【作者】 高燕；

【导师】 高祥；

【作者基本信息】 四川大学 ， 药学， 2022， 硕士

【摘要】 研究目的:目前,癌症作为全世界患病率最高的疾病之一,除了传统的化疗、化疗和手术治疗的方式,一直缺乏合适且有效的治疗手段。基因治疗为癌症提供了新的策略。肿瘤的基因治疗是指将有效的遗传物质（如RNA或者DNA）通过某种手段递送至靶细胞（如免疫细胞或者肿瘤细胞等）中,从而纠正异常的生理过程。目前,对于基因治疗的研究已经有着深入的探究,包括在结肠癌、胰腺癌、乳腺癌以及人类免疫缺陷病（HIV）等等疾病中有着广泛的应用。基因治疗具有以下优势:（1）可持续在局部给药;（2）调节基因表达;（3）毒性较低等等。但是限制基因治疗应用最大的问题在于:缺乏安全、高效的递送载体。目前使用最广泛的基因递送载体包括:病毒载体和非病毒载体,病毒载体尽管具有较高的递送效率,但是缺乏生物安全性,以及较容易产生免疫原性。目前,基于mRNA的基因治疗具有广泛的研究应用,mRNA是与基因序列互补的单链RNA分子,在蛋白质的翻译过程中能够直接作为核糖体转录的模板,从而表达出相应的蛋白分子。相比于质粒DNA而言,mRNA具有以下优点:（1）不整合进基因组;（2）免疫原性较小;（3）更容易规模化生产等等。但是由于mRNA具有较大的分子构象、不易压缩以及容易被核糖核酸酶降解的特点,使得mRNA比DNA更难递送。因此,本课题拟开发一种能安全有效的递送mRNA的非病毒载体,以用于mRNA的基因治疗。Bim又称为Bcl2l11,属于Bcl2家族。所编码的蛋白含有198个氨基酸并且能够形成二聚体,这些二聚体参与多种细胞的凋亡过程。Bim能够直接激活Bax和Bak,与Bcl2、Bcl-x L和Mcl-1产生拮抗作用,促进Bax/Bak的释放,同时向线粒体转移,进而使线粒体的细胞膜通透性发生改变,最终诱发细胞凋亡。因此,本课题选择了Bim基因作为mRNA分子模板,以期通过激活线粒体凋亡途径来治疗结肠癌。细胞穿膜肽（Cell-penetrating peptides,简称CPPs）,是由5到30个左右的氨基酸组成的短肽。由于富含赖氨酸或精氨酸,具有较强的正电性,因此CPPs能与带负电的细胞膜结合,能被细胞所摄取。CPPs通常分为三类,蛋白衍生肽、由两个天然序列融合形成的嵌合肽和合成肽。基于CPPs的穿膜特性,我们合成了两种穿膜肽:cRGD和R9。在本课题组前期研究的基础上,利用阳离子脂质体（2,3-二油酰基-丙基）-三甲基氯化铵（DOTAP）与甲氧基-聚乙二醇-聚己内酯（m PEG-PCL）嵌段聚合物自组装形成DOTAP-m PEG-PCL纳米胶束（DMP）,我们以DMP纳米胶束为骨架,将两种串联的穿膜肽cRGD-R9偶联于DMP上,从而制备一种能安全有效递送mRNA的功能化DMP纳米载体（DMP-cRGD-R9,简称DMP-039）。本课题中我们利用DMP-039纳米粒与Bim mRNA（简称mBim）进行复合形成mBim/DMP-039复合物。mBim/DMP-039复合物在局部给药后,能有效治疗小鼠CT26结肠癌腹腔和皮下移植肿瘤模型,同时研究了mBim/DMP-039复合物在体内的分布和代谢情况,mBim/DMP-039复合物在系统性给药后,能有效的治疗小鼠CT26结肠癌肺移植肿瘤模型。因此,DMP-039纳米粒具备安全有效的递送mRNA的能力,从而达到利用基因治疗的手段来治疗结肠癌的目的。研究方法:首先以自组装的方式制备了带有马来酰亚胺基团的DMP纳米粒（简称DMP-Mal）,将cRGD-R9（039）穿膜肽与DMP-Mal通过迈克尔加成反应偶联形成DMP-039纳米粒。我们对DMP-039纳米粒的粒径、电位和电镜进行定性测定,以及DMP-039纳米粒的细胞毒性进行研究;另外,采用体外转录的方法合成了含有Bim基因序列的Bim mRNA（mBim）,并且通过凝胶电泳实验验证了Bim mRNA的条带大小;然后,探究了DMP-039纳米粒与mRNA的结合能力、DMP-039纳米粒对mRNA的保护能力以及对EGFP编码的mRNA（Enhanced Green Fluorescent Protein,简称EGFP）在CT26细胞和293T细胞上的转染效率,同时测定了mBim/DMP-039复合物的粒径和电位;接下来,用多种通路抑制剂处理CT26细胞,探究细胞对mRNA/DMP-039复合物的摄取机制;随后通过实时荧光定量QPCR、蛋白质免疫印迹实验（Western Blot,简称WB）、细胞增殖实验、克隆形成实验以及细胞凋亡实验,验证Bim基因的体外作用机制;接下来我们建立了小鼠结肠癌CT26腹腔和皮下移植肿瘤模型,通过局部注射mBim/DMP-039复合物来进行治疗,并且对治疗后的五脏进行石蜡切片和苏木精-伊红染色（Hematoxylin-eosin staining,简称HE）,对肿瘤组织石蜡切片和用CD31、TUNEL、Bim、caspase 3和caspase 9抗体染色分析;为了实现系统性给药,我们研究了mBim/DMP-039复合物在小鼠体内的分布和体内代谢情况,进而建立了小鼠CT26结肠癌肺移植肿瘤模型,通过尾静脉注射mBim/DMP-039复合物来治疗,解剖后对肺组织进行石蜡切片,并用CD31、TUNEL、Bim、caspase 3和caspase 9抗体染色分析,同时用CD8、Ly-6C、CD206和IFN-γ抗体染色分析肺组织的免疫微环境情况。最后,通过尾静脉注射mBim/DMP-039复合物24小时后,取小鼠的血样进行血常规分析。研究结果:粒径电位结果显示DMP-039纳米粒的粒径为268.9±12.4 nm（PDI=0.382）,电位为17.4±0.5 mV。通过透射电镜TEM观察DMP-039纳米粒具有规则的球形形态,并且观察到形态的大小与粒径结果一致。我们通过MTT实验检测了DMP-039纳米粒的细胞毒性,结果显示DMP-039纳米粒的IC₅₀明显低于PEI25K,这说明DMP-039纳米粒的细胞毒性较小,拥有较高的安全性。体外转录得到的Bim mRNA,通过凝胶电泳实验验证其基因片段大小,结果显示条带为591nt,符合基因理论长度。与DMP-039纳米粒相比,mBim/DMP-039复合物的粒径为381.6±21.5 nm（PDI=0.213）,电位为13.1±0.5 mV,说明DMP-039纳米粒与Bim mRNA以静电相互用作之后粒径适当增大了。