【作者】 朱元方；

【导师】 胡丽娜；

【作者基本信息】 重庆医科大学 ， 妇产科学， 2009， 博士

【摘要】 卵巢癌死亡率居女性生殖系统恶性肿瘤之首,近20年来,尽管在对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%。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模式。靶向和高效是基因治疗的关键和瓶颈问题,也是基因治疗能否在临床上得到广泛应用的关键所在。癌组织特异性启动子介导的新奇靶向治疗策略的应用以及纳米粒、超声微泡等非病毒载体的出现为基因治疗卵巢癌展现出了一个全新的应用前景。本课题首先分析了超声微泡介导外源基因转染卵巢癌的可行性;然后构建了一个含有人端粒酶逆转录酶(hTERT)启动子和融合型自杀基因Fcy::Fur的重组真核表达质粒pGL3-hTERT-Fcy::Fur;最后,在体外培养的卵巢癌细胞和体内卵巢癌移植瘤动物模型上,用超声微泡作基因载体,介导pGL3-hTERT-Fcy::Fur质粒转染,探讨自杀基因治疗卵巢癌的疗效和靶向性,为卵巢癌基因治疗提供可靠的实验依据。本课题研究主要包括以下四个部分的内容:第一部分超声微泡介导基因转染卵巢癌细胞参数条件的筛选及可行性分析目的筛选超声微泡介导基因转染卵巢癌细胞的参数条件,并分析它作为基因转移技术在卵巢癌细胞中的转染效率,探讨它用于卵巢癌基因转移的可行性。方法(1)以0.5、0.75、1.0、1.25、1.5、2.0 W/cm2不同强度的超声作用于对数生长的卵巢癌SKOV3细胞60 s,通过台盼兰染色,筛选出对细胞活性无明显抑制的超声强度;(2)取自制脂质微泡一瓶,向瓶内注入5 ml PBS,用力摇匀至粉末完全溶解,检测其原始浓度,将微泡稀释5倍和10倍,制备出不同浓度的微泡。然后将筛选出的声强分别与不同浓度的微泡联合作用于SKOV3细胞,台盼兰染色后,筛选出对细胞活性无明显抑制的最适声强和微泡浓度的组合;(3)将SKOV3细胞分成5组,①空白组;②微泡组;③超声组;④超声+微泡组;⑤脂质体组,用上述不同基因转移方式转染pGL3-Promoter-EGFP质粒,通过观察、计数表达绿色荧光蛋白细胞,比较各组转染效率。结果(1)声强0.5、0.75 W/cm2的超声辐照,SKOV3细胞死亡率<10%,其他四种声强辐照后,SKOV3细胞死亡率均>10%;(2)微泡原始浓度约为4×108/ml ,其中0.8×108/ml或0.4×108/ml浓度的微泡联合0.5 W/cm2超声辐照,细胞死亡率<10%;(3)超声组和微泡组仅见很少的荧光细胞,两者比较无统计学差异,p=0.64。超声微泡组与脂质体组均见大量的绿色荧光细胞,各组均值分别为79.9±17.6和62.7±22.5,两者比较有统计学差异,p=0.041。超声微泡组明显高于单纯超声辐照组和单纯的微泡孵育组,P<0.001。结论(1)适当浓度微泡联合超声辐照对SKOV3细胞活性没有影响。(2)超声微泡能将外源基因在卵巢癌细胞中高效转移。(3)超声辐照载基因微泡是一种定位靶向释放系统。第二部分含hTERT启动子和融合型自杀基因重组质粒pGL3-hTERT-Fcy∷Fur的构建、鉴定及表达目的构建含端粒酶逆转录酶(hTERT)启动子和融合型自杀基因Fcy::Fur的重组真核表达质粒,对它进行鉴定,并检测它在卵巢癌细胞和正常人胚肺成纤维细胞中的表达。方法(1)设计并合成两端分别带有Nco I、Xba I酶切位点的一对引物,从质粒pORF5-Fcy::Fur中PCR扩增出Fcy::Fur基因片段,0.8%琼脂糖凝胶电泳鉴定PCR产物,DNA凝胶回收试剂盒快速纯化回收Nco I、Xba I双酶切后的PCR产物。将纯化后的Fcy::Fur PCR产物亚克隆进含hTERT启动子的荧光素酶基因表达质粒p2XEB,构建hTERT启动子驱动的Fcy::Fur基因重组真核表达质粒pGL3-hTERT-Fcy∷Fur。同理,将纯化后的Fcy::Fur PCR产物亚克隆进含猿猴病毒40(SV40)启动子的荧光素酶基因表达质粒pGL3-Promoter,构建SV40启动子驱动的Fcy::Fur基因重组对照质粒pGL3-Pro-Fcy::Fur,重组子转化感受态大肠杆菌JM109后,筛选阳性克隆菌落并培养,抽提质粒,重组子经Nco I、Xba I双酶切鉴定并送上海生工公司测序。(2)鉴定后的重组子通过超声微泡技术转染卵巢癌SKOV3细胞和人胚肺成纤维MRC-5后,RT-PCR和Westen-blot检测转染后的两种细胞中Fcy∷Fur mRNA和蛋白表达。结果(1) pGL3-hTERT-Fcy∷Fur和pGL3-Pro-Fcy::Fur两种重组质粒酶切和测序结果与预期完全相符,hTERT启动子和Fcy::Fur片段序列与GenBank报道一致,且插入方向正确。(2) RT-PCR和Westen-blot结果表明, hTERT启动子驱动的Fcy::Fur基因在卵巢癌SKOV3细胞中高表达,而在人胚肺成纤维MRC-5中几乎无表达,两者比较有统计学差异,P<0.01。结论(1)含端粒酶逆转录酶启动子和融合型自杀基因Fcy::Fur的重组真核表达质粒pGL3-hTERT-Fcy∷Fur成功构建。(2) hTERT启动子驱动的下游Fcy∷Fur基因能够实现在肿瘤细胞和正常细胞中的差异性表达,为下一步探讨基因靶向治疗奠定了实验基础。第三部分超声微泡介导pGL3-hTERT-Fcy∷Fur转染抑制卵巢癌细胞增殖及诱导其凋亡的体外实验目的探讨端粒酶逆转录酶启动子驱动自杀基因转录的重组真核表达质粒pGL3-hTERT-Fcy∷Fur对体外卵巢癌细胞的靶向杀伤作用。方法用前面筛选出的超声微泡作载体,将端粒酶逆转录酶启动子驱动的Fcy::Fur基因重组质粒pGL3-hTERT-Fcy∷Fur和SV40启动子驱动的Fcy::Fur重组对照质粒pGL3-Pro-Fcy::Fur以及pGL3-Promoter(空载质粒)3种质粒分别转染卵巢癌SKOV3细胞和人胚肺成纤维MRC-5细胞。与不同浓度的前药5-氟胞嘧啶(5-FC)共培养后,CCK-8法测定转染细胞的增殖抑制率;吖啶橙/溴化乙啶(AO/EB)染色观察凋亡细胞并计算凋亡率;电镜检测转染细胞后的超微结构变化;高效液相色谱法(HPLC)检测转染细胞内的5-FC的浓度变化;RT-PCR检测SKOV3和MRC-5中hTERT mRNA的表达,荧光素酶分析hTERT启动子在两种细胞中的活性。结果(1)pGL3-hTERT-Fcy∷Fur对卵巢癌SKOV3细胞表现出专一性的增殖抑制和凋亡诱导效应。CCK-8结果表明:转染了pGL3-Pro-Fcy::Fur质粒的SKOV3和MRC-5细胞的增殖抑制率无统计学差异,P=0.87,而转染pGL3-hTERT-Fcy::Fur质粒时,SKOV3的增殖抑制率明显高于MRC-5,P=0.036,当5-FC浓度低于250 mg/L时,pGL3-Promoter对SKOV3和MRC-5两种细胞的增殖抑制率均没有明显影响;AO/EB染色显示:转染pGL3-hTERT-Fcy::Fur质粒时,SKOV3凋亡率明显高于MRC-5,P=0.028;透射电镜见SKOV3细胞和线粒体肿胀,而MRC-5细胞未见超微结构改变。