【作者】 王岸聪；

【导师】 刘培淑；

【作者基本信息】 山东大学 ， 妇产科学， 2009， 博士

【摘要】 研究背景Toll样受体（toll-like receptors,TLRs）是一类跨膜蛋白,最初从果蝇胚胎发育过程中被发现。哺乳类动物与果蝇的TLRs具有高度的同源性。TLRs的主要特征是细胞外富含亮氨酸的重复结构,是白介素-1受体（IL-1R）超家族的一员,在宿主的天然免疫应答中起哨兵样作用,在人的细胞中发现有十种表达亚型。不同亚型识别不同配子,启动各自的信号传导。Toll样受体4（toll-like receptor 4,TLR4）是TLRs中的一个亚型,它是在哺乳动物中最早被发现的与果蝇Toll受体高度同源的TLR。TLR4存在于机体的免疫细胞中,TLR4通过激活免疫细胞使机体产生内源或外源性免疫应答。在TLR4信号传导途径中,髓样细胞分化蛋白（MyD88）是TLR4下游的重要的衔接因子。在免疫反应中TLR4可通过MyD88依赖通路（MyD88-dependent）和非MyD88依赖通路（MyD88-independent）进行信号传导。Adam等的研究表明TLR4在胃癌、肝细胞癌、前列腺癌、淋巴瘤等多种恶性肿瘤细胞中表达,特别是TLR4的Asp299Gly基因多态性与肿瘤的发生发展高度相关。此外,TLR4在肿瘤细胞的凋亡过程中起重要作用。Fukata等的研究表明TLR4在人和小鼠的结肠肿瘤细胞中过表达,TLR4基因正常的小鼠（C3H/HeN mice）比TLR4基因敲除小鼠（C3H/HeJ mice）更易患结肠癌。Cianchi等研究发现,TLR4通过抑制凋亡增强了肿瘤细胞的免疫逃逸。Yusuf等研究证实:TLR4可以促进树突状细胞（DC）的成熟,从而增强肿瘤细胞的宿主免疫防御,在促癌因子7,12-二甲基苯并蒽（DMBA）的作用下,C3H/HeJ小鼠较C3H/HeN小鼠更易诱发皮肤癌。TLR4是把双刃剑,对不同组织中的致癌/抑癌作用使得TLR4成为非免疫学领域特别是肿瘤学领域中的研究热点。卵巢癌（ovarian cancer）是女性生殖系统最常见的恶性肿瘤,严重威胁妇女的生命和健康。在卵巢恶性肿瘤中,近70%患者在确诊时已是进展期或已有转移。卵巢癌治疗的重要手段是手术治疗和以紫杉醇、铂类等为主的联合化疗。紫杉醇（Paclitaxel,Pac）是从太平洋紫杉属短叶（Taxus brevifolia）紫杉中提取的一种抗癌新药。Pac作用于β微管蛋白N端第31个氨基酸上,促进微管蛋白聚合并抑制其解聚,使细胞周期移行阻断在G₂/M期,促使细胞死亡;美国的临床研究表明Pac对卵巢癌及乳腺癌有突出疗效,被誉为近15年来最好的抗肿瘤新药。Pac作为一线的化疗药物主要用于治疗卵巢癌,而肿瘤细胞对Pac耐药性的产生导致肿瘤的复发,使卵巢癌患者总的五年生存率仅为40%左右。文献报道在卵巢上皮癌组织及细胞系中,TLR4为阳性表达。研究表明:Pac与TLR4信号传导存在内在联系,Pac是TLR4的一个潜在配体。TLR4对卵巢癌细胞中Pac治疗作用的影响,Kelly等的研究显示:Pac启动TLR4/MyD88信号传导,激活了蛋白激酶C（Akt/PKC）,并进一步导致了NF-κB活化。TLR4通路的下游信号因子TAB1（TAK1-binding protein 1）可促进X连锁的凋亡抑制蛋白（XIAP）水平升高,而XIAP通过下调caspase-8表达而抑制TRAIL的凋亡途径。RNA干扰（RNA interference,RNAi）是指在进化过程中高度保守的、由双链RNA（double-stranded RNA,dsRNA）诱发的、同源mRNA高效特异性降解的现象。RNAi主要通过转录后水平阻断基因的表达,可以在研究基因的功能方面应用。同时可以通过关闭非必需或致病基因而改变人体的基因表达。哺乳动物中通过RNAi机制实现基因表达的抑制有多种策略,其中以构建小干扰RNA（small intereferenceRNA,siRNA）表达载体的方法最为稳定和持久。为此,寻找合适的靶基因进行RNAi,可以更细致的探讨肿瘤细胞复杂的生物学行为。本研究拟通过运用RNAi技术沉默TLR4基因的表达,探讨干扰TLR4后卵巢癌细胞对Pac敏感性的影响和发生机制,为提高Pac治疗卵巢癌的疗效寻找新途径,并为卵巢癌的临床治疗提供新的靶点。本研究分两部分:一、构建TLR4 shRNA表达质粒,稳定转染卵巢癌细胞并鉴定TLR4基因的沉默效果。二、探讨TLR4对卵巢癌细胞紫杉醇敏感性影响的作用机制。第一部分RNAi表达载体的构建及沉默TLR4基因的效果鉴定研究目的:应用基因工程技术构建针对TLR4的shRNA真核表达载体并转染卵巢癌细胞,鉴定沉默TLR4基因的效果。研究方法:1.应用基因工程技术构建TLR4的shRNA真核表达载体（1）参阅已被文献证明有效的TLR4特异性siRNA序列:正义链为5’-CGATGATATTATTGACTTA-3’;反义链5′-TAAGTCAATAATATCATCG-3’。阴性对照选择与人类基因没有任何同源的干扰序列,正义链5’-GACTTCATAAGGCGCATGC-3’;反义链5’-GCATGCGCCTTATGAAGTC-3’。（2）siRNA插入模板的设计:根据TLR4siRNA设计针对其编码区的DNA寡核苷酸链,序列为:5′-GATCCCGATGATATTATTGACTTATTCAAGACGTAAGTCAATAATATCATCGTTTTTTGTCGACA-3’。以同样方式设计阴性对照引物,序列为:5’-GATCCGACTTCATAAGGCGCATGCTTCAAGACGGCATGCGCCTTATGAAGTCTTTTTTGTCGACA-3’。（3）载体与siRNA链连接:线性化质粒载体pGenesil-1与siRNA模板链连接,合成重组质粒分别命名为pGenesil-shTLR4（目的重组质粒）和pGenesil-shControl（阴性对照重组质粒）。（4）重组质粒的转化与提取:将重组体转化大肠杆菌DH5a,筛选卡那霉素（kana^r）抗性的阳性克隆,提取质粒。（5）酶切鉴定及DNA测序:对构建的表达载体质粒pGenesil-shTLR4和pGenesil-shControl分别用SalI做酶切鉴定及DNA测序。2.