当我们用不同比例的DMP-039纳米粒复合Bim mRNA时,凝胶电泳实验结果显示当Bim mRNA与DMP-039的比例为1:25（w/w）时,DMP-039纳米粒能通过静电相互作用力而完全结合Bim mRNA,并以此比例进行后续实验。在抗核糖核酸酶（RNase）实验中,DMP-039纳米粒能够保护Bim mRNA不被降解长达4小时之久。我们用EGFP/DMP-039复合物分别转染小鼠CT26结肠癌细胞和人胚肾细胞293T,其中对小鼠CT26结肠癌细胞的转染效率高达41.29%,对人胚肾细胞293T的转染效率高达98.06%,证明DMP-039纳米粒具有较强的细胞递送效率。在细胞摄取实验中,我们发现EGFP/DMP-039复合物主要通过脂筏通路（lipid raft,脂筏是质膜上富含胆固醇和鞘磷脂的微结构域,大小约70 nm左右,是一种动态结构）进入细胞内。随后验证了Bim基因在体外作用机制,我们发现Bim mRNA能被DMP-039纳米粒有效的递送并且在细胞内具有较高的表达水平,Bim mRNA能通过线粒体凋亡途径来抑制肿瘤细胞生长。mBim/DMP-039复合物通过局部给药后,能有效的抑制小鼠结肠癌CT26腹腔和皮下移植肿瘤模型中的生长,并且通过抑制肿瘤血管生成,促进癌细胞凋亡,达到治疗目的。我们探究了mRNA/DMP-039复合物在体内的分布和代谢情况,活体成像结果显示mBim/DMP-039复合物主要分布在小鼠的肺部,HPLC-MS检测结果显示mBim/DMP-039复合物主要通过尿液的形式代谢,这说明了mBim/DMP-039复合物具有较高的生物安全性,具有系统性治疗的潜力。随后我们建立了小鼠CT26结肠癌肺移植肿瘤模型,mBim/DMP-039复合物通过尾静脉给药后,结果显示治疗后小鼠的全肺组织中肿瘤面积明显降低,肺部表面肿瘤结节数明显减少,同时招募T细胞、单核细胞等免疫细胞参与反应,抑制肿瘤细胞的生长。与此同时,mBim/DMP-039复合物血常规检测指标正常,说明DMP-039纳米粒具有较高的生物安全性。研究结论:实验结果表明我们制备的DMP-039纳米粒具有较高的转染效率和生物安全性,能通过静电相互作用力有效的结合mRNA,并且保护mRNA不被核糖核酸酶降解;mRNA/DMP-039复合物能通过多种途径被细胞摄取,提高了细胞利用效率,并且能通过细胞线粒体凋亡途径抑制癌细胞的生长;mBim/DMP-039复合物能通过局部或系统性给药治疗小鼠结肠癌CT26细胞的腹腔、皮下以及肺肿瘤转移模型,说明DMP-039纳米粒具有递送mRNA并用于肿瘤基因治疗的潜力。更多还原

【Abstract】 Purpose:At present,cancer is one of the highest prevalence in the worldwide.Except chemotherapy and surgery,there is lacking suitable and effective treatment strategies for cancer therapy.However,gene therapy has provided a new strategy for cancer therapy.Cancer gene therapy refers to deliver effective genetic materials to target cells,such as immune cells or tumour cells,so that interrupt the anormal physiological processes.At present,there are a plenty of researches on gene therapy,including a wide range of applications in colon cancer,pancreatic cancer,breast cancer,and human immunodeficiency（HIV）and other diseases.Gene therapy has following advantages（1）sustain by local administration;（2）regulate of gene expression;（3）lower toxicity and so on.However,one the most important challenge in gene therapy is that deliver vectors.Traditional gene delivery vectors include viral and non-viral vectors.The viral vectors could easily deliver nuclear into cells,but they have safety concerns and immune reaction.At present,mRNA-based gene therapy has a wide range of research applications.mRNA is a single-stranded RNA molecule complementary to a gene sequence,which can be directly used as a template for ribosome reading during protein translation,and then express the corresponding protein.Compared with plasmid DNA,mRNA has following advantages:（1）not integrate into the genome;（2）less immunogenic;（3）easy to produce on a large scale,etc.However,mRNA is more difficult to deliver than DNA due to its large molecular