本文關(guān)鍵詞：細(xì)胞外鉀離子對體外培養(yǎng)神經(jīng)干細(xì)胞凋亡和分化的影響及其機(jī)制的研究　出處：《第四軍醫(yī)大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 鉀離子 神經(jīng)干細(xì)胞 細(xì)胞凋亡 分化 Hes1基因

【摘要】： 以往觀點(diǎn)認(rèn)為哺乳動物大腦的神經(jīng)形成和發(fā)生只存在于胚胎期和出生早期,而成體中樞中因神經(jīng)變性或損傷而缺失的神經(jīng)細(xì)胞則無法再生。近年來,在胚胎和成體大腦中發(fā)現(xiàn)了一類終末未分化細(xì)胞,稱為“神經(jīng)干細(xì)胞”,這類細(xì)胞終生保有自我更新能力,并具有分化成為星形膠質(zhì)細(xì)胞、少突膠質(zhì)細(xì)胞和神經(jīng)元的潛能。在生理狀態(tài)下,成年哺乳動物腦內(nèi)持續(xù)性神經(jīng)再生主要存在于側(cè)腦室的室管膜下區(qū)和海馬的顆粒細(xì)胞下層兩個(gè)的區(qū)域,而在腦缺血或腦外傷等病理狀態(tài)下,中樞神經(jīng)系統(tǒng)神經(jīng)再生能力增強(qiáng),這些再生的神經(jīng)細(xì)胞能夠與腦內(nèi)神經(jīng)回路進(jìn)行整合,參與神經(jīng)網(wǎng)絡(luò)重塑和修復(fù),但中樞神經(jīng)系統(tǒng)本身再生能力有限,因此神經(jīng)干細(xì)胞移植治療神經(jīng)系統(tǒng)疾病具有廣闊前景。研究發(fā)現(xiàn),由于病理狀態(tài)下微環(huán)境的改變,移植后細(xì)胞存活率下降并且神經(jīng)元分化率較低,所以研究調(diào)控神經(jīng)干細(xì)胞增殖、分化及凋亡的信號機(jī)制,對利用神經(jīng)干細(xì)胞治療神經(jīng)系統(tǒng)疾病具有重要意義。 鉀離子是細(xì)胞內(nèi)主要的陽離子,對維持細(xì)胞滲透壓和細(xì)胞膜的正常電活動等具有重要的意義。近年來的研究發(fā)現(xiàn),K+及K+通道在調(diào)節(jié)細(xì)胞增殖分化和凋亡上發(fā)揮著重要的功能,但目前關(guān)于K+對神經(jīng)干細(xì)胞分化及凋亡的影響及其機(jī)制還存在一定的爭議,國內(nèi)研究也較少。本研究利用體外培養(yǎng)模型獲得穩(wěn)定的胎鼠端腦神經(jīng)干細(xì)胞,并以此為基礎(chǔ)研究培養(yǎng)環(huán)境中K+及其濃度差異對體外神經(jīng)干細(xì)胞凋亡、增殖及分化的影響,初步探討了K+與神經(jīng)干細(xì)胞增殖分化相關(guān)基因Hes1的關(guān)系。通過本研究旨在進(jìn)一步探討細(xì)胞外K+對神經(jīng)干細(xì)胞的凋亡及分化的影響和其可能的機(jī)制,為神經(jīng)干細(xì)胞應(yīng)用提供理論基礎(chǔ)。 I小鼠神經(jīng)干細(xì)胞的原代培養(yǎng)、鑒定及不同濃度鉀離子對體外培養(yǎng)神經(jīng)干細(xì)胞凋亡的影響 第一部分小鼠神經(jīng)干細(xì)胞的體外培養(yǎng)及鑒定 目的通過穩(wěn)定可靠的神經(jīng)干細(xì)胞體外培養(yǎng)模型,獲得大量神經(jīng)干細(xì)胞。方法顯微下分離胎鼠端腦組織,培養(yǎng)液為無血清培養(yǎng)基中添加表皮生長因子和堿性成纖維生長因子,利用胰酶消化法培養(yǎng)并傳代。顯微鏡觀察細(xì)胞形態(tài),并利用免疫熒光法檢測培養(yǎng)細(xì)胞中巢蛋白(Nestin),及5%血清誘導(dǎo)分化7 d后細(xì)胞中神經(jīng)絲蛋白200(NF-200)和膠質(zhì)纖維酸性蛋白(GFAP)的表達(dá)。結(jié)果在無血清條件培養(yǎng)基條件下,分離培養(yǎng)的細(xì)胞持續(xù)增殖并形成神經(jīng)細(xì)胞球,免疫熒光顯示,培養(yǎng)細(xì)胞Nestin陽性,誘導(dǎo)后,可以分化為NF-200陽性或GFAP陽性的成熟神經(jīng)細(xì)胞。結(jié)論實(shí)驗(yàn)中原代培養(yǎng)細(xì)胞為神經(jīng)干細(xì)胞。 第二部分不同濃度鉀離子對神經(jīng)干細(xì)胞凋亡的影響及機(jī)制 目的研究不同濃度K+對神經(jīng)干細(xì)胞凋亡的影響及其機(jī)制的初步探討。方法體外培養(yǎng)獲得原代神經(jīng)干細(xì)胞,取第3～5代培養(yǎng)細(xì)胞,分為對照組(Control組),20 mM K+組,40 mM K+組和80 mM K+組,硝苯地平組(Nifedipin組)。對照組為基礎(chǔ)培養(yǎng)液(0.1% N2 +DMEM/F12),20 mM K+組、40 mM K+、80 mM K+為基礎(chǔ)培養(yǎng)液中添加KCL配制成相應(yīng)濃度,Nifedipin組為在80 mM K+組培養(yǎng)液基礎(chǔ)上添加硝苯地平配制20μmmol/L Nifedipin +80 mmol/L KCL培養(yǎng)液。利用MTT法檢測細(xì)胞活性,TUNEL染色分析各組細(xì)胞凋亡,Western blot法檢測Caspase-3活性片段表達(dá),Hoechst33342染色觀察凋亡細(xì)胞核變化。結(jié)果20 mM K+組、40 mM K+組和80 mM K+組細(xì)胞增殖活性與對照組相比均降低(0.455±0.006、0.44±0.007、0.226±0.017 VS 0.61±0.012,P0.01),其中80 mM K+組活性最低。