摘要
随着分子生物学及生物技术的迅速发展，近年来基因治疗的基础研究与应用得到了长足的进步。在基因疗法的研究中，基因传输方法是成功进行基因治疗的一个关键步骤，其中非侵袭、靶向的基因输送系统因其诸多优点成为基因疗法研究中的一个热点，在临床上具有重要的潜在应用价值。虽然传统的病毒基因载体具有较高的基因转染效率，但该类方法的安全性和长期疗效受到质疑，限制了其临床应用。近几年，非病毒基因载体引起了研究者的关注并得到了广泛研究。但目前的研究表明，单纯的非病毒载体基因转染率不高，导致基因治疗效果明显受限。超声靶向微泡破坏技术(Ultrasound-targetedmicrobubble destruction，UTMD)是一种靶向、高效、有广阔应用前景的非病毒基因输送新策略，其通过诱发细胞膜声孔作用，增加膜通透性，进而有效促进大分子物质的胞内传输，且可避免其他基因传输方法的副作用。在该类方法的研究中，UTMD的参数设置与优化组合对基因转染效率有至关重要的影响。本论文将对UTMD的参数进行详细分析与优化，为靶向、高效、无创的基因治疗研究提供一种可行的实验方案；同时尝试将物理(UTMD)和化学(PEI)的基因转染策略有机地结合起来，探究一种有效的基因传输与基因转染方法；尝试运用UTMD转染靶向Survivin基因的短发夹状RNA干扰质粒，分析基因沉默效应、凋亡诱导及增殖抑制作用，开创癌症基因治疗的新篇章。本文包括以下三部分：
第一部分超声靶向微泡破坏参数设置与优化增强基因转染的研究
目的①探讨不同超声及转染参数、细胞培养方式对细胞活力、基因传输和声孔作用的影响，优化超声靶向微泡破坏(UTMD)参数，减少对细胞活力利质粒完整性的影响；②比较不同声学微泡对体外基因转染的作用及其安全性；③探讨优化的UTMD参数对裸鼠人宫颈癌(Hela)皮下移植瘤基因的靶向传输效率。方法体外研究：①悬浮培养或贴壁培养Hela细胞，设置不同超声强度、占空比及辐照时间，比较其对细胞活力及红色荧光蛋白基因(DsRed)表达效率的影响；②显微镜和扫描电镜观察细胞形态及细胞膜表面的超微结构；③系统比较不同转染参数组合、不同质粒[DsRed利荧光素酶质粒(pCMV-LUC)]对细胞活力及基因表达效率的影响，分析UTMD对基因转染的增强作用，琼脂糖凝胶电泳分析质粒完整性；④分析脂质体微泡(LM)的增效作用，对微泡浓度、超声参数进行优化，运用最优参数对不同细胞系(HepG_2、Ishikawa、MCF-7和B16-F10)行超声处理，并与Sono Vue微泡、聚乙烯亚胺(PEI)介导的基因转染进行比较。体内研究：将两种报告质粒(EGFP、DsRed)经裸鼠尾静脉注入，对基因表达持续时间(1～7 d)、质粒用量(20～50μg)、靶向性和安全性进行分析。结果体外研究：①高声强和高占空比显著降低细胞存活率(P＜0．01)，导致细胞脱离。细胞培养方式和三种超声参数均有显著的交互作用(P＜0．01)；贴壁培养时，细胞活力随着占空比的增加、辐照时间的延长而逐步降低。悬浮培养时，经相同超声参数转染的细胞数目明显增加，存活率无明显下降；②电镜结果显示，适当条件的超声辐照能导致细胞表面出现可逆性孔道。悬液培养的细胞经1．0 W／cm~2、20％占空比的辐照3 min后，声孔作用最强；③当质粒浓度达到30μg／孔时，两种质粒转染率均最高。与单纯超声辐照相比，UTMD可显著提高基因转染效率(P＜0．01)。④电泳结果表明，质粒DNA结构的完整性不受优化参数影响；⑤超声辐照联合LM处理后基因转染率显著增加(P＜0．01)；当培养细胞暴露于最优条件时，不同细胞类型对超声的反应性不同。体内研究：P+UTMD组内荧光表达显著高于P组、P+US组或P+LM组，差异均有统计学意义(p＜0．01)，基因表达的最佳时间是第3 d(P＜0．01)，两种不同报告基因的转染率较一致，非靶向器官未见明显基因表达且未观察到明显的组织损伤，结论①不同超声和转染参数、培养方式对细胞活力和基因传输效率有较大影响，对其优化后可产生较强的声孔作用，减少细胞损伤；②声学微泡对超声介导基因转染有显著的增效作用，UTMD是一种有效的体外基因输送方法；③UTMD能显著增加体内报告基因转染，是一种高效、非侵袭性的基因转染方法。
第二部分超声靶向微泡破坏联合聚乙烯亚胺增强基因转染的研究
目的①分析UTMD联合PEI增强癌细胞基因转染的最优体外条件及协同作用；②探讨UTMD联合PEI增强裸鼠移植瘤基因输送的可行性和应用价值。方法体外研究：①制备PEI／pCMV-LUC复合物用于乳腺癌细胞(MCF-7)基因转染，以不同方式孵育微泡／转染复合物，通过荧光素酶活性和细胞存活率测定，对超声辐照参数进行优化，对质粒浓度、孵育时间、血清、溶媒类型、培养基体积等因素进行分析。②将不同质粒DNA(DsRed、pCMV-LUC)与不同分子量PEI(25 kDa、750 kDa)以不同的N／P比制备PEI／DNA复合物，利用凝胶阻滞实验分析PEI／DNA复合物的混合比例，MTT法测定PEI的细胞毒性，评价不同分子量PEI对基因转染的影响及超声辐照的增强作用。体内研究：经荷瘤裸鼠尾静脉分别注入PBS、质粒、质粒+Sono Vue微泡、PEI／DNA复合物+SonoVue微泡，仅对一侧肿瘤行超声辐照，另一侧肿瘤作为对照，对基因转染和组织学检查进行分析。结果体外研究：①适当条件的超声辐照可促进PEI／DNA复合物转染，UTMD联合PEI的转染效率显著高于单纯超声辐照和PEI转染(P＜0．01)。超声辐照前细胞与PEI／DNA复合物共孵育2 h时，荧光素酶活性显著增强(P＜0．01)。②琼脂糖凝胶电泳显示，N／P比≥3时，PEI可有效地缩合质粒DNA，两种PEI及两种质粒DNA的电泳情况相似。细胞毒性与PEI浓度相关。②单独超声辐照能提高裸质粒和PEI／DNA复合物的荧光素酶活性(P＜0．05)，但前者的增加幅度显著小于后者(P＜0．05)，25 kDa显著优于750 kDa(P＜0．01)。体内研究：UTMD联合PEI可显著增强基因转染，受辐照移植瘤的荧光素酶活性增加了10倍(P＜0．01)；与非联合PEI时比较，荧光素酶表达增加了111倍(P＜0．01)。无论有否超声照射，裸鼠其他器官组织中均无明显的基因表达(P＞0．05)，且未观察到明显的组织损伤。结论①UTMD联合PEI对基因转染有协同作用，是一种增强质粒DNA基因表达简单而有应用前景的方法，优化的超声和转染参数能显著提高体外癌细胞的基因表达效率。②UTMD联合PEI可显著增强报告基因在肿瘤组织的靶向传输利转染，是一种很有前景、新型而安全的体内基因输送方法，为基因治疗提供一种高效的新方法。
第三部分超声靶向微泡破坏联合RNAi沉默Survivin表达及诱导细胞凋亡的研究
目的①构建靶向Survivin基因的shRNA真核表达质粒，研究RNA干扰技术(RNAi)对凋亡抑制因子Survivin的降调节作用；②通过UTMD联合RNAi，观察体内外Survivin基因的沉默效应、细胞凋亡的诱导效应和增殖抑制作用。方法①构建三个靶向Survivin基因的shRNA真核表达质粒(pSIREN／S1／S2／S3)和对照质粒(pSIREN／con)。②体外研究：通过UTMD或脂质体转染筛选，将最优重组质粒进行详细研究，应用FITC-annexin V和7-AAD双染、DNA ladder分析细胞凋亡，RT-PCR和蛋白质印迹检测mRNA及蛋白表达的变化。③体内研究：将荷瘤裸鼠分三组：质粒+超声辐照组(P+US)，质粒+微泡+超声辐照组(P+UTMD)，对照组(C)。对组织样本行冰冻切片、组织学检查、采用免疫组化检测移植瘤Survivin、增殖细胞核抗原(PCNA)、Bcl-2、Bax、Caspase-3、Ki-67、核干细胞因子(NS)、p53蛋白在各组肿瘤标本中的表达，采用CD34标记、测定微血管密度(MVD)，应用TUNEL法分析凋亡指数(AI)。结果①酶切和测序分析证实重组质粒构建成功。②体外研究：RT-PCR和蛋白质印迹法表明，pSIREN／S3的抑制效果最显著。流式细胞分析显示，P+L组的细胞凋亡率(31．58％±3．12％)显著高于各对照组(P＜0．01)，但仍低于P+UTMD组(43．86％±4．44％，P＜0．01)；DNA ladder显示，脂质体或UTMD转染处理后可检测到明显凋亡条带；P+UTMD组的Survivin mRNA及蛋白表达抑制率为83．33％±2．73％和79．67％±3．55％，均显著高于其他各组(P＜0．01)。③体内研究：P+UTMD组的PCNA、Ki-67、Bcl-2、Survivin及NS蛋白表达下降，而Bax、Caspase-3和P53蛋白表达明显上调，MVD明显减少，AI明显增加，与C组及P+US组比较，差异均有统计学意义(P＜0．05)。结论Survivin可作为癌症基因治疗的理想靶标，UTMD联合shRNA干扰技术能显著阻抑靶基因Survivin的表达，有效诱导体内外细胞凋亡，抑制细胞增殖，为肿瘤基因治疗及研究提供有前景的新方法。
