基因工程制备的microRNA-34a前药应用于骨肉瘤的实验研究

发布时间：2018-04-15 11:11

本文选题：骨肉瘤 + miRNA-34a　；参考：《武汉大学》2016年博士论文

【摘要】：第一部分：基因T程方法制备的miR-34a前药通过诱导凋亡与细胞周期捕获抑制骨肉瘤生长背景：骨肉瘤是发生在儿童及青年的最常见的骨科恶性肿瘤,microRNA-34a(下文简称miR-34a)是一种基于微小RNA的新型抗癌药物。我们最近开发了一种用来合成miR-34a前药新的生物工程方法。实验第一部分的目的即为了评估所制备的miR-34a前药在原位裸鼠骨肉瘤模型中的抑癌安全性及有效性。方法：在体外实验中,利用miR-34a转染骨肉瘤细胞系,细胞的增殖活性采用MTT实验来评估；肿瘤的侵袭及转移能力采用细胞基质侵袭小室实验来量化；细胞凋亡及各细胞周期细胞的分布情况均采用商业化试剂盒处理后流式细胞仪检测；miR-34a作用于肿瘤细胞的靶靶蛋白表达采用western blot来检测；体内实验中,我们采用骨肉瘤的原位种植模型来评估miR-34a的体内安全性及有效性,miR-34a先用商业化的in vivo-jetPEI(以后简称PEI)来混合均匀,再通过静脉注射入负荷肿瘤的裸鼠,定期监测肿瘤的大小,在实验的最后获取裸鼠的血液并检测血液肝功能、肾功能及相关生化指标。结果：与对照组相比,miR-34a可以显著地抑制骨肉瘤细胞系(143B与MG-63)在体外的生长情况,这种抑制作用存在明显的量-效关系；在相应的流式细胞仪检测中,可以观测到与细胞增殖实验结果相一致的细胞凋亡现象与细胞周期捕获现象。进一步western-blot实验表明,与细胞凋亡和细胞周期捕获的相关miR-34a靶蛋白如SIRT1, BCL2, c-MET,和CDK6都相应降低。在动物实验中,经过两周,共6次经尾静脉给药可显著抑制骨肉瘤的生长,实验结束时的生化检查也表明,治疗剂量的miR-34a静脉给药不会带来明显的毒副反应；结论：基因工程获得的miR-34a前药可抑制骨肉瘤生长,并且不会带来明显的全身毒副作用：可能机制为miR-34a可相对特异地诱导骨肉瘤细胞凋亡与细胞周期捕获。第二部分：联合应用基因工程制得的miR-34a前药与多柔比星可协同抑制骨肉瘤增长目的：第一部分结果表明,应用基因工程方法制取的miR-34a在体内和体外实验中均能抑制骨肉瘤细胞或者瘤体的生长。在第二部分的实验中,我们我们主要来验证miR-34a在骨肉瘤的治疗中是否与传统化疗药物多柔比星存在协同作用；方法：在体外实验中,运用Chou-Talalay方法设计miR-34a与多柔比星药物联用的多种组合模式,运用MTT法测定联合用药后肿瘤细胞的增殖活性,原始数据导入Compusyn软件并通过计算药物联用指数来了解两种药物是否存在协同作用；然后,利用细胞基质侵袭小室实验来评估药物对肿瘤的侵袭及转移能力的影响；细胞凋亡及各细胞周期细胞采用流式细胞仪检测;利用Western-Blot技术检测药物相关靶点的表达改变；在体内实验中,首先建立骨肉瘤的原位种植模型,然后将miR-34a单独或者与多柔比星联合系统应用于载瘤裸鼠,监测肿瘤的生长,在实验的最后获取动物血液标本进行药物安全性分析,并切取肿瘤标本进行病理学检查。结果：结果显示,miR-34a与多柔比星联合应用存在协同作用,这种协同作用在早期、同时给药时达到最大；与单一用药相比,二者联合用药都能更有效地抑制143B细胞细胞增殖抑制、细胞周期捕获以及细胞侵袭等；与这些细胞活动相对应的miR-34a靶点SIRT1,c-MET,与CDK6也都相应降低；在体内实验中,联合应用miR-34a及多柔比星时对肿瘤的抑制效果也明显优于单一用药；在药物安全性方面,经过两周的给药,小鼠的肝功能及肾功能均未见明显损害,体内总体蛋白水平也没有明显降低；结论：上述结果表明,联合应用miR-34a及多柔比星在细胞及动物体内试验均能达到一定的肿瘤抑制协同作用,在治疗剂量下二者联用不会产生明显的毒副作用。
[Abstract]:The first part: the process method of T gene by miR-34a prodrug by inducing apoptosis and cell cycle arrest inhibited the growth of osteosarcoma background: osteosarcoma is occurring in children and youth of the most common malignant tumor in the Department of orthopedics, microRNA-34a (hereinafter referred to as miR-34a) is a new type of micro anticancer drugs based on RNA. We developed a used to synthesize miR-34a prodrugs of new biological engineering method recently. To the first part of the experiment is to evaluate the preparation before the miR-34a drug safety and efficacy of anti carcinoma in situ of osteosarcoma in nude mice model. Methods: in vitro, using human osteosarcoma cells miR-34a transfection, cell proliferation activity by MTT experimental evaluation; invasion and metastasis of tumor cells by stromal invasion assay was used to quantify; using commercial kits and the distribution of cell cycle and apoptosis After treatment by flow cytometry; expression of target protein in tumor cell miR-34a using Western blot to detect; in vivo, we used orthotopic osteosarcoma model to evaluate the safety and efficacy of miR-34a in vivo, miR-34a first commercial in vivo-jetPEI (later referred to as PEI) to mix evenly, then through the intravenous injection into the tumor load of nude mice, regular monitoring of tumor size, at the end of the experiment to obtain the blood and blood of nude mice liver function, renal function and related biochemical indicators. Results: compared with control group, miR-34a can significantly inhibit osteosarcoma cell lines (143B and MG-63) on the growth of in vitro. There was an obvious dose-response relationship in this inhibition; flow