【作者】 张波；

【导师】 何剪太；

【作者基本信息】 中南大学 ， 外科学， 2010， 博士

【摘要】 基因载体是基因治疗的关键之一。基因载体主要分为病毒载体和非病毒载体。病毒载体的基因转染率相对较高,但其安全性及机体对病毒的免疫反应是一个大难题；以脂质体为代表的非病毒载体较低的转染效率一定程度上影响了基因治疗的效果。超声微泡造影剂介导基因转运系统因其安全、高效和直接靶向性成为近年发展起来的新研究热点,其独特优点和发展潜力已被越来越多的研究者所证实。目前的超声造影剂微泡平均直径为数微米,但研究表明疾病状态中的血管内皮间隙至多允许直径小于700nm的颗粒穿过,因此微米级微泡不能穿透血管内皮间隙,大大减弱了其基因转运的能力。纳米技术的飞速发展为纳米级超声微泡介导基因治疗提供了理论基础。近年来肝癌的基因治疗成为生物科学和临床医学的研究热点之一,其中作用直接、合理、有效的自杀基因疗法备受关注。PNP/Fludarabine是一种新型的自杀基因系统,它的杀伤效应是目前为止各系统中最强的,其杀伤肿瘤的机制主要是能从多个环节杀伤肿瘤细胞,不论是对分裂期还是静止期的肿瘤细胞都具有强大的杀伤效应。而其前药经系统作用后的毒性产物可以穿过脂质膜,杀伤临近细胞,旁观者效应强大。[目的]本研究旨在研制一种直径<700nm的纳米级超声微泡；以GFP为目的基因、以超声介导纳米级超声微泡检测体外基因转染效率及相对表达量来验证其作为新型基因载体的可行性；自行构建自杀基因表达质粒pcDNA3.1(+)/PNP,以纳米级超声微泡作为基因载体介导PNP/Fludarabine自杀基因系统,探讨其对肝癌的治疗效果以及该系统的作用机理,为超声介导纳米级超声微泡系统未来能作为临床基因治疗手段提供实验基础。[方法]1、选择DPPC、DPPA混合磷脂以及全氟丙烷气体等材料采用高剪切分散法进行纳米级超声微泡的研制,并通过均匀设计法选择最优制备方法。2、采用MTT法检测不同浓度的纳米级超声微泡对HepG2肝癌细胞的细胞毒性,选择安全浓度范围；以GFP为目的基因,通过荧光显微镜、流式细胞仪以及实时荧光PCR法检测纳米级超声微泡与DNA质粒的结合效率和体外基因转运效率,并与相同条件下脂质体的转运效率进行比较,以及超声照射后纳米级超声微泡对目的基因转染效率的改变。3、选用真核基因表达系统pcDNA3.1 (+)和PNP自行构建自杀基因表达质粒pcDNA3.1(+)/PNP,基因克隆鉴定采用限制酶SalⅠ和EcoRV双酶切鉴定、PCR鉴定以及重组质粒核酸序列测定。4、以超声照射介导自制的纳米级超声微泡作为基因转运系统,以自行构建的自杀基因表达质粒pcDNA3.1(+)/PNP作为治疗基因,并以Fludarabine前药处理进行肝癌的基因治疗研究。先用RT-PCR方法检测两株细胞系中PNP的表达情况；再以AFP高表达的HepG2细胞与AFP低表达的SMMC7721细胞为实验材料,将重组质粒pcDNA3.1(+)/PNP转染入各组实验细胞,采用MTT法检测PNP/Fludarabine系统对两种肝癌细胞生长的影响以研究其对肝癌细胞的杀伤作用；同时采用Annexin V-FITC凋亡试剂盒对其作用后的细胞进行凋亡检测以了解PNP基因的凋亡效应；以0.75μg／ml的Fludarabine处理后,通过MTT法检测了空转PNP与转染PNP阳性的两种肝癌细胞以不同比例混合后的细胞生存率,观察其旁观者效应。[结果]1、以混合磷脂和全氟丙烷气体作基本原料,通过高剪切分散处理可成功制备全氟丙烷脂质超声微泡。通过均匀设计和多元回归分析获得制备纳米级超声微泡的最优制备条件为：高剪切速度为6档；高剪切时间为8min; DPPC浓度为20mg/50ml; T80/S60比值为1。最优条件下制备的纳米级超声微泡直径小、浓度高、均一性好,粒径分布208-416nm之间,平均粒径仅为(335±5)nm。2、在浓度≤5.0%时纳米级超声微泡对HepG2肝癌细胞无明显的细胞毒性；在该浓度下纳米级超声微泡与DNA质粒也具有较高的结合效率；在体外基因转运中,该纳米微泡可有效转运GFP目的基因表达质粒进入HepG2,其转运效率达32%,与相同条件下脂质体的转染效率34%无统计学意义的差异；加入超声照射后,纳米微泡的基因转染效率显著提高,达到79.9%。3、结果表明,酶切电泳结果显示约750bp附近有一特异条带,与目的基因的长度接近。再用特异性引物对重组质粒进行PCR反应,电泳结果显示约750p处有一特异条带,证明目的基因已正确插入pcDNA3.1 (+),并得到了真核表达载体pcDNA3.1(+)/PNP,测序鉴定结果与Genbank中登录的序列相比对完全正确。4、RT-PCR显示在750bp出现明显的条带,说明pcDNA3.1(+)/PNP质粒经过纳米级超声微泡已成功转染至人肝癌细胞中并高效表达。MTT法检测当Fludarabine浓度仅为0.75μg/ml, PNP/Fludarabine系统对HepG2细胞和SMMC7721细胞均具有明显的细胞毒效应；并且AFP高表达的HepG2细胞相对于AFP低表达的SMMC 7721细胞来说,只需要更低的Fludarabine浓度(0.5μg/ml)就可以达到同等的杀伤效果。Annexin V-FITC凋亡凋亡检测经0.75μg／ml的Fludarabine前药处理后,经pcDNA3.1/PNP转染的两组人肝癌细胞均出现明显凋亡现象,细胞凋亡率分别达到41.34%和53.15%。在0.75μg／ml的Fludarabine处理后,通过MTT法检测了空转PNP与转染PNP阳性的两种肝癌细胞以不同比例混合后的细胞生存率,发现只需要5%-10%的转染PNP阳性细胞就导致细胞存活率明显降低至10%-20%,说明PNP/Fludarabine系统对两种人肝癌细胞的杀伤作用都存在明显的旁观者效应。[结论]1、最优条件下制备的纳米级超声微泡粒径及分布范围小,分散均匀、稳定性好,是目前国际、国内文献中粒径较小的纳米级超声微泡,为后续研究提供了良好基础。2、超声介导自制的纳米级超声微泡系统无毒、性状稳定且基因转运效率较高,是一种非常有潜力的安全高效的新型基因转运系统,有望成为临床基因治疗的新途径。3、自行构建的真核表达载体pcDNA3.1(+)/PNP,测序鉴定结果与Genbank中登录的序列相比对完全正确,说明已成功扩增了大肠杆菌嘌呤核苷磷酸化酶基因的DNA序列,无碱基突变。4、超声介导的自制纳米级超声微泡能有效转染质粒进入肝癌细胞内；在较低浓度下,PNP/Fludarabine自杀基因系统能够成功抑制肿瘤细胞生长,对两种人肝癌细胞具有明显的诱导凋亡作用,且该系统对两种人肝癌细胞的杀伤作用都存在明显的旁观者效应,证明细胞凋亡和旁观者效应是PNP/Fludarabine自杀基因系统诱导肝癌细胞杀伤作用的重要机制。通过上述实验,我们认为自制的纳米级超声微泡可高效转染外源基因进入细胞,并获得有效表达。超声照射可以显著增加外源基因在细胞中高效、特异地表达。PNP/Fludarabine系统在纳米级超声微泡系统的介导下,通过直接细胞毒作用及良好的旁观者效应可有效杀死肿瘤细胞。这一新型纳米级超声微泡介导自杀基因靶向性治疗的策略将为肝癌及相关肿瘤的治疗提供新的思路和实验依据。更多还原

