二氢叶酸还原酶与卵巢上皮癌多药耐药关系的研究
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摘要
第一章卵巢癌多药耐药的研究进展(文献综述)
卵巢恶性肿瘤是女性生殖器三大恶性肿瘤之一。卵巢隐匿于盆腔的深部,早期病变时不易被发现,一旦出现症状时大多属于晚期,治疗后容易复发,死亡率亦一直位居妇科恶性肿瘤之首。目前卵巢癌的治疗目的是早期争取治愈、晚期控制复发、延长生存期及提高患者生活质量。治疗原则是以手术为主,联合化疗及放疗的综合治疗。大多数的情况下,手术是很难将卵巢癌的转移灶彻底的切除干净。对于那些残留的细小的转移和种植结节.都需要化学药物来进行治疗。因此,化疗在卵巢癌治疗的过程中具有极其重大的意义。晚期卵巢癌的治疗方法主要以肿瘤细胞减灭术及术后辅助化疗为主。但化疗失败常会导致卵巢癌的复发,其主要原因在于肿瘤细胞产生的多药耐药(multidrug resistance,MDR)。以顺铂为主的联合化疗对卵巢癌的有效率达40%-60%,由于疾病进展和机体对化疗产生耐药,晚期卵巢癌患者的5年生存率低于20%。所以,多药耐药是肿瘤化疗失败的主要因素之一。因此,肿瘤产生耐药机制的研究以及在临床上预防耐药、对抗耐药并尝试逆转耐药就成为了当前卵巢癌治疗中的热点和难点,成为提高卵巢癌的治疗效果及改善卵巢癌患者生存的重要目标之一。
第二章卵巢癌组织中二氢叶酸还原酶基因的表达及临床意义
目的探讨卵巢癌组织中二氢叶酸还原酶基因(DHFR)(?)的表达及其临床意义。方法采用实时荧光定量PCR法测定80例卵巢癌组织、50例良性卵巢组织及30例正常卵巢组织中的DHFR mRNA表达情况,分析其与卵巢癌临床病理及多药耐药的相关性。结果(1)DHFR mRNA表达量在止常卵巢,良性卵巢肿瘤和卵巢癌组织中分别为0.584±0.234,0.895±0.783和0.197±0.412,相比较差异有统计学意义(P<0.001,P=0.027,PO.05),但在上皮性癌中浆液性癌组织中DHFR mRNA表达量则明显高于粘液性和内膜样癌,相比较差异有统计学意义(P<0.001)。(3)卵巢癌组织中DHFR mRNA表达量与患者的腹水量,淋巴结转移和远处器官转移无明显相关(P>O.05),但大网膜转移者肿瘤组织中DHFR mRNA表达量则低于无转移者(P=0.044)。(4)在卵巢癌患者中治疗后缓解者组织中DHFR mRNA表达量低于肿瘤进展者(P 第三章二氢叶酸还原酶基因表达上调对卵巢上皮癌细胞系生物学特性影响的体外实验研究
目的构建携带人二氢叶酸还原酶(DHFR)基因的慢病毒表达载体pWPI。方法采用PCR方法扩增二氢叶酸还原酶cDNA全长,与EZ-T克隆载体连接,HindⅢ及BamHI-HF限制性内切酶双酶切回收的PCR片段并补平其缺口。慢病毒系统载体使用pWPI系统,采用PmeI酶切载体后回收片段,将其磷酸化,T4酶连接载体与目的基因。表达载体鉴定均采用核苷酸序列测定,重组质粒采用脂质体转染293T包装细胞后获得包装的病毒颗粒。通过流式细胞仪检测成功构建好的DHFR-pWPI-SKOV3细胞、空载pWPI-SKOV3细胞以及亲本SKOV3细胞在不同的顺铂浓度下(2.5ug/ml,5.0ug/ml,10.0ug/ml,20.0ug/m1)不同时间段(24h、48h及72h)的三种细胞的凋亡情况以及IC50顺铂浓度(6.0ug/m1)下三种细胞的周期变化,高效液相法(HPLC法)检测不同顺铂浓度(4.0ug/ml,6.0ug/ml,8.0ug/ml)不同时间段(24h、48h及72h)药物作用后三种细胞内的顺铂浓度,以及透射电镜观察IC50顺铂浓度(6.0ug/m1)下三种细胞的超微结构变化,以探讨DHFR基因的过表达对卵巢癌细胞的体外生物学行为影响。结果(1)成功扩增二氢叶酸还原酶全长并连接入pWPI载体构建成重组表达载体DHFR-pWPI,重组质粒测序结果显与DHFR基因的同源性达100%,按标准生产程序转染293T后有DHFR基因的表达,将其病毒液感染SKOV3细胞。(2)通过流式细胞仪检测转染DHFR基因的细胞的凋亡变化,发现DHFR-pWPI-SKOV3细胞在顺铂浓度(2.5ug/ml、5.0ug/m1)刺激下,其24h、48h及72h的凋亡率均低于pWPI-SKOV3及SKOV3细胞；DHFR-pWPI-SKOV3细胞在顺铂浓度(10.0ug/ml、20.0ug/ml)刺激下,其24h及48h的凋亡率均低于pWPI-SKOV3及SKOV3细胞,但在72h时,凋亡率却高于SKOV3细胞株。表明DHFR-pWPI-SKOV3细胞在浓度小于5.0ug/ml时,不管时间的改变(72小时内),其对顺铂的抵抗力高于其他两种细胞。(3)通过流式细胞仪检测不同时间段IC50顺铂浓度(6.0ug/m1)对三种不同处理卵巢癌细胞的周期变化,结果提示1C50(6.0ug/m1)顺铂浓度给药时,DHFR-pWPI-SKOV3、pWPI-SKOV3及SKOV3三种细胞在24h、48h时其G1期含量明显低于G2+S细胞：而在72h时,DHFR-pWPI-SKOV3细胞的G2+S期明显低于pWPI-SKOV3及SKOV3细胞。(4)采用HPLC法检测细胞内顺铂浓度,结果显示细胞培养液中顺铂浓度(4.0ug/m1)给药24h、48h后及顺铂浓度(6.0ug/m1)给药24h后,DHFR-pWPI-SKOV3细胞内的顺铂含量均明显低于pWPI-SKOV3及SKOV3细胞；顺铂浓度(6.0ug/m1)给药48h及顺铂浓度(8.0ug/ml)给药24h、48h后,DHFR-pWPI-SKOV3细胞内的顺铂含量明显高于pWPI-SKOV3及SKOV3细胞。(5)通过电镜观察1C50顺铂浓度在不同时间段刺激三种不同处理的细胞的超微结构改变,发现DHFR-pWPI-SKOV3细胞在给药24h及48h其微丝聚集,线粒体从数量上及结构上改变明显,而pWPI-SKOV3细胞微丝少见,线粒体数目减少,但结构改变不明显,SKOV3细胞亦很少见到微丝,但其线粒体的结构改变亦明显,扩张与固缩同时存在；三者均出现扩张的内质网；三者均少见正常的细胞器；在给药72h后,pWPI-SKOV3及SKOV3细胞可见微丝排列紊乱,核膜部分消失,核糖体大量的聚集,进入了明显的坏死阶段,而DHFR-pWPI-SKOV3细胞未见微丝,核膜几乎完全消失,胞质内有白色囊状物质,线粒体结构基本消失,大部分细胞濒临死亡。结论成功采用慢病毒载体系统构建了二氢叶酸还原酶重组慢病毒转基因,耐药性功能实验表明DHFR表达量增高时卵巢癌细胞系耐药性增加,可以认为DHFR与卵巢癌顺铂的耐药性相关,为探讨DHFR在肿瘤多药耐药过程中的分子机理奠定基础。
第一节RNA干扰质粒载体的构建及鉴定
目的构建二氢叶酸还原酶(DHFR, dyhidrofolate reductase)基因RNAi慢病毒载体,为探讨抑伟DHFR基因表达在卵巢癌铂类耐药治疗中的意义奠定基础。方法设计DHFR基因靶向的发夹状siRNA,筛选出最佳siRNA沉默片段,退火后连接入p GSCIL载体,转化后进行序列鉴定,脂质体转染SKOV3细胞株,并进行荧光摄像。结果将退火后的双链寡核苷酸片段连接至pGPU6/GFP/Neo载体,测序结果正确。转染SKOV3细胞后,PCR鉴定干扰效果。G418筛选稳定细胞株。结论成功构建针对DHFR基因的siRNA载体,找到了有效的干扰靶序列,转染SKOV3细胞后,DHFR基因的表达明显减弱。
第二节DHFR慢病毒干扰载体的构建及其在卵巢上皮癌细胞中生物学特性影响
