ECSOD基因体外转染对恒河猴骨髓间充质干细胞生物学功能的影响

发布时间：2018-03-29 12:54

本文选题：超氧化物歧化酶　切入点：间充质干细胞　出处：《苏州大学》2012年硕士论文

【摘要】：随着科学技术的高速发展，核能及核技术在工业、农业、医疗及军事等各领域的应用愈加广泛，拥有和正在发展核武器的国家越来越多，尤其是核电技术的开发和利用发展迅速。尽管人们在核能技术的开发和利用过程中注意了辐射安全与防护，但核与辐射突发事件仍常有发生，使受到高剂量电离辐射照射的人群发生急性放射病（acute radiation sickness，ARS）。根据受照射剂量大小和病程表现，，把急性放射病大体分为骨髓型、肠型和脑型三种类型ARS，而骨髓型又分为轻、中、重和极重度四度。目前，对ARS患者的主要救治措施有抗放药、造血干细胞移植和综合对症支持治疗，这些措施可成功救治重度以下的ARS患者，但对于极重度骨髓型ARS没有行之有效的治疗方法，现有的医疗水平仅能延长患者的存活时间。极重度骨髓型ARS病情严重、治疗困难，骨髓造血功能衰竭、免疫功能丧失，全身多脏器损伤是患者的主要的致死原因。众所周知，射线对机体造成的损伤主要有直接损伤和间接损伤两部分，直接损伤作用时间较短，损伤较快，主要导致机体内部分子结构的改变和生物活性的丧失，只能通过直接屏蔽的方法防护。间接损伤是一个复杂的过程，由于机体内含有大量的水（约占生物体干重的80%），射线作用于水分子后，引起水分子活化和自由基生成，然后通过自由基攻击机体内DNA分子中的碱基、核糖和磷酸二脂键造成碱基与核糖氧化、键断裂与蛋白质交联等多种类型的损伤，同时自由基还可通过一系列的反应破坏生物膜系统，从而使物质转运、能量转换、信息传递与识别等基本生物活动受到破坏，以上称之为间接损伤。机体内天然存在着一些清除自由基的酶类，例如：能分解H2O2的过氧化氢酶与过氧化物酶，催化O2-歧化反应的超氧化物歧化酶，清除羟基过氧化物的谷胱甘肽转硫酶等多种。但是这些酶类主要是清除内源性自由基，对于射线作用产生的大量外源性自由基作用相对较弱。虽然，这些自体的超氧化物歧化酶可通过清除自由基来抗辐射，但是，对于射线直接作用的脏器组织和辐射早期自由基对机体组织造成的损伤并没有良好的防治效果。寻找一种能够有效清除辐射早期产生的自由基同时能够对多组织脏器进行修复的新型治疗措施迫在眉睫。骨髓间充质干细胞是来源于骨髓的一类非造血干细胞，可以在体外扩增并具有多向分化潜能，在特定诱导条件下，可分化为成骨细胞、软骨细胞、脂肪细胞、肌腱细胞、肌管、神经细胞与支持造血干细胞的基质等。已有多项研究证实，将骨髓间充质干细胞植入体内后，他们能够向多个受损组织部位归巢并分化成为相应的组织细胞，并且能够与各种病毒载体相结合，可进行多种基因的转染，它有望成为治疗急性放射病的新型细胞药物。基于以上背景，本研究拟采用腺病毒表达载体，通过腺病毒载带胞外分泌型超氧化物歧化酶（extracellular superoxide dismutase,ECSOD）基因和报告基因EGFP转染恒河猴骨髓间充质干细胞，研究ECSOD对恒河猴骨髓间充质干细胞（rhesusmacaque bone marrow mesenchymal stem cells, R-BMSC）生物学性质的影响，旨在为极重度骨髓型急性放射病的临床救治提供实验依据。首先，应用梯度密度离心法和贴壁培养法分离、培养并纯化R-BMSC，培养细胞呈纤维状贴壁生长。第三代R-BMSC用流式细胞仪检测其表面抗原，结果证明为非造血细胞，符合文献报道的间充质干细胞表面标志。通过成功诱导R-BMSC向成骨及成脂方向分化，证明了其具有多向分化的能力。第二，应用gateway技术体外构建载带ECSOD和EGFP基因的腺病毒表达载体，在293细胞中包装并扩增，获得了活力较好的腺病毒。应用不同滴度腺病毒体外感染R-BMSC，然后应用荧光显微镜和流式细胞仪检测，发现感染效率可达到90%以上；用ELISA法检测感染后R-BMSC细胞培养上清液，可检测到有ECSOD蛋白的表达。最后，研究了ECSOD基因转染对R-BMSC生物学性质的影响。通过流式细胞仪对转染了ECSOD基因的R-BMSC（MSC-ECSOD）细胞表型进行鉴定，结果显示MSC-ECSOD与未转染的R-BMSC的表型相似。进一步分析ECSOD基因转染对R-BMSC的分化潜能的影响，发现MSC-ECSOD成脂诱导14天后，用油红O染色可见有被染成红色的脂肪油滴；MSC-ECSOD成骨诱导17天后，茜素红染色可见被染成红色的骨结节。这些结果说明ECSOD转染对R-BMSC分化潜能没有影响。MTT法观察感染前后细胞增殖能力也没有统计学差异。综上所述，本研究采用梯度密度离心和贴壁培养法培养的R-BMSC，具有与人骨髓间充质干细胞相似的生物学特性。并且成功鉴定了R-BMSC的表型和分化能力。用腺病毒载体体外感染R-BMSC，感染效率可达90%以上。ECSOD对R-BMSC的表型和生物学性质没有影响。本研究为ECSOD基因修饰的R-BMSC对极重度骨髓型急性放射病的救治提供实验基础。
[Abstract]:With the rapid development of science and technology, nuclear energy and nuclear technology in industry, agriculture, medical and military applications in various fields such as more widely, more and more countries have and are developing nuclear weapons, especially nuclear power development and utilization technology developed rapidly. Although people pay attention to the radiation safety and protection in the development and utilization of nuclear technology in the process, but the nuclear and radiological incidents still occur, the incidence of acute radiation sickness by high dose of ionizing radiation (acute radiation sickness, group ARS). According to the size and duration of irradiation dose is the acute radiation sickness can be divided into bone marrow, intestinal and brain type three types of ARS however, bone marrow type is divided into light, heavy and severe four degrees. At present, there is resistance to put the medicine on the main approaches to the treatment of patients with ARS, hematopoietic stem cell transplantation and symptomatic treatment, these measures can successful treatment of severe The following ARS patients, but for the extremely severe bone marrow type ARS has no effective treatment methods, the current medical standards can only prolong survival time. Extremely severe bone marrow type ARS severity, treatment difficulties, bone marrow failure, loss of immune function, multiple organ injury is the main cause of death of patients.
As everyone knows, ray on the body caused by injury include direct damage and indirect damage to the two part, direct injury for a short period of time, the main damage quickly, resulting in loss of biological activity of the body and change the internal molecular structure, only through the method of direct shielding protection. Indirect injury is a complex process, because the body contains a large number of the water (about 80% of the dry weight of the organism), irradiation effects on water molecules, caused by activation of water molecules and free radicals, and then through the free radical attack in the bases in a DNA molecule, DNA and two bases and ribose phosphate bonds caused by lipid oxidation, protein cross-linking bond breaking and many other types of injury. At the same time, free radicals can also through a series of reaction to destroy the biomembrane system, so that the material transport, energy conversion, basic information