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。
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 SKOV3
Objective: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 lines
Objective: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 cells
Objective: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.
引文
[1] Casado E, Nettelbeck DM, Gomez-Navarro J,et al. Transcriptional targeting for ovarian carcinoma gene therapy[J]. Gynecologic Oncology, 2001, 82(2):229-37.
[2] Gubbels JA, Claussen N, Kapur AK,et al.The detection, treatment, and biology of epithelial ovarian carcinoma[J]. J Ovar Res. 2010, 3(1):8.
[3] Rudi Bao, Muthu Selvakumaran, Thomas C. Hamilton, et al. Targeted Gene Therapy of Ovarian Carcinoma Using an Ovarian-Specific Promoter[J]. Gynecologic Oncology, 2002, 84(3), 228–234.
[4]Nicholas B. Berry, Yong Mee Cho, Maureen A, et al. Transcriptional targeting in ovarian carcinoma cells using the human epididymis protein 4 promoter[J]. Gynecologic Oncology, 2004, 92(2), 896–904.
[5]贾雪梅.卵巢癌的自杀基因治疗[J].中国肿瘤生物治疗杂志,2003,10(3): 216-18.
[6] Alvarez RD, Gomez-Navarro J, Wang M, et al.Adenoviral-mediated suicide gene therapy for ovarian carcinoma [J].Mol Ther,2000,2(5):524-530.
[7] Kaur T, Slavcev RA, Wettig SD.Addressing the challenge: current and future directions in ovarian carcinoma therapy [J]. Curr Gene Ther, 2009,9(6):434-58.
[8]金萍,孔北华等.多聚乙烯基亚胺介导的pOSP1-HSVtk基因对卵巢癌的抗瘤研究.中国肿瘤生物治疗杂志[J].2005, 12(2):111-5.
[9]张虹,崔恒.卵巢癌的基因治疗研究.实用医学杂志[J].2003,19(6),579-81。
[10] Brian G. Gentry, Paul D. Boucher ,Donna S. Shewach, et al. Hydroxyurea Induces Bystander Cytotoxicity in cocultures of Herpes Simplex Virus Thymidine Kinase-Expressing and Nonexpressing HeLa Cells Incubated with Ganciclovir[J]. Carcinoma Res, 2006, 66(2): 3845-51.
[11]Zhang Al, Wang QS, Han ZQ, et al. Relationship between the expression of connexin43 and bystander effect of suicide gene therapy in ovarian carcinoma. Journal of Huazhong University of Science and Technology [J], 2004, 24(5): 476-79.
[12]王国慧,何军芳,樊卫等.重组Ad-Cp-CDglyTK双自杀基因腺病毒载体构建及体外抑制鼻咽癌细胞的实验研究[J].中国病理生理杂志,2008,24(2):215-9.
[13] Mesnil M, Yamasaki H,et al. Bystander effect in herpes simplex virus-thymidine kinase/ganciclovir carcinoma gene therapy: role of gap-junctional intercellular communication.[J]. Carcinoma Res, 2000, 60(15):3989-99.
[14]冉海涛,任红,王志刚等.超声空化效应对体外培养细胞膜作用的实验研究[J].中华超声影像学杂志,2003,12(8):499-501.
[15]凌智瑜,王志刚,冉海涛等。超声微泡造影剂对心肌组织毛细血管通透性影响的实验研究[J].中国超声医学杂志.2004, 20(5):327-30.
[16]Zhigang W, Zhiyu L, Haitao R, et al. Ultrasound-mediated microbubble destruction enhances VEGF gene delivery to the infarcted myocardium in rats [J]. Clin Imaging. 2004, 28(6):395-98.
[17]Vancraeynest D, Havaux X, Pouleur AC, et al. Myocardial delivery of colloid nanoparticles using ultrasound-targeted microbubble destruction[J]. Eur Heart J. 2006, 27(2):237-45.
[18] Li X, Wang Z, Ran H, et al. Experimental research on therapeutic angiogenesis induced by Hepatocyte Growth Factor directed by ultrasound-targeted microbubble destruction in rats [J]. J Ultrasound Med, 2008, 27: 439-46.
[19]Korpanty G, Chen S, Shohet RV, et al. Targeting of VEGF-mediated angiogenesis to rat myocardium using ultrasonic destruction of microbubbles. Gene Ther [J]. 2005, 12 (17):1305-12.