目的构建二氢叶酸还原酶(DHFR, dyhidrofolate reductase)基因RNA干扰慢病毒载体,研究其耐药性功能影响,为探讨抑制DHFR基因表达在卵巢癌铂类耐药治疗中的意义奠定基础。方法设计DHFR基因靶向的发夹状siRNA,筛选出最佳siRNA沉默片段,退火后连接入pGSCIL/GFP载体,转化后进行序列鉴定,脂质体转染SKOV3细胞株,并进行荧光摄像。Western blot验证构建成功通过流式细胞仪检测成功构建好的DHFR-pGSCIL-SKOV3细胞、空载pGSCIL-SKOV3细胞以及亲本SKOV3细胞在不同的顺铂浓度下(2.5ug/m1,5.0ug/ml,10.0ug/ml,20.0ug/m1)不同时间段(24h、48h及72h)的三种细胞的凋亡情况以及IC50顺铂浓度(4.4ug/ml)下三种细胞的周期变化,高效液相法(HPLC法)检测不同顺铂浓度(2.5ug/ml,5.0ug/ml,7.5ug/ml)不同时间段(24h、48h及72h)药物作用后三种细胞内的顺铂浓度,以及透射电镜观察IC50顺铂浓度(4.4ug/m1)下三种细胞的超微结构变化,以探讨DHFR基因的沉默对卵巢癌细胞的体外生物学行为影响。结果(1)将退火后的双链寡核苷酸片段连接至pGSCIL/GFP载体,测序结果正确。转染SKOV3细胞后,Western blot鉴定十扰效果。(2)在给药24h、48h及72h,DHFR-pGCSIL-SKOV3、pGCSIL-SKOV3及SKOV3细胞的凋亡率随着顺铂浓度(2.5ug/ml、5.0ug/ml、10.0ug/ml、20.0ug/m1)的增大而升高,且DHFR-pGCSIL-SKOV3细胞给药48h及72h后的凋亡率明显高于pGCSIL-SKOV3及SKOV3细胞。(3)在IC50(4.4ug/ml)顺铂浓度给药24h、48h后,DHFR-pGCSIL-SKOV3、pGCSIL-SKOV3及SKOV3细胞均以G1期为主。DHFR-pGCSIL-SKOV3在48h时G1期的细胞比例明显减少,S期和G2/M期比例增多,72h后三种细胞G1期的含量均明显降低,S期和G2/M期的含量均增高。(4)采用高效液相检测细胞内顺铂浓度时,当2.5ug/m1、5.0ug/ml顺铂浓度作用三种不同处理的卵巢癌细胞时,DHFR-pGCSIL-SKOV3细胞在给药后24h、48h其细胞内顺铂浓度均明显高于pGCSIL-SKOV3及SKOV3细胞。而DHFR-pGCSIL-SKOV3细胞在7.5ug/ml顺铂浓度给药后24h其细胞内顺铂浓度均明显低于PGCSIL-SKOV3及SKOV3细胞,而在48h却又高于pGCSIL-SKOV3及SKOV3细胞。(5)在IC50(4.4ug/m1)给药24h、48h三种不同处理细胞的微丝均少见,DHFR-pGCSIL-SKOV3细胞在24h时可见线粒体肿胀、脊消失、空泡化、萎缩,内质网扩张明显,有聚集的核糖体,末见高尔基体,48h时其有些线粒体溶解消失,且出现大量的白色囊泡；而pGCSIL-SKOV3及SKOV3细胞24h、48h内质网扩张少见,线粒体结构改变不明显,甚至有2-3个高尔基体存在；DHFR-pGCSIL-SKOV3细胞至72h时,微丝明显的聚集、增多,线粒体结构亦消失。余两种细胞微丝排列紊乱,线粒体形态多样化。结论成功构建针对DHFR基因的siRNA慢病毒载体,找到了有效的干扰靶序列,转染SKOV3细胞后,DHFR基因的表达明显减弱。通过对DHFR下调后耐药性功能研究证实DHFR基因的下调与经过铂类药物作用的体外卵巢癌细胞的药物敏感性有一定的联系,抑制DHFR基因表达能增加顺铂药物敏感性,因此,抑制卵巢癌细胞DHFR基因的表达可以考虑作为顺铂多药耐药的逆转机制。
CHAPTER I RESEARCH PROGRESS FOR OVARIAN CANCER MULTIDRUG-RESISTANT
Ovarian cancer is female genitals one of three major malignant tumor. Ovarian hidden in the deep of the pelvic cavity, early lesions will not be discovered, once appear when symptoms are belong to late, after treatment, the easy relapse, mortality also is always in the lethal gynecological malignancy. At present the aim of treatment for ovarian cancer early, late recurrence control for cure, prolonging the survival time and improve the quality of life of the patients. Treatment is mainly surgery, chemotherapy and radiation treatment. Most of the cases, surgery is difficult to ovarian cancer metastases thoroughly clean the resection. For those who remain the tiny transfer and planting nodules. All need chemical drugs to treat them. Therefore, the chemotherapy treatment in the process of oarian cancer have extremely great significance. Adanced oarian cancer treatments mainly by tumor cells was reduced and postoperative adjuvant chemotherapy out to give priority to. But often cause ovarian cancer chemotherapy failed the relapse, the main reason is that the tumor cells to produce more of drug-resistant (multidrug resistance, MDR). With the combination chemotherapy cisplatin primarily for ovarian cancer rate was40%-60%, because disease progression and the body's resistance to chemotherapy produce, patients with adanced oarian cancer of the5year's survival rates below20%. So. many drug-resistant tumor chemotherapy is one of the main factors of failure. Therefore, tumours produce mechanisms of resistance in clinical research and prevent resistance against drug resistance, resistance to try and reverse became the current ovarian cancer treatment of hot and difficult, which become improve ovarian cancer cure effect and improve ovarian cancer survival one of the important targets.