Developments of techniques in molecular biology have led to the progress of genetherapy which is mainly dependent on the development of the methods of gene delivery.Meanwhile,noninvasive delivery systems that could target specific anatomical sites wouldbe hotspot for gene therapy and would be valuable clinical tools.Viral vector has beendeveloped as highly efficient methods for gene delivery to a variety of tissues,but thesafety of this kind of delivery method and immune responses are still concerned in theclinical applications.The effectiveness of nonviral gene vectors for delivering genes intocells has attracted a great deal of attention in recent years.Nonviral vectors are safer,butthe transfection efficiencies are too low to be of clinical value.Among non-viral techniques,ultrasound-targeted microbubble destruction (UTMD)has evolved as a new promising toolfor organ-specific gene delivery in in vitro and in vivo targeting delivery via a processwhich is called sonoporation,allowing for the macromolecule transfering into the cellsefficiently.To make such technology suitable to the clinical setting,parameters of UTMDtechnique should be optimized systematically.Moreover,we assessed whether the novelcombination of UTMD and polyethylenimine (PEI)was available and useful tool for genedelivery and transfection.Furhtermore,a detailed study of the combination of UTMD andshRNA targeting human Survivin gene in vitro and in vivo was carried out in this work andelucidated the effects of gene silencing cell apoptosis and proliferation,laying thefoundation for cancer gene therapy.This study includes three parts as follows.Part 1 Optimization of Ultrasound-targeted Microbubble Destruction Enhanced GeneTransfection In Vitro
Objective 1.To investigate different parameters of ultrasound andtransfectionas well as culture conditions that would affect cell viability,sonoporation andtransfection rate of gene in cancer cells.2.To establish the optimal parameters ofultrasound-mediated microbubble destruction (UTMD)which enhanced gene transfection,3.To evaluate the transfection efficiency and safety of different acoustic microbubbles invitro in order to obtain higher transfection efficiency with minimal effect on cell viabilityand structural integrity of plasmid DNA.4.To evaluate the feasibility of delivery andlocalization of plasmid in subcutaneous transplantation tumors of human cervicalcarcinoma (Hela)in nude mice facilitated by UTMD and to optimize the correlatedparameters in vivo.Methods HeLa cells were cultured in vitro using two different protocolsin two groups,Group A:24 hrs culture for complete adherence;Group S:cell suspension.Subsequently,cells were transfected by different ultrasound intensity,duty cycle andexposure time.Gene transfection (DsRed)and cell viability were evaluated.Usingmicroscope and scanning electron microscopy (SEM),the changes of shape and thesonoporation on cell membrane induced by UTMD were observed.The influence ofparameters on transfection efficiency and cell viability of different cell lines (Hela andIshikawa)using two different DNA plasmids,DsRed and luciferase recombinant plasmid(pCMV-LUC),were studied.The different parameters of UTMD were systematicallystudied to optimize gene