[20]Juffermans LJ, Meijering DB, Wamel A, et al.Ultrasound and microbubble targeted delivery of therapeutic compounds: ICIN Report Project 49: Drug and gene delivery through ultrasound and microbubbles[J]. Neth Heart J. 2009, 17(2):82-6.
[21] Liu X, Wu J.Acoustic microstreaming around an isolated encapsulated microbubble[J]. J Acoust Soc Am. 2009, 125(3):1319-30.
[22]Chomas JE, Dayton P, May D, et al. Threshold of fragmentation for ultrasonic contrast agents[J]. J Biomedical Optics 2001, 6:141–50.
[1] Moolten FL. Tumor chemosensitivity conferred by inserted herpes thymidine kinase genes: paradigm for a prospective carcinoma control strategy. Carcinoma Research , 1986 , 46(10) : 5276-81.
[2]王国慧,何军芳,樊卫等.重组Ad-Cp-CDglyTK双自杀基因腺病毒载体构建及体外抑制鼻咽癌细胞的实验研究[J].中国病理生理杂志,2008,24(2):215-9.
[3] Alvarez RD, Gomez-Navarro J, Wang M, et al.Adenoviral-mediated suicide gene therapy for ovarian carcinoma[J].Mol Ther,2000,2(5):524-530.
[4] Shimomura O. The discovery of aequorin and green fluorescent protein[J]. Journal of Microscopy,2005,217(1):1-15.
[5] Persons DA, Allay JA, Riberdy JM, et al. Use of the green fluorescent protein as a marker to identify and track genetically modified hematopoietic cells[J]. Nature Medicine, 1998, 4(10): 1201-5.
[6] Kalil RA,Teixeira LA,Mastalir ET,et a1.Experimental model of gene transfection in healthy canine myocardium: perspectives of gene therapy for ischemic heart disease[J]. Arq Bras Cardiol,2002,79(3):223-32.
[7] Okabe S,Arai T,Yamashita H,et a1.Adenovirus-mediated prodrug-enzyme therapy for CEA-producing colorectal carcinoma cells[J]. Journal of CarcinomaResearch and Clinic Oncology,2003,129(6):367-73.
[8]陈勇,江军,张尧等.含rPB-DR启动子和TK基因的重组腺病毒的构建和鉴定[J].第三军医大学学报,2006,28(10):1043-45.
[9]刘晓平,李宝金,张超等. HSV-tk基因治疗靶向载体的构建及特异性表达分析[J].癌症,2006,25(2):179 - 84.
[10] Culver KW,Ram Z,Wallbridge S,et a1.In vivo gene transfer with retroviral vector-producer cells for treatment of experimental brain tumors[J]. Science, 1992, 256(5063):1550-52.
[11] Freeman SM, Abboud CN, Whartenby KA, et al. The "bystander effect": tumor regression when a fraction of the tumor mass is genetically modified[J]. Carcinoma Research,1993 ,53(21):5274-83.
[12] Zhang Al, Wang QS, Han ZQ, et al. Relationship between the Expression of Connexin43 and Bystander Effect of Suicide Gene Therapy in Ovarian Carcinoma[J]. Journal of Huazhong University of Science and Technology [J], 2004, 24(5): 476-9.
[13] Tong X, Shine DH, Agoulnik I, et al . Adenovirus mediated thymidine kinase gene therapy may enhance sensitivity of ovarian carcinoma cells to chemotherapeutic agents[J]. Anticarcinoma Res, 1998, 18 (5A) : 3421-26.
[1]樊萍,杨竹,胡接力,等。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[J]。生物技术通报,2009,204(7):122-127。
[2]郑元义,张茂惠,彭剑萍,等。计算机图像识别技术在超声微泡粒径分析中的应用[J]。中国超声医学杂志,2007,23(17):727-729。
[3] Aeynest D, Havaux X, Pouleur AC, et al. Myocardial delivery of colloid nanoparticles using ultrasound-targeted microbubble destruction[J]. Eur Heart J, 2006, 27(2):237-245.
[4] Guzman HR, McNamara AJ, Nguyen DX, et al. Bioeffectscaused by changes in acoustic cavitation bubble density and cellconcentration: a unified explanation based on cell-to-bubble ratio andblast radius[J]. Ultrasound Med. Biol. 2003, 29(4):1211–1222.