CHAPTER II THE EXPRESSION OF DIHYDROFOLATE REDUCTASE GENE AND ITS CLINICAL SIGNIFICANCE IN OVARIAN CANCER
Objective To investigate the expression of dihydrofolate reductase (DHFR) mRNA in ovarian cancer and to elucidate the relationship between the clinical pathologic parameters and the expression of DHFR mRNA in ovarian cancer. Method The expression of DHFR in80cases of ovarian carcinoma and50cases of benign ovarian and30cases of normal control were detected by real-time fluorescent quantitative polymerase chain reaction-and the relationship between them was further demonstrated. Results (1)The content of DHFR mRNA expressed in ovarian carcinoma, benign ovarian tumor and normal ovarian tissues was0.584±0.234,0.895±0.783and0.197±0.412. There were significant differences between them (P<0.001,P=0.027, P<0.001).(2)There was not relationship between the content of DHFR mRNA expressed and the stage and pathological grade in ovarian cancer, but DHFR mRNA expression in serous carcinoma was significantly higher than that in mucinous and endometrioid carcinoma (P<0.001).(3)The expression of DHFR mRNA in ovarian cancer was not related significantly and the amount of ascites-lymph node metastasis and distant metastasis in the patients with ovarian cancer(P>0.05), but the expression of DHFR mRNA in patients with the omentum metastasis was down-regulated compared with that without the omentum metastasis, there was difference statistics significantly (P=0.044).(4)The expression content of DHFR mRNA in patients with CR for treatment was lower than that with SD or PD or PR (P<0.001), and also the expression of DHFR mRNA in patients with cisplatinum-sensitive was down-regulated compared with those of cisplatinum multidrug-resistant (0.130±0.103vs0.341±0.701P=0.011).(5)Youden index of the ROC curve for DHFR mRNA expression was0.331, which would be used to determine the nature of the ovarian tumors. The median survival time of patients with DHFR mRNA expression up-regulated (less than0.331) was16.4months, but the median survival time of patients with DHFR mRNA expression down-regulated (higher than0.331) was44.5months, there was difference statistical significantly (P<0.001), and COX multivariate analysis also showed DHFR mRNA expression was an independent prognostic factor in ovarian cancer(P=0.018). Conclusions It was suggested that DHFR would be to play in physiology metabolism in normal cells. There was a relationship between DHFR mRNA up-regulated expression and cisplatinum multidrug-resistant in ovarian cancer, and the expression of DHFR mRNA could be used to determine whether there was cisplatinum multidrug-resistant in ovarian cancer as a potential marker.
CHAPTER III THE BIOLOGICAL ACTIONS EFFECT OF DIHYDROFOLATE REDUCTASE GENE EXPREESION UPREGULATION IN EPITHELIAL OVARIAN CANCER IN VITRO EXPERIMENTS
Objective To construct a lentiviral expressing vector harboring human dihydrofolate reductase (DHFR) gene. Methods The cDNA length of DHFR gene was amplified by PCR and was connected to cloned vector EZ-T. The recovered PCR fragment was obtained by digesting with restriction enzymes named HindⅢ and BamHI-HF, then blunted at the gap. Lentiviral vector system adopted pWPI system.The vector pWPI was digested with PmeI enzyme, and then was recovered fragment and phosphorylated. The phosphorylated vector was connected with DHFR gene by T4enzyme. Recombinant plasmid was confirmed by PCR and sequencing nucleotide. The recombinant retroviral vector pWPI was selected to transfect packaging cell293T to gain virus particles with infection ability,which were infected SKOV3cell. Adopting the flow cytometric to detect the cell apoptosis of DHFR-pWPI-SKOV3cells, pWPI-SKOV3cells and SKOV3cells in different cisplatin concentrations (2.5ug/ml,5.0ug/ml,10.0ug/ml.20.0ug/ml) and different time period (24h,48h and72h),and the of cell periodic changes of theres cells under IC50cisplatin concentration (6.0ug/ml), we also used highly effective liquid phase method (HPLC) to test intracellular cisplatin concentration in different induction of cisplatin concentration (2.5ug/ml,5.0ug/ml,10.0ug/ml,20.0ug/ml) and different time period (24h.48h and72h),what's more.we further adopted transmission electron microscope to observe ultrastructural changes of these kinds of cells under induction of IC50cisplatin concentration (6.0ug/ml).The aim of this study is investigate how the upregulated DHFR gene expression affects biological actions of ovarian cancer cell in vitro.Results (1)The recombinant plasmid, named DHFR-pWPI, which was constructed and identified Packaged by packaging cell293T. The homology between sequencing results and DHFR gene sequence was up to100%, and the expression of DHFR in293T cells was determined by RT-PCR.(2)Through the flow cytometric to detect apoptosis changes cells carried DHFR gene,we found that DHFR-pWPI-SKOV3cell in the stimulation of cisplatin concentration (2.5ug/ml,5.0ug/ml), its apoptosis rate at24h.48h and72h was lower than pWPI-SKOV3and SKOV3cells; DHFR-pWPI-SKOV3cells in the induction of cisplatin concentration (10.0ug/ml,20.0ug/ml), whose apoptosis rate at24h and48h was lower than pWPI-SKOV3and SKOV3cells, but at72h, its apoptosis rate was higher than SKOV3cell line.The results showed that DHFR-pWPI-SKOV3cells in the concentration less than5.0ug/ml, regardless of the time change (≥72hours).whose cisplatin drug resistance is higher than the other two kinds of cells.(3) We adopted the flow cytometric to test different time period under IC50cisplatin concentration (6.0ug/ml) for three kinds of ovarian cancer cells cycle, and the results indicated that G1stage rate of DHFR-pWPI-SKOV3, pWPI-SKOV3and SKOV3cells at24h and48h were obviously lower than those of G2and S stage rates:However, at72h, DHFR-pWPI-SKOV3cells G2+S phase rates were significantly lower than the pWPI-SKOV3and SKOV3cells.(4) This experiment used HPLC method to detect intracellular cisplatin concentration, and the results showed that the cells in the cisplatin concentration (4.0ug/ml) at24h,48h and cisplatin concentration (6.0ug/ml) at24h after the medicine induction, the intracellular cisplatin content of DHFR-pWPI-SKOV3cell were significantly lower than pWPI-SKOV3and SKOV3cells; cisplatin concentration (6.0ug/ml) at48h and cisplatin concentration (8.0ug/ml) at24h and48h. the intracellular cisplatin content of DHFR-pWPI-SKOV3cell were obviously higher than pWPI-SKOV3and SKOV3cells.(5)The experiment obeserved ultrastructural changes of three different cells induced by IC50cisplatin concentration in different time period through the electron microscope.which found that for DHFR-pWPI-SKOV3cells, the microwire gathered together at24h and48h, and the number and structure of mitochondria had obvious change, for pWPI-SKOV3cells,which had rare microfilament, the number of mitochondria decreased but structure change is not apparent, we also saw seldom microfilament and obvious mitochondrial structure changes in SKOV3cells, expand and pyknotic mitochondrial structure existed at the same time; all of hese cells appeared expansion of endoplasmic reticulum and had rare normal organelles; at72h,there are inordinate microfilament, a part of nuclear membrane disappeared, a lot of ribosomes gathered together in pWPI-SKOV3and SKOV3cells, and there were rare microfilament in DHFR-pWPI-SKOV3cells,nuclear membrane completely disappeared, many white cystic matter were seen in cytoplasm, mitochondrial structure disappeared completely, which seems most cells is on the verge of death.Conclusion The lentiviral expressing vector harboring human DHFR had been constructed successfully, DHFR expression increased when the cell was drug-resistant in ovarian cancer cell, which suggested DHFR gene expression is related to cisplatin drug resistance in ovarian cancer,The results made the foundation to investigate the molecular mechanism of multidrug-resistance in tumor.
