载体。结论：构建携带针对Plk1基因shRNA真核表达载体成功。第三章靶向Plk1的siRNA对肝癌BEL-7402细胞株生物学行为的影响目的：利用RNA（RNAi）干扰技术，研究表达Plk1的短发夹RNA（shRNA）载体对肝癌细胞株BEL-7402生物学行为的影响。方法：根据靶基因的不同区域设计两组shRNA以及一个阴性对照，利用电穿孔法转染入肝癌细胞株BEL-7402，获得稳定表达的克隆后，半定量RT-PCR检测肝癌细胞株BEL-7402的Plk1、Cdc25C、p53mRNA的变化，Western blot检测Plk1蛋白的表达，MTT法检测细胞增殖，流式细胞仪检测细胞周期变化。结果：利用电穿孔将载体转入肝癌细胞株BEL-7402，利用G418筛选，获得稳定表达的克隆。半定量RT-PCR检测转染shRNA载体的BEL—7402细胞的Plk1表达情况，Plk1siRNA-251，Plk1siRNA-1396，Plk1siRNA-hk转染组与对照组分别为0.184±0.014；0.218±0.043；0.683±0.071；0.729±0.053。检测p53mRNA分别为0.425±0.052；0.400±0.056；0.180±0.027；0.174±0.027。检测Cdc25C mRNA分别为0.387±0.083：0.353±0.059；0.816±0.052；0.837±0.065。Western blot检测BEL—7402细胞转染shRNA载体后的Plk1蛋白的表达分别为0.032±0.007、0.040±0.008、0.272±0.041、0.278±0.053。MTT检测发现转染shRNA载体后BEL—7402细胞增殖明显减慢。细胞周期检测证实转染Plk1siRNA可以引起BEL-7402细胞G0／G1期减少，G₂／M期增高，而对S期影响不大。结论：1．成功筛选出两条能特异而高效抑制Plk1基因表达的shRNA，为进一步研究该基因的功能和作用机制提供了新的手段；2．用电穿孔法筛选出稳定表达Plk1 siRNA的载体的BEL-7402细胞克隆，发现其可以显著抑制Plk1 mRNA的表达；并能引起p53mRNA的升高与Cdc25C mRNA的下降；3．用电穿孔法筛选出稳定表达Plk1 siRNA的载体的BEL-7402细胞克隆，发现其可以显著抑制Plk1蛋白的表达；4．BEL-7402细胞转染Plk1 siRNA载体后，可明显抑制细胞增殖；改变细胞周期分布。第四章靶向Plk1的siRNA对肝癌BEL-7402细胞株化疗敏感性影响的实验研究目的：利用RNA（RNAi）干扰技术，研究表达靶向Plk1的短发夹RNA（shRNA）载体对肝癌细胞株BEL-7402化疗敏感性的影响。方法：将Plk1siRNA-251，Plk1siRNA-1396，Plk1siRNA-hk转染组与对照组的BEL-7402细胞作为研究对象，用长春新碱处理细胞后，用MTT法检测四组细胞的生长抑制率，计算IC₅₀值，流式细胞仪和Hoechest染色法检测各组细胞凋亡。结果：长春新碱处理后，Plk1siRNA-251组和Plk1siRNA-1396组与BEL7402组之间的生长抑制率有显著性差异（P＜0.01）。Plk1siRNA-hk组与BEL7402组之间的生长抑制率无显著性差异（P＞0.05）。Plk1siRNA-251组的IC₅₀为14.541ug／ml，较Plk1siRNA-hk组IC₅₀22.380ug／ml降低35.02％，较BEL7402组IC₅₀21.176 ug／ml降低31.33％；而Plk1siRNA-1396组的IC₅₀为13.413ug／ml，较Plk1siRNA-hk组IC₅₀降低40.07％，较BEL7402组IC₅₀降低36.66％。5ug／ml浓度长春新碱作用24h后，Plk1siRNA-251组和Plk1siRNA-1396组凋亡指数分别为19.09％±3.97％、19.80％±5.47％明显高于Plk1siRNA-hk组和BEL7402组（P＜0.01）。结论：利用RNA干扰沉默肝癌细胞Plk1基因能增加长春新碱的化疗敏感性，值得进一步研究。更多还原

【Abstract】 Operation is the main treatment to deal with primary hepatocellularcarcinoma（PHC）which is one of the most common malignant tumors inChina at present. Chemotherapy and radiotherapy play an important role inthe systemic treatment of PHC. However, the effects of chemotherapy areoverall dissatisfying because PHC is not sensitve to that. Therefore,how todegrade the toxicity of chemotherapeutics and strengthen the curative effectof chemotherapy deserve study and research thoroughly. With thedevelopment of molecular biology technique and gene research, genetictherapy has been applied on the liver cancer, which lead to a new treatmentfor PHC.Plk1（Polo-like kinase 1）,a member of the polo-like kinase family whichis the mammalian serine/threonine protein kinase, plays pivotal roles in theregulation of cell cycle progression as a important DNA damage checkpointkinase in G₂/M phase of the mitosis. Plk1 activity is necessary for entry intomitosis, centrosome maturation, bipolar spindle formation and cytokinesis.Plk1contributes to the pathways which positively trigger entry into andprogression through mitosis and accelerate cellular proliferation. In multiplekind of tumors, Overexpression of Plk1 is observed and the level of Plk1expression has prognostic value for predicting outcomes in patients. PHCusually presented with simultaneouly underlying liver disease, eg, liver cirrhosis and chronic hepatitis which cause eontinueous liver damage andsubsequent