本文選題：神經(jīng)干細(xì)胞 + 施萬(wàn)細(xì)胞��； 參考：《中國(guó)醫(yī)科大學(xué)》2009年博士論文

【摘要】： 前言 施萬(wàn)細(xì)胞(Schwann cells, SCs)是周圍神經(jīng)系統(tǒng)結(jié)構(gòu)和功能的主要細(xì)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(wàn)細(xì)胞進(jìn)行增殖、遷移,并能產(chǎn)生多種神經(jīng)營(yíng)養(yǎng)因子,促進(jìn)和引導(dǎo)遠(yuǎn)側(cè)神經(jīng)斷端的生長(zhǎng),向不同的周圍神經(jīng)支架移植施萬(wàn)細(xì)胞能加快神經(jīng)再生,因而施萬(wàn)細(xì)胞已經(jīng)應(yīng)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(wàn)細(xì)胞的來(lái)源有限,培養(yǎng)周期長(zhǎng),并且容易受增殖更快的成纖維細(xì)胞污染和排斥,所以施萬(wàn)細(xì)胞的分離、純化并達(dá)到治療的數(shù)量十分困難,臨床上的應(yīng)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(wàn)細(xì)胞來(lái)源,對(duì)周圍神經(jīng)損傷的修復(fù)治療具有重要的意義。 神經(jīng)干細(xì)胞(neural stem cells, NSCs)存在于中樞神經(jīng)系統(tǒng)的多個(gè)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(zhì)細(xì)胞、少突膠質(zhì)細(xì)胞等中樞神經(jīng)系統(tǒng)細(xì)胞以及血細(xì)胞、肌細(xì)胞等其他系統(tǒng)細(xì)胞。神經(jīng)干細(xì)胞因具有很高的增殖活性并能進(jìn)行長(zhǎng)期培養(yǎng)、在分化為其他細(xì)胞之前一直保持其表型不變、免疫原性低等優(yōu)點(diǎn)被用于中樞神經(jīng)系統(tǒng)疾病的細(xì)胞治療,由于其具有優(yōu)秀的重塑神經(jīng)組織的潛能,被認(rèn)為是神經(jīng)系統(tǒng)再生合適的供體細(xì)胞。有報(bào)道,脂肪干細(xì)胞源性神經(jīng)球能分化為施萬(wàn)樣細(xì)胞,但是體外培養(yǎng)的中樞神經(jīng)系統(tǒng)來(lái)源的神經(jīng)干細(xì)胞是否能分化為施萬(wàn)細(xì)胞尚未見(jiàn)報(bào)道。 促分裂原活化蛋白激酶(mitogenic activated protein kinase pathway, MAPK)通路對(duì)生長(zhǎng)和分化極為重要,哺乳動(dòng)物體內(nèi)存在的主要的三條MAPK通路：細(xì)胞外信號(hào)調(diào)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(diào)節(jié)細(xì)胞生長(zhǎng)增殖、分化、和凋亡的最基本信號(hào)途徑,JNK、P38通路可被應(yīng)激刺激、細(xì)胞因子、生長(zhǎng)因子等激活。在不同的細(xì)胞系中,MAPK通路起到何種作用一直存在爭(zhēng)議,此通路是否在神經(jīng)干細(xì)胞誘導(dǎo)為施萬(wàn)樣細(xì)胞過(guò)程中起到作用目前尚未見(jiàn)報(bào)道。 本研究采用無(wú)血清培養(yǎng)技術(shù)培養(yǎng)新生大鼠海馬神經(jīng)干細(xì)胞,通過(guò)單細(xì)胞克隆培養(yǎng)、Nestin免疫熒光染色及誘導(dǎo)分化能力進(jìn)行鑒定。通過(guò)向培養(yǎng)液中添加heregulin-β1、堿性成纖維細(xì)胞生長(zhǎng)因子(basic fibroblast growth factor, bFGF)、血小板源性生長(zhǎng)因子(platelet-derived growth factor-AA, PDGF-AA)等誘導(dǎo)劑,新生大鼠海馬神經(jīng)干細(xì)胞可以分化為施萬(wàn)樣細(xì)胞,進(jìn)一步實(shí)驗(yàn)應(yīng)用細(xì)胞外信號(hào)調(diào)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(jìn)行誘導(dǎo)分化干預(yù),探討這3條通路在神經(jīng)干細(xì)胞向施萬(wàn)樣細(xì)胞誘導(dǎo)中的作用,對(duì)獲得大量的施萬(wàn)樣細(xì)胞及臨床上進(jìn)行神經(jīng)缺損的細(xì)胞治療提供理論和實(shí)驗(yàn)基礎(chǔ)。 