本文選題：神經(jīng)干細胞 + 施萬細胞�。� 參考：《中國醫(yī)科大學》2009年博士論文

【摘要】： 前言 施萬細胞(Schwann cells, SCs)是周圍神經(jīng)系統(tǒng)結構和功能的主要細胞,在周圍神經(jīng)的發(fā)育和再生中起著重要的作用。周圍神經(jīng)損傷后,施萬細胞進行增殖、遷移,并能產(chǎn)生多種神經(jīng)營養(yǎng)因子,促進和引導遠側神經(jīng)斷端的生長,向不同的周圍神經(jīng)支架移植施萬細胞能加快神經(jīng)再生,因而施萬細胞已經(jīng)應用于臨床神經(jīng)系統(tǒng)再生和脫髓鞘疾病模型的治療。然而,體外培養(yǎng)施萬細胞的來源有限,培養(yǎng)周期長,并且容易受增殖更快的成纖維細胞污染和排斥,所以施萬細胞的分離、純化并達到治療的數(shù)量十分困難,臨床上的應用受到限制。因此,尋找一種容易獲取、增殖周期短且免疫原性小的施萬細胞來源,對周圍神經(jīng)損傷的修復治療具有重要的意義。 神經(jīng)干細胞(neural stem cells, NSCs)存在于中樞神經(jīng)系統(tǒng)的多個部位,有自我更新和多向分化能力,能分化為神經(jīng)元、星形膠質細胞、少突膠質細胞等中樞神經(jīng)系統(tǒng)細胞以及血細胞、肌細胞等其他系統(tǒng)細胞。神經(jīng)干細胞因具有很高的增殖活性并能進行長期培養(yǎng)、在分化為其他細胞之前一直保持其表型不變、免疫原性低等優(yōu)點被用于中樞神經(jīng)系統(tǒng)疾病的細胞治療,由于其具有優(yōu)秀的重塑神經(jīng)組織的潛能,被認為是神經(jīng)系統(tǒng)再生合適的供體細胞。有報道,脂肪干細胞源性神經(jīng)球能分化為施萬樣細胞,但是體外培養(yǎng)的中樞神經(jīng)系統(tǒng)來源的神經(jīng)干細胞是否能分化為施萬細胞尚未見報道。 促分裂原活化蛋白激酶(mitogenic activated protein kinase pathway, MAPK)通路對生長和分化極為重要,哺乳動物體內(nèi)存在的主要的三條MAPK通路：細胞外信號調節(jié)激酶(extracellular signal regulated kinase, ERK)通路、c-jun氨基末端激酶(c-jun N-terminal kinase, JNK)通路、P38通路。其中,ERK是調節(jié)細胞生長增殖、分化、和凋亡的最基本信號途徑,JNK、P38通路可被應激刺激、細胞因子、生長因子等激活。在不同的細胞系中,MAPK通路起到何種作用一直存在爭議,此通路是否在神經(jīng)干細胞誘導為施萬樣細胞過程中起到作用目前尚未見報道。 本研究采用無血清培養(yǎng)技術培養(yǎng)新生大鼠海馬神經(jīng)干細胞,通過單細胞克隆培養(yǎng)、Nestin免疫熒光染色及誘導分化能力進行鑒定。通過向培養(yǎng)液中添加heregulin-β1、堿性成纖維細胞生長因子(basic fibroblast growth factor, bFGF)、血小板源性生長因子(platelet-derived growth factor-AA, PDGF-AA)等誘導劑,新生大鼠海馬神經(jīng)干細胞可以分化為施萬樣細胞,進一步實驗應用細胞外信號調節(jié)激酶(extracellular signal regulated kinase, ERK)通路、c-jun氨基末端激酶(c-jun N-terminal kinase, JNK)通路、P38通路通路抑制劑進行誘導分化干預,探討這3條通路在神經(jīng)干細胞向施萬樣細胞誘導中的作用,對獲得大量的施萬樣細胞及臨床上進行神經(jīng)缺損的細胞治療提供理論和實驗基礎。 實驗方-法 本實驗通過無血清培養(yǎng)技術體外培養(yǎng)新生大鼠海馬神經(jīng)干細胞,應用單細胞克隆技術對培養(yǎng)的神經(jīng)干細胞進行純化,應用免疫熒光染色對所培養(yǎng)細胞進行神經(jīng)干細胞鑒定,應用免疫熒光染色和Western Blot技術測定分化后神經(jīng)干細胞S-100和P75蛋白的表達情況,RT-PCR技術檢測P0、Krox-20、Oct-6 mRNA表達；通過與神經(jīng)元共培養(yǎng)的方法檢測施萬樣細胞的功能,應用Western Blot、免疫熒光染色技術檢測ERK、JNK、P38通路抑制劑對神經(jīng)干細胞誘導分化的影響。 實驗結果 1、新生大鼠海馬神經(jīng)干細胞的分離培養(yǎng)和鑒定 新生大鼠海馬分離的神經(jīng)干細胞呈神經(jīng)球樣,懸浮生長,折光性強,傳代后細胞較原代培養(yǎng)細胞增殖加快,單細胞克隆培養(yǎng)形成的克隆球表達Nestin,誘導分化1w后細胞表達NSE、GFAP及Galc。 2、神經(jīng)干細胞誘導分化結果 向培養(yǎng)液中添加HRG、RA、FSK、PDGF-AA等誘導劑后,新生大鼠海馬神經(jīng)干細胞的形態(tài)發(fā)生改變,免疫熒光染色及Western Blot檢測顯示分化后細胞表達膠質細胞特異性標志：S-100和P75。應用RT-PCR檢測P0、Krox-20和Oct-6mRNA在SCs、dNSCs及NSCs中的表達情況。