【Abstract】 Gene vector is one of the keys to gene therapy. Gene vector is divided into two categories, viral vector and non-viral vector. Gene transfer rate of viral vector is relatively high, but its safety and overcoming body’s immune response against virus are difficult problems. Lower transfection efficiency of non-viral vector as liposome affects the effectiveness of gene therapy to a certain extent. Ultrasound microbubbles contrast agent-mediated gene transfer system is a newly developing hot spot in recent years. And its unique advantages and development potential have been confirmed by more and more researchers. The average diameter of current ultrasound contrast agents is several microns. However, research has shown that the vascular endothelial gap in disease state only allows the particle whose diameter less than 700nm across, so that micron microbubbles can not pass through the vascular endothelial gap. Therefore it limits its gene transfer capabilities greatly. The rapid development of nanotechnology provides a theoretical basis for nanoscale ultrasound microbubbles mediated gene therapy. In recent years, gene therapy for liver cancer, of which suicide gene therapy is direct, reasonable and effective for concern, is becoming one of hot spots in life sciences and clinical medicine. PNP/Fludarabine is a novel suicide gene system, and its killing effect is the strongest of the systems by far. Its anti-tumor mechanism mainly is that killing tumor cells in many steps, and having a powerful killing effect both for division and resting stage of tumor cells. And toxicity product after the effects of prodrug can pass through the lipid membrane, kill nearby cells, and have a strong bystander effect.[Objective] The research aims to develop a nanoscale ultrasound microbubbles which diameter less than 700nm. To verify the feasibility of that it as a novel gene vector by detection of gene transfer efficiency and relative expression levels in vitro using nanoscale ultrasound microbubbles as media and GFP as target gene. Suicide gene expression plasmid pcDNA3.1(+)/PNP was built, and nanoscale ultrasound microbubbles as gene vector mediated PNP/Fludarabine suicide gene system. To explore its therapeutic effect and the mechanism on therapy for liver cancer, and to provide an experimental basis for nanoscale ultrasound microbubbles-mediated system as clinical gene therapy method in the future.[Methods] 1. Mixture phospholipid of DPPC and DPPA, perfluoropropane gas and other materials were selected, high shear dispersion method was used for nanoscale ultrasound microbubbles development, and the best preparation method was chosen through the uniform design.2. The cytotoxicity of different concentrations of nanoscale ultrasound microbubbles to HepG2 hepatoma cells was tested by MTT assays. And safe concentration range was chosen. The combining efficiency of nanoscale ultrasound microbubbles with the DNA plasmid and gene transfer efficiency in vitro were tested by fluorescence microscopy, flow cytometry and real-time fluorescence PCR methods, with GFP as the target gene. And results were compared to the gene transfer efficiency of liposomes under the same conditions, as well as gene transfection efficiency changes after ultrasound irradiation.3. Eukaryotic gene expression system pcDNA3.1 (+) and the PNP were selected to build the suicide gene expression plasmid pcDNA3.1 (+) /PNP, Gene cloning was identified by restriction enzymes SalI and EcoRV double-enzyme digestion, PCR, and recombinant plasmid nucleic acid sequencing. 