超支化纳米载体转基因修饰骨骼肌成肌细胞治疗缺血性心脏病的研究

发布时间：2018-05-14 09:16

本文选题：骨骼肌成肌细胞 + 纯化　；参考：《复旦大学》2012年博士论文

【摘要】：第一部分小鼠成体骨骼肌成肌细胞提取及纯化方法的改进研究背景作为成体种子细胞及基因治疗工程细胞,高纯度骨骼肌成肌细胞(skeletal myoblasts, SkMs)在缺血性心脏病的治疗中展示了良好的临床应用前景。如何快速、高效、经济地获取足量的高纯度SkMs一直在不断地进行摸索、改进。研究目的建立一种快速、高效的小鼠成体SkMs提取、纯化的实验方法。研究方法通过布比卡因预处理激活肌骨骼肌中的SkMs,48h后,配制优化混合酶,通过一步消化分离法及改良纯化法,从成年骨骼肌中分离高纯度SkMs,并与普通差速贴壁法、反复差速贴壁法及Percoll非连续密度梯度离心法等传统纯化方法分离的SkMs的纯度相比较。研究结果通过改良提取、纯化法所得到的SkMs纯度可达到92.59±1.29%,明显高于普通差速贴壁法、反复差速贴壁法及Percoll非连续密度梯度离心法(P0.05),并具有很好的增殖及分化能力。研究结论通过采用改良成体SkMs提取及纯化法,能快速高效地获取高纯度SkMs。研究背景基因治疗正逐步被应用于多种疾病的防治,前景十分广阔。而在基因转染的过程中,最关键的核心问题是如何选择理想的基因载体。研究目的探讨一种新型的非病毒基因载体——超支化聚酰胺胺(hyperbranched polyamidoamine, h-PAMAM)纳米颗粒在基因转染中的效率、毒性及转染后的基因表达情况。研究方法以“改良一锅法”合成h-PAMAM纳米颗粒,将核酸(deoxyribonucleic acid, DNA)及h-PAMAM以不同的质量比复合构建h-PAMAM-DNA复合物,观察其粒径大小及Zeta电位情况；在核酸内切酶的作用下,观察纳米颗粒对质粒的保护作用；以h-PAMAM-DNA复合物转染COS7及HEK293细胞系,测定在不同质量比情况下的转染效率及细胞存活率,得出最优化的转染方案：h-PAMAM介导的人血管内皮生长因子165(human vascular endothelial growth factor165, hVEG165)基因转染后,测定hVEG165基因在转染后的表达情况。研究结果 h-PAMAM-DNA复合物的粒径及zeta电位随着h-PAMAM/DNA质量比的增加而提高,h-PAMAM能保护质粒不受核酸内切酶的消化,保持其完整性和稳定性长达2小时。h-PAMAM介导质粒转染时,最佳转染条件下,转染效率可达47A7±1.42%(COS7细胞)及40.8±0.98%(HEK293细胞),此时细胞毒性较轻微,细胞存活率较高(COS7:91.38±0.46%；HEk293:92.38±O.61％)；h-PAMAM介导的hVEG165基因转染后,hVEG165能表达14天之久,峰值出现在第2天。研究结论作为一种新型非病毒基因载体,h-PAMAM介导了一种经济、高效、安全的基因治疗方法。研究背景骨骼肌成肌细胞(skeletal myoblasts, SkMs)移植协同血管内皮生长因子(vascular endothelial growth factor, VEGF)基因治疗是一种有潜力的治疗缺血性心脏病的方案。然而,有待优化的基因载体及不可调控的血管内皮生长因子的表达阻碍了基因治疗的应用。因此,寻求一种经济、高效、可控的基因转染平台非常是有必要的。研究目的本实验中,我们探讨了超支化聚酰胺胺(hyperbranched polyamidoamine, h-PAMAM)纳米颗粒介导的低氧可调控性人血管内皮生长因子165(hypoxia regulated human vascular endothelial growth factor, HRE-hVEGF165)基因转染协同SkMs移植治疗缺血性心脏病的可行性及有效性。研究方法将核酸(deoxyribonucleic acid, DNA)及h-PAMAM纳米颗粒以不同的质量比复合构建h-PAMAM-DNA复合物,以复合物转染SkMs,测定在不同质量比情况下的转染效率及细胞存活率,得出最优化的转染方案；免疫染色、Realtime-PCR及Elisa法测定其基因转染后的表达情况。将h-PAMAM-HRE-hVEGF165复合物修饰的SkMs移植至C57/BL6小鼠的缺血心肌中,探讨心肌细胞凋亡情况,移植干细胞存活率,心梗面积及胶原沉积,血管密度及心功能。研究结果 h-PAMAM介导质粒转染SkMs时,最佳转染条件下,转染效率可达43.47士2.22%,此时细胞毒性较轻微,细胞存活率较高(91.38±0.48%)。在低氧情况下,h-PAMAM介导的HRE-hVEG165基因转染时能表达hVEGF165基因18天之久。h-PAMAM-pHRE-hVEG165修饰的SkMs移植至缺血心肌后,过表达的VEGF可导致心肌凋亡的减少,移植成肌细胞存活率的增加,梗死面积及胶原沉积的减少,血管密度的增加,从而抑制左室的重构,提高了心功能。研究结论 h-PAMAM介导的HRE-hVEGF165基因转染SkMs是可行、有效的,并且是一种新型的有前景的缺血性心脏病的基因治疗策略。
[Abstract]:Part one
Improvement of extraction and purification methods of adult skeletal muscle myoblasts in mice
Research background
As the adult seed cell and gene therapy engineering cells, the high purity skeletal myoblasts (SkMs) shows a good clinical prospect in the treatment of ischemic heart disease. How to quickly, efficiently and economically obtain a full and high purity SkMs has been constantly groping and improving.
Objective: to establish a rapid and efficient method for the extraction and purification of SkMs from adult mice.
research method
After pretreatment with bupivacaine to activate SkMs and 48h in musculoskeletal muscles, the mixed enzyme was optimized, the high purity SkMs was separated from the adult skeletal muscle by one step digestion and purification method, and the SkMs was separated from the conventional differential adherence method, the repeated differential adherence method and the Percoll discontinuous gradient centrifugation method. The purity is compared.
Research results
The purity of SkMs obtained by improved extraction and purification can reach 92.59 + 1.29%, which is obviously higher than that of ordinary differential adhesion method, repeated differential adherence method and Percoll discontinuous density gradient centrifugation (P0.05), and has a good ability of proliferation and differentiation.
research conclusion
By means of improved adult SkMs extraction and purification, high purity SkMs. can be obtained quickly and efficiently.
Research background
Gene therapy is being gradually applied to the prevention and control of various diseases, and the prospect is very broad. In the process of gene transfection, the key problem is how to choose the ideal gene carrier.
research objective
To investigate the efficiency, toxicity and gene expression of a new non viral gene vector, hyperbranched polyamidoamine (h-PAMAM) nanoparticles, in gene transfection.
research method