結果顯示,dNSCs和SCs中均有PO、Krox-20和Oct-6 mRNA的表達,SCs中的mRNA表達與相關報道相符。 3、不同濃度ERK1/2、P38、JNK抑制劑對神經(jīng)干細胞增殖和凋亡影響 當抑制劑濃度為5μM時,各組間未見明顯差異。而當抑制劑濃度為10、15μM時,P38組可見干細胞球逐漸增大,與對照組相比有明顯差異。3w時球中心細胞壞死,多個克隆球連接成片狀。ERK組、JNK組2w時全部死亡。TUNEL法檢測細胞凋亡,當抑制劑濃度為10μM時,與對照組相比, P38組細胞凋亡比例明顯減低,而ERK、JNK組細胞凋亡比例明顯升高。當抑制劑濃度為5μM時,與對照組相比,此3組細胞凋亡比例無明顯變化。 4、應用誘導劑后磷酸化及總ERK、P38和JNK的表達情況 Western blot顯示,神經(jīng)干細胞在加入誘導劑后1h即可見磷酸化ERK1/2水平升高,并持續(xù)增加,8h左右達到高峰,此后逐漸降低,恢復正常。 加入抑制劑后,與加入前相比,相應各組的ERK、P38和JNK的磷酸化水平顯著降低,并長時間維持在較低水平。 5、加入抑制劑后,神經(jīng)干細胞向施萬細胞誘導分化結果 3w后,與其它組相比,ERK組施萬樣細胞所占百分比明顯減少(P0.01)而P38組和JNK組與對照組相比,施萬樣細胞百分比未見明顯變化(P0.05) 結論 1、FSK、RA、HRG、PDGF-AA、bFGF誘導劑能誘導新生大鼠海馬神經(jīng)干細胞分化為施萬樣細胞。 2、神經(jīng)干細胞源性施萬樣細胞表達膠質細胞標志性蛋白：S-100和P75,表達P0、Krox-20、Oct-6等施萬細胞標志性mRNA;分泌促進神經(jīng)元軸突生長的可溶性營養(yǎng)因子。 3、神經(jīng)干細胞分化為施萬樣細胞過程中磷酸化ERK表達增強,ERK信號轉導通路被激活。 4、在神經(jīng)干細胞分化為施萬樣細胞過程中,加入ERK信號轉導通路抑制劑U0126,能夠抑制神經(jīng)干細胞分化為施萬樣細胞。P38、JNK信號轉導通路抑制劑SB203580和SP600125對神經(jīng)干細胞分化為施萬樣細胞無明顯作用。
[Abstract]:Preface
Schwann cells (SCs) is the main cell of the structure and function of the peripheral nervous system. It plays an important role in the development and regeneration of the peripheral nerve. After the peripheral nerve injury, Schwann cells proliferate, migrate, and can produce a variety of neurotrophic factors to promote and guide the growth of the distal nerve broken ends and to the different surrounding gods. Schwann cells have been applied to the treatment of the regenerative and demyelinating disease models of the clinical nervous system. However, the source of Schwann cells in vitro is limited, the culture cycle is long, and the proliferation and rejection of fibroblasts are easy to grow, so the isolation and purification of Schwann cells It is very difficult to achieve the number of treatment, and the clinical application is limited. Therefore, it is of great significance to find a source of Schwann cells, which is easy to obtain, the proliferation cycle is short and the immunogenicity is small, and it is of great significance for the repair and treatment of peripheral nerve injury.
