本文選題：脂肪間充質(zhì)干細胞 + 鞘氨醇激酶1��； 參考：《鄭州大學》2017年碩士論文

【摘要】：背景和目的:多發(fā)性硬化(multiple sclerosis,MS)是一種以中樞神經(jīng)系統(tǒng)白質(zhì)炎性脫髓鞘為主要病理特點的自身免疫疾病,最常累及部位為腦室周圍白質(zhì)、視神經(jīng)、脊髓等。其病因及發(fā)病機制迄今不明,目前觀點認為,攜帶先天遺傳易感基因的個體,在后天環(huán)境中,因病毒感染、外傷等外因的作用下,誘導中樞髓鞘成分發(fā)生異常自身免疫應答而致病。目前傳統(tǒng)的治療方法如糖皮質(zhì)激素、干擾素β等,雖然可以減緩MS的進展,但是,長期服用這些藥物往往會導致嚴重的副作用。因此,尋找療效好,副作用低的藥物迫在眉睫。間充質(zhì)干細胞(mesenchymal stem cells,MSCs)是一種多能、具有抗炎和再生能力的成體干細胞,普遍存在于骨髓和其它組織中,如臍帶(UC),胎兒肝臟和脂肪組織等。近年來,隨著對間充質(zhì)干細胞的研究不斷深入,發(fā)現(xiàn)其不僅具有突破譜系屏障分化為神經(jīng)細胞的功能,而且還可以通過抑制免疫反應、促進髓鞘再生和誘導神經(jīng)損傷修復來治療一些神經(jīng)退行性疾病。本實驗通過建立自身免疫性腦脊髓炎(experimental autoimmune encephalomyelitis,EAE)小鼠模型,分別采用不同來源的間充質(zhì)干細胞(mesenchymal stem cells,MSC)對EAE模型小鼠進行治療,觀察各組小鼠在治療過程中病情輕重的變化,并通過一些實驗室檢查來探究其可能的機制,從而為MS的治療提供新的思路和方法。方法:1.采用髓鞘少突膠質(zhì)細胞糖蛋白(MOG35-55)作為抗原與完全弗氏佐劑混合免疫C57BL/6小鼠制作EAE模型。在小鼠發(fā)病后隨機分為對照組,模型組,UCMSC組、ADMSC組和UCMSC-SPK1組,通過尾靜脈給予不同來源的間充質(zhì)干細胞治療,從發(fā)病開始后每隔七天注射一次,共3次;正常對照組和模型組用相同劑量的生理鹽水替代治療。2.采用雙盲法每天對各組小鼠進行神經(jīng)功能缺損評分,持續(xù)33天。3.免疫后第33天處死小鼠,取出腦和脊髓放入㧟80℃冰箱凍存。4.通過電子顯微鏡觀察各組小鼠脊髓中髓鞘脫失情況。5.通過蛋白印跡法來檢測各組小鼠脊髓中GFAP和MBP的表達。6.研磨脾臟細胞收集脾臟細胞,通過流式細胞儀檢測脾臟中調(diào)節(jié)性T細胞(CD4+CD25+和foxp3+)和NK(NK1.1+CD3-)細胞的比例。7.通過攜帶SPK1基因的腺病毒(Ad-SPK1)轉(zhuǎn)染UCMSC,運用westernblot檢測UCMSC、ADMSC和UCMSC-SPK1中SPK1的表達。結(jié)果:1.MOG35-55和完全弗氏佐劑混合免疫成功制備了EAE小鼠模型。2.Westernblot結(jié)果顯示,相比UCMSC,ADMSC中SPK1的表達量更高,而經(jīng)過攜帶SPK1基因的腺病毒(Ad-SPK1)的轉(zhuǎn)染,UCMSC-SPK1中SPK1的表達量比ADMSC更高。3.雙盲法對各組小鼠神經(jīng)功能缺損評分結(jié)果顯示,與模型組相比,UCMSC組、ADMSC組和UCMSC-SPK1組小鼠的神經(jīng)功能缺損評分明顯降低;將這三個細胞治療組進行比較發(fā)現(xiàn),ADMSC組比UCMSC組的神經(jīng)評分降低的更加明顯,而相較ADMSC組,UCMSC-SPK1組小鼠的神經(jīng)缺損評分下降的趨勢更為顯著。4.電鏡下觀察小鼠脊髓髓鞘脫失情況,結(jié)果表明:與正常對照組小鼠相比,模型組中小鼠脊髓的髓鞘大面積脫失;而經(jīng)過細胞治療后髓鞘脫失面積明顯較少,程度顯著減輕;對比三個細胞治療組之間髓鞘脫失情況發(fā)現(xiàn),相比UCMSC組,ADMSC組和UCMSC-SPK1組小鼠脊髓的改善更加明顯,而這種趨勢在UCMSC-SPK1組中表現(xiàn)的更加顯著。5.Westernblot檢測小鼠脊髓中星形膠質(zhì)細胞活化標記蛋白GFAP的表達發(fā)現(xiàn),與對照組相比,模型組小鼠脊髓中GFAP的表達明顯增加,而與模型組相比,UCMSC組,ADMSC組和UCMSC-SPK1組小鼠脊髓中GFAP的表達明顯降低;將這三組細胞治療組進行比較發(fā)現(xiàn),相比UCMSC組,ADMSC組和UCMSC-SPK1組中GFAP的表達降低的更加明顯,而相較ADMSC組,UCMSC-SPK1組小鼠脊髓中GFAP的表達減少的更加顯著。6.Westernblot檢測各組小鼠脊髓中MBP的表達結(jié)果顯示,與正常組相比,模型組小鼠脊髓中MBP的表達明顯減少,而經(jīng)過MSC治療后,各治療組小鼠脊髓中MBP的表達明顯增高;將這三個細胞治療組進行比較發(fā)現(xiàn),ADMSC組小鼠脊髓中MBP的表達要明顯多于UCMSC組,而這種趨勢在UCMSC-SPK1組中表現(xiàn)的更為顯著。7.流式細胞儀對各組小鼠脾臟中的調(diào)節(jié)性T細胞和NK細胞的比例進行測定,結(jié)果顯示,相較對照組,模型組小鼠脾臟中調(diào)節(jié)性T細胞的比例明顯降低,而NK細胞的比例卻顯著升高;經(jīng)過細胞治療后,UCMSC組,ADMSC組和UCMSC-SPK1組小鼠脾臟中調(diào)節(jié)性T細胞的比例顯著升高,而NK細胞的比例顯著降低;相較UCMSC組,ADMSC組和UCMSC-SPK1組中這種趨勢的表現(xiàn)更為明顯,而與ADMSC組相比,UCMSC-SPK1組中調(diào)節(jié)性T細胞升高和NK細胞降低的表現(xiàn)更為顯著。結(jié)論:1.不同來源的間充質(zhì)干細胞治療都可以顯著減輕EAE模型小鼠神經(jīng)功能缺損評分,其機制可能是通過調(diào)節(jié)免疫反應,抑制神經(jīng)系統(tǒng)炎癥反應和促進髓鞘再生來實現(xiàn)的。2.經(jīng)過SPK1基因的轉(zhuǎn)染,UCMSC-SPK1在治療EAE中表現(xiàn)出了比ADMSC更好的療效,而原始的UCMSC卻比ADMSC療效差。3.不同來源的干細胞,其表達的SPK1的量不同,這可能導致了它們在治療EAE模型小鼠療效上的差異。