hepatocyte regeneration result in a tendency for the control geneof cell cycle to mutate, and the intensified hepatocyte regeneration keep theDNA mutation from repair and make it multiplied in the regenerationprocess, and finally leading to the occurrence. Thus, We select Plk1 as thepotential gene therapy target to regulation the function of the DNA damagecheckpoint of G₂/M phase so as to develop a anticancer approach.RNA interference （RNAi） is a new powerful tool to inhibit the targetgene expression by post-transcriptional gene silencing, which mediated bydouble-stranded RNA （dsRNA） destructing the endogenous target mRNAssequence-specificly. The technical application has been utilized widely in theresearch of functional genome and tumor gene therapy. So silencing theexpression Plk1 in hepatocellular cells and strengthen the sensitive ofchemotherapeutics by RNA interference maybe bring a new pathway to treatPHC. PartⅠExpression and clinical significance of Polo-like kinase 1 inprimary hepatocellular carcinomaObjective To investigate the expressions of Polo-like kinase 1 mRNAand protein in primary hepatocellular carcinoma with regard to the clinicalsignificance.Methods Expressions of Pik1 mRNA and protein were detected bysemi-quantitative RT-PCR、Western blot technique in 21 cases ofPHC,7cases of hepatic cirrhosis and 7cases of normal liver tissues ascontrols. The relevance between the expressions of Plk1 mRNA and theclinical pathology of the patients of PHC were studied.Results Semi-quantitative RT-PCR demonstrated that the expressionratio of Plk1 mRNA were 0.572±0.144 in the tissues of PHC, whichsignificantly up-regulated compared with that in hepatic cirrhosis as0.250±0.057 and normal hepatic tissues as 0.218±0.073（P＜0.01）. Theexpression of Plk1 mRNA was significantly positive correlated with the sizeof the tumor and the portal vein embolism. Western blot demonstrated thatthe expression of Plk1 protein also were higher than that in hepatic cirrhosisand normal hepatic tissues.Conclusion1. The expressions of Plk1 mRNA and protein were significantlyincreased in PHC tissues.2. The expressions of Plk1 mRNA was in high relevance with the tumorsize and the portal vein embolism of PHC. PartⅡConstuction and identificantion of Eukaryotic expressionvector of siRNA targeting Plk1 geneObjective To construct the eukaryotic expression vector of siRNAtargeting Plk1 gene.Methods One pair of short hairpin RNA targeting Plk1 and anegative control nonsense short hairpin RNA were designed and synthesizedand inserted into plasmid pGenesil-2 to generate siRNA eukaryoticexpression vector. Their correct construction were tested by PCR、releasedby Barn H and HindⅢand recombinatant plasmid sequencing.Results The short hairpin RNA motifs of targeting Plk1 gene 251—269 site and 1396—1417 site and the negative control short hairpin RNAmotifs were synthesized and inserted into plasmid pGenesil-2.Therecombinatant siRNA plasmid were successfully constructed by directbacteria solution PCR、double digestion and nucleic acid sequencingidentification.Conclusion Construct the eukaryotic expression vector of siRNAtargeting Plk1 gene successfully PartⅢEffect of siRNA targeting Plk1 gene on the biological behaviorof