transmission and identification of biological activities have been destroyed, above called between The damage in the body. There are some natural free radical scavenging enzymes, such as: H2O2 decomposition of catalase and peroxidase, superoxide dismutase catalytic disproportionation of O2-, a variety of scavenging hydroxyl peroxide and glutathione S-transferase. But these enzymes mainly remove endogenous free radicals, a large number of exogenous free the ray produced radical effect is relatively weak. Although superoxide dismutase of these autologous by scavenging free radicals to resist radiation, but no good control effect for early organ ray direct action and radiation free radical to tissue injury. To find an effective clearance model the treatment measures of early radiation generated free radicals also can repair the multiple organs is imminent.
Bone marrow mesenchymal stem cells derived from bone marrow is a kind of non hematopoietic stem cells may have the potential of multi-directional differentiation in vitro and in specific conditions induced to differentiate into osteoblasts, chondrocytes, adipocytes, tendon cells, myotubes, nerve cells and support hematopoietic stem cells in stroma. A number of studies have confirmed that bone marrow mesenchymal stem cells in vivo, they are able to a position of the damaged tissue homing and differentiation into corresponding cells, and can be combined with a variety of viral vectors, for a variety of gene transfection, it is expected to become the new drug in the treatment of acute radiation sickness.
Based on the above background, this study intends to use adenovirus expression vector by adenovirus carrying the extracellular superoxide dismutase (extracellular superoxide, dismutase, ECSOD) gene and reporter gene EGFP transfected Ganges RIver monkey bone marrow mesenchymal stem cells, the ECSOD study of Ganges RIver monkey bone marrow mesenchymal stem cells (rhesusmacaque bone marrow mesenchymal stem cells, R-BMSC) affect the biological properties, for clinical treatment of extremely severe bone marrow form of acute radiation sickness and provide experimental basis.
First of all, the application of density gradient centrifugation and adherent culture method of separation, cultivation and purification of R-BMSC, cultured cells showed fibrous adherent growth. The third generation of R-BMSC flow cytometry was used to detect the surface antigen, proved to be non hematopoietic cells, consistent with the reported mesenchymal stem cell surface marker R-BMSC into induction. Bone and adipogenic differentiation by success, proved its ability of multi-directional differentiation.
Second, the application of gateway technology in construction of adenovirus expression vector carrying ECSOD gene and EGFP gene in 293 cells, packaging and amplification, obtained good activity of adenovirus. The application of different titer adenovirus infected R-BMSC in vitro, and then the application of fluorescence microscopy and flow cytometry, found that the infection efficiency can reach above 90%; ELISA was used to detect the infected R-BMSC cell culture supernatant, detect the expression of ECSOD protein.
Finally, the effect of ECSOD gene transfection on R-BMSC biological properties. Based on the ECSOD gene transfer by flow cytometry (MSC-ECSOD) R-BMSC cell phenotype was identified. Results showed that the phenotype of MSC-ECSOD and untransfected R-BMSC similar. Further analysis on the differentiation potential of ECSOD gene transfection on R-BMSC, found that MSC-ECSOD induced adipogenesis 14 days later, oil red O staining was dyed red fatty oil drops; MSC-ECSOD after 17 days of osteogenic differentiation, alizarin red staining was dyed red bone nodules. These results demonstrate that ECSOD transfection differentiation potential of R-BMSC had no effect on.MTT cell proliferation was observed before and after infection was not statistically significant.
In summary, this study by density gradient centrifugation and adherent culture medium R-BMSC with human bone marrow mesenchymal stem cell biological characteristics. And successfully identified similar phenotype and differentiation ability of R-BMSC. Use adenovirus infected R-BMSC in vitro, did not affect the infection efficiency of more than 90%.ECSOD of the R-BMSC phenotype and biological nature. In this study, ECSOD gene modified R-BMSC for the treatment of extremely severe bone marrow form of acute radiation disease provide experimental basis.

【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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