[5]朱元方,胡丽娜,李攀,等。自制脂质微泡联合超声辐照介导pGL3-Promoter-–EGFP质粒转染卵巢癌细胞的实验研究。中国超声医学杂志,2008,24(4):303-306。
[6] Ryo Suzuki Tumor specific ultrasound enhanced gene transfer in vivowith novel liposomal bubbles[J].Journal of Controlled Release,2008, 125 (5): 137–144.
[7]苏林,王志刚,李兴升等。超声辐照微泡声学造影剂增强脂质体介导肝细胞基因转染的体外实验研究[J]。中国超声医学杂志2007,23(10):721-723。
[8] Ultrasonic gene and drug delivery to the cardiovascular system[J].Advanced drug delivery reviews,2008,60(10),1177-1192.
[9] Suzuki R ,Takizawa T,Negishi Y,et al.Gene delivery by combination of novel liposomal bubbles with perfluoropropane and ultrasound[J].J Control Release, 2007,117(1):130-136.
[10] Vandenbroucke RE, Lentacker I, Demeester J, et al. Ultrasound assisted siRNA delivery using PEG-siPlex loaded microbubbles[J]. J Control Release. 2008, 85 (3):265-73.
[1]丰有吉,沈铿主编。妇产科学[ M]。人民卫生出版社,2005年8月第1版:330-331。
[2] Rocconi RP, Numnum TM, Stoff-Khalili M, et al. Targeted gene therapy for ovarian carcinoma[J]. Current Gene Therapy, 2005, 5(6):643-53.
[3] Chien J, Narita K, Rattan R, et al. A role for candidate tumor-suppressor gene TCE-AL7 in the regulation of c-Myc activity, cyclin D1 levels and cellular transforma-tion. [J]. Oncogene. 2008, 27(58):7223-34.
[4] Chua AC, Klopcic B, Lawrance IC, et al.Iron: an emerging factor in colorectal carcinogenesis.[J]. World J Gastroenterology,2010,16(6):663-72.
[5] Esteller M. Aberrant DNA methylation as a carcinoma-inducing mechanism [J]. Annual Review Pharmacology Toxicology, 2005, 45:629-56.
[6] Hong SH, Kim HG, Chung WB, et al. DNA hypermethylation of tumor-relatedgenes in gastric carcinoma[J]. J Korean Med Sci, 2005, 20(2):236-41.
[7] Fillat C, CarrióM, Cascante A, et al. Suicide gene therapy mediated by the Herpes Simplex virus thymidine kinase gene/Ganciclovir system: fifteen years of application [J]. Current Gene Therapy. 2003, 3(1):13-26.
[8] Evan GI, Vousden KH. Proliferation, cell cycle and apoptosis in carcinoma[J]. Nature,2001, 411(6835):342-48.
[9] Mesnil M, Yamasaki H, et al. Bystander effect in herpes simplex virus-thymidine kinase/ganciclovir carcinoma gene therapy: role of gap-junctional intercellular communication[J]. Carcinoma Res,2000,60(15):3989-99.
[10] Freeman SM, Abboud CN, Whartenby KA, et al. The "bystander effect": tumor regression when a fraction of the tumor mass is genetically modified [J].Carcinoma Res,1993, 53(21):5274-83.
[11] Rivera E, Lee J, Davies A, et al.Clinical development of ixabepilone and other epothilones in patients with advanced solid tumors[J]. Oncologist. 2008,13 (12):1207-23.
[12]樊萍,杨竹,王志刚等.携自杀基因微泡转染SKOV3的超声参数及转染效率.第四军医大学学报[J].2009,30(22):2511-14.
[13] Chen Z, Liang K, Xie M, et al. Novel ultrasound-targeted microbubble destruction mediated short hairpin RNA plasmid transfection targeting survivin inhibits gene expression and induces apoptosis of HeLa cells.[J]. Molecular Biology Reports. 2009,36(8):2059-67.
[14] Aoi A, Watanabe Y, Mori S,et al.Herpes simplex virus thymidine kinase-mediated suicide gene therapy using nano/microbubbles and ultrasound.[J]. Ultrasound in Medecin& Biology. 2008,34(3):425-34.
[15] Shen ZP, Brayman AA, Chen L, et al.Ultrasound with microbubbles enhancesgene expression of plasmid DNA in the liver via intraportal delivery [J]. Gene Ther. 2008,15(16):1147-55.
[16] Chen BA, Meng QQ, Wu W, et al. Enhancement of reversing drug resistance of K562/A02 cells to adriamycin by ultrasound-induced cavitation [J]. Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2008,16(6):1283-87.