hepatocellular carcinoma cell line BEL-7402Objective To study the influence of shRNA targeting Plk1 on thebiological behavior of hepatocellular carcinoma cell line BEL-7402.Methods The recombinant eukaryotic plasmids were transmittedinto BEL-7402 cells by electroporation. After gained stable expressedclones,the biological behabiors of the Plk1 siRNA transfected cells wereobserved by the experiments such as semi-quantitive RT-PCR、Westernblot、MTr and flow cytometry.Results The stable expressed Plk1 siRNA cell clone were gainedselected by G418 after electroporation transfection into BEL-7402successfully. We identified cell sublines stably transfected byPlk1siRNA-251, Plk1siRNA-1396, Plk1siRNA-hk. Semi-quanfitive RT-PCRdemonstrated that the expression of Plk1 and Cdc25C mRNA decreased andthe expression of p53 mRNA increased in Plk1siRNA-251,Plk1siRNA-1396groups significantly compared with that in Plk1siRNA-hk and controlgroups. Western blot demonstrated that the expression ratio of Plk1 proteinwere 0.032±0.007、0.040±0.008、0.272±0.041、0.278±0.053 in Plk1siRNA-251,Plk1siRNA-1396, Plk1siRNA-hk and control groups separately. Comparedwith other two groups, Plk1siRNA-251 and Plk1siRNA-1396 groups grewmore slowly and had a higher proportion of cells in G₂/M phase whereas lesscells in G₀/G₁ phase.Conclusion:1. Selected a pair of shRNA targeting Plk1 gene, which provide a new meas of the functional and mechanism to research Plk1 gene.2. The expressions of Plk1 mRNA and protein were inhibited which tocause the expression of p53 mRNA to elevate and Cdc25C mRNA todescend in stable expressed Plk1 siRNA cell done.3. The transfection of targeting Plk1 siRNA vector could inhabit theproliferation of BEL-7402 cells and change the cell cycle.. PartⅣStudy of the siRNA targeting Plk1 gene inducing sensitivity ofhepatocellular carcinoma cell line BEL-7402 to chemotherapyObjective To study the influence of chemotherapy sensitivity ofshRNA targeting Plk1 to hepatocellular carcinoma cell line BEL-7402.Methods Using the hepatocellular carcinoma cells constructed inprevious experiment, MTT assay was used to detect the cell growthinhibiting rate of transgene tumor cells affected by vincristine and IC₅₀ werecalculated.Flow cytometry and Hoechst dyeing was used to demonstrate theapoptosis index of different cells.Results Observed by MTT assay to hepatocellular carcinoma cellsaffected by vincristine, the growth inhibiting rate of Plk1siRNA-251 andPlk1siRNA-1396 group cells were higher significantly than that ofPlk1siRNA-hk and control group cells（P＜0.01）.The IC₅₀ of Plk1siRNA-251group was 14.541 ug/ml and cut down 35.02%, 31.33% compared withPlk1siRNA-hk group and control group. The IC₅₀ of Plk1siRNA-1396 groupwas 13.413ug/ml and cut down 40.07 %,36.66 % compared withPlk1siRNA-hk group and control group. Afer given 5ug/ml vincristine 24hlater, the apoptosis index of Plk1siRNA-251 and Plk1siRNA-1396 groupwere 19.09%±3.97 %,19.80%±5.47% and increased significantlycompared with Plk1siRNA-hk group and control group（P＜0.01）.Conclusion The siRNAs targeting Plk1 can not only suppress theexpression of Plk1 in hepatocellular cell, but can also enhance thechemotherapy sensitivity,which deserve to be studied further.更多还原