【作者】 樊萍；

【导师】 杨竹；

【作者基本信息】 重庆医科大学 ， 妇产科学， 2010， 硕士

【摘要】 卵巢癌是病死率较高的妇科肿瘤,近20年来,尽管在其治疗方面取得诸多进展,但晚期患者5年生存率一直徘徊在15%-30%。卵巢癌是机体在各种发病高危因素下,在基因水平上失去了对局部组织细胞生长的正常调控,导致异常增生而形成的新生物,其具体发病原因仍不明确,通常一些学者认为机体细胞生长要受原癌基因和抑癌基因的双重调控,正常情况下,两类基因协同调控,使细胞生长处于平衡状态。抑癌基因功能减弱,或者原癌基因被激活,都会打破这一平衡,导致癌症的发生。随着分子生物学和基因技术的发展,基因治疗为卵巢癌患者提供了一种新的治疗策略,但目前由于缺乏一种安全、稳定、高效的基因转染方法,而极大地限制了基因治疗在临床上的应用。新近研究发现,超声靶向破坏微泡定位释放技术(UTMD)应用于基因治疗是一种新型的无创性基因转移技术。开展UTMD介导目的基因靶向治疗的深入研究可望为卵巢癌的基因治疗提供一种新型、无创、高效、靶向、安全的新途径。本课题首先构建增强型绿色荧光蛋白(EGFP)与HSV1TK共表达载体;然后分析了超声微泡介导外源基因转染卵巢癌的可行性并筛选出合适的超声参数;最后,应用UTMD技术将HSV1TK转染入卵巢癌细胞,观察转染后HSV1TK基因在卵巢癌细胞内的表达情况及自杀基因对卵巢癌细胞的杀伤效应,探讨超声微泡介导自杀基因系统对卵巢癌细胞抗瘤效应的机理,为研究和评价UTMD介导的自杀基因转染卵巢癌提供可靠的实验依据,为卵巢癌基因治疗提供一个新的途径和手段。本课题研究主要包括以下三个部分的内容:第一部分增强型绿色荧光蛋白(EGFP)与I型单纯疱疹病毒胸苷激酶(HSV1TK)共表达载体的构建及其在卵巢癌细胞SKOV3的表达鉴定目的:构建单纯疱疹病毒I型胸苷激酶(HSV1TK)的真核表达载体pcDNA3.1-EGFP/HSV1TK,鉴定其在真核细胞中的表达和功能。方法:以pORF-HSV1TK为模板,PCR扩增的目的基因HSV1TK片段与pMD18-T载体相连接构建重组克隆pMD18-T/HSV1TK。将双酶切的HSV1TK片段,插入pcDNA3.1-EGFP多克隆位点,构建pcDNA3.1-EGFP/ HSV1TK真核表达载体并进行酶切、测序鉴定。分别用荧光显微镜观察和RT-PCR方法检测脂质体介导pcDNA3.1-EGFP/ HSV1TK在卵巢癌细胞SKOV3的表达;分别用MTT法和光镜检测胸苷激酶/丙氧鸟苷(HSV1TK/GCV)系统对SKOV3体外杀伤作用及旁观者效应。结果:重组载体酶切鉴定结果与预期结果一致,基因序列与GENEBANK上报道的HSV1TK基因序列完全一致。荧光显微镜观察转染后的细胞发出绿色荧光;RT-PCR结果表明HSV1TK基因能在SKOV3内有效表达。MTT和光镜结果显示转染HSV1TK基因的SKOV3细胞,加入前体药物GCV处理后对其有明显的杀伤作用和旁观者效应。结论:成功构建的真核表达载体pcDNA3.1-EGFP/ HSV1TK,能在SKOV3细胞中稳定表达,HSV1TK对卵巢癌细胞SKOV3体外有强大的杀伤作用和旁观者效应。第二部分超声微泡介导基因转染卵巢癌细胞SKOV3的超声参数及转染效率研究目的:探究超声介导携Ⅰ型单纯疱疹病毒胸苷激酶(HSV1TK)自杀基因微泡转染SKOV3细胞的超声参数及转染效率。方法:超声在不同微泡浓度与辐照时间(8,15,30,60 s间隔1 s)组合下辐照SKOV3细胞,MTT法筛选最适辐照时间和微泡浓度。将SKOV3分成6组分别进行以下处理:超声辐照组,脂质体+超声辐照组,脂质体+微泡+超声辐照组,微泡+超声辐照组,阴性对照组(裸质粒),阳性对照组(脂质体),分别转染pcDNA3.1-EGFP/HSV1TK质粒后,用荧光显微镜、流式细胞技术对各组转染效率进行定性和定量检测,最后采用逆转录聚合酶链反应(RT-PCR)方法检测HSV1TK基因的表达。结果: MTT检测在超声强度0.5 W/cm2,频率1 MHz,微泡浓度5.6×107个/ml,辐照时间8 s间隔1 s条件下,超声辐照微泡转染组与对照组比较(P>0.05),超声辐照微泡对细胞活性无明显抑制。经此条件转染后荧光显微镜下可观察到脂质体+微泡+超声辐照组的绿色荧光强度最强;流式细胞技术检测表明,微泡+超声辐照组转染效率为(11.74±0.19)% ,比超声辐照组(2.19±0.22)%高,而脂质体+微泡+超声辐照组(25.62±0.08)%均高于其他各组(P<0.05); RT-PCR检测HSV1TK基因mRNA表达结果示,在脂质体+微泡+超声辐照组的SKOV3细胞中表达最高。结论:在最适的超声参数下,超声介导微泡不仅能单独促进HSV1TK基因转染并有效在SKOV3细胞中表达,而且还能够增强脂质体的转染效率。第三部分超声微泡介导自杀基因系统对卵巢癌细胞抗瘤效应实验研究目的:探讨超声微泡介导的自杀基因系统对卵巢癌细胞抗瘤效应的机理。方法:使用最适的超声转染参数,超声介导微泡将pORF-HSV1TK质粒转染SKOV3细胞后,RT-PCR检测SKOV3中HSV1TK mRNA的表达;转染的SKOV3细胞与不同浓度的前药丙氧鸟苷(GCV)共培养后,MTT法测定各组转染细胞的增殖抑制率及在最适GCV浓度下各组转染细胞增殖抑制率随时间的变化情况;光镜观察给予GCV后不同组转染细胞的数量、形态变化;流式细胞术(FCM)检测各组SKOV3细胞早期凋亡率及细胞周期。结果:RT-PCR检测HSV1TK基因的mRNA在各组转染细胞中均有表达(阴性对照组除外),其中在脂质体+微泡+超声辐照组的SKOV3细胞中表达最高;MTT检测到脂质体+微泡+超声辐照组的HSV1TK/GCV在GCV浓度为10-100μg/ml范围内时,对SKOV3的增殖抑制率明显强于其它组(P<0.05);同时检测了在GCV(浓度为100μg/ml)作用24-48 h内,各组细胞增殖抑制率也显著增高,并且脂质体+微泡+超声辐照组细胞增殖抑制率明显强于其它组(P<0.05);光镜下可见脂质体+微泡+超声辐照组经HSV1TK/GCV处理后SKOV3的数量显著减少,形态明显异常;FCM检测脂质体+微泡+超声辐照组SKOV3细胞凋亡率为(49.13±0.82)%,且大多数细胞周期被阻断于G1期,和其他各组比较有显著差异, P <0.05。结论:超声介导微泡能增强HSV1TK/ GCV系统抑制SKOV3增殖和诱导SKOV3凋亡的作用,并且SKOV3细胞周期被阻断在G1期,从而发挥抗瘤效应。更多还原

