基于干细胞的新型基因靶向传递系统的构建及在肿瘤治疗中的体内外研究

发布时间：2018-06-26 02:50

  本文选题：基因治疗 + 骨髓间充质干细胞　； 参考：《浙江大学》2016年博士论文

【摘要】：作为一种极具前景的新型肿瘤辅助治疗手段,基因疗法近年来已被证明有着极大的应用潜力,然而,如何安全、有效地将这些治疗基因传递至肿瘤部位,以及跟踪传递至转移瘤却是当前肿瘤基因治疗领域一直面临的一个瓶颈问题。例如,对于抗肿瘤自杀基因疗法,就需要有一种载体,它既要能克服体内的种种生理障碍将治疗基因传递至肿瘤细胞,并促使相关功能蛋白在肿瘤区域获得高表达,又要在抵达肿瘤细胞前能有效保护所携载的治疗基因,避免其在血液循环中被降解,这就对相应的载体系统提出了很高的要求。本课题针对这一肿瘤基因治疗中的重要问题,基于靶向药物传递系统(Target Drug Delivery System, TDDS)的概念,提出了一种以生物活性细胞构建基因靶向传递系统的设想。研究采用非病毒基因转染系统对骨髓间充质干细胞(BMSC)进行基因重组以携载治疗基因,进而利用BMSC的肿瘤归巢性和免疫豁免性等特性,实现将治疗基因靶向运输至肿瘤细胞的目的。并通过体内外研究全面考察了这一基于BMSC的基因靶向传递系统在小鼠肺肿瘤模型中的抗肿瘤治疗效果。同时,对静脉注射的BMSC向肿瘤组织的迁移和穿透以及BMSC作为基因传递载体的安全性进行了探讨。为了使BMSC能高效携载治疗基因,本课题首先对BMSC的体外基因转染系统进行了考察。通过对课题前期工作中合成的一些新型非病毒基因载体以及部分常用的商品化转染试剂在BMSC上的基因转染效率和细胞毒性的筛选,发现精胺修饰的阳离子化普鲁兰多糖(spermine-pullulan, SP)在BMSC上表现了高效的转染效率和较低的细胞毒性。进一步,本课题对SP的合成及转染条件分别进行了优化以获得在大鼠BMSC上最佳的基因转染效率。课题同时还对SP/pDNA复合物入胞后在细胞内的分布和SP的降解过程进行了考察,研究了该载体在干细胞上应用的安全性。随后,本课题分别在体内外考察了BMSC对黑色素转移瘤细胞(B16F10)的趋向性(归巢性),探讨利用BMSC作为载体向肿瘤组织靶向传递治疗基因的可行性。课题首先通过体外Transwell实验确认了BMSC对B16F10肿瘤细胞的趋向性。进一步在体内通过荧光标记的方式观察BMSC静脉注射后在小鼠主要脏器中的分布及在荷瘤肺部的局部分布,确认了BMSC向肺部黑色素肿瘤细胞的归巢特性。为了研究构建的基于BMSC的靶向基因传递系统在肿瘤基因治疗中的疗效,本课题选择了经典的基于疱疹病毒胸苷激酶／更昔洛韦(Herpes simplex virus thymidine kinase/Ganciclovir, HSV-TK/GCV)的自杀基因治疗系统,考察了以BMSC作为HSV-TK的传递载体结合GCV治疗后在黑色素瘤肺转移小鼠模型上的治疗效果。体外研究结果显示,利用本课题构建的基于SP的非病毒基因转染系统可以有效重组BMSC表达自杀基因HSV-TK,并引起BMSC的自杀,且这种自杀作用与GCV呈明显的剂量依赖关系。体外旁观者效应的考察发现重组自杀基因后的BMSC在一定GCV浓度下能有效抑制B16F10肿瘤细胞的生长。但这种旁观者效应需要BMSC与肿瘤细胞的良好接触才能有效发挥,且对肿瘤细胞的杀伤效率与BMSC和肿瘤细胞的细胞数目比有关,BMSC所占的细胞数比例越高,相应的旁观者效应就越强。体内抑瘤实验进一步证明以BMSC作为HSV-TK的靶向肿瘤的传递载体在GCV存在的条件下可以显著抑制黑色素转移瘤的发展,显示了良好的肿瘤抑制效果。进一步地,为了实现自杀基因和前体药物对于荷瘤组织的同步靶向,提高这一自杀基因治疗策略的抗肿瘤效果,本课题设计了一种可以将基因和前体药物分别靶向到肿瘤组织的组合传递系统。该系统在采用BMSC传递自杀基因HSV-TK靶向至肿瘤细胞的同时利用脂质体载体系统将前体药物GCV共传递至荷瘤肺部。通过同时提高基因在肿瘤组织的表达水平和前体药物在肿瘤负荷脏器的局部浓度来获得更好的抑瘤效果,并降低潜在的毒副反应。课题通过逆向蒸发法制备了具有肺被动靶向功能的GCV脂质体,结果显示该脂质体静脉注射后能显著提高GCV在肺部的药物浓度。体内共靶向研究发现,静脉注射该脂质体后,能将携载的药物有效传递至BMSC与肿瘤细胞所在的区域。体外在三维肿瘤球模型上的治疗结果表明,重组自杀基因HSV-TK的BMSC结合GCV脂质体进行治疗后,能有效抑制肿瘤球体积的增大。体内在黑色素瘤肺转移小鼠模型上的治疗结果也表明,采用该共靶向传递策略进行抗肿瘤治疗亦显示了更好地肿瘤杀伤效率,其对肺肿瘤结节的抑制效果是单用GCV溶液和重组自杀基因HSV-TK的BMSC的两倍,同时小鼠的生存时间相比单用GCV溶液和重组BMSC也得到了明显的延长。TUNEL细胞凋亡染色实验结果也显示使用该共靶向传递策略进行治疗后能引起更多肿瘤细胞的凋亡。为了考察BMSC作为治疗基因的靶向传递载体在趋向肿瘤组织后是否还会产生向肿瘤组织深层穿透的能力。本研究构建了体外三维肿瘤球模型以模拟体内的微小癌巢结构,利用激光扫描共聚焦显微镜观察BMSC对三维肿瘤球模型的穿透能力。结果显示BMSC具有良好的肿瘤球穿透性。在与肿瘤球共孵育48 h后即可观察到BMSC向肿瘤球内部的穿透,在共孵育72h后,则可发现BMSC已分布于肿瘤球的核心区域。而对于其他非干细胞的细胞系,如HEK293细胞,即使孵育72 h后也未观察到其对肿瘤球的穿透。进一步地,研究采用基因重组绿色荧光蛋白的BMSC (GFP-BMSC)观察其在体内向肿瘤组织的穿透能力。结果发现,在BMSC注射后第15天,在肿瘤组织的深层可以观察到明显的绿色荧光信号。为了更好地示踪BMSC向肿瘤组织的迁移和穿透,课题在前期研究的基础上制备了乙二胺-普鲁兰多糖修饰的氧化铁纳米粒,并用该纳米粒标记BMSC。标记后的BMSC用普鲁士蓝(Prussian Blue)染色法特异性显色。对染色后的肺部肿瘤切片的观察证明,BMSC通过静脉注射并被肺部截留后,会逐渐向肺部的肿瘤组织迁移并穿透入肿瘤组织深层。在获得体内外良好的抗肿瘤效果基础上,本课题对BMSC作为基因靶向传递载体对小鼠的潜在毒副作用进行了初步考察。研究结果显示,未经基因重组的BMSC本身并不具备致瘤性,但是在荷瘤小鼠模型上注射大量未经基因重组的BMSC(每只小鼠注射超过1×106个细胞)会一定程度地促进肿瘤生长。对经重组自杀基因的TK-BMSC联合GCV溶液或GCV脂质体治疗后的小鼠体重观察结果显示,该疗法并未造成小鼠体重的急剧变化。研究还对肝和肺这两个BMSC静脉注射后主要的分布脏器在治疗后的毒副作用进行了考察。对血液中谷丙转氨酶(ALT)和谷草转氨酶(AST)的检测结果显示自杀基因治疗前后小鼠血液中的ALT和AST水平并未发生显著变化。但肺和肝的组织切片显示在治疗后会有轻微的炎症反应,但未见对正常的组织结构形态造成破坏。进一步地,本课题对经自杀基因重组的BMSC和GCV脂质体治疗后的小鼠进行了初步地短期和长期的毒副作用观察。对肺部切片的观察结果显示治疗后短期内(治疗结束后第2天)正常肺部细胞存在着轻微的损伤。但这种损伤是可逆的,在治疗结束后的第90天肺部细胞已完全恢复至正常形态,小鼠亦未见有整体毒性反应。上述结果初步证明本课题构建的基于BMSC的自杀基因治疗策略是相对安全和低毒的。本课题为发展基于干细胞载体的基因靶向传递系统及其在肿瘤基因治疗中的研究提供了理论与实验的基础。