(2)HPLC检测结果示:转染pGL3-hTERT-Fcy∷Fur质粒并孵育48小时后,SKOV3内5-FC浓度明显低于MRC-5内浓度。(3) SKOV3和MRC-5中hTERT mRNA表达分别为阳性和阴性,hTERT启动子在卵巢癌细胞中活性为26.2%,在MRC-5细胞为0.65%。结论(1)端粒酶逆转录酶启动子驱动的Fcy::Fur基因重组质粒pGL3-hTERT-Fcy∷Fur对端粒酶逆转录酶阳性的卵巢癌SKOV3细胞具有专一性的增殖抑制和凋亡诱导效应,而对端粒酶逆转录酶阴性的正常人胚肺成纤维细胞几乎无杀伤作用。(2)pGL3-hTERT-Fcy∷Fur对卵巢癌SKOV3细胞的专一性杀伤作用是由于hTERT启动子在端粒酶逆转录酶阳性的SKOV3细胞中活性高导致Fcy∷Fur高表达所致。第四部分超声微泡介导pGL3-hTERT-Fcy∷Fur转染抑制卵巢癌裸鼠移植瘤的体内实验目的利用重组质粒pGL3-hTERT-Fcy∷Fur作治疗基因,应用超声定位辐照载基因微泡技术,探讨对裸鼠卵巢癌皮下移植瘤的转导和转录双重靶向的抑瘤效应。方法(1)超声微泡介导的转导靶向探讨:将荷瘤鼠随机分成5组,每组5只,分别给予不同方法处理。①超声辐照载基因微泡治疗组,将载有pGL3-hTERT-Fcy∷Fur基因的0.2 ml微泡经尾静脉注射裸鼠后,用超声探头辐照肿瘤部位,功率为0.5 W/cm2,辐照10 S,停5 S,反复6次;②超声辐照基因治疗组,尾静脉注射含同样基因量的培养液0.2 ml,再予超声辐照;③超声辐照微泡治疗组,尾静脉注射不含基因的微泡0.2 ml后,再予超声辐照;④载基因微泡治疗组,注射载相同剂量的基因微泡,但不予超声辐照;⑤对照组,注射等量的生理盐水。各组上述处理每天1次,连续7 d。各治疗组第2天开始,每只每天腹腔注射生理盐水稀释的5-FC药物,连续7 d,对照组则注射等量生理盐水。末次处理后5天剖取瘤块测体积、重量,计算抑瘤率;TUNEL检测肿瘤细胞凋亡情况。(2) hTERT启动子介导的转录靶向探讨:将荷瘤鼠随机分成4组,每组5只,①载pGL3-hTERT-Fcy∷Fur基因微泡组;②载pGL3-Pro-Fcy::Fur基因微泡组;③载pGL3-Promoter空载基因微泡组;④生理盐水对照组。载基因微泡和生理盐水均通过尾静脉注射,超声辐照部位为肿瘤和肝脏组织,其余处理同上。剖取瘤块测体积、重量,计算抑瘤率;TUNEL检测肿瘤细胞和肝脏凋亡情况。结果(1)转导靶向探讨结果:①与对照组比较,载基因微泡治疗组的肿瘤体积、重量、抑瘤率及凋亡率均无统计学差异,P>0.05;②超声辐照基因治疗组与超声辐照微泡治疗组比较,无统计学差异,P>0.05,而两者与对照组比较,均有统计学差异,P<0.05;③超声辐照载基因微泡治疗组与其他组两两比较,肿瘤体积、重量最小,抑瘤率最大,凋亡率最高,均有显著性差异,P<0.05。(2)转录靶向探讨结果:①pGL3-Promoter空载组与载pGL3-hTERT-Fcy∷Fur基因微泡组、载pGL3-Pro-Fcy::Fur基因微泡组比较,肿瘤体积、重量、抑瘤率及肿瘤细胞凋亡率均有统计学差异,P<0.05。②载pGL3-hTERT-Fcy∷Fur基因微泡组和载pGL3-Pro-Fcy::Fur基因微泡组比较,肿瘤体积、重量、抑瘤率及肿瘤细胞凋亡率无统计学差异,P>0.05,而肝脏细胞凋亡率有统计学差异,前者明显低于后者。结论(1)超声辐照载基因微泡在体内能实现基因定位释放,有望成为基因治疗的常规新型靶向载体技术。(2)hTERT启动子驱动的基因在体内对卵巢癌有靶向性杀伤作用,而对正常组织杀伤作用及小。(3)载自杀基因微泡在超声定位辐照下,体内抑瘤效果好。更多还原

【Abstract】 Ovarian cancer is a common female genital system malignant tumor type with the highest mortality rate. Despite the fact that the clinical treatment for it has been developing dramatically in the past two decades, the average 5-year survival rate for a patient is less than 30%. Gene therapy has become a new treatment modality for ovarian cancer with the development of molecular biology and gene technology. Targeted expression of remedial genes and high transfection efficiency are the key points as well as the emphasis and nodus of gene therapy. As a new treatment modality for ovarian cancer, gene therapy has sparked a renewed interest with the emergence of novel targeted therapeutic strategies and the development of non-viral gene carriers such as nanoparticles and ultrasound-microvesicle.In the present study, we first analyzed the feasibility and transfection efficiency of ectogenous plasmid transfected into ovarian cancer SKOV3 cells by ultrasound-mediated microvesicle destruction. Then, we constructed a recombinant expression plasmids pGL3-hTERT-Fcy::Fur which contained hTERT promoter and Fcy∷Fur suicide gene and analyzed its expression in human ovarian cancer cells and human embryonic lung fibroblast cells. Eventually, we explored the therapeutic effect and the targeting of suicide gene using ultrasound-mediated microvesicle destruction as genetic carrier to transfect the recombinant plasmids pGL3-hTERT-Fcy::Fur into SKOV3 cells in vitro and transplanted tumor in vivo with the hope of providing an faithful experimental data for gene therapy for ovarian cancer. 