structure,not easy to condense and can destroy by RNase.Therefore,this work aims to develop a non-viral vector with high safety and high delivery efficiency for mRNA-based cancer gene therapy.Bim,also known as Bcl2l11,belongs to the Bcl2 family.Bim gene can encode protein with 198 amino acids and forms heterodimers or homodimers,which act as“suicide gene”to involve in the regulation of apoptosis in various cellular activities.Bim gene can directly activate Bax and Bak,antagonize Bcl2,Bcl-x L and Mcl-1,promote the release of Bax/Bak.Then they can transfer to mitochondria,change the permeability of cell membrane,and induce cell apoptosis.Therefore,this work choose Bim gene as mRNA template in order to treat colon cancer.Cell-penetrating peptides（CPPs）are consist of short peptides with 5-30 amino acids.As they have a plenty of lysine and arginine,they can interact with cell membranes by electrostatic interaction,and enter cells by endocytosis.CPPs are generally classified into three categories,including protein-derived peptides,chimeric peptides,and synthetic peptides.Based on the penetrating properties of CPPs,we synthesized two penetrating peptides:cRGD and R9.Based on our previous research,we used N-[1-（2,3-dioleoyloxy）propyl]-N,N,N-trimethylammonium methyl-sulfate（DOTAP）and m PEG-PCL polymer self-assemble into DOTAP-m PEG-PCL（DMP）micelles.Then,we used DMP as the backbone to fuse cRGD-R9,forming a mRNA delivery system（DMP-039）.The prepared DMP-039 nanoparticles have the advantages of low cytotoxicity,high transfection efficiency and high safety.In this work,we used DMP-039 nanoparticles to deliver Bim mRNA（mBim）forming mBim/DMP-039 complex.After local administration,mBim/DMP-039 complex could efficiently inhibit the CT26 abdominal cavity metastatic model and the CT26subcutaneous xenograft model.The distribution and metabolism of the mBim/DMP-039complex in vivo were also investigated.After systemic administration,mBim/DMP-039complex could efficiently inhibit the CT26 pulmonary metastases model.Method:First,DMP nanoparticles with maleimide groups（DMP-Mal）were prepared and then fused with 039 peptide by Michael addition,forming DMP-039 nanoparticles.The particle size and zeta potential of DMP-039 were determined by dynamic light scattering.The morphology of the DMP-039 nanoparticles was observed by transmission electron microscopy（TEM）.In addition,mRNA encoding Bim gene was synthesized by in vitro transcription（IVT）,and the particle size of Bim mRNA was verified by gel retarding assay.Then,we used different molecular ratios of DMP-039nanoparticles to evaluate the binding ability of mRNA,and investigated the ability of DMP-039 to protect