[17] Chen ZY, Liang K, Xie MX, et al. Induced apoptosis with ultrasound-mediated microbubble destruction and shRNA targeting survivin in transplanted tumors [J] .Advance in therapy. 2009,26(1):99-106.
[18] Sellers WR, Fisher DE. Apoptosis and carcinoma drug targeting [J]. J Clin Invest 1999; 104(12):1655-61.
[19] O'Brien TA, Tuong DT, Basso LM, et a1. Co-expression of the uracil phosphori- bosyltransferase gene with a chimeric human nerve growth factor receptor cytosine daminase fusion gene, using a single retroviral vector, augments cytotoxicity of transduced human T cells exposed to 5-fluorocytosine[J]. Hum Gene Thera. 2006,17(5):518-30.
[20] Chung-Faye GA,Chen MJ, et a1. In vivo gene therapy for colon carcinoma using adenovirus-mediated transfer of the fusion gene cytosine deaminase and uracil phosphoribosyl transferase[J]. Gene Therapy 200l,8(20):l547-54.
[21] Fujiwara T, Urata Y, et al. Telomerase-specific oncolytic virotherapy for human carcinoma with the hTERT Pro[J]. Current Carcinoma Drug Targets. 2007,7 (2):191 -201.
[1] Verma IM, Somia N. Gene therapy--promises, problems and prospects [J].Nature, 1997, 389(6648):239-42.
[2] Tomanin R, Scarp M. Why do we need new gene therapy viral vectors? Characteristics, limitations and future perspectives of viral vector transduction [J]. Cur Gene Ther, 2004, 4(4):357-72.
[3] Ritter T, Lehmann M, Volk HD. Improvements in gene therapy: averting the immune response to adenoviral vectors [J]. Bio Drugs 2002,16(1):3-10.
[4] Li SD, Huang L. Targeted delivery of siRNA by nonviral vectors: lessons learned from recent advances[J]. Current opinion in investigational drugs. 2008,9(12):1317-23.
[5] Gramiak R, Shah PM. Echocardiography of the aortic root[J]. Investigative radiology,1968,3(5):356-66.
[6] Schneider.Contrast media in ultrasonography:from synthesis to clinical use.The SonoVue example[J].Ann Cardiol Angeiol,2002,51(4):218-20.
[7] Alexander L. Klibanov.Targeted delivery of gas-filled microspheres,contrast agents for ultrasound imaging[J].Advanced Drug Delivery Renews,1999,37:139-57.
[8] Rapoport N, Gao Z, Kennedy A. Multifunctional nanoparticles for combining ultrasonic tumor imaging and targeted chemotherapy[J]. J Natl Carcinoma Inst. 2007, 99(14):1095-106.
[9] Chen BA, Meng QQ, Wu W,et al. Enhancement of reversing drug resistance of K562/A02 cells to adriamycin by ultrasound-induced cavitation[J]. Zhongguo Shi Yan Xue Ye Xue Za Zhi,2008,16(6):1283-87.
[10] Meijering BD, Juffermans LJ, van Wamel A, et al.Ultrasound and microbubble-targeted delivery of macromolecules is regulated by induction of endocytosis andpore formation [J]. Circulation Res,2009,104(5):679-87.
[11] Liang HD, Tang J, Halliwell M, et al. Sonoporation, drug delivery, and genetherapy[J].Proceedings of Institution Mechanical Engineers H,2010,224(2):343- 61.
[12] RyoSuzuki. Tumor specific ultrasound enhanced gene transfer in vivo with novel liposomal bubbles. Journal of Controlled Release 125 (2008) 137-44.
[13] N. Sakaguchi, C. Kojima, A. Harada, et al. Enhancement of transfect ion activity of lipoplexes by complexationwith transferrin-bearing fusogenic polymer-modified liposome’s, Int. JPharm. 2006, 325: 186-90.
[14] Lentacker I, Wang N, Vandenbroucke RE, et al. Ultrasound exposure of lipoplex loaded microbubbles facilitates direct cytoplasmic entry of the lipoplexes [J]. Molecular Pharmaceutics. 2009, 6(2):457-67.
[15] Li T, Tachibana K, Kuroki M, et al. Gene transfer with echo-enhanced contrast agents: comparison between Albunex, Optison, and Levovist in mice--initial results [J]. Radiology, 2003, 229(2):423-8.