[Abstract]:Background and objective: multiple sclerosis (MS) is a kind of autoimmune disease characterized by white plasma demyelinating in the central nervous system, which is most often involved in the white matter, optic nerve, and spinal cord around the ventricle. Its etiology and pathogenesis are not so far. Individuals, in the environment, induced by external causes such as virus infection and trauma, induce abnormal autoimmune responses in the central myelin sheath. Traditional therapies such as glucocorticoid and interferon beta can slow the progress of MS, but long-term use of these drugs often leads to serious side effects. Therefore, Mesenchymal stem cells (MSCs) is a kind of pluripotent stem cells with anti-inflammatory and regenerative ability, which is commonly found in bone marrow and other tissues, such as umbilical cord (UC), fetal liver and adipose tissue. It is found that it not only has the function of breaking through the pedigree barrier to differentiate into nerve cells, but also can be used to treat some neurodegenerative diseases by inhibiting the immune response, promoting the regeneration of myelin sheath and inducing the repair of nerve injury. This experiment is based on the establishment of autoimmune encephalomyelitis (experimental autoimmune encephalomyelitis, EAE) mice Mesenchymal stem cells (MSC) was used to treat EAE model mice respectively. The changes of the severity of the disease during the treatment were observed, and some laboratory tests were used to explore the possible mechanisms, so as to provide new ideas and methods for the treatment of MS. Methods: 1. the myelin sheath was less. The EAE model of C57BL/6 mice was immunized with MOG35-55 as antigen and complete Freund adjuvant. The mice were randomly divided into the control group, the model group, the UCMSC group, the ADMSC group and the UCMSC-SPK1 group, and the caudal vein was given to different sources of mesenchymal stem cells, and the injection was given every seven days from the onset of the onset of the disease. 3 times, the normal control group and the model group were treated with the same dose of saline replacement therapy for.2. with double blind method of nerve function defect score in each group every day. After 33 days of.3. immunization, the mice were killed and the brain and spinal cord were taken out, and the cryopreservation.4. at 80 C was used to observe the loss of myelin sheath in the spinal cord of each group through the electron microscope. .5. was used to detect the expression of GFAP and MBP in the spinal cord of mice by Western blot, and.6. was used to grind spleen cells to collect spleen cells. The proportion of T cells (CD4+CD25+ and foxp3+) and NK (NK1.1+CD3-) cells in the spleen was detected by flow cytometry, and.7. was transfected by adenovirus carrying SPK1 gene (Ad-SPK1). Results: the expression of SPK1 in CMSC, ADMSC and UCMSC-SPK1. Results: 1.MOG35-55 and complete Freund adjuvant immunization successfully prepared the EAE mouse model.2.Westernblot results, and the expression of SPK1 was higher in ADMSC than UCMSC, and the expression of the SPK1 was higher than that of the adenovirus carrying the SPK1 gene. The neurological deficit scores of each group showed that the neurological deficit scores of the UCMSC, ADMSC and UCMSC-SPK1 groups were significantly lower than those in the model group; compared with the three cell groups, the ADMSC group was significantly lower than the UCMSC group, compared with the ADMSC group and the UCMSC-SPK1 group. The demyelination of the spinal cord in mice was observed under the.4. electron microscope. The results showed that the myelin sheath of the mouse