4 parts are included in the present study.Part one Parameters screening and feasibility analyzing of the ectogenous plasmids transfected into skov3 cells using ultrasound-mediated microvesicle destruction as genetic carrierObjective To screen the optimal parameters of ultrasound intensity and microvesicle concentration and to investigate the feasibility of ectogenous plasmid transfected into SKOV3 cells (Human ovarian cancer cell line) using ultrasound-mediated microvesicle destruction as genetic carrier.Methods⑴6 groups SKOV3 cells were exposed continuously to different ultrasound intensity of 0.5,0.75,1.0,1.25,1.5 and 2.0 W/cm2 for 60 s respectively in order to screen out the optimal ultrasound intensity that do no conspicuous harm to SKOV3 cells by trypan blue staining.⑵A bottle of self-made lipid microvesicle was lysised into 5 ml PBS and its initial concentration was detected, then, the microvesicle,s initial concentration was diluted for 5 and 10 times by PBS. SKOV3 cells were under the impact of different combinations of the screened ultrasound intensity with microvesicle of different concentration so as to screen out the optimal parameters combination.⑶SKOV3 cells were divided into 5 groups: naked plasmid group (NP) , plasmid with microvesicle group (MV), plasmid with ultrasound group(US), plasmid with ultrasound and microvesicle group(MV & US), plasmid with liposome group(LP). pGL3-Promoter-EGFP plasmid were transfected into them via each mediator mentioned above, the transfection efficiency of each group was calculated.Results⑴The cells death rate is<10% when the ultrasound intensity is below 0.75 W/cm2.⑵When exposed to combination of the microvesicle concentration of 0.4×108/ml or 0.8×108/ml with the ultrasound intensity of 0.5 W/cm2, the cells death rate is <10%.⑶Few fluorescence cells were observed in either MB group or US group and no significant difference was found between them (p=0.64). However, large quantities of fluorescence cells were observed in both MB&US group and LP group, with an average number of 79.9±17.6 and 62.7±22.5 respectively, and significant difference was found between that of the two groups (p=0.041). The transfection efficiency of MB&US group is the highest than that of any of the others.Conclusions⑴The SKOV3 cells can tolerate the ultrasound at a 0.5 W/cm2 intensity and the microvesicle at a 0.8×108/ml or 0.4×108/ml concentration.⑵Ectogenous plasmid can be transfected into SKOV3 cells efficiently by ultrasound-mediated microvesicle destruction.⑶ultrasound-mediated microvesicle destruction is an targeting gene delivery system.Part two Construction, verification and expression of the recombinant Plasmid containing hTERT promoter and Fcy∷Fur suicide geneObjective To construct, verify the recombinant plasmid containing hTERT promoter and Fcy∷Fur suicide gene and detect its expression in SKOV3 [human ovarian cancer cell line, human telomerase reverse transcriptase (hTERT positive)] and MRC-5 (human embryonic lung fibroblast cell line, hTERT negative).Methods⑴The cDNA sequence (CDS) encoding the whole open reading frame of the Fcy::Fur gene was generated by a PCR strategy taking the pORF5-Fcy::Fur plasmid containing the complete CDS for Fcy::Fur as a template. A 5’ primer containing the NCO I and a 3’ primer containing the Xba I site were used to generate a 1139bp PCR product from BamH I to EcoR I. After being subcloned and completely sequenced to verify the correct reading frame and lack of other mutations, the Fcy::Fur cDNA was then introduced as an Nco I to Xba I fragment into the unique Nco I to Xba I sites of the P2XEB plasmid, from which Fcy::Fur can be expressed under the regulation of the hTERT promoter, thus acquiring the pGL3-hTERT- Fcy::Fur recombinant plasmid, which was further identified by restriction enzyme digestions. The control recombinant plasmid pGL3-SV40-Fcy::Fur (containing SV40 constitutive promoter, non tissue-specific) was constructed in much the same way through insertion of the Fcy::Fur PCR product into plasmid pGL3-promoter. All the recombinant plasmids were routinely transformed into Escherichia coli JM109, amplified, extracted, and finally identified by restriction enzyme analysis and gene sequencing by Shanghai Sangong technological Limited company.⑵After being identified and sequenced to verify the correct reading frame and lack of other mutations, the recombinant plasmids were transfected into SKOV3 and MRC-5 to detect its expression by reversed transcriptive-polymerase chain reaction and Westen-blot analysis.Results⑴pGL3-hTERT-Fcy::Fur and pGL3-SV40-Fcy::Fur were constructed successfully. Furthermore, DNA sequencing results showed that both the sequence of hTERT promoter and Fcy::Fur gene are identical with the array published in GeneBank and their insertion direction was correct.⑵Both the results of RT-PCR and Westen-blot indicated Fcy::Fur gene driven by hTERT promoter was expressed higher in SKOV3 than in MRC-5, significant difference was found between them.Conclusion The recombinant plasmid pGL3-hTERT-Fcy::Fur containing hTERT promoter and Fcy∷Fur suicide gene was constructed successfully, which has different expression levels in ovarian cancer cell and normal cell.Part three Ultrasound-microvesicle-mediated transfection of pGL3-hTERT-Fcy::Fur exhibits specific proliferation inhi- bition and apoptosis induction effects on SKOV3 ovarian cancer cells in vitroObjective To explore the specific lethal effects of Fcy::Fur gene driven by the hTERT promoter on SKOV3 ovarian cancer cells in vitro by ultrasound-microbubble-mediated transfection.Methods Both SKOV3 and MRC-5 Cells were transfected with pGL3-hTERT-Fcy::Fur, pGL3-SV40-Fcy::Fur or pGL3-Promoter plasmid (not containing the Fcy::Fur gene) respectivly using the ultrasound- microvesicle as gene vector. 