【摘要】： 目的：探討無血清環(huán)境下聯(lián)合應用bFGF、PDGF、Ra、HRG、Forskolin對大鼠神經(jīng)干細胞誘導分化的作用。方法：用無血清培養(yǎng)技術從14-16天孕齡胚鼠腦中分離、培養(yǎng)神經(jīng)干細胞；用免疫細胞化學方法對神經(jīng)干細胞進行鑒定，并通過流式細胞儀檢測神經(jīng)干細胞的增殖能力；用傳2-3代的神經(jīng)干細胞接種于多聚賴氨酸包被的玻片，分以下四組，分別為兩步法組、一步法組、血清組和神經(jīng)干細胞培養(yǎng)組(bFGF組)；通過免疫細胞化學技術，用Map2和S100抗體鑒定分化的細胞；通過圖像分析系統(tǒng)來測量神經(jīng)元樣細胞的最長突起長度、胞體周長、胞體面積，神經(jīng)元細胞計數(shù)并計算各組Map2陽性的細胞比例。用全細胞膜片鉗技術記錄分化后細胞的鈉電流；并且用統(tǒng)計分析軟件對數(shù)據(jù)進行統(tǒng)計分析。結果：1、神經(jīng)干細胞的分離、培養(yǎng)、觀察和鑒定：從孕14—15天的胎鼠大腦皮層分離的細胞呈圓形，折光性好；培養(yǎng)6—7天后可以傳代，細胞球邊界清晰，折光性好，立體感強；免疫細胞化學方法鑒定細胞球內有較多Nestin陽性細胞。流式細胞儀檢測各代細胞球內均有較多細胞處于G1／M和S期。2．神經(jīng)干細胞的誘導分化：Map2的免疫熒光技術顯示一步法組Map2陽性神經(jīng)元樣細胞的比例最高，為43.35±13.75μm，，且一步法誘導出的神經(jīng)元樣細胞中有相當多細胞表達TH陽性。圖像分析結果顯示：一步法組與兩步法組細胞胞體較大，胞體面積分別為136.22±33.08μm~2、131.45±24.58μm~2，胞體周長分別為48.63±5.5μm、49.3±7.71μm，細胞突起長，最長突起分別為123.63±31.48μm、116.51±38.34μm。統(tǒng)計結果顯示：①Map2陽性神經(jīng)元樣細胞所占比例：一步法組與血清組有顯著差異(P＜0.05)，其它各組之間沒有顯著差異。②胞體面積及周長：一步法組與兩步法組之間沒有顯著差異，但它們和其余各組之間都有顯著差異。③突起的長度：一步法組與血清組、bFGF組有顯著差異，與兩步法組之間沒有顯著差異：兩步法組與bFGF組有顯著差異。電生理檢測顯示：一步法組中大部分形態(tài)成熟的神經(jīng)元樣細胞(80％)能誘發(fā)電壓依賴性的Na~+通道電流，兩步法僅有少量細胞(20％)能誘發(fā)電壓依賴性的Na~+通道電流，而未分化細胞及其它兩組細胞均未記錄到內向電流。結論：1．培養(yǎng)的原代及傳代后的大鼠胚胎皮層NSCs表達Nestin，并具有自我更新和多向分化的能力。2．在體外無血清條件下聯(lián)合應用bFGF、PDGF、RA、HRG、Forskolin對大鼠NSCs向神經(jīng)元定向誘導分化是可行的，能促進NSCs分化成神經(jīng)元。3．在體外無血清條件下，聯(lián)合應用5種因子的一步法和分兩步進行誘導的兩步法相比，一步法既能促進NSCs分化成更多的神經(jīng)元，也可促進分化后神經(jīng)元的成熟
[Abstract]:Aim: to investigate the effect of combined use of bFGF,PDGF,Ra,HRG,Forskolin in serum-free environment on differentiation of neural stem cells (NSCs) in rats. Methods: the neural stem cells were isolated from the brains of 14-16 days gestational embryos by serum-free culture technique, the neural stem cells were identified by immunocytochemistry, and the proliferative ability of neural stem cells was detected by flow cytometry. Two to three generations of neural stem cells were inoculated on the polylysine coated slides. They were divided into the following four groups: two-step group, one-step group, serum group and neural stem cell culture group (bFGF group). The differentiated cells were identified by Map2 and S100 antibodies by immunocytochemistry. The longest process length, cell body perimeter, cell body area, neuronal cell count and Map2 positive cell proportion were measured by image analysis system. The sodium currents of differentiated cells were recorded by whole-cell patch clamp technique, and the data were analyzed by statistical analysis software. Results: 1. Isolation, culture, observation and identification of neural stem cells: the cells isolated from fetal rat cerebral cortex were round and good refraction; After 6-7 days of culture, the boundary of the cell ball was clear, the refractive index was good, the stereosensitivity was strong, and many Nestin positive cells were identified by immunocytochemistry. Flow cytometry analysis showed that more cells were in G 1 / M and S phase in each generation. 2. The differentiation of neural stem cells: the proportion of Map2 positive neuron-like cells in Map2 group was the highest (43.35 鹵13.75 渭 m). Many of the neuron-like cells induced by one step method expressed TH positive. The results of image analysis showed that the cell bodies in the one-step group and the two-step group were larger, the cell body area was 136.22 鹵33.08 渭 m ~ 2131.45 鹵24.58 渭 m ~ (-2), the cell body circumference was 48.63 鹵5.5 渭 m ~ (-1) and 49.3 鹵7.71 渭 m, respectively. The longest protuberances were 123.63 鹵31.48 渭 m and 116.51 鹵38.34 渭 m respectively. The results showed that the proportion of 1Map2 positive neuron-like cells in the one-step group was significantly different from that in the serum group (P < 0. 05). There was no significant difference in cell body area and perimeter between other groups. 2 there was no significant difference between one-step group and two-step group, but there was significant difference between them and other groups. There was significant difference between bFGF group and two-step group. There was significant difference between two-step group and bFGF group. Electrophysiological examination showed that most of the mature neuron-like cells (80%) in the one-step group could induce voltage-dependent Na~ channel currents, and only a few cells (20%) could induce voltage-dependent Na~ channel currents in the two-step method. However, no inward currents were recorded in undifferentiated cells and in other two groups. Conclusion: 1. Primary and passage rat embryonic cortex NSCs express Nestin, and have the ability of self-renewal and multi-differentiation. 2. Combined use of bFGF,PDGF,RA,HRG, in serum-free condition in vitro It is feasible to induce the differentiation of rat NSCs into neurons by Forskolin, which can promote the differentiation of NSCs into neurons. 3. Under the condition of serum-free in vitro, the one-step method of five kinds of factors was used in combination with the two-step method of two-step induction. One-step method can not only promote the differentiation of NSCs into more neurons, but also promote the maturation of differentiated neurons.
【學位授予單位】：南通大學
【學位級別】：碩士
【學位授予年份】：2006
【分類號】：R329.1

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