CHAPTER Ⅳ THE BIOLOGICAL IMPACT OF DIHYDROFOLATE REDUCTASE GENE EXPRESSION DOWNREGULATION IN EPITHELIAL OVARIAN CANCER CELLS IN VITRO EXPERIMENTS
THE FIRST QUARTER:CONSTRUCTION AND IDENTIFICATION OF DIHYDRIFOLATE REDUCTASE GENE siRNA-EXPRESSION PLASMID
Objective To construct dyhidrofolate reductase gene(DHFR) small interfering RNA(siRNA)and its expression vector,and explore the significance of DHFR inhibition in the treatment of platinum multidrug-resistance in ovarian cancer. Methods We designed DHFR gene-targeted hairpin siRNA and then screened the best siRNA silence fragment, and inserted into pGPU6/GFP/Neo plasmid,which was identified by sequencing. Human malignant ovarian cancer SKOV3cells were transfected with the constructed vector using lipofectin transfection method. Fluorescence photographs were taken. Then adopting G418method to screen the stable cell line. Results Three groups of DHFR gene-targeted hairpin siRNA were designed,and inserted into pGPU6/GFP/Neo vector after annealing. Vectors containing siRNA were right by sequencing. Fluorescence photographs showed that SKOV3cells were transfected successfully by lipofectin transfection method,and PCR method identified its interference effect. Conclusion DHFR gene-targeted siRNA and its vector was successfully constructed. And one sequence with the highest inhibition efficiency was screened out. DHFR gene expression declined obviously after the constructed plasmid was transfected into SKOV3cell.
THE SECOND QUARTER:THE CONSTRUCTION OF siRNA DHFR LENTIVIRUS INTERFERENCE CARRIER AND ITS BIOLOGICAL FUNNCTION AND INFLUENCE IN OVARIAN CANCER
Objective To construct dyhidrofolate reductase (DHFR) gene RNAi interference lentivirus carrier, to explore the relationship between the inhibition of DHFR gene and platinum drug resistance in ovarian cancer,which made the foundation for Resistance mechanisms in the therapy of ovarian cancer. Methods To design targeting hairpin siRNA of DHFR gene, select the best siRNA silent sequence, join to pGSCIL/GFP vector after anneal, adopt PCR to identify recombination sequence, transfect into SKOV3cell lines through liposomes, finally use fluorescence camera. Western blotting identify the interferential DHFR gene of ovarian cancer SKOV3cell was constructed successfully,named DHFR-pGSCIL-SKOV3cell. Adopting the flow cytometric to detect the cell apoptosis of DHFR-pGSCIL-SKOV3cells, pGSCIL-SKOV3cells and SKOV3cells in different cisplatin concentrations (2.5ug/ml,5.0ug/ml,10.0ug/ml,20.0ug/ml) and different time period (24h,48h and72h),and the of cell periodic changes of theres cells under IC50cisplatin concentration (4.4ug/ml), we also used highly effective liquid phase method (HPLC) to test intracellular cisplatin concentration in different induction of cisplatin concentration (2.5ug/ml,5.0ug/ml,10.0ug/ml,20.0ug/ml) and different time period (24h.48h and72h),what's more.we further adopted transmission electron microscope to observe ultrastructural changes of these kinds of cells under induction of IC50cisplatin concentration (4.4ug/ml).The aim of this study is investigate how the downregulated DHFR gene expression affects biological actions of ovarian cancer cell in vitro.Results (1)Let double-strand nucleotide after annealing connect to pGSCIL/GFP vector, sequence result is correct. Transfect Virus particles into SKOV3cell, using western blotting identifed interference effect.(2)Through the flow cytometric to detect apoptosis changes of cells knocked DHFR gene,we found that DHFR-pGCSIL-SKOV3, pGCSIL-SKOV3and SKOV3cell apoptosis rate increased with the raised cisplatin concentration (2.5ug/ml.5.0ug/ml,10.0ug/ml,20.0ug/ml) at24h、48h and72h,and the apoptosis rate of DHFR-pGCSIL-SKOV3was obvious higher than pGCSIL-SKOV3and SKOV3cells at24h and48h.(3)We adopted the flow cytometric to test different time period under IC50cisplatin concentration (4.4ug/ml) for three kinds of ovarian cancer cells cycle, and the results indicated that G1stage rate of DHFR-pGCSIL-SKOV3, pGCSIL-SKOV3and SKOV3cells at24h、48h and72h were obviously higher than those of G2and S stage rates; However, at24h and48h, DHFR-pGCSIL-SKOV3cells G2/M and S phase rates were significantly increased while G1phase rates decreased,at72h, G2/M and S phase rates were significantly increased while G1phase rates decreased for all of three kinds of cells.(4)This experiment used HPLC method to detect intracellular cisplatin concentration, and the results showed that the cells in the cisplatin concentration (2.5ug/ml、5.0ug/ml) at24h and48h,the intracellular cisplatin content of DHFR-pGCSIL-SKOV3cell were significantly higher than pGCSIL-SKOV3and SKOV3cells; but cisplatin concentration (7.5ug/ml) at24h, the intracellular cisplatin content of DHFR-pGCSIL-SKOV3cell were obviously lower than pGCSIL-SKOV3and SKOV3cells.while higher than pGCSIL-SKOV3and SKOV3cells at48h.(5)The experiment obeserved ultrastructural changes of three different cells induced by IC50cisplatin concentration(4.4ug/ml) in different time period through the electron microscope,which found that for DHFR-pGCSIL-SKOV3at24h, visible when mitochondria swelling, its ridge disappear, the empty bubble and atrophy, obvious endoplasmic reticulum expansion.ribosomes gathered together, rare golgiapparatus.At48h,some mitochondria were dissolve and disappeared, and a large number of white vesicles were appeared; at72h, the micro filament obvious increased and gathered together, mitochondrial structure also disappear.But the microfilament of the other two cells arranged disorderly, mitochondrial form diversification.However,for pGCSIL-SKOV3and SKOV3cells at24h and48h rare endoplasmic reticulum expansion, mitochondrial structure change is not obvious, and even existed2-3golgi apparatus; the microwire gathered together at24h and48h, and the number and structure of mitochondria had obvious change, for pWPI-SKOV3cells,which had rare microfilament, the number of mitochondria decreased but structure change is not apparent, we also saw seldom microfilament and obvious mitochondrial structure changes in SKOV3cells, expand and pyknotic mitochondrial structure existed at the same time; all of hese cells appeared expansion of endoplasmic reticulum and had rare normal organelles; at72h,there are inordinate microfilament, a part of nuclear membrane disappeared, a lot of ribosomes gathered together in pWPI-SKOV3and SKOV3cells, and there were rare microfilament in DHFR-pWPI-SKOV3cells,nuclear membrane completely disappeared, many white cystic matter were seen in cytoplasm, mitochondrial structure disappeared completely, which seems most cells is on the verge of death.Conclusions We are succeed in constructing DHFR gene siRNA carrier, screen the effective target sequence, and then transfect into SKOV3cell.DHFR gene expression were significantly weakened. The research indicated thatknock out DHFR gene is related to cisplatin drug resistance in ovarian cancer,The results made the foundation to investigate the molecular mechanism of multidrug-resistance in tumor.