實(shí)驗(yàn)方-法 本實(shí)驗(yàn)通過(guò)無(wú)血清培養(yǎng)技術(shù)體外培養(yǎng)新生大鼠海馬神經(jīng)干細(xì)胞,應(yīng)用單細(xì)胞克隆技術(shù)對(duì)培養(yǎng)的神經(jīng)干細(xì)胞進(jìn)行純化,應(yīng)用免疫熒光染色對(duì)所培養(yǎng)細(xì)胞進(jìn)行神經(jīng)干細(xì)胞鑒定,應(yīng)用免疫熒光染色和Western Blot技術(shù)測(cè)定分化后神經(jīng)干細(xì)胞S-100和P75蛋白的表達(dá)情況,RT-PCR技術(shù)檢測(cè)P0、Krox-20、Oct-6 mRNA表達(dá)；通過(guò)與神經(jīng)元共培養(yǎng)的方法檢測(cè)施萬(wàn)樣細(xì)胞的功能,應(yīng)用Western Blot、免疫熒光染色技術(shù)檢測(cè)ERK、JNK、P38通路抑制劑對(duì)神經(jīng)干細(xì)胞誘導(dǎo)分化的影響。 實(shí)驗(yàn)結(jié)果 1、新生大鼠海馬神經(jīng)干細(xì)胞的分離培養(yǎng)和鑒定 新生大鼠海馬分離的神經(jīng)干細(xì)胞呈神經(jīng)球樣,懸浮生長(zhǎng),折光性強(qiáng),傳代后細(xì)胞較原代培養(yǎng)細(xì)胞增殖加快,單細(xì)胞克隆培養(yǎng)形成的克隆球表達(dá)Nestin,誘導(dǎo)分化1w后細(xì)胞表達(dá)NSE、GFAP及Galc。 2、神經(jīng)干細(xì)胞誘導(dǎo)分化結(jié)果 向培養(yǎng)液中添加HRG、RA、FSK、PDGF-AA等誘導(dǎo)劑后,新生大鼠海馬神經(jīng)干細(xì)胞的形態(tài)發(fā)生改變,免疫熒光染色及Western Blot檢測(cè)顯示分化后細(xì)胞表達(dá)膠質(zhì)細(xì)胞特異性標(biāo)志：S-100和P75。應(yīng)用RT-PCR檢測(cè)P0、Krox-20和Oct-6mRNA在SCs、dNSCs及NSCs中的表達(dá)情況。結(jié)果顯示,dNSCs和SCs中均有PO、Krox-20和Oct-6 mRNA的表達(dá),SCs中的mRNA表達(dá)與相關(guān)報(bào)道相符。 3、不同濃度ERK1/2、P38、JNK抑制劑對(duì)神經(jīng)干細(xì)胞增殖和凋亡影響 當(dāng)抑制劑濃度為5μM時(shí),各組間未見(jiàn)明顯差異。而當(dāng)抑制劑濃度為10、15μM時(shí),P38組可見(jiàn)干細(xì)胞球逐漸增大,與對(duì)照組相比有明顯差異。3w時(shí)球中心細(xì)胞壞死,多個(gè)克隆球連接成片狀。ERK組、JNK組2w時(shí)全部死亡。TUNEL法檢測(cè)細(xì)胞凋亡,當(dāng)抑制劑濃度為10μM時(shí),與對(duì)照組相比, P38組細(xì)胞凋亡比例明顯減低,而ERK、JNK組細(xì)胞凋亡比例明顯升高。當(dāng)抑制劑濃度為5μM時(shí),與對(duì)照組相比,此3組細(xì)胞凋亡比例無(wú)明顯變化。 4、應(yīng)用誘導(dǎo)劑后磷酸化及總ERK、P38和JNK的表達(dá)情況 Western blot顯示,神經(jīng)干細(xì)胞在加入誘導(dǎo)劑后1h即可見(jiàn)磷酸化ERK1/2水平升高,并持續(xù)增加,8h左右達(dá)到高峰,此后逐漸降低,恢復(fù)正常。 加入抑制劑后,與加入前相比,相應(yīng)各組的ERK、P38和JNK的磷酸化水平顯著降低,并長(zhǎng)時(shí)間維持在較低水平。 5、加入抑制劑后,神經(jīng)干細(xì)胞向施萬(wàn)細(xì)胞誘導(dǎo)分化結(jié)果 3w后,與其它組相比,ERK組施萬(wàn)樣細(xì)胞所占百分比明顯減少(P0.01)而P38組和JNK組與對(duì)照組相比,施萬(wàn)樣細(xì)胞百分比未見(jiàn)明顯變化(P0.05) 結(jié)論 1、FSK、RA、HRG、PDGF-AA、bFGF誘導(dǎo)劑能誘導(dǎo)新生大鼠海馬神經(jīng)干細(xì)胞分化為施萬(wàn)樣細(xì)胞。 2、神經(jīng)干細(xì)胞源性施萬(wàn)樣細(xì)胞表達(dá)膠質(zhì)細(xì)胞標(biāo)志性蛋白：S-100和P75,表達(dá)P0、Krox-20、Oct-6等施萬(wàn)細(xì)胞標(biāo)志性mRNA;分泌促進(jìn)神經(jīng)元軸突生長(zhǎng)的可溶性營(yíng)養(yǎng)因子。 3、神經(jīng)干細(xì)胞分化為施萬(wàn)樣細(xì)胞過(guò)程中磷酸化ERK表達(dá)增強(qiáng),ERK信號(hào)轉(zhuǎn)導(dǎo)通路被激活。 4、在神經(jīng)干細(xì)胞分化為施萬(wàn)樣細(xì)胞過(guò)程中,加入ERK信號(hào)轉(zhuǎn)導(dǎo)通路抑制劑U0126,能夠抑制神經(jīng)干細(xì)胞分化為施萬(wàn)樣細(xì)胞。P38、JNK信號(hào)轉(zhuǎn)導(dǎo)通路抑制劑SB203580和SP600125對(duì)神經(jīng)干細(xì)胞分化為施萬(wàn)樣細(xì)胞無(wú)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(xué)位授予單位】：中國(guó)醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R329.28

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