重组质粒pGenesil-shTLR4、pGenesil-shControl分别转染SKOV3及A2780细胞,检测TLR4基因的沉默效果（1）采用脂质体（Lipofectamin）2000介导重组质粒pGenesil-shTLR4、pGenesil-shControl转染SKOV3、A2780细胞,G418筛选阳性克隆。（2）RT-PCR及Western blot检测稳定转染TLR4 shRNA质粒的SKOV3和A2780中TLR4和MyD88的mRNA及蛋白表达水平。结果:1.TLR4shRNA重组质粒载体酶切鉴定及DNA测序（1）构建的pGenesil-shTLR4和pGenesil-shControl的重组质粒,经琼脂糖凝胶电泳观察到被SalI酶切出一条约400bp的阳性带。pGenesil-shTLR4和pGenesil-shControl的重组质粒均符合设计要求。（2）重组质粒pGenesil-shTLR4和pGenesil-shControl的DNA测序结果通过DNAssist 2.2软件进行比对,与预先设计结果完全相符。2.TLR4基因的沉默效果鉴定（1）经G418筛选,4周后获得稳定转染pGenesil-shTLR4和pGenesil-shControl的SKOV3及A2780的阳性克隆。（2）RT-PCR,Westernblot检测结果表明:在亲代的SKOV3及A2780细胞中,TLR4在mRNA及蛋白水平均阳性表达,而MyD88在SKOV3中阳性表达（MyD88⁺）,在A2780中几乎不表达（MyD88^-）;在目的质粒（pGenesil-shTLR4）转染卵巢癌细胞株SKOV3和A2780后,两种细胞中的TLR4mRNA及蛋白的表达水平较转染前均明显降低。结论:1.应用基因工程技术构建的重组质粒pGenesil-shTLR4和pGenesil-shControl,经酶切鉴定及DNA测序证实构建成功。2.pGenesil-shTLR4的质粒载体可以稳定的干扰卵巢癌细胞株SKOV3、A2780中TLR4基因的表达。第二部分TLR4对卵巢癌细胞紫杉醇敏感性影响的实验研究研究目的:1.检测卵巢癌细胞SKOV3、A2780中caspase3/7活性的表达、细胞生长抑制率、细胞周期的变化,观察干扰TLR4基因前后SKOV3、A2780细胞对紫杉醇作用敏感性的变化。2.检测SKOV3、A2780细胞凋亡通路中相关蛋白的表达水平,探讨干扰TLR4基因后卵巢癌细胞紫杉醇作用敏感性变化的相关发生机制。研究方法:1.采用Caspase-Glo 3/7荧光检测法检测紫杉醇作用24h后,转染目的质粒、阴性对照质粒的SKOV3细胞（SKOV3/shTLR4、SKOV3/shControl）及A2780细胞（A2780/shTLR4、A2780/shControl）中Caspase-3/7活性的表达。2.采用四甲基偶氮唑盐（Dimethylthiazol-2-yl-diphenyl tetrazalium bromide,MTT）法,检测紫杉醇对转染目的质粒、阴性对照质粒的SKOV3细胞（SKOV3/shTLR4、SKOV3/shControl）及A2780细胞（A2780/shTLR4、A2780/shControl）的生长抑制率（Growth inhibitory rate,GIR）的影响。3.流式细胞术（FCM）检测紫杉醇作用24h后,卵巢癌细胞SKOV3/shTLR4、SKOV3/shControl及A2780/shTLR4、A2780/shControl的细胞周期的分布情况。4.Western blot检测紫杉醇作用24h后,卵巢癌细胞SKOV3/shTLR4、SKOV3/shControl及A2780/shTLR4、A2780/shControl细胞中凋亡相关蛋白XIAP、Akt的表达。结果:1.紫杉醇对卵巢癌细胞SKOV3及A2780细胞Caspase-3/7活性的影响:紫杉醇作用24h后,TLR4基因沉默的SKOV3细胞（SKOV3/shTLR4）较阴性对照细胞（SKOV3/shControl）其Caspase-3/7的活性显著性升高（p＜0.001）;紫杉醇作用24h后,TLR4基因沉默的A2780细胞（A2780/shTLR4）较阴性对照细胞（A2780/shControl）其Caspase-3/7的活性无显著性变化（p=0.242）。2.紫杉醇对卵巢癌细胞SKOV3及A2780的生长抑制作用:在SKOV3/shTLR4细胞中,紫杉醇作用24h后,其生长抑制率（GIR）为59%,显著性高于未干扰组（29%）及阴性对照组（31%）（p＜0.001）;相同紫杉醇作用24h,A2780/shTLR4、为干扰组、阴性对照细胞三者生长抑制率（GIR）无显著性变化（p=0.054）。3.紫杉醇对卵巢癌细胞SKOV3及A2780细胞生长周期的影响:SKOV3/shTLR4细胞生长周期较阴性对照组（SKOV3/shControl）明显停滞于S期（p＜0.01）;相同作用下,A2780/shTLR4细胞生长周期较A2780/shControl细胞无显著性变化（p=0.06）。4.紫杉醇对卵巢癌细胞SKOV3及A2780细胞中凋亡相关蛋白表达的影响:在SKOV3/shTLR4细胞中,紫杉醇作用6h后pAkt的表达明显降低;12h后,XIAP的表达也出现明显降低。而A2780/shTLR4（MyD88阴性表达）细胞在紫杉醇作用的24h中pAkt、XIAP蛋白表达均无明显变化。结论:1.TLR4抑制紫杉醇对卵巢癌细胞SKOV3（MyD88阳性表达）的促凋亡作用,沉默TLR4的表达可以增强SKOV3细胞对紫杉醇的敏感性。2.TLR4基因的沉默可以抑制Akt的信号通路传导而促进SKOV3细胞的凋亡,增强SKOV3细胞对紫杉醇的敏感性。更多还原

