本文選題：超氧化物歧化酶　切入點(diǎn)：間充質(zhì)干細(xì)胞　出處：《蘇州大學(xué)》2012年碩士論文

【摘要】：隨著科學(xué)技術(shù)的高速發(fā)展，核能及核技術(shù)在工業(yè)、農(nóng)業(yè)、醫(yī)療及軍事等各領(lǐng)域的應(yīng)用愈加廣泛，擁有和正在發(fā)展核武器的國家越來越多，尤其是核電技術(shù)的開發(fā)和利用發(fā)展迅速。盡管人們在核能技術(shù)的開發(fā)和利用過程中注意了輻射安全與防護(hù)，但核與輻射突發(fā)事件仍常有發(fā)生，使受到高劑量電離輻射照射的人群發(fā)生急性放射病（acute radiation sickness，ARS）。根據(jù)受照射劑量大小和病程表現(xiàn)，，把急性放射病大體分為骨髓型、腸型和腦型三種類型ARS，而骨髓型又分為輕、中、重和極重度四度。目前，對ARS患者的主要救治措施有抗放藥、造血干細(xì)胞移植和綜合對癥支持治療，這些措施可成功救治重度以下的ARS患者，但對于極重度骨髓型ARS沒有行之有效的治療方法，現(xiàn)有的醫(yī)療水平僅能延長患者的存活時間。極重度骨髓型ARS病情嚴(yán)重、治療困難，骨髓造血功能衰竭、免疫功能喪失，全身多臟器損傷是患者的主要的致死原因。 眾所周知，射線對機(jī)體造成的損傷主要有直接損傷和間接損傷兩部分，直接損傷作用時間較短，損傷較快，主要導(dǎo)致機(jī)體內(nèi)部分子結(jié)構(gòu)的改變和生物活性的喪失，只能通過直接屏蔽的方法防護(hù)。間接損傷是一個復(fù)雜的過程，由于機(jī)體內(nèi)含有大量的水（約占生物體干重的80%），射線作用于水分子后，引起水分子活化和自由基生成，然后通過自由基攻擊機(jī)體內(nèi)DNA分子中的堿基、核糖和磷酸二脂鍵造成堿基與核糖氧化、鍵斷裂與蛋白質(zhì)交聯(lián)等多種類型的損傷，同時自由基還可通過一系列的反應(yīng)破壞生物膜系統(tǒng)，從而使物質(zhì)轉(zhuǎn)運(yùn)、能量轉(zhuǎn)換、信息傳遞與識別等基本生物活動受到破壞，以上稱之為間接損傷。機(jī)體內(nèi)天然存在著一些清除自由基的酶類，例如：能分解H2O2的過氧化氫酶與過氧化物酶，催化O2-歧化反應(yīng)的超氧化物歧化酶，清除羥基過氧化物的谷胱甘肽轉(zhuǎn)硫酶等多種。但是這些酶類主要是清除內(nèi)源性自由基，對于射線作用產(chǎn)生的大量外源性自由基作用相對較弱。雖然，這些自體的超氧化物歧化酶可通過清除自由基來抗輻射，但是，對于射線直接作用的臟器組織和輻射早期自由基對機(jī)體組織造成的損傷并沒有良好的防治效果。尋找一種能夠有效清除輻射早期產(chǎn)生的自由基同時能夠?qū)Χ嘟M織臟器進(jìn)行修復(fù)的新型治療措施迫在眉睫。 骨髓間充質(zhì)干細(xì)胞是來源于骨髓的一類非造血干細(xì)胞，可以在體外擴(kuò)增并具有多向分化潛能，在特定誘導(dǎo)條件下，可分化為成骨細(xì)胞、軟骨細(xì)胞、脂肪細(xì)胞、肌腱細(xì)胞、肌管、神經(jīng)細(xì)胞與支持造血干細(xì)胞的基質(zhì)等。已有多項(xiàng)研究證實(shí)，將骨髓間充質(zhì)干細(xì)胞植入體內(nèi)后，他們能夠向多個受損組織部位歸巢并分化成為相應(yīng)的組織細(xì)胞，并且能夠與各種病毒載體相結(jié)合，可進(jìn)行多種基因的轉(zhuǎn)染，它有望成為治療急性放射病的新型細(xì)胞藥物。 