mRNA against RNase A by gel retarding assay.The transfection efficiency of DMP-039 nanoparticles evaluated by transfecting EGFP mRNA（Enhanced Green Fluorescent Protein,EGFP）on CT26 or 293T cell lines.At the same time,the particle size and zeta potential of mBim/DMP-039 complex were determined.The cellular uptake mechanism of DMP-039/mRNA complex was determined by different inhibitors.We then studied the anti-cancer mechanism of the mBim/DMP-039 complex on CT26 cells in vitro,such as Quantitative Real-Time PCR（QPCR）,cell proliferation assay,clonogenic assay,in vitro apoptosis assay,and western blot analysis.Next,we established the CT26 abdominal cavity metastatic model and the CT26 subcutaneous xenograft model to assess the therapeutic effect of the mBim/DMP-039 complex by local administration.After treatment,the main organs of each groups were detected by H&E,and tumor slice was analyzed by stained with CD31,TUNEL,caspase9 and caspase3.We further intended to evaluate the therapeutic potential of the mBim/DMP-039complex through systemic administration.We first analyzed the in vivo distribution behavior of the DMP-039 nanoparticles.Then we investigated the therapeutic potential of the mBim/DMP-039 complex on CT26 pulmonary metastatic tumor model by i.v.administration.After treatment,the lung slices were stained with CD31,TUNEL,caspase9,and caspase3,and also analyzed the immune cells by CD8,Ly-6C,CD206,and IFN-γ.After treatment with mBim/DMP-039 complex,the blood was collected and analyzed for evaluating the safety of biomaterials in vivo.Results:The results showed that the particle size of DMP-039 nanoparticles was 268.9±12.4 nm（PDI=0.382）with the zeta potential of 17.4±0.5 mV.The morphology of DMP-039 nanoparticles was observed by TEM,which was monodispersed with a diameter of nearly 250 nm.We assessed the cytotoxicity of DMP-039 nanoparticles by MTT,and the results showed that the IC₅₀ DMP-039 nanoparticles were significantly lower than that of PEI25K.To prepare the mBim/DMP-039 complex,mRNA molecules were first synthesized using an in vitro transcription method followed by 5＇end-capping and 3＇end-poly（A）reaction.The purified murine Bim mRNA was then electrophoresed for verification.We could obverse a band of approximately 591 nt on the gel,which was the expected gene length.Compared with DMP-039 nanoparticles,the particle size of mBim/DMP-039 complex was 381.6±21.5 nm（PDI=0.213）,and the zeta potential was 13.1±0.5 mV,indicating that DMP-039 nanoparticles and Bim mRNA interact electrostatically.We then investigated the binding ability of DMP-039 to mRNA,the results showed that there was no obvious mRNA band with a molecular ratio