TUNEL陽性細(xì)胞數(shù)統(tǒng)計(jì)分析結(jié)果顯示,80 mM K+組明顯高于對照組[(27.3±5.3)% VS (7±1.4)%,P0.01],20 mM K+組較對照組相比降低[(4.8±1.2)% VS (7±1.4)%,P0.05],40 mmol/L K+組無顯著變化[(5.4±1.5)% VS (7±1.4)%,P0.05],80 mmol/L K+組Caspase-3活性片段表達(dá)高于其余各組。硝苯地平組TUNEL陽性細(xì)胞率與80 mmol/L K+組相比顯著降低[(10.4±2.1)% VS (26.2±5.7)%,P0.01]。結(jié)論升高細(xì)胞外濃度K+可以抑制神經(jīng)干細(xì)胞活性,且過高濃度K+可以誘導(dǎo)神經(jīng)干細(xì)胞凋亡,而膜Ca2+通道的開放和細(xì)胞內(nèi)Ca2+的增加可能是誘導(dǎo)凋亡發(fā)生的機(jī)制之一。 II細(xì)胞外鉀離子對神經(jīng)干細(xì)胞增殖分化的影響 目的探討細(xì)胞外K+濃度對神經(jīng)干細(xì)胞增殖分化的影響,并分析Hes1基因表達(dá)的差異。方法體外培養(yǎng)獲得原代神經(jīng)干細(xì)胞,取第3～5代培養(yǎng)細(xì)胞,分為正常培養(yǎng)組(Normal組),普通誘導(dǎo)分化組(Control組),20mM K+組和40mM K+組。正常對照組為基礎(chǔ)培養(yǎng)液(DMEM/F12、0.1% N2),普通誘導(dǎo)分化組為普通誘導(dǎo)分化液(DMEM/F12、5% FBS、0.1% N2),20mM K+和40mM K+組培養(yǎng)液分別為20 mM /40 mM KCL、0.1% N2、DMEM/F12。MTT法和臺盼蘭排除實(shí)驗(yàn)檢測細(xì)胞活性,免疫熒光法檢測細(xì)胞Nestin及NF-200表達(dá),RT-PCR法檢測Hes1基因mRNA表達(dá)水平。結(jié)果與正常培養(yǎng)組相比,36 h后20 mM K+組和40 mM K+組細(xì)胞活性均降低(0.379±0.006,0.372±0.007 VS 0.435±0.012,P0.01),而死亡細(xì)胞數(shù)檢測無明顯差異[(8.2±1.3)%, (9.7±2.0)% VS (9.0±1.5)%,P0.05]。與普通誘導(dǎo)分化組相比,干預(yù)2 d后20 mM K+組和40 mM K+組Nestin陽性細(xì)胞百分比降低[(51.4±7.2)%, (49.1±5.6)% VS (75.7±8.2)%,P0.01)],分化7 d后NF-200表達(dá)升高[(65.1±5.5)%, (62.2±6.1) % VS (39.4±4.3)%, P0.01)],RT-PCR結(jié)果顯示,與普通分化組相比20 mM K+組和40 mM K+組細(xì)胞Hes1mRNA水平上表達(dá)降低。結(jié)論細(xì)胞外K+抑制神經(jīng)干細(xì)胞增殖并促進(jìn)神經(jīng)干細(xì)胞向神經(jīng)元分化,對Hes1基因表達(dá)的抑制可能是其影響分化的機(jī)制之一。
[Abstract]:The previous view and exists only in the embryonic and early postnatal formation of the mammalian brain nerve, and adult central nerve degeneration or injury due to lack of nerve cells cannot regenerate. In recent years, in the embryonic and adult brain found one terminal at the end of the differentiation of cells, called neural stem cells "these cells maintain life-long self-renewal capacity, and could differentiate into astrocytes, oligodendrocytes and neurons potential. Under physiological condition, the adult mammalian brain persistent nerve regeneration mainly exists in the subventricular zone and the subgranular zone of the hippocampus under two area, and in the state of cerebral ischemia or cerebral injury and other pathological conditions, enhance the regeneration of Central nerve regeneration, these nerve cells in the brain and neural circuits can be integrated in the network remodeling and nerve repair, but The central nervous system itself regeneration ability is limited, so neural stem cell transplantation in the treatment of diseases of the nervous system has broad prospects. The study found that due to the pathological state of micro environment changes after transplantation, cell survival rate decreased and neuronal differentiation rate is low, so the study on the regulation of neural stem cell proliferation, differentiation and apoptosis signaling mechanism, has important significance the use of neural stem cells in treatment of nervous system diseases.