4. Self-made nanoscale ultrasound microbubbles after ultrasound irradiation was used as gene transfer system; self-constructed suicide gene expression plasmid pcDNA3.1(+)/PNP was used as the therapeutic gene, and Fludarabine was processed as prodrug, in gene therapy research for liver cancer. RT-PCR method was used for detection of the expression of PNP in two cell lines. HepG2 cells of AFP high expression and SMMC7721 cells of AFP low expression were used as experimental materials, and the recombinant plasmids pcDNA3.1(+)/PNP were transfected into each set of cells. To investigate the killing effect of two kinds of human liver cancer cells, MTT assays were used for testing the effect of the PNP/Fludarabine system on two kinds of liver cancer cell growth. Annexin V-FITC apoptosis kits for cell apoptosis test were used after the effects of the suicide gene system in order to understand apoptosis effects of the PNP gene. After treatment of 0.75μg/ml Fludarabine, MTT assays were tested for cell survival rates of two kinds of liver cancer cells, idling PNP and positive transfection PNP cells, mixing in different proportions. Bystander effect was observed.[Results] 1. Using mixed phospholipid and perfluoropropane gas as a basic raw material, perfluoropropane lipid ultrasound could be successfully prepared through high-shear distributed processing. By uniform design and multiple regression analysis, the optimal preparation conditions of preparation of nanoscale ultrasound microbubbles were:6 files of high-shear rate,8min of high-shear time,20mg/50ml of DPPC concentration,1 of T80/S60 ratio. Nanoscale ultrasound microbubbles that prepared under optimal conditions was small in diameter, at high concentrations and good uniformity. Particle size distributed in 208-416nm, with an average particle size of only (335±5) nm.2. It had no significant cell toxicity that nanoscale ultrasound microbubbles to HepG2 liver cancer cells in the concentration of≤5.0%. In that concentration nanoscale microbubbles combined with the DNA plasmid with high efficiency; in vitro gene dilivery, the nanoscale microbubbles could effectively transfer GFP gene expression plasmid into HepG2, its transfer efficiency up to 32%. It had no significant difference from the liposome transfection efficiency-34% under the same conditions. After ultrasound irradiation, gene transfection efficiency of nanoscale microbubbles significantly increased, reaching 79.9%.3. Restriction enzyme digestion and electrophoresis results showed that there was a specific band about 750bp, which was nearly the length of the target gene. Then the recombinant plasmid was amplified using specific primers by PCR method. Electrophoresis results showed that a specific band about 750bp and proved that the target gene had been correctly inserted pcDNA3.1(+), and the eukaryotic expression vector pcDNA 3.1(+)/PNP had been constructed. Sequencing results comparing to the sequences in Genbank was entirely correct.4. RT-PCR results showed that there was a clear band about 750bp, indicating