【Abstract】 BackgroundTLRs(toll-like receptors) are the transmembrane proteins,which were initially found in Drosophila during embryogenesis.There is high homologous for TLRs between mammalian and Drosophila.The main features of TLRs are that it containing leucine-rich repeat structure in Extracellular.TLRs are belonging to the members of IL-1R superfamily 1,which worked as a sentine at the host’s natural immune response.There are about 10 kind subtypes of TLRs found in human cells.TLR4 is a subtype of TLRs that was initially discovered in mammals,which acquires high homologous to TLRs in Drosophila.Myeloid differentiation factor 88(MyD88) was first described as a myeloid differentiation primary response gene.It was later suggested to be the critical adaptor in TLR4 signalling pathway.Further advances in the understanding of TLR4 show that TLR4 signaling is divided into MyD88-dependent and MyD88-independent(TRIF-dependent) pathways.The immune system recognizes the presence of bacterial pathogens through the expression of TLRs.Recent studies found TLR4 was overexpressed in the cells of gastric carcinoma,hepatocellular,human prostate carcinoma,and lymphomas etc.Moreover,it had been reported that the Asp299Gly variant of TLR4 was positively associated with the tumor pathogenesis and progression.Fukata et al.reported that TLR4 was overexpressed in human and murine colorectal neoplasia.And TLR4-normal mice(C3H/HeN mice) were more susceptible to colon carcinogenesis than TLR4-deficient mice(C3H/HeJ mice).Cianchi et al. reported that TLR4 induced the immune escape in tumor cells by inhibiting its apoptosis.In the research of cutaneous skin carcinogenesis,it showed that the C3H/HeJ mice developed more tumors by treatment of DMBA than that of C3H/HeN mice.The results indicate that TLR4 plays an important role in the prevention of skin tumor induced by DMBA.TLR4 is a double-edged sword for carcinogenicity in different tissues,which becomes a research hotspot in non-immune areas,especially in the field of tumor.Ovarian cancer is the most common malignant tumors in women,which is a serious threat to the lives and health of women.In ovarian cancer,nearly 70%of the patients is suffered in advanced or has been transferred at the time of diagnosis. Cytoreductive surgery with concomitant paclitaxel and platinum-based chemotherapy is the main treatment method for ovarian cancer.Paclitaxel is a product of the Pacific yew and its anti-mitotic actions are due to its ability to bind and stabilize microtubules, which prevented proper cell division during mitosis.Paclitaxel is a firstline chemotherapeutic agent used to the treatment of ovarian cancer.But the emergence of paclitaxel resistance make the tumor recurrence.And the total five-year survival rate is only around 40%in ovarian cancer patients.It has been reported that TLR4 is expressed in some