[Abstract]:As a promising new type of cancer adjuvant therapy, gene therapy has been proved to have great potential in recent years. However, how to safely, effectively transfer these therapeutic genes to tumor sites, and track the transfer to metastatic tumor is a bottleneck problem in the field of tumor gene therapy. For example, In the case of antitumor suicide gene therapy, a carrier is needed. It can overcome the physiological obstacles in the body, transfer the gene to the tumor cells, promote the high expression of the related functional proteins in the tumor area, and protect the carrying therapeutic genes effectively before reaching the tumor cells, and avoid it in the blood circulation. Degradation, this is a very high requirement for the corresponding carrier system. This subject is based on the concept of Target Drug Delivery System (TDDS), which is an important problem in this tumor gene therapy, and proposes an idea of constructing a gene targeting transfer system with bioactive cells. The transfection system recombines the gene of bone marrow mesenchymal stem cells (BMSC) to carry the gene, and then uses the characteristics of the homing and immunity immunity of BMSC to target the target of the gene delivery to the tumor cells. The BMSC based gene targeting transmission system in the lung of mice is thoroughly investigated through the study in vitro and in vivo. The effect of antitumor therapy in the tumor model is also discussed. The migration and penetration of intravenous BMSC to the tumor tissue and the safety of BMSC as a gene delivery carrier are discussed. In order to enable BMSC to carry the therapeutic gene efficiently, this topic first examined the gene transfection system of BMSC in vitro. The gene transfection efficiency and cytotoxicity of some new non viral gene vectors and some commonly used commercialized reagents on BMSC showed that the spermine modified cationic pullulan polysaccharide (spermine-pullulan, SP) showed high efficient transfer efficiency and low cytotoxicity on BMSC. The synthesis and transfection conditions of SP were optimized to obtain the best gene transfection efficiency on rat BMSC. The subject also investigated the distribution of SP/pDNA complex in cell and the degradation process of SP, and studied the safety of the application of the carrier on the stem cells. Then, the subject investigated the B in vivo and in vitro, respectively. MSC's tendency (homing) to melanin metastatic tumor cells (B16F10), and to explore the feasibility of using BMSC as a carrier to target the tumor tissue targeting gene delivery. The subject first confirmed the tendency of BMSC to B16F10 tumor cells by in vitro Transwell experiment. Further observation of BMSC intravenous injection by fluorescence labeling in vivo was carried out. The distribution of the main organs in the mice and the local distribution of the tumor bearing lungs confirmed the homing characteristics of BMSC to the lung melanoma cells. In order to study the therapeutic effect of the constructed BMSC based gene delivery system on the tumor gene therapy, the subject selected the classic Herpes s based on the herpes virus thymidine kinase / Guecy Lowe (Herpes s) Implex virus thymidine kinase/Ganciclovir, HSV-TK/GCV) system of suicide gene therapy, which examined the therapeutic effect of BMSC as