Potassium is the major intracellular cation, plays an important role in the maintenance of cell osmotic pressure and membrane normal electrical activity. Recent studies have found that K+ and K+ channels play an important function in the regulation of cell proliferation differentiation and apoptosis, but there are still some controversy about the effect of K+ on cell differentiation and apoptosis of neural stem and its mechanism, the domestic study is less. This study culture model to obtain stable fetal rat telencephalon neural stem cells in vitro, and on the basis of research on K+ and its concentration in the environment of culture differences on stem cell apoptosis in vitro proliferation and differentiation of nerve, the relationship between K+ and proliferation of neural stem cells differentiation gene Hes1 was discussed. The aim of this study was to further explore the extracellular K+ on neural stem cell differentiation and apoptosis effect and its possible mechanism, to provide for the application of neural stem cells Theoretical basis.
Primary culture of neural stem cells in I mice and identification of the effects of different concentrations of potassium ions on the apoptosis of neural stem cells in vitro
In vitro culture and identification of mouse neural stem cells in vitro
Objective to cultivate a stable and reliable model of the neural stem cells in vitro, to obtain a large number of neural stem cells in fetal rat brain. The separation method of the microscopic end, the medium was in serum-free medium containing epidermal growth factor and basic fibroblast growth factor, cultured and passaged by trypsin digestion method. Cell morphology was observed by microscope, and the detection of cell culture nestin by immunofluorescence (Nestin), and 5% serum induced differentiation in cells after 7 d neurofilament 200 (NF-200) and glial fibrillary acidic protein (GFAP) expression. Results in serum-free medium conditions, cultured cells continue to proliferate and form neural cells, immunofluorescence showed that cultured cells were Nestin positive after induction can differentiate into NF-200 positive or GFAP positive mature nerve cells. Conclusion the primary cultured cells into neural stem cells.