of 25:1（w/w）for DMP-039:Bim mRNA.These results suggested that DMP-039 could effectively bind with mRNA and the ratio used to for further assay.In a RNase protection assay,DMP-039 nanoparticles could protect mRNA from degradation for up to 4 hours.We then transfected CT26 or 293T cells with EGFP/DMP-039 complex,the transfection efficiency was up to 41.29%on CT26 cells and 98.06%on 293T cells,which proved that DMP-039 nanoparticles had the high capability of delivery efficiency.In the cellular uptake assay,we observed that EGFP/DMP-039 complex could enter cellular mainly through lipid rafts pathway.Moreover,we observed that Bim mRNA could be efficiently delivered by DMP-039 nanoparticles.We then studied the anti-cancer mechanism of the mBim/DMP-039 complex with respect to CT26 cells in vitro.These findings suggested that the mBim/DMP-039 complex have an ability to inhibit the growth of cancer cells in vitro by mitochondrial apoptosis pathway.Furthermore,after local treatment with mBim/DMP-039 complex,the growth of CT26 cells in the CT26 abdominal cavity metastatic model and the CT26 subcutaneous xenograft model was significantly inhibited.The mechanism of mBim/DMP-039 complex was that inhibited tumor angiogenesis and promoted tumor cell apoptosis through mitochondrial pathway.We then analyzed the in vivo distribution behavior and metabolic behavior of the DMP-039 nanoparticles.The results showed that mBim/DMP-039 complex could be sustained accumulation at lung,and DMP-039 nanoparticles can be degraded and eliminated from the body by urine.Therefore,we further investigated the therapeutic potential of the mBim/DMP-039 complex to treat a CT26 pulmonary metastatic tumor model by i.v.administration.The results showed that mBim/DMP-039 complex group exhibited anti-cancer effects after 13 doses.There were no pathology changes on the surface of lung tissues from the mBim/DMP-039 complex group.Additionally,there were no significant changes and toxicity in the blood test.Our results indicated that i.v.administration of the mBim/DMP-039 complex could suppress the growth of tumour cells by inducing apoptosis and immune reactions on a CT26 lung metastatic tumor model.Conclusion:This work showed that DMP-039 nanoparticles had high transfection efficiency and safety,could effectively bind mRNA and protect mRNA fromRNase.At the same time,mBim/DMP-039 complex could enter cells mainly by lipid raft pathway.mBim/DMP-039 complex had the capability of therapeutic effect in multiple colon cancer models by inducing mitochondrial apoptosis.DMP-039 nanoparticles were an available single-vector for mRNA-based gene therapy in clinical cancer applications更多还原