卵巢恶性肿瘤是女性生殖器三大恶性肿瘤之一。卵巢隐匿于盆腔的深部,早期病变时不易被发现,一旦出现症状时大多属于晚期,治疗后容易复发,死亡率亦一直位居妇科恶性肿瘤之首。目前卵巢癌的治疗目的是早期争取治愈、晚期控制复发、延长生存期及提高患者生活质量。治疗原则是以手术为主,联合化疗及放疗的综合治疗。大多数的情况下,手术是很难将卵巢癌的转移灶彻底的切除干净。对于那些残留的细小的转移和种植结节.都需要化学药物来进行治疗。因此,化疗在卵巢癌治疗的过程中具有极其重大的意义。晚期卵巢癌的治疗方法主要以肿瘤细胞减灭术及术后辅助化疗为主。但化疗失败常会导致卵巢癌的复发,其主要原因在于肿瘤细胞产生的多药耐药(multidrug resistance,MDR)。以顺铂为主的联合化疗对卵巢癌的有效率达40%-60%,由于疾病进展和机体对化疗产生耐药,晚期卵巢癌患者的5年生存率低于20%。所以,多药耐药是肿瘤化疗失败的主要因素之一。因此,肿瘤产生耐药机制的研究以及在临床上预防耐药、对抗耐药并尝试逆转耐药就成为了当前卵巢癌治疗中的热点和难点,成为提高卵巢癌的治疗效果及改善卵巢癌患者生存的重要目标之一。
第二章卵巢癌组织中二氢叶酸还原酶基因的表达及临床意义
目的探讨卵巢癌组织中二氢叶酸还原酶基因(DHFR)(?)的表达及其临床意义。方法采用实时荧光定量PCR法测定80例卵巢癌组织、50例良性卵巢组织及30例正常卵巢组织中的DHFR mRNA表达情况,分析其与卵巢癌临床病理及多药耐药的相关性。结果(1)DHFR mRNA表达量在止常卵巢,良性卵巢肿瘤和卵巢癌组织中分别为0.584±0.234,0.895±0.783和0.197±0.412,相比较差异有统计学意义(P<0.001,P=0.027,P
目的构建携带人二氢叶酸还原酶(DHFR)基因的慢病毒表达载体pWPI。方法采用PCR方法扩增二氢叶酸还原酶cDNA全长,与EZ-T克隆载体连接,HindⅢ及BamHI-HF限制性内切酶双酶切回收的PCR片段并补平其缺口。慢病毒系统载体使用pWPI系统,采用PmeI酶切载体后回收片段,将其磷酸化,T4酶连接载体与目的基因。表达载体鉴定均采用核苷酸序列测定,重组质粒采用脂质体转染293T包装细胞后获得包装的病毒颗粒。通过流式细胞仪检测成功构建好的DHFR-pWPI-SKOV3细胞、空载pWPI-SKOV3细胞以及亲本SKOV3细胞在不同的顺铂浓度下(2.5ug/ml,5.0ug/ml,10.0ug/ml,20.0ug/m1)不同时间段(24h、48h及72h)的三种细胞的凋亡情况以及IC50顺铂浓度(6.0ug/m1)下三种细胞的周期变化,高效液相法(HPLC法)检测不同顺铂浓度(4.0ug/ml,6.0ug/ml,8.0ug/ml)不同时间段(24h、48h及72h)药物作用后三种细胞内的顺铂浓度,以及透射电镜观察IC50顺铂浓度(6.0ug/m1)下三种细胞的超微结构变化,以探讨DHFR基因的过表达对卵巢癌细胞的体外生物学行为影响。结果(1)成功扩增二氢叶酸还原酶全长并连接入pWPI载体构建成重组表达载体DHFR-pWPI,重组质粒测序结果显与DHFR基因的同源性达100%,按标准生产程序转染293T后有DHFR基因的表达,将其病毒液感染SKOV3细胞。(2)通过流式细胞仪检测转染DHFR基因的细胞的凋亡变化,发现DHFR-pWPI-SKOV3细胞在顺铂浓度(2.5ug/ml、5.0ug/m1)刺激下,其24h、48h及72h的凋亡率均低于pWPI-SKOV3及SKOV3细胞；DHFR-pWPI-SKOV3细胞在顺铂浓度(10.0ug/ml、20.0ug/ml)刺激下,其24h及48h的凋亡率均低于pWPI-SKOV3及SKOV3细胞,但在72h时,凋亡率却高于SKOV3细胞株。表明DHFR-pWPI-SKOV3细胞在浓度小于5.0ug/ml时,不管时间的改变(72小时内),其对顺铂的抵抗力高于其他两种细胞。(3)通过流式细胞仪检测不同时间段IC50顺铂浓度(6.0ug/m1)对三种不同处理卵巢癌细胞的周期变化,结果提示1C50(6.0ug/m1)顺铂浓度给药时,DHFR-pWPI-SKOV3、pWPI-SKOV3及SKOV3三种细胞在24h、48h时其G1期含量明显低于G2+S细胞：而在72h时,DHFR-pWPI-SKOV3细胞的G2+S期明显低于pWPI-SKOV3及SKOV3细胞。(4)采用HPLC法检测细胞内顺铂浓度,结果显示细胞培养液中顺铂浓度(4.0ug/m1)给药24h、48h后及顺铂浓度(6.0ug/m1)给药24h后,DHFR-pWPI-SKOV3细胞内的顺铂含量均明显低于pWPI-SKOV3及SKOV3细胞；顺铂浓度(6.0ug/m1)给药48h及顺铂浓度(8.0ug/ml)给药24h、48h后,DHFR-pWPI-SKOV3细胞内的顺铂含量明显高于pWPI-SKOV3及SKOV3细胞。(5)通过电镜观察1C50顺铂浓度在不同时间段刺激三种不同处理的细胞的超微结构改变,发现DHFR-pWPI-SKOV3细胞在给药24h及48h其微丝聚集,线粒体从数量上及结构上改变明显,而pWPI-SKOV3细胞微丝少见,线粒体数目减少,但结构改变不明显,SKOV3细胞亦很少见到微丝,但其线粒体的结构改变亦明显,扩张与固缩同时存在；三者均出现扩张的内质网；三者均少见正常的细胞器；在给药72h后,pWPI-SKOV3及SKOV3细胞可见微丝排列紊乱,核膜部分消失,核糖体大量的聚集,进入了明显的坏死阶段,而DHFR-pWPI-SKOV3细胞未见微丝,核膜几乎完全消失,胞质内有白色囊状物质,线粒体结构基本消失,大部分细胞濒临死亡。结论成功采用慢病毒载体系统构建了二氢叶酸还原酶重组慢病毒转基因,耐药性功能实验表明DHFR表达量增高时卵巢癌细胞系耐药性增加,可以认为DHFR与卵巢癌顺铂的耐药性相关,为探讨DHFR在肿瘤多药耐药过程中的分子机理奠定基础。
第一节RNA干扰质粒载体的构建及鉴定
目的构建二氢叶酸还原酶(DHFR, dyhidrofolate reductase)基因RNAi慢病毒载体,为探讨抑伟DHFR基因表达在卵巢癌铂类耐药治疗中的意义奠定基础。方法设计DHFR基因靶向的发夹状siRNA,筛选出最佳siRNA沉默片段,退火后连接入p GSCIL载体,转化后进行序列鉴定,脂质体转染SKOV3细胞株,并进行荧光摄像。结果将退火后的双链寡核苷酸片段连接至pGPU6/GFP/Neo载体,测序结果正确。转染SKOV3细胞后,PCR鉴定干扰效果。G418筛选稳定细胞株。结论成功构建针对DHFR基因的siRNA载体,找到了有效的干扰靶序列,转染SKOV3细胞后,DHFR基因的表达明显减弱。
第二节DHFR慢病毒干扰载体的构建及其在卵巢上皮癌细胞中生物学特性影响
目的构建二氢叶酸还原酶(DHFR, dyhidrofolate reductase)基因RNA干扰慢病毒载体,研究其耐药性功能影响,为探讨抑制DHFR基因表达在卵巢癌铂类耐药治疗中的意义奠定基础。方法设计DHFR基因靶向的发夹状siRNA,筛选出最佳siRNA沉默片段,退火后连接入pGSCIL/GFP载体,转化后进行序列鉴定,脂质体转染SKOV3细胞株,并进行荧光摄像。Western blot验证构建成功通过流式细胞仪检测成功构建好的DHFR-pGSCIL-SKOV3细胞、空载pGSCIL-SKOV3细胞以及亲本SKOV3细胞在不同的顺铂浓度下(2.5ug/m1,5.0ug/ml,10.0ug/ml,20.0ug/m1)不同时间段(24h、48h及72h)的三种细胞的凋亡情况以及IC50顺铂浓度(4.4ug/ml)下三种细胞的周期变化,高效液相法(HPLC法)检测不同顺铂浓度(2.5ug/ml,5.0ug/ml,7.5ug/ml)不同时间段(24h、48h及72h)药物作用后三种细胞内的顺铂浓度,以及透射电镜观察IC50顺铂浓度(4.4ug/m1)下三种细胞的超微结构变化,以探讨DHFR基因的沉默对卵巢癌细胞的体外生物学行为影响。结果(1)将退火后的双链寡核苷酸片段连接至pGSCIL/GFP载体,测序结果正确。转染SKOV3细胞后,Western blot鉴定十扰效果。(2)在给药24h、48h及72h,DHFR-pGCSIL-SKOV3、pGCSIL-SKOV3及SKOV3细胞的凋亡率随着顺铂浓度(2.5ug/ml、5.0ug/ml、10.0ug/ml、20.0ug/m1)的增大而升高,且DHFR-pGCSIL-SKOV3细胞给药48h及72h后的凋亡率明显高于pGCSIL-SKOV3及SKOV3细胞。(3)在IC50(4.4ug/ml)顺铂浓度给药24h、48h后,DHFR-pGCSIL-SKOV3、pGCSIL-SKOV3及SKOV3细胞均以G1期为主。DHFR-pGCSIL-SKOV3在48h时G1期的细胞比例明显减少,S期和G2/M期比例增多,72h后三种细胞G1期的含量均明显降低,S期和G2/M期的含量均增高。(4)采用高效液相检测细胞内顺铂浓度时,当2.5ug/m1、5.0ug/ml顺铂浓度作用三种不同处理的卵巢癌细胞时,DHFR-pGCSIL-SKOV3细胞在给药后24h、48h其细胞内顺铂浓度均明显高于pGCSIL-SKOV3及SKOV3细胞。而DHFR-pGCSIL-SKOV3细胞在7.5ug/ml顺铂浓度给药后24h其细胞内顺铂浓度均明显低于PGCSIL-SKOV3及SKOV3细胞,而在48h却又高于pGCSIL-SKOV3及SKOV3细胞。(5)在IC50(4.4ug/m1)给药24h、48h三种不同处理细胞的微丝均少见,DHFR-pGCSIL-SKOV3细胞在24h时可见线粒体肿胀、脊消失、空泡化、萎缩,内质网扩张明显,有聚集的核糖体,末见高尔基体,48h时其有些线粒体溶解消失,且出现大量的白色囊泡；而pGCSIL-SKOV3及SKOV3细胞24h、48h内质网扩张少见,线粒体结构改变不明显,甚至有2-3个高尔基体存在；DHFR-pGCSIL-SKOV3细胞至72h时,微丝明显的聚集、增多,线粒体结构亦消失。余两种细胞微丝排列紊乱,线粒体形态多样化。结论成功构建针对DHFR基因的siRNA慢病毒载体,找到了有效的干扰靶序列,转染SKOV3细胞后,DHFR基因的表达明显减弱。通过对DHFR下调后耐药性功能研究证实DHFR基因的下调与经过铂类药物作用的体外卵巢癌细胞的药物敏感性有一定的联系,抑制DHFR基因表达能增加顺铂药物敏感性,因此,抑制卵巢癌细胞DHFR基因的表达可以考虑作为顺铂多药耐药的逆转机制。
CHAPTER I RESEARCH PROGRESS FOR OVARIAN CANCER MULTIDRUG-RESISTANT
