【摘要】： 神經(jīng)干細(xì)胞(neural stem cells, NSCs)是一種具有自我更新能力的細(xì)胞,可增殖分化為中樞神經(jīng)系統(tǒng)內(nèi)的3種細(xì)胞:神經(jīng)元、星形膠質(zhì)細(xì)胞和少突膠質(zhì)細(xì)胞。因其具有一定的自我更新和分化能力,從而在臨床上有廣闊的應(yīng)用前景。新近研究表明,脊髓損傷后,在損傷局部移植NSCs能促進(jìn)成年動物模型的功能恢復(fù),但移植的NSCs主要分化為膠質(zhì)細(xì)胞,形成膠質(zhì)瘢痕,阻礙了神經(jīng)功能進(jìn)一步恢復(fù)。腦源性神經(jīng)營養(yǎng)因子(brain -derived neurotrophic factor BDNF)是Barde等1982年從豬腦中提取的含量較低的堿性蛋白,是神經(jīng)營養(yǎng)因子家族中最具有代表性的成員之一,許多的研究顯示,腦源性神經(jīng)營養(yǎng)因子能夠促進(jìn)神經(jīng)干細(xì)胞向神經(jīng)元方向分化。 因此我們選擇神經(jīng)干細(xì)胞和腦源性神經(jīng)營養(yǎng)因子作為研究對象,主要探討了腦源性神經(jīng)營養(yǎng)因子對神經(jīng)干細(xì)胞增殖及其分化的影響,尋找合適的誘導(dǎo)時間和誘導(dǎo)濃度。同時研究了腦源性神經(jīng)營養(yǎng)因子的兩個受體TrkB和P75在腦源性神經(jīng)營養(yǎng)因子促進(jìn)神經(jīng)干細(xì)胞向神經(jīng)元方向分化中的作用。 本文主要分為兩部分 第一部分神經(jīng)干細(xì)胞的體外培養(yǎng)、分化及其鑒定 目的 掌握新生SD大鼠神經(jīng)干細(xì)胞體外分離培養(yǎng)的方法,并對其生長特性和光、電鏡結(jié)構(gòu)進(jìn)行觀察。 方法 1從出生1-3d的SD大鼠腦組織中分離神經(jīng)干細(xì)胞進(jìn)行原代培養(yǎng)。 2采用不同的接種密度、不同的傳代方法,在體外培養(yǎng)神經(jīng)干細(xì)胞,觀察細(xì)胞的生長情況。 3 Nestin、Brdu、NSE、GFAP免疫細(xì)胞化學(xué)對神經(jīng)干細(xì)胞進(jìn)行鑒定。 4透射電子顯微鏡觀察神經(jīng)球的超微結(jié)構(gòu)。 結(jié)果和結(jié)論 1培養(yǎng)的細(xì)胞Nestin陽性,具有多向分化和自我增殖能力(Brdu、NSE、GFAP陽性),是神經(jīng)干細(xì)胞。本方法簡單、易操作,可行性強(qiáng)。 2體外培養(yǎng)神經(jīng)干細(xì)胞接種密度為1×106/ml時,細(xì)胞增殖速度快,克隆球生成的數(shù)量多。傳代的方法應(yīng)視情況而定,傳代時間為7d左右。 3透鏡結(jié)果:未分化的NSCs,細(xì)胞核漿比例比較大,核圓形或橢圓形。胞漿內(nèi)的細(xì)胞器比較少,核糖體豐富。分化的NSCs,核漿比例縮小,胞漿內(nèi)細(xì)胞器開始增多,可見到高爾基復(fù)合體、線粒體、微絲微管等。 第二部分腦源性神經(jīng)營養(yǎng)因子對神經(jīng)干細(xì)胞增殖和分化的影響 目的 探討B(tài)DNF對NSCs增殖與分化的影響,尋找合適的誘導(dǎo)時間和誘導(dǎo)濃度,同時研究BDNF兩個受體TrkB和P75在促NSCs分化為神經(jīng)元中的作用。 方法 1 BDNF對NSCs增殖的影響。實(shí)驗(yàn)分為4個組,各組按照BDNF的濃度不同分為對照組0ng/ml實(shí)驗(yàn)組2ng/ml、20ng/ml、200ng/ml。MTT法檢測實(shí)驗(yàn)組和對照組在不同的時間點(diǎn)(1d、3d、5d、7d)對細(xì)胞的增殖效應(yīng),并繪制生長曲線。 2觀察BDNF對NSCs分化的影響,主要是采用免疫細(xì)胞化學(xué)技術(shù)。在體外誘導(dǎo)分化NSCs,分別在誘導(dǎo)后的1d、3d、5d、7d取出細(xì)胞,行免疫細(xì)胞化學(xué)染色,并計算陽性率。對比分析各組在各個時間段的細(xì)胞分化為神經(jīng)元的能力,探索促神經(jīng)元分化的最佳濃度和最佳時間。實(shí)驗(yàn)分組同上MTT測定。 3使用RT-PCR技術(shù)檢測TrkB、p75 mRNA水平的變化。實(shí)驗(yàn)分為三組:未分化組,分化后未加干預(yù)組和分化后加入20ng/mlBDNF組。未分化組在傳代后第三天,分化組在誘導(dǎo)分化后的第1 d、3d、5d分別檢測上述三個指標(biāo)的變化。探討在BDNF的干預(yù)下,NSCs分化為神經(jīng)元過程中,BDNF的兩個受體的表達(dá)。 結(jié)果 1 MTT的統(tǒng)計結(jié)果表明:不同濃度的BDNF在不同的時間,所測得490nm處的OD值與對照組相比均無顯著性差異,P0.05。 2隨著BDNF濃度的增加,神經(jīng)干細(xì)胞分化為神經(jīng)元的比率也呈增高的趨勢,差異顯著,P0.05。在分化的時間上,各組均是在第3天時達(dá)到最高,其中,實(shí)驗(yàn)組最高接近80%,而對照組僅為35%。 3 RT-PCR的結(jié)果顯示:BDNF的兩個受體TrkB和p75在未分化的NSCs均有所表達(dá)。BDNF在促NSCs分化的過程中,在分化后的1d、3d、5d,TrkB表達(dá)與對照組相比均有所上升,差異顯著P0.05。p75表達(dá)與對照組相比則無顯著性差異P0.05。 結(jié)論 1 BDNF并不能明顯的促進(jìn)NSCs的增殖。 2 BDNF能顯著的促進(jìn)NSCs向神經(jīng)元方向分化,其分化的比率呈劑量依賴性,并且在分化后的第三天達(dá)到最高。 3 BDNF在促NSCs向神經(jīng)元方向分化過程中,主要是通過上調(diào)TrkB受體發(fā)揮作用,p75無明顯作用。
[Abstract]:Neural stem cells (NSCs) are a kind of cell with self-renewal ability, and can proliferate and differentiate into three kinds of cells in the central nervous system: neurons, astrocytes and oligodendrocytes. because of its self-renewal and differentiation ability, it has a wide application prospect in clinic. Recent studies have shown that, after spinal cord injury, NSCs can promote the functional recovery of adult animal models after spinal cord injury, but the transplanted NSCs mainly differentiate into the glial cells to form the glial scar, which is an obstacle to the further recovery of the neurological function. Brain-derived neurotrophic factor (BDNF) is a low-content basic protein extracted from pig brain in 1982, and is one of the most representative members in the family of neurotrophin. The brain-derived neurotrophic factor can promote the differentiation of neural stem cells into the neurons. So we selected the neural stem cells and the brain-derived neurotrophic factor as the research object, mainly discussed the effects of the brain-derived neurotrophic factor on the proliferation and differentiation of the neural stem cells, and looked for the appropriate induction time and induction. At the same time, the two receptors, TrkB and P75 of the brain-derived neurotrophic factor, were studied to promote the