transfection,i.e.such as DNA concentration,duty cycle and theduration of exposure.The enhancement of SonoVue microbubble under UTMD conditionwas analyzed.The effects of sonication on plasmid DNA were investigated.Enhancedeffects of liposome microbubble (LM)under UTMD conditions were studied.Differentconcentrations of LM and ultrasound parameters were optimized.Treatment parametersoptimized in HeLa cells were applied in 4 other cell lines (HepG_2,Ishikawa,MCF-7 andB 16-F10)and compared with the transfection of PEI.In transplanted tumors in vivo study,LM and two different plasmids (EGFP and DsRed)were injected by tail vein into the nudemice followed by ultrasound exposure.Duration of gene expression (1～7 d),plasmid usage(20～50μg),localization and safety were evaluated.Results Low intensity and duty cyclehave no great impact on cell viability.Cell injury were found to increase progressively withhigh intensity and duty cycle (P＜0.01),and cell detachment was significantly accompaniedby ultrasound exposure in adherent HeLa cells.Results of factorial design showed that the culture conditions and the ultrasound parameters had interaction between them (P＜0.01).SEM demonstrated that the phenomenon of transient pores in the cell membrane undersuitable ultrasound irradiation was observed in details.The ideal sonoporation conditionsthat cell viability was above 80% and more membrane holes were noted to be at 1.0 W/cm~2exposured 3 min with a duty cycle of 20% in cell suspension.The increased transfectionefficiency was related to plasmid concentration and the highest transfection efficiency wasobtained when DNA concentration was 30μg/well.Compared with the ultrasoundirradiation alone,transfection efficiency of optimal UTMD was significantly increased(P＜0.01).The results of agarose gel electrophoresis indicated that the structural integrity ofplasmid DNA was unaffected by the optimal ultrasound parameters.LM and ultrasoundexposure increased transfection efficiency in cultured Hela cells significantly (P＜0.01).Transfection efficiency was the most prominent under the condition of 6% LM.Noapparent cell damage was found in the all groups.These experiments also revealed thatresponses to ultrasound treatment were different for all tested cell lines,dead andtransfected cells in the treated groups were different from the non-irradiated groupssignificantly.In in vivo study,strong fluorescence expression was seen in P+UTMD groupin which gene expression was significantly higher than that in any other groups (P＜0.01).Gene expression level at third day post ultrasound exposure was significantly higher thanany other time points (P＜0.01).There was not significantly expression level between twodifference reporter genes (P=I.000).No tissue damage was seen histologically.Conclusions Ultrasound and transfection parameters,culture conditions have a greatimpact on gene delivery efficiency and cell viability.The optimized parameters andconditions could decrease the cell injury and have a great impact on the sonoporation.Acoustic microbubbles by UTMD could enhance effectively without apparently adverseeffect in vitro and in vivo.UTMD would be an effective,noninvasive gene transfectionmethod and provide a novel,safe non-viral alternative to current gene therapy.
Part 2 Study of Ultrasound-Targeted Microbubble Destruction Enhanced GeneDelivery Accompanied with PEI