ovarian tumors and ovarian cancer cells.Data show that TLR4 signalling is correlated with paclitaxel resistance. And paclitaxel is a known TLR4 ligand.In the study of Kelly et al,it supposes that paclitaxel evokes TLR4/MyD88 signalling,activating protein kinase C(Akt/PKC), which further activated NF-κB.And the downstream signal factor TAB1 of TLR4 pathway(TAK1-binding protein 1) can increase the expression level of XIAP which inhibit TRAIL apoptosis pathway through down-regulating of caspase-8 expression.RNA interference(RNAi) refers to the efficient specific degradation of homologous mRNA phenomenon which is induced by double-stranded RNA(dsRNA).RNAi mainly blocks gene expression in post-transcriptional level,and we can use this technique to investigate the function of some genes.At the same time,we can turn off non-essential gene or change gene expression in the human body.In mammals,we have a variety of strategies to knock down the expression of mRNA.And siRNA expression vector is the most stable and durable method.Therefore,we can find more appropriate target genes for RNAi interference.And it can help us to explore in detail the complex biological behavior of tumor cells.In the research,RNAi technique was used to knock down the expression of TLR4 in ovarian cancer cell lines SKOV3(MyD88~+) and A2780(MyD88~-) in order to find out whether TLR4 RNAi could increase paclitaxel chemosensitivity in MyD88~+ cells.We hope that the RNAi might provide a new target for clinical treatment of ovarian cancer. The research is divided into two parts:the one is to construct RNAi expression vector to knock down TLR4 expression in human epithelial ovarian cancer cells;the other is to investigate the role of TLR4 for paclitaxel chemotherapy in ovarian cancer cells.PARTⅠTHE CONSTRUCTION OF THE RNAi EXPRESSION VECTOR AND ITS EFFECTS ON THE TRANSFECTED OVARIAN CANCER CELLSObjective:To construct the RNAi expression vector for TLR4,and study its inhibition role on TLR4 gene in the ovarian cancer cells.Methods:1.The construction of RNAi expression vector for TLR4(1) Design of siRNA sequence:Sequence specifically targeting TLR4 were confirmed to be valid by a previous report with following:5’-CGATGATATTATTGACTTA-3’ (sense) and 5’-TAAGTCAATAATATCATCG-3’(antisense).And the negative control was used in all the experiments.The sequence of the "interference" target site is 5’-GACTTCATAAGGCGCATGC-3’,which bears no homology to any sequences in the human genome database.Therefore,the transcript-hairpin siRNA is expected to have no interference on human genes.