基于以上背景，本研究擬采用腺病毒表達(dá)載體，通過腺病毒載帶胞外分泌型超氧化物歧化酶（extracellular superoxide dismutase,ECSOD）基因和報告基因EGFP轉(zhuǎn)染恒河猴骨髓間充質(zhì)干細(xì)胞，研究ECSOD對恒河猴骨髓間充質(zhì)干細(xì)胞（rhesusmacaque bone marrow mesenchymal stem cells, R-BMSC）生物學(xué)性質(zhì)的影響，旨在為極重度骨髓型急性放射病的臨床救治提供實(shí)驗(yàn)依據(jù)。 首先，應(yīng)用梯度密度離心法和貼壁培養(yǎng)法分離、培養(yǎng)并純化R-BMSC，培養(yǎng)細(xì)胞呈纖維狀貼壁生長。第三代R-BMSC用流式細(xì)胞儀檢測其表面抗原，結(jié)果證明為非造血細(xì)胞，符合文獻(xiàn)報道的間充質(zhì)干細(xì)胞表面標(biāo)志。通過成功誘導(dǎo)R-BMSC向成骨及成脂方向分化，證明了其具有多向分化的能力。 第二，應(yīng)用gateway技術(shù)體外構(gòu)建載帶ECSOD和EGFP基因的腺病毒表達(dá)載體，在293細(xì)胞中包裝并擴(kuò)增，獲得了活力較好的腺病毒。應(yīng)用不同滴度腺病毒體外感染R-BMSC，然后應(yīng)用熒光顯微鏡和流式細(xì)胞儀檢測，發(fā)現(xiàn)感染效率可達(dá)到90%以上；用ELISA法檢測感染后R-BMSC細(xì)胞培養(yǎng)上清液，可檢測到有ECSOD蛋白的表達(dá)。 最后，研究了ECSOD基因轉(zhuǎn)染對R-BMSC生物學(xué)性質(zhì)的影響。通過流式細(xì)胞儀對轉(zhuǎn)染了ECSOD基因的R-BMSC（MSC-ECSOD）細(xì)胞表型進(jìn)行鑒定，結(jié)果顯示MSC-ECSOD與未轉(zhuǎn)染的R-BMSC的表型相似。進(jìn)一步分析ECSOD基因轉(zhuǎn)染對R-BMSC的分化潛能的影響，發(fā)現(xiàn)MSC-ECSOD成脂誘導(dǎo)14天后，用油紅O染色可見有被染成紅色的脂肪油滴；MSC-ECSOD成骨誘導(dǎo)17天后，茜素紅染色可見被染成紅色的骨結(jié)節(jié)。這些結(jié)果說明ECSOD轉(zhuǎn)染對R-BMSC分化潛能沒有影響。MTT法觀察感染前后細(xì)胞增殖能力也沒有統(tǒng)計學(xué)差異。 綜上所述，本研究采用梯度密度離心和貼壁培養(yǎng)法培養(yǎng)的R-BMSC，具有與人骨髓間充質(zhì)干細(xì)胞相似的生物學(xué)特性。并且成功鑒定了R-BMSC的表型和分化能力。用腺病毒載體體外感染R-BMSC，感染效率可達(dá)90%以上。ECSOD對R-BMSC的表型和生物學(xué)性質(zhì)沒有影響。本研究為ECSOD基因修飾的R-BMSC對極重度骨髓型急性放射病的救治提供實(shí)驗(yàn)基礎(chǔ)。
[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.

【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R329

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本文編號：1681233