48h after incubation with 5-FC at various concentrations (0, 32, 65, 130, 250 and 500 mg/L), cell counting kit-8 (CCK-8) assay was employed to analyze the proliferation inhibitory rate of cells in each group. Acridine orange/ethidium bromide (AO/EB) staining and transmission electron microscopy were used for the analysis of cell apoptosis. High performance liquid chromatogram(HPLC)was introduced to analyze the concentration change of 5-FC. hTERT expressions and its promoter activity in SKOV3 and MRC-5 cells were detected respectively by reversed transcriptive-polymerase chain reaction (RT-PCR) and luciferase analysis.Results⑴Transfection of pGL3-hTERT-Fcy::Fur Plasmid led to specific proliferation inhibition and induced specific apoptosis in SKOV3. The SKOV3 and MRC-5 cells that were transfected with pGL3-SV40-Fcy::Fur showed an proliferation inhibitory rate that was not significantly different (P=0.87), nevertheless, the proliferation inhibitory rate of pGL3-hTERT-Fcy::Fur/SKOV3 is higher than that of pGL3-hTERT-Fcy::Fur/MRC-5 (P=0.036). pGL3-Promoter showed no dramatic influence on proliferation of either SKOV3 or MRC-5 cells co-cultured at a 5-FC concentration that was below 250 mg/L (the proliferation inhibitory rates were below 10%). when co-cultured with 5-FC at a concentration of 250 mg/L, apoptosis rate was significantly higher in pGL3-hTERT-Fcy::Fur/SKOV3 cells than in pGL3-hTERT- Fcy::Fur/MRC-5 cells, P=0.028, yet, there was no significant difference between the apoptosis rate in pGL3-SV40-Fcy::Fur/SKOV3 cells and pGL3-SV40-Fcy::Fur/MRC-5 cells, P = 0.82. Moreover, swollen mitochondria and apoptotic body appeared in pGL3-hTERT-Fcy::Fur/ SKOV3 cells, while no significant changes were detected in pGL3-hTERT-Fcy::Fur/ MRC-5 cells.⑵The result of HPLC indicated that 5-FC concentration was lower in SKOV3 than that in MRC-5 cells 48 hour after transfection by pGL3-hTERT-Fcy::Fur.⑶Positive and negative expression of hTERT mRNA were deteched in SKOV3 and MRC-5. The SKOV3 cells showed a more potent promoter activity(26.2%)that was 40 times higher than that in MRC-5 cells(0.65%).Conclusions⑴Fcy::Fur suicide gene driven by the hTERT promoter exhibits specific proliferation inhibition and apoptosis induction effects on hTERT positive SKOV3 while has little lethal effects on hTERT negative MRC-5.⑵The specific proliferation inhibition and apoptosis induction effects of the pGL3-hTERT-Fcy::Fur plasmid in SKOV3 cells was due to its potent hTERT promoter activity which resulted in much higher transcription of the Fcy::Fur gene, on the contrary, the low hTERT promoter activity led to its lower transcription in MRC-5 cells. Part four Treatment of transplanted ovarian cancer by ultrasound-mediated pGL3-hTERT-Fcy∷Fur-loaded microv- esicle destructionObjective To explore the targeting anti-tumor effects on transplanted ovarian cancer using ultrasound-microvesicle as gene transfer to mediate pGL3-hTERT-Fcy∷Fur gene transfection.Method⑴Transduction targeting mediated by ultrasound-mediated microvesicle destruction. 25 nude mice modle of ovarian cancer were assigned randomly to 5 groups, each group received different treatment.①Ultrasound targeted destroy the gene-loaded microvesicle group (US&MV), 0.2 ml pGL3-hTERT-Fcy∷Fur-loaded microvesicle was injected into nude mice through tail vein, then, ultrasound irradiation was released to destroy the microvesicle on the region of tumor.