【Abstract】 The ovarian carcinoma’s case fatality is more higher than other gynecology tumors, in the latest 20 years, although it made much progress in the therapy for this carcinoma, the 5-year survival rate of patient in advanced stage remained in 15%-30%. The ovarian carcinoma is a neoplasm which formed by the cells lost the normal control from the gene level and lead to the paraplasm induced by many kinds of high risk factors. Its etiopathogenisis is still unknown. Usually the growth of organism cell is regulated dually by the proto-oncogene and antioncogene. Under the normal conditions, the growth of cells keeps balance synergistically regulated by these two kinds of gene. This balance is broken if the function of antioncogene is weaken or the proto-oncogene is activated, then the carcinomas occur. With the development of molecular biology and gene technology, the gene therapy has provided one kind of new treatment strategy for the ovarian carcinoma patient. But it is limited greatly to apply the gene therapy on clinical because still lacked one kind of safe,stable,high effective gene transfection method at present. Recently, study suggested that ultrasound-targeted microbubble destruction (UTMD) is a new type of non-invasive gene transfection technology. The profound study on UTMD mediated gene target theropy is expected to supply a new,non-invasive,high effective,targeted and safe method.Firstly, we construct a co-expression vector of enhanced green fluorescent protein(EGFP)and HSV1TK;then, we analyzed the possibility of microbubble carrying suicide gene to transfect ovarian carcinoma cell lines SKOV3 and screened the optimal ultrasound parameters;finally, the vitro expriment is performed to study inhibition the proliferation and induction the apoptosis of microbubble carrying suicide gene to transfect ovarian carcinoma cell,the expression of HSV1TK gene after transfection in ovary carcinoma cells was obsered and the casualty effect of suicide gene on ovary carcinoma cells was investigated, which can provide the reliable experiment basis for studying and evaluating the application of UTMD induced suicide gene to transfect ovarian carcinoma cells, Thus providing a new way and method for the ovarian carcinoma gene therapy。Three parts are included in the present study. Part one Construction co-expression vector of enhanced green fluorescent protein and HSV1TK and expression in ovarian carcinoma cells SKOV3Objective:To construct EGFP and HSV1TK co-expression vector and to detect its expression and function in eukaryocyte ovarian carcinoma cells SKOV3.Methods:The PCR products HSV1TK were inserted into cloning vector pMD18-T to construct the plasmid of pMD18/ HSV1TK.The HSV1TK gene fragment obtained from pMD18T/ HSV1TK that was digested,and then inserted into pcDNA3.1-EGFP. The recombinant pcDNA3.1-EGFP/ HSV1TK was identified with restriction analysis and DNA sequence. The expression plasmid pcDNA3.1-EGFP/ HSV1TK was transfected into ovarian carcinoma cells SKOV3 mediated by liposome reagent, then the expressions of EGFP in cells were observed by fluorescence microscopy and