Ovarian cancer is female genitals one of three major malignant tumor. Ovarian hidden in the deep of the pelvic cavity, early lesions will not be discovered, once appear when symptoms are belong to late, after treatment, the easy relapse, mortality also is always in the lethal gynecological malignancy. At present the aim of treatment for ovarian cancer early, late recurrence control for cure, prolonging the survival time and improve the quality of life of the patients. Treatment is mainly surgery, chemotherapy and radiation treatment. Most of the cases, surgery is difficult to ovarian cancer metastases thoroughly clean the resection. For those who remain the tiny transfer and planting nodules. All need chemical drugs to treat them. Therefore, the chemotherapy treatment in the process of oarian cancer have extremely great significance. Adanced oarian cancer treatments mainly by tumor cells was reduced and postoperative adjuvant chemotherapy out to give priority to. But often cause ovarian cancer chemotherapy failed the relapse, the main reason is that the tumor cells to produce more of drug-resistant (multidrug resistance, MDR). With the combination chemotherapy cisplatin primarily for ovarian cancer rate was40%-60%, because disease progression and the body's resistance to chemotherapy produce, patients with adanced oarian cancer of the5year's survival rates below20%. So. many drug-resistant tumor chemotherapy is one of the main factors of failure. Therefore, tumours produce mechanisms of resistance in clinical research and prevent resistance against drug resistance, resistance to try and reverse became the current ovarian cancer treatment of hot and difficult, which become improve ovarian cancer cure effect and improve ovarian cancer survival one of the important targets.
CHAPTER II THE EXPRESSION OF DIHYDROFOLATE REDUCTASE GENE AND ITS CLINICAL SIGNIFICANCE IN OVARIAN CANCER
Objective To investigate the expression of dihydrofolate reductase (DHFR) mRNA in ovarian cancer and to elucidate the relationship between the clinical pathologic parameters and the expression of DHFR mRNA in ovarian cancer. Method The expression of DHFR in80cases of ovarian carcinoma and50cases of benign ovarian and30cases of normal control were detected by real-time fluorescent quantitative polymerase chain reaction-and the relationship between them was further demonstrated. Results (1)The content of DHFR mRNA expressed in ovarian carcinoma, benign ovarian tumor and normal ovarian tissues was0.584±0.234,0.895±0.783and0.197±0.412. There were significant differences between them (P<0.001,P=0.027, P<0.001).(2)There was not relationship between the content of DHFR mRNA expressed and the stage and pathological grade in ovarian cancer, but DHFR mRNA expression in serous carcinoma was significantly higher than that in mucinous and endometrioid carcinoma (P<0.001).(3)The expression of DHFR mRNA in ovarian cancer was not related significantly and the amount of ascites-lymph node metastasis and distant metastasis in the patients with ovarian cancer(P>0.05), but the expression of DHFR mRNA in patients with the omentum metastasis was down-regulated compared with that without the omentum metastasis, there was difference statistics significantly (P=0.044).(4)The expression content of DHFR mRNA in patients with CR for treatment was lower than that with SD or PD or PR (P<0.001), and also the expression of DHFR mRNA in patients with cisplatinum-sensitive was down-regulated compared with those of cisplatinum multidrug-resistant (0.130±0.103vs0.341±0.701P=0.011).(5)Youden index of the ROC curve for DHFR mRNA expression was0.331, which would be used to determine the nature of the ovarian tumors. The median survival time of patients with DHFR mRNA expression up-regulated (less than0.331) was16.4months, but the median survival time of patients with DHFR mRNA expression down-regulated (higher than0.331) was44.5months, there was difference statistical significantly (P<0.001), and COX multivariate analysis also showed DHFR mRNA expression was an independent prognostic factor in ovarian cancer(P=0.018). Conclusions It was suggested that DHFR would be to play in physiology metabolism in normal cells. There was a relationship between DHFR mRNA up-regulated expression and cisplatinum multidrug-resistant in ovarian cancer, and the expression of DHFR mRNA could be used to determine whether there was cisplatinum multidrug-resistant in ovarian cancer as a potential marker.