pcDNA 3.1(+)/PNP plasmid had been successfully transfected into liver cancer cells and highly expressed through the nanoscale ultrasound microbubbles. In MTT assays it was found when the concentration of Fludarabine was only 0.75g/ml, PNP/Fludarabine system had obvious cytotoxic effects on HepG2 cells and SMMC7721 cells; and compared to SMMC 7721 cells of AFP low expression HepG2 cells of AFP high expression that only needed lower concentrations of Fludarabine (0.5 g/ml) could achieve the same killing effect. In Annexin V-FITC apoptosis test, after treatment of the prodrug,0.75μg/ml Fludarabine, two groups of transfected human hepatoma cells by pcDNA 3.1(+)/PNP were apparent apoptosis. Apoptosis rates were 41.34% and 53.15% respectively. After the 0.75g/ml of Fludarabine treatment, cell survival rates were observed by MTT assays of two kinds of liver cancer cells, idling PNP and positive transfection PNP cells, mixing in different proportions. The results showed that only 5%-10% of the PNP-positive transfection cells caused survival rate significantly to reduce 10%-20%, indicating that PNP/Fludarabine system had obviously bystander effects on the killing effect of two kinds of human liver cancer cells.[Conclusion] 1. Nanoscale ultrasound microbubbles that prepared under optimal conditions was small in size and distribution, uniformly dispersed, and good stability. It was a relatively small nanoscale ultrasound microbubbles in international and domestic literature, and it provided a good basis for follow-up study.2. Ultrasound-mediated self-made nanoscale ultrasound microbubbles system was non-toxic, stable, and highly efficient in gene transfer. It was a very promising, safe, efficient and novel gene transfer system. And it was expected to be a new method of clinical gene therapy.3. The self-constructed eukaryotic expression vector pcDNA3.1(+) /PNP was sequenced. Sequencing results comparing to the sequences in Genbank was entirely correct, that indicated successful amplification of the E.coli purine nucleoside phosphorylase gene DNA sequences with no mutation.4. Ultrasound-mediated self-made nanoscale ultrasound microbubbles could effectively transfected plasmid into the liver cells; at low concentration the suicide gene PNP/Fludarabine system had inhibited tumor cell growth successfully and induced apoptosis significantly for two kinds of human liver cancer cells. It was proved that apoptosis and bystander effect were important mechanisms for liver cancer cell killing effect induced by the PNP/Fludarabine suicide gene system.Through these experiments, we believed that self-made nanoscale ultrasound microbubbles could highly transfect exogenous genes efficiently into cells and obtain a valid expression. Ultrasonic irradiation could significantly increase efficiency and specificity of expression of exogenous genes in cells. The PNP/Fludarabine system mediated by the nanoscale ultrasound microbubbles system, could kill tumor cells effectively by direct cytotoxicity and good bystander effects. This novel nanoscale ultrasound microbubbles-mediated suicide gene targeting therapy strategies provided new ideas and experimental evidence for liver cancer and related tumors.更多还原