Neural stem cells (NSCs) exists in many parts of the central nervous system and has the ability of self renewal and multidirectional differentiation. It can differentiate into neurons, astrocytes, oligodendrocytes and other central nervous system cells, as well as blood cells, and muscle cells. Neural stem cells have high proliferation activity. It can be cultured for a long time and keep its phenotype unchanged before differentiation into other cells. The advantages of low immunogenicity are used in the cell therapy of central nervous system disease. Because of its excellent potential of remolding the nerve tissue, it is considered to be a suitable donor cell for the regeneration of the nervous system. It can differentiate into Schwann like cells, but it is not reported whether neural stem cells derived from central nervous system can differentiate into Schwann cells in vitro.
The mitogenic activated protein kinase pathway (MAPK) pathway is very important for growth and differentiation. There are three main MAPK pathways in mammals: extracellular signal regulated kinase (kinase, ERK) pathway, and amino terminal kinase. JNK) pathway, P38 pathway. In which, ERK is the most basic signaling pathway to regulate cell proliferation, differentiation, and apoptosis. JNK, P38 pathway can be stimulated by stress, cytokines, growth factors, etc. in different cell lines, the role of MAPK pathway has been controversial, whether this pathway is induced by neural stem cells for Schwann like cells. The role of the course has not yet been reported.
In this study, the rat hippocampal neural stem cells were cultured with serum-free culture. Nestin immunofluorescence staining and differentiation ability were identified by single cell clone culture. Heregulin- beta 1, basic fibroblast growth factor (basic fibroblast growth factor, bFGF), and platelet derived growth factors were added to the culture medium. Platelet-derived growth factor-AA (PDGF-AA) and other inducers, neonatal rat hippocampal neural stem cells can differentiate into Schwann like cells, and further experimental application of extracellular signal regulated kinase (extracellular signal regulated kinase, ERK) pathway, c-jun amino terminal kinase (c-Jun N-terminal) pathway, pathway pathway The inhibitors were induced and differentiated to explore the role of these 3 pathways in the induction of neural stem cells to Schwann cells, and to provide theoretical and experimental basis for obtaining a large number of Schwann cells and the clinical treatment of neural defects.