a carrier of HSV-TK and GCV treated mice model of melanoma lung metastasis. The results of the study in vitro showed that the system based on this topic was effective for the non viral gene transfection system based on SP. The recombinant BMSC expressed the suicide gene HSV-TK and caused the suicide of BMSC, and the suicide effect was in a dose-dependent manner with GCV. In vitro bystander effect found that the BMSC of the recombinant suicide gene could effectively inhibit the growth of B16F10 tumor cells in a certain GCV concentration. However, this bystander effect requires the good of BMSC and tumor cells. Good contact can be used effectively, and the killing efficiency of the tumor cells is related to the ratio of BMSC to the cell number of the tumor cells. The higher the number of cells in BMSC is, the stronger the bystander effect is. In vivo tumor inhibition experiment further proves that the carrier of BMSC as the target tumor of HSV-TK can be significantly suppressed under the presence of GCV. The development of melanin metastases shows a good tumor inhibition effect. Further, in order to achieve the synchronization target of the suicide gene and precursor drug to the tumor bearing tissue and improve the antitumor effect of this suicide gene therapy strategy, a combination of the base and the precursor drugs is designed to be targeted to the tumor tissue, respectively. The system uses the BMSC to transfer the suicide gene HSV-TK to the tumor cells and simultaneously uses the liposome carrier system to transfer the precursor drug GCV to the tumor bearing lung. GCV liposomes with passive targeting of the lung were prepared by reverse evaporation. The results showed that the liposomes could significantly increase the drug concentration of GCV in the lungs after intravenous injection. In vivo co targeting study found that after intravenous injection of the liposome, the drug could be effectively transferred to BMSC and tumor finely after the injection of the liposome. The treatment results on the three-dimensional tumor ball model in vitro show that the BMSC combined with the GCV liposome of the recombinant suicide gene HSV-TK can effectively inhibit the increase of the tumor ball volume. The treatment results on the mice model of melanoma lung metastasis also show that the common targeting transfer strategy is used to treat the tumor. The treatment also showed a better tumor killing efficiency, and its inhibitory effect on lung tumor nodules was two times as much as the BMSC of the GCV solution and the recombinant suicide gene HSV-TK. At the same time, the survival time of the mice was significantly longer than the GCV solution and the recombinant BMSC. The result of the apoptosis staining experiment of.TUNEL cells also showed the use of the co targeting delivery. The strategy can induce more tumor cells to apoptosis. In order to investigate the ability of BMSC as a target delivery carrier to the tumor tissue to penetrate the tumor tissue deep through the tumor tissue, this study constructed a three-dimensional tumor ball model in vitro to simulate the microcarcinoma nest structure in the body and use laser scanning copolymerization. The penetration ability of BMSC to the three-dimensional tumor