differentiation of the neural stem cells to the neuron in the brain-derived neurotrophic factor. The present invention The paper is divided into two parts: the first part of the neural stem cells body Method for culturing and culturing new SD rat neural stem cells in vitro for external culture, differentiation and identification purposes and The growth characteristics and the structure of light and electron microscope were observed in this paper. primary culture of isolated neural stem cells from SD rat brain tissue of 1-3d with different inoculation Density, different passage methods, cultured neural stem cells in vitro to observe the growth of the cells. estin, Brdu, NSE, GFAP immunocytochemical the identification of neural stem cells. The ultrastructures of the neurospheres were observed by a transmission electron microscope. Nestin-positive, with multiple-orientation The method is simple and easy to operate, strong feasibility. In vitro culture of neural stem cells. In the case of 1-106/ ml, the rate of cell proliferation is fast, and the number of cloned balls is much higher. The method shall be determined according to the condition and the passage time is 7days left. The results of the right. 3 lens: the non-differentiated NSCs, the proportion of the cytoplasm of the nucleus is large, the nucleus is circular or elliptical, and the organelles in the cytoplasm The number of NSCs and the cytoplasm of the differentiated NSCs were reduced, and the cytoplasm and the cytoplasm of the NSCs were reduced. inner fine The effects of the second part of the brain-derived neurotrophic factor on the proliferation and differentiation of neural stem cells are discussed in this paper. The NF The effect of the proliferation and differentiation of NSCs and the finding The effect of the two receptors TrkB and P75 on the proliferation of NSCs was studied. The effect of BDNF on the proliferation of NSCs was studied. The experiment was divided into 4 groups. Test group: 2ng/ ml, 20ng/ ml, 200ng/ ml. MTT assay the proliferation of the cells was observed at different time points (1d, 3d, 5d, 7d) in the experimental group and the control group, and the growth curve was plotted. The effect of DNF on the differentiation of NSCs is mainly the use of immunocytochemistry. after the guide 1d, 3d, 5d, 7. The cells were taken out, the immunocytochemical staining was performed, and the positive rate was calculated. The cell differentiation of each time period is the ability of the neuron to explore the optimal concentration and the best time for the differentiation of the neuron. The changes of TrkB and p75 mRNA levels were detected by RT-PCR. The experiment was divided into three groups: non-differentiation group, no intervention group after differentiation, and after differentiation, 20ng/ ml of BDNF group was added. Not divided In the third day after passage, the differentiation group detected the changes of the three indices in the first day, the third day and the third day after induction of differentiation. In the process of neurons, the expression of the two receptors of BDNF. At different time, the OD value at 490nm was not significantly different from that of the control group, P <0.05. 2. With the increase of the concentration of BDNF, the ratio of the neural stem cells to the neurons was also increased, the difference was significant, P The results of RT-PCR showed that the two receptors, TrkB and p, were the highest in the experimental group and 35% in the control group. 75 Expression of BDNF in undifferentiated NSCs. In the process of NSCs differentiation, the expression of TrkB in the differentiated 1d, 3d, 5d, and TrkB was significantly higher than that of the control group (P0.05). The expression of p75 was no significant difference between the control group and the control group (P <0.05). Conclusion 1BDNF does not significantly promote the NS.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R329

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