Experimental recipe - Method
In this experiment, the rat hippocampal neural stem cells were cultured in vitro by serum-free culture, and the cultured neural stem cells were purified by single cell cloning technology. Immunofluorescence staining was used to identify the neural stem cells in the cultured cells. Immunofluorescence staining and Western Blot technique were used to determine the S-100 of neural stem cells after differentiation. And the expression of P75 protein, RT-PCR technique was used to detect the expression of P0, Krox-20, Oct-6 mRNA, and the function of the Schwann cells was detected by co culture with neurons. The effects of ERK, JNK, P38 pathway inhibitor on the differentiation of neural stem cells were detected by Western Blot and immunofluorescence staining.
experimental result
1, isolation, culture and identification of neural stem cells from hippocampus of neonatal rats
The neural stem cells isolated from the hippocampus of the newborn rats were neuroglobule, suspended and refracted, and the proliferation of the cells was faster than that of the primary cultured cells. The cloned cells formed by single cell clone culture expressed Nestin, and the cells expressed NSE, GFAP and Galc. after the induction of 1W differentiation.
2, neural stem cells induce differentiation
After adding the inducers of HRG, RA, FSK, and PDGF-AA to the culture medium, the morphological changes of the neural stem cells in the hippocampus of the newborn rats were changed. The immunofluorescence staining and the Western Blot detection showed that the cells expressed the specific markers of glial cells after the differentiation: S-100 and P75. used RT-PCR detection P0, Krox-20 and Oct-6mRNA were expressed and expressed. The results showed that PO, Krox-20 and Oct-6 mRNA were expressed in dNSCs and SCs, and mRNA expression in SCs was consistent with relevant reports.
3, the effects of different concentrations of ERK1/2, P38 and JNK inhibitors on proliferation and apoptosis of neural stem cells
When the inhibitor concentration was 5 M, there was no significant difference between each group. When the inhibitor concentration was 10,15 M, the P38 group showed that the stem cell balls gradually increased. Compared with the control group, there was a significant difference between the cells of the central cell necrosis at.3w and the multiple cloned spheres connected to a group of.ERK groups. The total death.TUNEL method was used to detect the apoptosis when the JNK group 2W, when the concentration of the inhibitor concentration was determined. When compared with the control group, the percentage of apoptotic cells in the P38 group was significantly lower than that of the control group, while the proportion of apoptosis in the group of ERK and JNK was significantly higher than that of the control group. When the inhibitor concentration was 5 mu M, there was no significant change in the percentage of apoptotic cells in the 3 groups compared with the control group.
4, phosphorylation and the expression of total ERK, P38 and JNK after induction.
Western blot showed that after adding inducer to the neural stem cells, the level of phosphorylated ERK1/2 increased and continued to increase, and the 8h reached the peak at about 8h, and then gradually decreased and returned to normal.
After adding inhibitors, the phosphorylation levels of ERK, P38 and JNK in the corresponding groups decreased significantly, and maintained at a low level for a long time.
5, after the inhibitor was added, the neural stem cells differentiated into Schwann cells.
After 3W, compared with other groups, the percentage of Schwann cells in group ERK decreased significantly (P0.01), but there was no significant change in the percentage of Schwann cells in the P38 and JNK groups (P0.05).
conclusion
1, FSK, RA, HRG, PDGF-AA and bFGF inducers can induce neural stem cells to differentiate into Schwann cells in neonatal rats.
2, neural stem cells derived Schwann cells express glial cell marker proteins, S-100 and P75, to express the marker mRNA of P0, Krox-20, Oct-6 and other Schwann cells, and secrete soluble nutrient factors that promote the growth of neuron axons.
3, the expression of phosphorylated ERK increased during the differentiation of neural stem cells into Schwann like cells, and the ERK signal transduction pathway was activated.
4, during the differentiation of neural stem cells into Schwann like cells, the addition of ERK signal transduction pathway inhibitor U0126 can inhibit the differentiation of neural stem cells into Schwann like cells.P38, and the JNK signal transduction pathway inhibitor SB203580 and SP600125 have no obvious effect on the differentiation of neural stem cells into Schwann like cells.
【學位授予單位】：中國醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2009
【分類號】：R329.28

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