CHAPTER III THE BIOLOGICAL ACTIONS EFFECT OF DIHYDROFOLATE REDUCTASE GENE EXPREESION UPREGULATION IN EPITHELIAL OVARIAN CANCER IN VITRO EXPERIMENTS
Objective To construct a lentiviral expressing vector harboring human dihydrofolate reductase (DHFR) gene. Methods The cDNA length of DHFR gene was amplified by PCR and was connected to cloned vector EZ-T. The recovered PCR fragment was obtained by digesting with restriction enzymes named HindⅢ and BamHI-HF, then blunted at the gap. Lentiviral vector system adopted pWPI system.The vector pWPI was digested with PmeI enzyme, and then was recovered fragment and phosphorylated. The phosphorylated vector was connected with DHFR gene by T4enzyme. Recombinant plasmid was confirmed by PCR and sequencing nucleotide. The recombinant retroviral vector pWPI was selected to transfect packaging cell293T to gain virus particles with infection ability,which were infected SKOV3cell. Adopting the flow cytometric to detect the cell apoptosis of DHFR-pWPI-SKOV3cells, pWPI-SKOV3cells and SKOV3cells in different cisplatin concentrations (2.5ug/ml,5.0ug/ml,10.0ug/ml.20.0ug/ml) and different time period (24h,48h and72h),and the of cell periodic changes of theres cells under IC50cisplatin concentration (6.0ug/ml), we also used highly effective liquid phase method (HPLC) to test intracellular cisplatin concentration in different induction of cisplatin concentration (2.5ug/ml,5.0ug/ml,10.0ug/ml,20.0ug/ml) and different time period (24h.48h and72h),what's more.we further adopted transmission electron microscope to observe ultrastructural changes of these kinds of cells under induction of IC50cisplatin concentration (6.0ug/ml).The aim of this study is investigate how the upregulated DHFR gene expression affects biological actions of ovarian cancer cell in vitro.Results (1)The recombinant plasmid, named DHFR-pWPI, which was constructed and identified Packaged by packaging cell293T. The homology between sequencing results and DHFR gene sequence was up to100%, and the expression of DHFR in293T cells was determined by RT-PCR.(2)Through the flow cytometric to detect apoptosis changes cells carried DHFR gene,we found that DHFR-pWPI-SKOV3cell in the stimulation of cisplatin concentration (2.5ug/ml,5.0ug/ml), its apoptosis rate at24h.48h and72h was lower than pWPI-SKOV3and SKOV3cells; DHFR-pWPI-SKOV3cells in the induction of cisplatin concentration (10.0ug/ml,20.0ug/ml), whose apoptosis rate at24h and48h was lower than pWPI-SKOV3and SKOV3cells, but at72h, its apoptosis rate was higher than SKOV3cell line.The results showed that DHFR-pWPI-SKOV3cells in the concentration less than5.0ug/ml, regardless of the time change (≥72hours).whose cisplatin drug resistance is higher than the other two kinds of cells.(3) We adopted the flow cytometric to test different time period under IC50cisplatin concentration (6.0ug/ml) for three kinds of ovarian cancer cells cycle, and the results indicated that G1stage rate of DHFR-pWPI-SKOV3, pWPI-SKOV3and SKOV3cells at24h and48h were obviously lower than those of G2and S stage rates:However, at72h, DHFR-pWPI-SKOV3cells G2+S phase rates were significantly lower than the pWPI-SKOV3and SKOV3cells.(4) This experiment used HPLC method to detect intracellular cisplatin concentration, and the results showed that the cells in the cisplatin concentration (4.0ug/ml) at24h,48h and cisplatin concentration (6.0ug/ml) at24h after the medicine induction, the intracellular cisplatin content of DHFR-pWPI-SKOV3cell were significantly lower than pWPI-SKOV3and SKOV3cells; cisplatin concentration (6.0ug/ml) at48h and cisplatin concentration (8.0ug/ml) at24h and48h. the intracellular cisplatin content of DHFR-pWPI-SKOV3cell were obviously higher than pWPI-SKOV3and SKOV3cells.(5)The experiment obeserved ultrastructural changes of three different cells induced by IC50cisplatin concentration in different time period through the electron microscope.which found that for DHFR-pWPI-SKOV3cells, the microwire gathered together at24h and48h, and the number and structure of mitochondria had obvious change, for pWPI-SKOV3cells,which had rare microfilament, the number of mitochondria decreased but structure change is not apparent, we also saw seldom microfilament and obvious mitochondrial structure changes in SKOV3cells, expand and pyknotic mitochondrial structure existed at the same time; all of hese cells appeared expansion of endoplasmic reticulum and had rare normal organelles; at72h,there are inordinate microfilament, a part of nuclear membrane disappeared, a lot of ribosomes gathered together in pWPI-SKOV3and SKOV3cells, and there were rare microfilament in DHFR-pWPI-SKOV3cells,nuclear membrane completely disappeared, many white cystic matter were seen in cytoplasm, mitochondrial structure disappeared completely, which seems most cells is on the verge of death.Conclusion The lentiviral expressing vector harboring human DHFR had been constructed successfully, DHFR expression increased when the cell was drug-resistant in ovarian cancer cell, which suggested DHFR gene expression is related to cisplatin drug resistance in ovarian cancer,The results made the foundation to investigate the molecular mechanism of multidrug-resistance in tumor.
CHAPTER Ⅳ THE BIOLOGICAL IMPACT OF DIHYDROFOLATE REDUCTASE GENE EXPRESSION DOWNREGULATION IN EPITHELIAL OVARIAN CANCER CELLS IN VITRO EXPERIMENTS
THE FIRST QUARTER:CONSTRUCTION AND IDENTIFICATION OF DIHYDRIFOLATE REDUCTASE GENE siRNA-EXPRESSION PLASMID
Objective To construct dyhidrofolate reductase gene(DHFR) small interfering RNA(siRNA)and its expression vector,and explore the significance of DHFR inhibition in the treatment of platinum multidrug-resistance in ovarian cancer. Methods We designed DHFR gene-targeted hairpin siRNA and then screened the best siRNA silence fragment, and inserted into pGPU6/GFP/Neo plasmid,which was identified by sequencing. Human malignant ovarian cancer SKOV3cells were transfected with the constructed vector using lipofectin transfection method. Fluorescence photographs were taken. Then adopting G418method to screen the stable cell line. Results Three groups of DHFR gene-targeted hairpin siRNA were designed,and inserted into pGPU6/GFP/Neo vector after annealing. Vectors containing siRNA were right by sequencing. Fluorescence photographs showed that SKOV3cells were transfected successfully by lipofectin transfection method,and PCR method identified its interference effect. Conclusion DHFR gene-targeted siRNA and its vector was successfully constructed. And one sequence with the highest inhibition efficiency was screened out. DHFR gene expression declined obviously after the constructed plasmid was transfected into SKOV3cell.
