發(fā)布時(shí)間：2018-05-04 15:10

  本文選題：脊髓損傷 + 骨髓間充質(zhì)干細(xì)胞　； 參考：《南方醫(yī)科大學(xué)》2017年碩士論文

【摘要】：背景:脊髓損傷(spinal cord injury,SCI)是生產(chǎn)生活中時(shí)常發(fā)生的一種后果比較嚴(yán)重的損傷。隨著工業(yè)時(shí)代的飛速發(fā)展,交通事故、運(yùn)動(dòng)損傷和高處墜落等導(dǎo)致SCI患者數(shù)量逐年上升,且多發(fā)生于青壯年人群中。脊髓損傷后,神經(jīng)元不能再生,常常容易發(fā)生截癱,治療難效果不理想,至今人們沒有找到好的方法去解決這一難題。一般臨床上采取手術(shù)減壓,結(jié)合藥物治療。藥物治療方面用甲強(qiáng)龍沖擊療法,但是治療效果不理想。因此,我們一直在探索應(yīng)用一些新的技術(shù)手段提高SCI的療效,使得脊髓損傷后損傷的軸突能再生,重建脊髓功能,盡快將之應(yīng)用于臨床治療中。近年來隨著組織工程技術(shù)研究的興起以及對(duì)SCI后的病理改變、機(jī)理認(rèn)識(shí)不斷深入的研究,科學(xué)家們提出采用細(xì)胞移植,另外結(jié)合基因工程技術(shù),使得脊髓損傷的治療成為嶄新的有效的方法變得可能。目的:探討聯(lián)合用腺病毒轉(zhuǎn)染目的基因Shootin1的骨髓間充質(zhì)干細(xì)胞移植治療脊髓損傷的療效,為進(jìn)一步研究治療脊髓損傷的方法作出有益的探索。方法:1.全骨髓貼壁培養(yǎng)法分離培養(yǎng)骨髓間充質(zhì)干細(xì)胞,觀察細(xì)胞形態(tài),并進(jìn)行流式細(xì)胞學(xué)鑒定。2.以腺病毒為載體,體外轉(zhuǎn)染Shootin1進(jìn)入BMSC,檢測Shootin1蛋白的表達(dá)情況;3.在體外進(jìn)行神經(jīng)誘導(dǎo)分化,觀察細(xì)胞是否向神經(jīng)元樣細(xì)胞分化;4.建立大鼠脊髓撞擊損傷模型,在脊髓損傷后半小時(shí)內(nèi)在原位分別注射移植Shootin1-BMSC和BMSC來進(jìn)行脊髓損傷后修復(fù)治療,每周對(duì)大鼠運(yùn)動(dòng)功能進(jìn)行BBB評(píng)分,5周后處死大鼠,取脊髓標(biāo)本進(jìn)行冰凍切片,進(jìn)行免疫熒光檢測神經(jīng)元相關(guān)標(biāo)記蛋白。結(jié)果:1.同未轉(zhuǎn)染Shootin1的骨髓間充質(zhì)干細(xì)胞相比,攜帶Shootin1的慢病毒感染BMSCs能持續(xù)穩(wěn)定高水平表達(dá)Shootin1蛋白;2.在體外經(jīng)過神經(jīng)誘導(dǎo)分化7天,Shootin1轉(zhuǎn)染的BMSC與正常BMSC細(xì)胞形態(tài)上均發(fā)生了變化,我們通過IF檢測神經(jīng)前體細(xì)胞特異性標(biāo)記Nestin和神經(jīng)元特異性核蛋白NeuN的發(fā)現(xiàn),Shootin1-BMSC和BMSC兩組Nestin與NeuN蛋白的表達(dá)并無明顯差異;3.骨髓間充質(zhì)干細(xì)胞移植后,進(jìn)行運(yùn)動(dòng)功能評(píng)分,發(fā)現(xiàn)細(xì)胞移植組與對(duì)照組相比,其運(yùn)動(dòng)功能恢復(fù)有明顯的變化,并且Shootin1-BMSC移植組的BBB評(píng)分最高,但Shootin1-BMSC移植組與BMSC組的評(píng)分相比(P0.05)沒有統(tǒng)計(jì)學(xué)意義。結(jié)論:1.本實(shí)驗(yàn)證明了 BMSCs細(xì)胞移植可以有效地促進(jìn)脊髓損傷后運(yùn)動(dòng)功能恢復(fù);2.Shootin1蛋白可以促進(jìn)脊髓損傷后損傷區(qū)域神經(jīng)再生和功能恢復(fù),但其作用較BMSCs直接移植組并不明顯。
[Abstract]:Background: spinal cord injury (sci) is a serious injury in life. With the rapid development of the industrial age, traffic accidents, sports injuries and falling from height lead to an increase in the number of SCI patients, and most of them occur among the young and middle-aged people. After spinal cord injury, the neurons can not regenerate, and are prone to paraplegia. So far, people have not found a good way to solve this problem. General clinical use of surgical decompression, combined with drug treatment. The medicine treatment aspect uses the nail strong dragon pulse therapy, but the treatment effect is not ideal. Therefore, we have been exploring some new techniques to improve the efficacy of SCI, so that the injured axons can regenerate after spinal cord injury, reconstruct spinal cord function, and apply it to clinical treatment as soon as possible. In recent years, with the rise of tissue engineering technology and the pathological changes after SCI, the understanding of mechanism has been deeply studied. Scientists have proposed the use of cell transplantation, in addition to the combination of genetic engineering technology. This makes the treatment of spinal cord injury a new and effective method. Objective: to investigate the effect of bone marrow mesenchymal stem cell transplantation combined with adenovirus transfected target gene Shootin1 in the treatment of spinal cord injury (sci). Method 1: 1. Bone marrow mesenchymal stem cells were isolated and cultured by whole bone marrow adherent culture method. Using adenovirus as vector, Shootin1 was transfected into BMSC in vitro to detect the expression of Shootin1 protein. Neural differentiation was conducted in vitro to observe whether the cells were differentiated into neuron-like cells. The spinal cord impingement injury model was established in rats. Shootin1-BMSC and BMSC were injected into the spinal cord injury within half an hour after spinal cord injury to repair the spinal cord injury. The rats were killed 5 weeks later with the BBB score of motor function every week. The spinal cord specimens were taken for frozen section and immunofluorescence was used to detect the neuron-related marker protein. The result is 1: 1. Compared with untransfected bone marrow mesenchymal stem cells (BMSCs), lentivirus infected with Shootin1 could maintain high and stable expression of Shootin1 protein in BMSCs. The morphologic changes of BMSC transfected with Shootin1 and normal BMSC cells were observed on the 7th day after neuronal induction and differentiation in vitro. We found that there was no significant difference in the expression of Nestin and NeuN protein between Shootin 1-BMSC and BMSC groups by if detection of neuronal specific Nestin and neuron-specific nucleoprotein NeuN. After bone marrow mesenchymal stem cell transplantation, the motor function score was evaluated. It was found that the motor function recovery of the cell transplantation group was significantly different from that of the control group, and the BBB score of the Shootin1-BMSC transplantation group was the highest. However, the score of Shootin1-BMSC transplantation group was not significantly higher than that of BMSC group (P 0.05). Conclusion 1. This study demonstrated that BMSCs cell transplantation can effectively promote motor function recovery after spinal cord injury. 2. Shootin1 protein can promote nerve regeneration and functional recovery in injured area after spinal cord injury, but its effect is not obvious compared with BMSCs direct transplantation group.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R651.2

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本文編號(hào)：1843465