ball model was observed by the focal microscope. The results showed that BMSC had good tumor penetration. After incubating 48 h with the tumor ball, the penetration of BMSC into the tumor sphere was observed. After incubation of 72h, it was found that BMSC was distributed in the core area of the tumor ball. HEK293 cells, such as HEK293 cells, did not observe the penetration of the tumor cells even after incubation for 72 h. Further, we studied the penetration ability of the recombinant green fluorescent protein (GFP-BMSC) to the tumor tissue in the body. The results showed that the obvious green fluorescence could be observed at the deep level of the tumor tissue at the fifteenth day after BMSC injection. Signal. In order to better trace the migration and penetration of BMSC into the tumor tissue, we prepared the ethylenediamine prulonide modified iron oxide nanoparticles on the basis of previous studies, and the BMSC was marked with the BMSC. staining method after the labeling of the nanoparticles with Prussian blue (Prussian Blue) staining. It has been shown that after intravenous injection and being intercepted by the lungs, BMSC will gradually migrate into the tumor tissue of the lungs and penetrate into the deep tumor tissue. On the basis of good antitumor effect in vivo and in vivo, the potential toxic and side effects of BMSC as a gene targeting carrier in mice are preliminarily investigated. The recombinant BMSC itself does not have a tumorigenicity, but a large number of ungenetically modified BMSC (injected more than 1 * 106 cells per mouse) in the tumor bearing mice model will promote tumor growth to a certain extent. The observation of the body weight of the mice after the combined GCV solution or GCV liposome treated by the recombinant suicidal gene and the GCV liposomes The treatment did not cause a sharp change in the weight of the mice. The study also examined the toxic and side effects of the main distributed organs after the two BMSC intravenous injection of the liver and lung. The results of the detection of ALT and AST in the blood showed the level of ALT and AST in the blood of mice before and after the gene therapy. There was no significant change in the lung and liver tissue sections, but there was a slight inflammatory response after treatment, but no damage to normal tissue structure was not found. Further, this subject was preliminary to the short-term and long-term side effects of the BMSC and GCV liposomes treated by suicide gene recombination. The observation of the section showed that there was a slight injury in the normal lung cells in the short term after treatment (second days after the end of the treatment). But the damage was reversible. The lung cells were completely restored to the normal form on the ninetieth day after the end of the treatment, and there was no overall toxicity in the mice. The strategy of suicide gene therapy at BMSC is relatively safe and low toxic. This topic provides a theoretical and experimental basis for the development of gene targeting delivery system based on stem cell carriers and its research in tumor gene therapy.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R730.5
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本文编号：2068732