HSV1TK was detected with RT-PCR. The killing SKOV3 effect and bystander effect of HSV1TK/GCV is observed by MTT and light microscope.Results:The sequence of the cloned DNA fragment was identical to HSV1TK that was reported on Gene bank, and the HSV1TK gene was inserted into eukaryotic expression vector pcDNA3.1-EGFP correctly. The recombinant expression plasmid was successfully transferred into ovarian carcinoma cells SKOV3,then observed by fluorescent microscope and effective expression of HSV1TK was also testified by RT-PCR. We can observe the killing SKOV3 effect and bystander effect of HSV1TK/GCV obviously by MTT and light microscope.Conclusion:The recombinant eukaryotic co-expression vector of EGFP and HSV1TK was successfully constructed and effectively expressed in ovarian carcinoma cells SKOV3. HSV1TK /GCV system has better killing effects and bystander effect in SKOV3 cells.Part two Ultrasound parameters and transfection efficiency of microbubble carrying suicide gene for transfecting ovarian carcinoma cell linesObjective:To investigate the ultrasound parameters and transfection efficiency of microbubble carrying herpes simplex virus thymidine kinase suicide gene(HSV1TK)for transfecting SKOV3 cells mediated by the ultrasound.Methods:Under the combination conditions of different ultrasound exposure times (8,15,30,and 60s with the interval as 1s) and microbubbles at different concentrations, SKOV3 cells were exposed to the ultrasound so as to screen out the optimal ultrasound exposure time and microbubble concentration by MTT assays. The SKOV3 cells were divided into 6 groups and treated by ultrasound, ultrasound combined with liposome, ultrasound combined with microbubbles and liposome, and ultrasound combined with microbubbles respectively. The groups of negative control and positive contro1 were treated by naked plasmid and liposome respectively, and then they were used to transfect plasmids pcDNA3.1-EGFP/ HSV1TK. The transfection efficiencies were observed qualitatively by fluorescence microscope and quantitatively by flow cytometry (FCM) .The expression of HSV1TK was detected by reverse transcription polymerase chain reaction (RT-PCR).Results:MTT assays showed that the transfection had no significant inhibition to cell viability under the conditions of the microbubble concentration (0.5 W/cm2), frequency (1 MHz), microbubble concentration (5.6×107/ml), and the interval of every 8 s ultrasound exposure time (1s). Under the fluorescence microscope, the green fluorescence intensity for the group of ultrasound combined with microbubbles and liposome was the greatest. The analysis of FCM showed that the transfection efficiency in group of ultrasound and microbubbles was higher than that in group of ultrasound〔(11.74±0.19)% vs(2.19±0.22)%〕.Furthermore, the transfection efficiency in group of ultrasound combined with microbubbles and liposome was(25.62±0.08)%, which was the highest among all groups (P<0.05). The detection of RT-PCR showed the expression of HSV1TK in group of ultrasound combined with microbubbles and liposome was the highest among the groups (P<0.05).Conclusion: Under the conditions of the optimal ultrasound parameters,ultrasound- mediated microbubbles can not only independently promote the hsv1tk gene transfection, but also strengthen the transfection efficiency of liposome. Meanwhile it can also be effectively expressed in SKOV3.PART three Study of anti-tumor effects in ultrasound microbubbles mediated suicide gene system on ovarian carcinoma cellsObjective:To study the anti-tumor effects in ultrasound microbubbles mediated suicide gene system on ovarian carcinoma cells.Methods:under the conditions of optimal ultrasound transfection parameters,single ultrasound or ultrasound combined with liposome mediated carrying suicide gene microbubbles,pORF-HSV1TK plasmid was transfected into the SKOV3 cells. RT-PCR was used to detect the expression of HSV1TK mRNA in SKOV3; After transfected SKOV3 cells co-culture with different concentrations of the prodrug ganciclovir (GCV), MTT was used to assay inhibition rates of proliferation in transfected cells,and the inhibition rate changes of proliferation over time at optimal concentration of GCV. Light microscope was used to observe changes of quantity and morphology of transfected cells at optimal concentration of GCV for each group; Flow cytometry (FCM) was used to detect the rate of early apoptosis and cell cycle of SKOV3 cells in each group.Results:The expression of HSV1TK mRNA was found in each transfected group by RT-PCR (except the negative control group), which is most in group of the ultrasound irradiation microbubbles combined with liposome; The inhibition rates of proliferation induced by HSV1TK/GCV to SKOV3 cells is stronger in group of the ultrasound irradiation microbubbles combined with liposome than that in other groups between the concentration of 10-100μg/ml of GCV;In each group, the larger GCV dosage was, the higher inhibition rates of proliferation was. After administrating GCV of 100μg/ml, during the course of 24-48h the inhibition rates of proliferation increased with the increase of time in each group,which is stronger in group of the ultrasound irradiation microbubbles combined with liposome than others; Compare with other groups, SKOV3 cells in group of the ultrasound irradiation microbubbles combined with liposome decreased significantly and abnomal cells are more by light microscope. The rate of early apoptosis of SKOV3 cells in group of the ultrasound irradiation microbubbles combined with liposome is (49.13±0.82)%, most of their cells cycle are in G1 phase.Conclusion : Ultrasound-mediated microbubbles can carry out anti-tumor effect on transfected SKOV3 cells in HSV1TK / GCV system by promoting to suppress the proliferation and induce early apoptosis of the cells, and the cells cycle was blocked in the G1 phase.更多还原