[16] Rahim A, Taylor SL, Bush NL, et al. Physical parameters affecting ultrasound/microbubble-mediated gene delivery efficiency in vitro [J]. Ultrasound Med Biol. 2006, 32(8):1269-79.
[17] Murata R, Nakagawa K, Ohtori S,et al. The effects of radial shock waves on gene transfer in rabbit chondrocytes in vitro [J]. Osteoarthritis Cartilage, 2007,15(11):1275-82.
[18] H. Mizuguchi, T. Nakagawa, M. Nakanishi, S. et al.Efficient gene transfer into mammalian cells using fusogenicliposome [J]. Biochem Biophys Res Commun, 1996,218:402–7.
[19]杨莉,谭开彬等.阴离子脂膜超声微泡的制备及评价.临床超声医学杂志,2006, 8(4):193-5.
[20] Schroeder A, Kost J, Barenholz Y. Ultrasound, liposomes, and drug delivery: principles for using ultrasound to control the release of drugs from liposomes. Chem Phys Lipids. 2009,162(1-2):1-16.
[21] Maruyama K, Suzuki R, TakizaWa T, et al. Drug and gene delivery by "bubble liposome’s" and ultrasound[J]. Yakugaku Zasshi 2007; 127(5):781-7.
[22] Nie F, Xu HX, Tang Q, et a1. Microbubble-enhanced ultrasound exposure improves gene transfer in vascular endothelial cells[J].World J Gastroenterology, 2006,12 (46):7508-13.
[23] Company G, Chen S, Sheet RV et al. Targeting of VEGF-mediated angiogenesis to rat myocardium using ultrasonic destruction of microbubbles [J]. Gene Thera ,2005,12(17):1305-12.
[24] Vancraeynest D, Havaux X, Pouleur AC, et al. Myocardial delivery of colloid nano-particles using ultrasound-targeted microvesicle destruction[J]. Eur Heart J, 2006,27(2):237-45.
[25] Bian AH ,Gao YH,Tan KB, et al.Preparation of human hepatocellular carcinoma - targeted liposome microbubbles and their immunological properties. World J Gastroenterol ,2004 ,10(23) :3424-27.
[26] Ellegala DB, Leong-Poi H, Garpenter JE, et al. Imaging tumor angiogenesis with contrast ultrasound and microbubbles targetedαvβ3. Circulation, 2003, 108(3):336-41.
[27] Cherkaoui S, Bettinger T, Hauwel M, et al. Tracking of antibody reduction fragments by capillary gel electrophoresis during the coupling to microparticles surface[J]. Journal of Pharmaceutical and Biomedical Analysis. 2010 Feb 1.
[28] Lindner JR.Microbubbles in medical imaging: current applications and future directions.[J]. Nature Review Drug Discovery. 2004 , 3(6):527-32.
[29] Tsunoda S, Mazda O, Oda Y, et al. Sonoporation using microbubble BR14 promotes pDNA/siRNA transduction to murine heart.Biochem Biophys. Res. Commun. 2005,336, 118–27.
[30] H. Takeuchi, K. Ohmori, I. Kondo, Interaction with leukocytes:phospholipids- -stabilized versus albumin-shell microbubbles, Radiology[J]. 2004,230:735–42.
[31] Delalande A, Bureau MF, Midoux P, et al.Ultrasound-assisted microbubbles gene transfer in tendons for gene therapy [J]. Ultrasonics. 2010,50(2):269-72.
[32] Hiromi Koike An efficient gene transfer method mediated by ultrasound and microbubbles into the kidney [J].The Journal of Gene Medicine, 7(1):108–16.
[33] Zhang QX. Enhanced Gene Delivery into Skeletal Muscles with Ultrasound and Microbubble Techniques.Acad Radiol[J]. 2006,13(3):363-7.
[34] Li X, Wang Z, Ran H, et al. Experimental research on therapeutic angiogenesis induced by Hepatocyte Growth Factor directed by ultrasound-targeted microbubble destruction in rats[J]. J Ultrasound Med. 2008, 27: 439-46.
[35] Ren JL, Wang ZG, Zhang Y, et al. Transfection efficiency of TDL compound in HUVECenhancedby ultrasound-targeted microbubble destruction [J].Ultrasound Med Biol. 2008, 34(11):1857-67.
[36] Zhang QX,W ang ZG.Ran HT,et a1.Enhanced gene transfection in tumor cells by ultrasound and mierobubbles[J].J Ultrasound in Clin Med(Chinese),2005,7(2):78-80.