Objective 1.To study the optimized condition of transfection efficiency enhanced by UTMD combined with PEI.2.To observe whether the combination can have asynergistic effect to increase gene transfection.3.To determine whether it could enhancegene delivery in vivo in tumor xenografts.Methods In in vitro study,MCF-7 cells weretransfected with the compounds prepared by the plasmids encoding luciferase(pCMV-LUC)and PEI.SonoVue microbubble was added to the cell suspension or culturedwith PEI/DNA before ultrasound irradiation.The strategy of ultrasound irradiation wasoptimized.The luciferase expression and cell viability were evaluated.Furthermore,theinfluencing factor,such as the plasmid concentration,incubation time,serum,the type ofsolvent and the volume of culture media,were examined.Moreover,two differentmolecular weights (MWs,25 kDa,750 kDa)of PEI were incubated with two differentplasmids (DsRed,pCMV-LUC)to prepare the cationic compounds (PEI/DNA),accordingto the N/P ratio (nitrogen/phosphate ratio).Formation of PEI/DNA complexes wereconfirmed by the gel retardation assay.Cell viability was assessed by MTT Assay.Theeffects of different MWs on their gene expression and the enhancement of ultrasound wereevaluated.In in vivo study,Hela cells were planted subcutaneously in both flanks of femalenude mice.Tumor-bearing mice were administered by tail vein with PBS,plasmid,plasmidand SonoVue microbubble,PEI/DNA and SonoVue microbubble.One tumor was exposedto ultrasound irradiation,while the other served as control.The feasibility of targeteddelivery and tissue specificity facilitated by UTMD and PEI was investigated.Geneexpressions and histology analysis were detected.Results Ultrasound irradiation under anappropriate condition could enhance the gene transfection of PEI/DNA complexes.Incontrast to the PEI/DNA complex alone without ultrasound irradiation or ultrasoundirradiation alone,the combination of UTMD and PEI had a significantly enhancedluciferase activity (P＜0.01).The 2 h pre-irradiation incubation with PEI/DNA complexexhibited a significantly enhanced luciferase activity (P＜0.01).Besides,serum,type ofsolvent and the volume of culture media could affect the transfection efficiency.Electrophoresis experiments revealed that PEI could condense plasmid DNA efficientlywhen the N/P ratio was 3 or higher,and two different plasmids were similar in thiscondition.Cytotoxicity was related to the concentration of PEI.Ultrasound irradiation,evenwithout microbubbles,could significantly enhance luciferase activity of naked plasmid orcationic compound (P＜0.05).The enhancement was significant for the PEI/DNA compound when compared with the naked plasmid (P＜0.05).Gene transfection of 25 kDawas significantly better than 750 kDa (P＜0.01).In in vivo study,the increase of transgeneexpression was related to UTMD with the presence of PEI dramatically.At least 10-foldincrease of luciferase gene transfer was obtained in irradiated tumors compared tonon-irradiated controls (P＜0.01),111-fold increase compared to UTMD alone (P＜0.01).There was not significantly gene expression in other organs or tissues regardless ofultrasound exposure (P＞0.05).No tissue damage was seen histologically.ConclusionsThese results suggest that the combination of UTMD and PEI has a synergistic effect ongene transfection and optimized ultrasound and transfection parameters provide efficientgene delivery in cancer cells.This is a simple and promising method to enhance the geneexpression.Meanwhile,the novel combination can enhance targeted gene delivery andreporter gene expression in tumors at intravenous administration.It is a promising,new andsafe method for gene therapy in vivo.