THE SECOND QUARTER:THE CONSTRUCTION OF siRNA DHFR LENTIVIRUS INTERFERENCE CARRIER AND ITS BIOLOGICAL FUNNCTION AND INFLUENCE IN OVARIAN CANCER
Objective To construct dyhidrofolate reductase (DHFR) gene RNAi interference lentivirus carrier, to explore the relationship between the inhibition of DHFR gene and platinum drug resistance in ovarian cancer,which made the foundation for Resistance mechanisms in the therapy of ovarian cancer. Methods To design targeting hairpin siRNA of DHFR gene, select the best siRNA silent sequence, join to pGSCIL/GFP vector after anneal, adopt PCR to identify recombination sequence, transfect into SKOV3cell lines through liposomes, finally use fluorescence camera. Western blotting identify the interferential DHFR gene of ovarian cancer SKOV3cell was constructed successfully,named DHFR-pGSCIL-SKOV3cell. Adopting the flow cytometric to detect the cell apoptosis of DHFR-pGSCIL-SKOV3cells, pGSCIL-SKOV3cells and SKOV3cells in different cisplatin concentrations (2.5ug/ml,5.0ug/ml,10.0ug/ml,20.0ug/ml) and different time period (24h,48h and72h),and the of cell periodic changes of theres cells under IC50cisplatin concentration (4.4ug/ml), we also used highly effective liquid phase method (HPLC) to test intracellular cisplatin concentration in different induction of cisplatin concentration (2.5ug/ml,5.0ug/ml,10.0ug/ml,20.0ug/ml) and different time period (24h.48h and72h),what's more.we further adopted transmission electron microscope to observe ultrastructural changes of these kinds of cells under induction of IC50cisplatin concentration (4.4ug/ml).The aim of this study is investigate how the downregulated DHFR gene expression affects biological actions of ovarian cancer cell in vitro.Results (1)Let double-strand nucleotide after annealing connect to pGSCIL/GFP vector, sequence result is correct. Transfect Virus particles into SKOV3cell, using western blotting identifed interference effect.(2)Through the flow cytometric to detect apoptosis changes of cells knocked DHFR gene,we found that DHFR-pGCSIL-SKOV3, pGCSIL-SKOV3and SKOV3cell apoptosis rate increased with the raised cisplatin concentration (2.5ug/ml.5.0ug/ml,10.0ug/ml,20.0ug/ml) at24h、48h and72h,and the apoptosis rate of DHFR-pGCSIL-SKOV3was obvious higher than pGCSIL-SKOV3and SKOV3cells at24h and48h.(3)We adopted the flow cytometric to test different time period under IC50cisplatin concentration (4.4ug/ml) for three kinds of ovarian cancer cells cycle, and the results indicated that G1stage rate of DHFR-pGCSIL-SKOV3, pGCSIL-SKOV3and SKOV3cells at24h、48h and72h were obviously higher than those of G2and S stage rates; However, at24h and48h, DHFR-pGCSIL-SKOV3cells G2/M and S phase rates were significantly increased while G1phase rates decreased,at72h, G2/M and S phase rates were significantly increased while G1phase rates decreased for all of three kinds of cells.(4)This experiment used HPLC method to detect intracellular cisplatin concentration, and the results showed that the cells in the cisplatin concentration (2.5ug/ml、5.0ug/ml) at24h and48h,the intracellular cisplatin content of DHFR-pGCSIL-SKOV3cell were significantly higher than pGCSIL-SKOV3and SKOV3cells; but cisplatin concentration (7.5ug/ml) at24h, the intracellular cisplatin content of DHFR-pGCSIL-SKOV3cell were obviously lower than pGCSIL-SKOV3and SKOV3cells.while higher than pGCSIL-SKOV3and SKOV3cells at48h.(5)The experiment obeserved ultrastructural changes of three different cells induced by IC50cisplatin concentration(4.4ug/ml) in different time period through the electron microscope,which found that for DHFR-pGCSIL-SKOV3at24h, visible when mitochondria swelling, its ridge disappear, the empty bubble and atrophy, obvious endoplasmic reticulum expansion.ribosomes gathered together, rare golgiapparatus.At48h,some mitochondria were dissolve and disappeared, and a large number of white vesicles were appeared; at72h, the micro filament obvious increased and gathered together, mitochondrial structure also disappear.But the microfilament of the other two cells arranged disorderly, mitochondrial form diversification.However,for pGCSIL-SKOV3and SKOV3cells at24h and48h rare endoplasmic reticulum expansion, mitochondrial structure change is not obvious, and even existed2-3golgi apparatus; the microwire gathered together at24h and48h, and the number and structure of mitochondria had obvious change, for pWPI-SKOV3cells,which had rare microfilament, the number of mitochondria decreased but structure change is not apparent, we also saw seldom microfilament and obvious mitochondrial structure changes in SKOV3cells, expand and pyknotic mitochondrial structure existed at the same time; all of hese cells appeared expansion of endoplasmic reticulum and had rare normal organelles; at72h,there are inordinate microfilament, a part of nuclear membrane disappeared, a lot of ribosomes gathered together in pWPI-SKOV3and SKOV3cells, and there were rare microfilament in DHFR-pWPI-SKOV3cells,nuclear membrane completely disappeared, many white cystic matter were seen in cytoplasm, mitochondrial structure disappeared completely, which seems most cells is on the verge of death.Conclusions We are succeed in constructing DHFR gene siRNA carrier, screen the effective target sequence, and then transfect into SKOV3cell.DHFR gene expression were significantly weakened. The research indicated thatknock out DHFR gene is related to cisplatin drug resistance in ovarian cancer,The results made the foundation to investigate the molecular mechanism of multidrug-resistance in tumor.
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2、Levy AS, Sather HN, Steinherz PG, et al.Reduced folate carrier and dihydrofolate reductase expression in acute lymphocytic leukemia may predict outcome:a Children's Cancer Group Study.J Pediatr Hematol Oncol [J].2003,25(9):688-695.
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4、Al-Shakfa F, Dulucq S, Brukner I, et al.DNA variants in region for noncoding interfering transcript of dihydrofolate reductase gene and outcome in childhood acute lymphoblastic leukemia [J]. Clin Cancer Res.2009,15(22):6931-6938.
5、Matsuo K, Lin YG, Roman LD, Overcoming platinum resistance in ovarian carcinoma [J]. Expert Opin Investig Drugs.2010,19(11):1339-1354.
6、于明东,李书忠.实时荧光定量PCR法检测人骨肉瘤耐MTX细胞系中RFC、DHFR、GST-π的mRNA表达[J].中国矫形外科杂志,2009,17(11)：8.58-861.
7、黄瀚,李兵,欧阳林旗,等.叶酸受体及二氢叶酸还原酶与人乳腺癌细胞MCF-7/ADR多要耐药关系[J].中国药理学通报,2011,27(1)：99-103
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10、Hagner N, Joerger M. Cancer chemotherapy:targeting folic acid synthesis[J].Cancer Manag Res.2010; 2:293-301.