(2) Design and construction of siRNA oligonucleotides:According to the design,the whole oligonucleotides template chain is as follow:5’-GATCCCGATGATATTATTGACTTATTCAAGACGTAAGTCAATAAT ATCATCGTTTTTTGTCGACA-3’.And the negative control sequence is as follow: 5’-GATCCGACTTCATAAGGCGCATGCTTCAAGACGGCATGCGCCTTATGAAGT CTTTTTTGTCGACA-3’.(3) The connecting of template strand siRNA and the vector: The vector and siRNA template chain was connected according to reaction system instructions.The new synthetic recombinant plasmid was named to be pGenesil-sh TLR4(target gene) and pGenesil-shControl(negative control).(4) Recombinant plasmid transformation and extraction:The recombinant plasmid was transformed into E coli.DH5a.After screening kanamycin-resistant(kanar~r) clones,the plasmids were collected.(5) Enzyme digestion and DNA sequencing:The recombinant plasmid vectors(pGenesil-shTLR4 and pGenesil-shControl) were identified by SalI enzyme digestion.And the positive bacilli were sent to Wuhan Crystal Biotech Company for sequencing.2.Investigate the effect of TLR4 gene silencing(1) pGenesil-shTLR4,pGenesil-shControl were transfected into SKOV3 and A2780 cells.Subclone cells(SKOV3/shTLR4,SKOV3/shControl,A2780/shTLR4 and A2780/ shControl cells) were obtained by G418 selection.(2) The expression level of TLR4 and MyD88 in SKOV3 and A2780 cells were analyzed by RT-PCR and Western blot methods.Results:1.The construction of the TLR4 RNAi vector(1) The pGenesil-shTLR4 and pGenesil-shControl was digested by SalI enzyme and prepared for agarose gel electrophoresis,A 400bp of DNA band was observed under ultraviolet.The recombinant plasmids are in line with the design requirements.(2) The sequencing results showed that the two constructing eukaryotic expression vectors have been identifed to contain the designed siRNA fragments,which comply with design requirements.2.Identification of TLR4-specific siRNA plasmid transfected in ovarian cancer cells.(1) Stable subclone cells(SKOV3/shTLR4,SKOV3/shControl,A2780/shTLR4 and A2780/shControl cells) were obtained after selected with G418 for 4 weeks.(2) The results by RT-PCR and Westemblots showed that in the parental SKOV3 and A2780 cells,TLR4 were expression at both mRNA and protein levels.MyD88 expressed in SKOV3 cells(MyD88~+),but it hardly expressed in A2780 cells(MyD88o);The TLR4 RNAi expression vector transfected into SKOV3 and A2780 ovarian cancer cells were confirmed to own the interference effects both in mRNA and protein expression level.Conclusions:1.We have succeed in building the TLR4 gene siRNA plasmid expression vector with the genetic engineering techniques.2.The pGenesil-shTLR4 was demonstrated to have inhibiting effect on the expression of endogenous TLR4 gene levels in SKOV3 