Part 3 Study of UTMD Associated with RNAi Techniques to Silence SurvivinExpression and Induce Cell Apoptosis
Objective 1.To construct the recombinant expression vectors targetedSurvivin gene and to analyze the silencing effect by RNAi in cervical cancer (HeLa)cellline.2.To investigate the gene silencing,apoptosis induction and the suppression ofproliferation in vitro and in vivo transfected by UTMD techniques associated with RNAiTechniques.Methods Three survivin-shRNA expression vectors (pSIREN/S1/S2/S3)and anegative control vector was constructed (pSIREN/con)were constructed.The shRNAvectors were added to Hela cells transfected by Lipofectamine or mediated by UTMD.Theoptimal recombinant plasmid was used in the systematic optimization study of UTMD,while plasmid group (P),ultrasound exposure group (US),SonoVue microbubble group (S),plasmid and SonoVue microbubble group (P+S),plasmid and ultrasound exposure group(P+US)were used as controls,respectively.Moreover,the cells were transfected byLipofectamine (P+L).The expressions of Survivin mRNA and protein were detected byRT-PCR and western blot analysis,respectively.Apoptosis index of transfected cells wasquantified by flow cytometry marked with FITC-annexin V/FITC and 7-AAD.In the in vivo study,nude mice were randomly arranged into 3 groups:control group,plasmidinjection and ultrasound (P+US),P+UTMD group.Frozen section,transfection efficiencyand histological examination were evaluated.Protein expressions of Survivin andproliferating cell nuclear antigen (PCNA),Bcl-2,Bax,Caspase-3,Ki-67,nucleostemin(NS)、p53 were investigated by immunohistochemistry.Furthermore,microvessel density(MVD)was detected by CD34 protein expressions and apoptotic index (AI)was measuredby TUNEL method.Results Three recombinant plasmids were successfully constructed.Inthe in vitro study,the plasmids were showed to efficiently and specifically inhibit theexpression levels of Survivin gene mRNA and protein except for the pSIREN/con,whilepSIREN/S3 was significantly higher than other recombinant plasmids (P＜0.05).Theapoptosis rate of P+L group [(31.58±3.12)%] was higher than control groups,but remainedlower than P+UTMD group [(43.86±4.44)%].Apoptosis index in P+UTMD group wassignificantly increased as compared with other groups (P＜0.01).There were apparentapoptosis and DNA ladder in P+UTMD and P+L group.After transfected with pSIREN/S3for 48 h,the inhibition ratio of survivin mRNA and protein in P+UTMD group were83.33%±2.73% and 79.67%±3.55%,respectively.The differences were significant whencompared with other groups (P＜0.01).In transplanted tumors experiment,comparing withthose in C and P+US groups,protein expressions of PCNA,Ki-67,Bcl-2,Survivin,NSwere down-regulated markedly,while those of Bax,Caspase-3 and P53 were up-regulatedsignificantly (P＜0.05).MVD decreased significantly,whereas AI increased remarkably(P＜0.05).Conclusions We suggest that Survivin can be regarded as an ideal target foranticancer therapy.UTMD combined with shRNA technique can induce apoptosis andinhibit proliferation significantly,without causing any apparently adverse effect,representing a new,promising technology that can be used in the tumor gene therapy andresearch.
引文
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