②Ultrasound irradiated gene group (US), 0.2 ml culture fluid containing gene was injected into nude mice, then, ultrasound irradiation was released on the region of tumor.③Ultrasound irradiated void microvesicle group(US&VMV), 0.2 ml microvesicle not containing gene was injected into nude mice, then, ultrasound irradiation was released.④Gene-loaded microvesicle group (MV), gene-loaded microvesicle was injected into nude mice, but ultrasound irradiation was not released.⑤Control group (CT), 0.2 ml physiological saline was injected.⑵Transcription targeting mediated by hTERT promoter. 20 nude mice modle of ovarian cancer were assigned randomly to 4 groups.①pGL3-hTERT-Fcy∷Fur-loaded microvesicle group.②pGL3-Pro-Fcy::Fur-loaded microvesicle group.③pGL3- Promoter loaded microvesicle.④Control group. Both gene-loaded microvesicle and physiological saline were injected into nude mice through tail vein, ultrasound irradiation was released on the region of tumor and liver. Each group,s treatment was performed everyday and last for 7 days. 0.3 ml 5-FC dilution administering(500 mg/kg)through intraperitoneal injection followed the treatment from the 2nd day on, 5 days after the last treatment, all the mice were sacrificed and the tumors were harvested to measure volumes, masses and calculate the tumor inhibition rate. Apoptotic cells were detected by TUNEL analysis.Results⑴The results of transduction targeting.①There is no significant difference about the mean tumor volume, mass and cell apoptosis rate between MV and CT group, P>0.05.②There is no significant difference about the mean tumor volume, mass and cell apoptosis rate between US and US&VMV group, P>0.05. Nevertheless, significant difference about the index mentioned above were found in the two groups when compared with CT group.③Compared with any group of the others, the mean tumor volume and mass was the lowest, while its tumor inhibition rate and cell apoptosis rate was the highest in US&MV group, P<0.05.⑵The results of transcription targeting.①Compared with pGL3-Promoter loaded microvesicle group, there is significant difference about the mean tumor volume, mass and cell apoptosis rate in both the pGL3-hTERT-Fcy∷Fur-loaded microvesicle group and pGL3-Pro- Fcy::Fur-loaded microvesicle group, P<0.05.②There is no significant difference about the tumor cell apoptosis rate between pGL3-hTERT- Fcy∷Fur-loaded microvesicle group and pGL3-Pro-Fcy::Fur-loaded microvesicle group, P>0.05. Nevertheless, there is significant difference about the liver cell apoptosis rate between them, the apoptos rate is higher in the latter.Conclusions①Ultrasound-mediated gene-loading microvesicle destruction is a targeting gene vector. The technique is possible to be adopted as a novel tool gene transfer for gene therapy.②Fcy∷Fur gene driven by hTERT promoter has targeting lethal effects on transplanted ovarian cancer while do little harm to normal cells.③Ultrasound- mediated Fcy∷Fur-loading microvesicle destruction exhibits efficient anti-cancer effect on transplanted ovarian cancer, the therapeutic mode is possible to be adopted as a novel tool for ovarian cancer.更多还原