A modified one pot method was used to synthesize h-PAMAM nanoparticles, and nucleic acid (deoxyribonucleic acid, DNA) and h-PAMAM were constructed with different mass ratio compound to construct h-PAMAM-DNA complex, and the size of particle size and Zeta potential were observed. Under the action of nucleic acid endonuclease, the protective effect of nanoparticles on plasmids was observed and transfected with h-PAMAM-DNA complex. COS7 and HEK293 cell lines were used to determine the transfection efficiency and cell survival rate under different mass ratio conditions. The optimal transfection scheme was obtained: h-PAMAM mediated human vascular endothelial growth factor 165 (human vascular endothelial growth factor165, hVEG165) gene was transfected to determine the expression of hVEG165 gene after transfection.
Research results
The particle size and zeta potential of the h-PAMAM-DNA complex increased with the increase of the mass ratio of h-PAMAM/DNA. H-PAMAM could protect the plasmid from the digestion of endonuclease, and maintain its integrity and stability for 2 hours.H-PAMAM mediated plasmid transfection. Under the optimal transfection condition, the transfection efficiency was 47A7 + 1.42% (COS7 cells) and 40.8 + 0.98% (HEK293). At this time, the cell viability was slight and the cell survival rate was higher (COS7:91.38 + 0.46%; HEk293:92.38 + O.61%). After transfection of hVEG165 gene mediated by h-PAMAM, hVEG165 could be expressed for a long time and the peak value appeared in second days.
research conclusion
As a new non viral gene vector, h-PAMAM mediates an economical, efficient and safe gene therapy.
Research background
Skeletal myoblasts (SkMs) transplantation and vascular endothelial growth factor (vascular endothelial growth factor, VEGF) gene therapy is a potential treatment for ischemic heart disease. However, the expression of the genes to be optimized and the expression of the non regulated vascular endothelial growth factor hinders gene therapy. Therefore, it is necessary to find an economical, efficient and controllable gene transfection platform.
research objective
In this experiment, we explored the feasibility and feasibility of hyperbranched hyperbranched polyamidoamine (h-PAMAM) nanoparticles mediated hypoxia regulated human vascular endothelial growth factor 165 (hypoxia regulated human vascular endothelial growth factor, HRE-hVEGF165) gene transfer and SkMs transplantation in the treatment of ischemic heart disease. Validity.
research method
Deoxyribonucleic acid (DNA) and h-PAMAM nanoparticles were combined to construct h-PAMAM-DNA complex with different mass ratio. Transfection efficiency and cell survival rate under different mass ratio were measured by transfection of h-PAMAM-DNA complex. The optimal transfection scheme was obtained. Immunostaining, Realtime-PCR and Elisa method were used to determine the gene transfection. The expression of h-PAMAM-HRE-hVEGF165 complex modified SkMs was transplanted into the ischemic myocardium of C57/BL6 mice. The apoptosis of myocardial cells, the survival rate of transplanted stem cells, the area of myocardial infarction and the deposition of collagen, blood vessel density and cardiac function were investigated.
Research results
When h-PAMAM mediated plasmid transfection to SkMs, the transfection efficiency of the transfection was 43.47, 2.22%, and the cytotoxicity was mild and the cell survival rate was higher (91.38 + 0.48%). Under the condition of hypoxia, h-PAMAM mediated HRE-hVEG165 gene transfection could express the 18 day.H-PAMAM-pHRE-hVEG165 modified SkMs transplantation to the ischemic heart. After muscle, overexpression of VEGF can lead to the decrease of myocardial apoptosis, increase the survival rate of myocytes, decrease of infarct area and collagen deposition, increase the density of blood vessels, inhibit the reconstruction of left ventricle and improve cardiac function.
research conclusion
H-PAMAM mediated HRE-hVEGF165 gene transfection of SkMs is feasible and effective, and is a new promising gene therapy strategy for ischemic heart disease.

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R329

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