The effect and mechanism of different concentrations of potassium ion on neural stem cell apoptosis in the second part
Objective to study the effect of different concentration of K+ on apoptosis of neural stem cells and its mechanism. The primary neural stem cells cultured in vitro cell culture, from third to 5, divided into control group (Control group), 20 mM K+ 40 mM group, K+ group and 80 mM K+ group, nifedipine group (Nifedipin group). The control group culture fluid based (0.1% N2 +DMEM/F12), mM 20 K+ group, 40 mM K+, 80 mM K+ based medium supplemented with KCL prepared by the corresponding concentration, Nifedipin group as the culture medium added with 20 mmol/L Nifedipin +80 nifedipine mmol/L KCL cultured in 80 mM K+ group. Cell viability was measured by MTT assay, cell apoptosis was analyzed by TUNEL staining, detect the expression of Caspase-3 fragment Western blot method, to observe the apoptotic changes of Hoechst33342 staining. Results of the 20 mM K+ 40 mM group, K+ group and 80 mM group K+ cell proliferation activity was decreased compared with the control group Low (0.455 + 0.006,0.44 + 0.007,0.226 + 0.017 VS 0.61 + 0.012, P0.01), of which 80 mM K+ group was the lowest number of.TUNEL positive cells in statistics, 80 mM in group K+ was significantly higher than the control group [(27.3 + 5.3)% VS (7 + 1.4)%, P0.01], mM 20 K+ group than in the control group lower than [(4.8 + 1.2)% VS (7 + 1.4)%, P0.05] 40, mmol/L K+ group had no significant change [(5.4 + 1.5)% VS (7 + 1.4)%, P0.05] 80, mmol/L K+ in Caspase-3 group was higher than that of the other groups. The expression of TUNEL positive cells compared with nifedipine group with the 80 mmol/L K+ group was significantly reduced [(10.4 + 2.1)% VS (26.2 + 5.7)%, P0.01]. conclusion elevated extracellular concentration of K+ can inhibit the activity of neural stem cells, and high concentration of K+ can induce apoptosis of neural stem cells, and the cell membrane and open Ca2+ channels in the increase of Ca2+ may be one of the induction machine for the occurrence of apoptosis.
Effect of II extracellular potassium on the proliferation and differentiation of neural stem cells
Objective to investigate the effects of extracellular K+ concentration on the proliferation and differentiation of neural stem cells, and analyze the difference of Hes1 gene expression. The primary neural stem cells cultured in vitro cell culture, from third to 5, divided into the normal group (Normal group), normal differentiation group (Control group), 20mM group and K+ the 40mM K+ group. Normal control group based medium (DMEM/F12,0.1% N2), the common differentiation group is a common differentiation medium (DMEM/F12,5% FBS, 0.1% N2), 20mM K+ and 40mM K+ were cultured respectively 20 mM /40 mM KCL, 0.1% N2, DMEM/F12.MTT assay and trypan blue exclusion cell activity assay. The expression of Nestin cells were detected by immunofluorescence and NF-200 to detect the expression of Hes1 gene mRNA RT-PCR method. Results compared with the normal culture group, after 36 h mM 20 K+ group and 40 mM group K+ cells activity were significantly decreased (0.379 + 0.006,0.372 + 0.007 VS 0.435 + 0.012, P0.01), and the death of fine No significant differences in cell number detection [(8.2 + 1.3)% and (9.7 + 2)% VS (9 + 1.5)%, compared with ordinary P0.05]. induced group, intervention after 2 D 20 mM 40 mM K+ group and K+ group the percentage of Nestin positive cells decreased [(51.4 + 7.2)%, (49.1 + 5.6)% VS (75.7 + 8.2)%, P0.01), 7 d after differentiation increased expression of NF-200 [(65.1 + 5.5)% and (62.2 + 6.1)% VS (39.4 + 4.3)%, P0.01), RT-PCR results showed that compared with the ordinary differentiation group 20 K+ group and 40 mM mM K+ cells Hes1mRNA expression level decreased. Conclusion K+ inhibits neural stem cells and neural stem cells into neurons and promote cell proliferation and differentiation, inhibit the expression of Hes1 gene may be one of the mechanism of the differentiation of its effects.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R329

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