and A2780 cells.PARTⅡROLE OF TLR4 FOR PACLITAXEL CHEMOTHERAPY IN HUMAN EPITHELIAL OVARIAN CANCER CELLSObjective:1.To observe the role of TLR4 for paclitaxel chemotherapy by detecting caspase3/7 activity,the changes of cells growth inhibition rate and cell cycle,in human epithelial ovarian cancer cells SKOV3 and A2780.2.To explore the mechanism of TLR4 on paclitaxel responsiveness in SKOV3 and A2780 cells by analyzing of some proteins in apoptosis pathway.Methods:1.The caspase-3/7 activity was analyzed by Caspase-Glo 3/7 detection kit after inhibition of TLR4 in SKOV3 and A2780 cells in the presence of paclitaxel.2.MTT assay were used to evaluate the cell growth inhibition rate(GIR) in SKOV3/shTLR4,SKOV3/shControl,A2780/shTLR4 and A2780/shControl cells in the presence of paclitaxel.3.The cell cycle distribution was investigated by Flow cytometry(FCM) in pre-and post-RNAi in SKOV3 and A2780 cells.4.The expression of XIAP and Akt proteins were detected by Western blot in SKOV3/shTLR4,SKOV3/shControl and A2780/shTLR4,A2780/shControl cells.Results:1.The caspase-3/7 activity was increased significantly in MyD88~+ cell line SKOV3/ shTLR4 treated by paclitaxel,compared with that of SKOV3/shControl cells.There was on significant difference on caspase-3/7 activity between A2780/shControl, A2780/shTLR4 cells.2.The GIR of SKOV3/shTLR4 cells was 59%in the presence of 2μM Pac for 24h, which was significantly higher than those of the parental SKOV3(29%) and SKOV3/ shControl cells(31%)(p＜0.001).No significant difference was observed on the proliferation among the three types of A2780 cells.3.The cells entry into the S phase was strongly inhibited in SKOV3/shTLR4 cells compared with the SKOV3/ shControl cells(p＜0.01).In contrast,no difference in spontaneous apoptosis was detected between A2780/shTLR4 and A2780/shControl cells.4.Moreover,the expression of XIAP and phosphorylated Akt(pAkt) in SKOV3/ shTLR4 cells were evaluated on different time of paclitaxel treatment.The results showed that the expression of pAkt was decreased significantly after 6 hours of paclitaxel treatment.XIAP levels was decreased 12 hours after treatment.In contrast, no change on the levels of total Akt was observed.But in A2780/shTLR4 cells,there was no significant change in expression of pAkt and XIAP.Conclusions:1.TLR4 inhibited SKOV3 cells(MyD88 expression positive) apoptosis,and the downregulation of TLR4 can enhance the sensitivity of SKOV3 cells to paclitaxel.2.The knockdown of TLR4 inhibited the expression of XIAP and pAkt in SKOV3/ shTLR4 cells,indicating that the knockdown of TLR4 might depress Akt pathway,and enhanced paclitaxel chemosensitivity.更多还原