cytometry corresponding detection, apoptosis can be observed is consistent with the experimental results of cell proliferation and cell cycle arrest For that phenomenon. Further Western-blot experiment, and cell apoptosis and cell cycle arrest of miR-34a target proteins such as SIRT1, BCL2, c-MET, and CDK6 were all decreased. In animal experiments, after two weeks, a total of 6 times after intravenous injection can significantly inhibit the growth of osteosarcoma, biochemical examination at the end of the experiment it showed that the therapeutic dose of intravenous miR-34a has obvious side reactions to drugs not; conclusion: the recombinant miR-34a prodrug can inhibit the growth of osteosarcoma, and will not cause significant systemic toxicity: a possible mechanism for miR-34a can be relatively specific induced tumor cell apoptosis and cell cycle arrest in osteosarcoma. The second part: the combined application of gene engineering prepared miR-34a prodrug of doxorubicin can inhibit the growth of osteosarcoma Objective: in the first part, the results show that the preparation of application of gene engineering methods in vivo and miR-34a In vitro can inhibit osteosarcoma cells or tumor growth. In the second part of the experiment, we mainly to verify whether miR-34a in the treatment of osteosarcoma with traditional chemotherapy drug doxorubicin has a synergistic effect; methods: in vitro, using a variety of combination mode of miR-34a and the design of multi Chou-Talalay method epirubicin combined with other drugs, determination of proliferation activity of tumor cells after combination with MTT method, the original data into Compusyn software and through the calculation of drug use index to understand whether there is a synergistic effect of two kinds of drugs; then, using cell matrix invasion assay to evaluate the drug to the tumor invasion and metastasis; and the cell cycle cell apoptosis was detected by flow cytometry; using Western-Blot technology to detect the expression of drug target change; in vivo First of all, to establish orthotopic osteosarcoma, then miR-34a alone or combined with doxorubicin loaded system in nude mice, tumor growth was monitored, analysis of drug safety in the end of the experiment to obtain animal blood samples, and the tumor pathology. Results: the results showed that there are synergistic effect of miR-34a combined with doxorubicin, this synergistic effect in the early stage, and reached the maximum when administered; compared with monotherapy, the combination of the two drugs can effectively inhibit the proliferation of 143B cell inhibition, cell cycle arrest and cell invasion; miR-34a target SIRT1, corresponding to these cell activities c-MET and CDK6 are reduced; in vivo, combination of miR-34a and doxorubicin on tumor inhibition effect was significantly better than the single drug; the drug safety, the After two weeks of administration, liver function and renal function of mice showed no obvious damage, has not significantly reduced the overall protein levels in vivo; conclusion: These results show that the combination of miR-34a and doxorubicin in vitro and in vivo animal experiment can achieve some antitumor synergy, at therapeutic doses of two combined with no obvious side effects.

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R738

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