spinal cord in the model group was greatly lost in comparison with the normal control group, while the myelin loss area was significantly less and the degree of myelin was significantly reduced after the cell treatment. The myelin sheath was compared between the three cell therapy groups. It was found that the improvement of the spinal cord in the ADMSC and UCMSC-SPK1 groups was more obvious than that in the UCMSC group, and the trend in the group UCMSC-SPK1 was more significant in.5.Westernblot detection of the expression of the astrocyte activation marker protein GFAP in the spinal cord of the mouse spinal cord, and the expression of GFAP in the spinal cord of the model mice was compared with the control group. The expression of GFAP in the spinal cord of the UCMSC, ADMSC and UCMSC-SPK1 groups decreased significantly compared with the model group. Compared with the group UCMSC, the expression of GFAP in the group ADMSC and the UCMSC-SPK1 group decreased more significantly than that in the UCMSC group, and the GFAP expression in the spinal cord of the UCMSC-SPK1 group was less than that in the group of ADMSC. The expression of MBP in the spinal cord of each group of mice by.6.Westernblot showed that the expression of MBP in the spinal cord of the model group decreased significantly compared with the normal group, and the expression of MBP in the spinal cord of each group of mice increased significantly after the treatment of MSC, and the three cell treatment groups were compared to the MBP in the spinal cord of the ADMSC group. The proportion of the regulatory T cells and NK cells in the spleen of each group was measured by the more significant.7. flow cytometer in group UCMSC-SPK1. The results showed that the proportion of the regulatory T cells in the spleen of the model group was significantly lower than that of the control group, while the proportion of NK cells was significantly higher than that of the control group. After cell therapy, the proportion of regulatory T cells in the spleen of the UCMSC, ADMSC and UCMSC-SPK1 groups increased significantly, while the proportion of NK cells decreased significantly; compared with the UCMSC group, the trend in the ADMSC group and the UCMSC-SPK1 group was more obvious, and the regulatory T cells in the UCMSC-SPK1 group and the NK cells in the UCMSC-SPK1 group were higher than those in the ADMSC group. Conclusion: 1. the treatment of mesenchymal stem cells from different sources can significantly reduce the score of neural function defect in EAE model mice. The mechanism may be the expression of.2. through SPK1 gene transfection by regulating the immune response, inhibiting the inflammatory response of the nervous system and promoting the regeneration of myelin sheath, and the expression of UCMSC-SPK1 in the treatment of EAE. The effect of ADMSC was better than that of the original UCMSC, but the stem cells from different sources of.3. were less effective than ADMSC, and the amount of SPK1 expressed differently, which may lead to their difference in the treatment of EAE model mice.

【學位授予單位】：鄭州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R744.51;R-332

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本文編號：1790894