【摘要】： 目的： 研究大鼠骨髓間充質(zhì)干細(xì)胞(BMSCs)體外分離、培養(yǎng)和擴(kuò)增方法,使其保持干細(xì)胞特性--自我更新和多向分化潛能；并探討腦活素及法舒地爾體外誘導(dǎo)骨髓間充質(zhì)干細(xì)胞增殖分化為神經(jīng)元樣細(xì)胞的可能性和條件,為神經(jīng)系統(tǒng)損傷和神經(jīng)退行性疾病的治療提供良好的理論基礎(chǔ)和實(shí)驗(yàn)依據(jù)。 方法： 本課題選取150g左右SD大鼠股骨和脛骨骨髓做骨髓間充質(zhì)干細(xì)胞的分離培養(yǎng),采用全骨髓直接貼壁法獲得原代大鼠骨髓BMSCs,差速貼壁結(jié)合消化控制法純化細(xì)胞。MTT法檢測BMSCs生長最佳血清濃度及生長曲線。流式細(xì)胞儀細(xì)胞檢測BMSCs生長周期及表面抗原CD34、CD44、CD29、CD90、CD45和CD31的表達(dá)。BMSCs經(jīng)bFGF預(yù)誘導(dǎo)后,使用腦活素和法舒地爾誘導(dǎo)骨髓間充質(zhì)干細(xì)胞,并用倒置相差顯微鏡觀察記錄細(xì)胞的形態(tài)學(xué)變化；細(xì)胞免疫組織化學(xué)法檢測誘導(dǎo)后各組細(xì)胞巢蛋白(Nestin)、神經(jīng)絲蛋白(NF),神經(jīng)元特異烯醇化酶(NSE)、膠質(zhì)纖維酸性蛋白(GFAP)等神經(jīng)細(xì)胞特異性標(biāo)志物的表達(dá)情況。 結(jié)果： 1.大鼠BMSCs的分離、培養(yǎng)和鑒定 (1)通過貼壁法能獲得純度較高的BMSCs,原代培養(yǎng)3天后細(xì)胞呈紡錘形。7天后細(xì)胞呈團(tuán)簇、集落狀生長。細(xì)胞傳代3代后仍增殖旺盛呈梭形。 (2)10%血清濃度為BMSCs生長最佳濃度。 (3)從傳代細(xì)胞生長曲線可見：第1-2天為細(xì)胞生長潛伏期,第3-4天為對(duì)數(shù)增長期,6天以后細(xì)胞的生長進(jìn)入平臺(tái)期,符合干細(xì)胞的生長規(guī)律。 (4)細(xì)胞周期分析顯示：處于G0/G1期的細(xì)胞為93.99%,處于非增殖狀態(tài),S期的細(xì)胞為5.46%,G2/M0期的細(xì)胞為0.54%。G0/G1期占整個(gè)細(xì)胞群的比例達(dá)93.99%,同時(shí)也說明了BMSCs的高分化潛能。 (5)流式細(xì)胞儀檢測細(xì)胞表面標(biāo)志：表達(dá)CD90、CD29、CD44,不表達(dá)CD34、CD45、CD31。 2.體外誘導(dǎo)BMSCs后的形態(tài)學(xué)變化 (1)腦活素體外誘導(dǎo)BMSCs增殖分化 以10ng/mlbFGF預(yù)誘導(dǎo)24小時(shí)后,細(xì)胞形態(tài)較預(yù)誘導(dǎo)前未發(fā)生改變。接著以腦活素誘導(dǎo)后,細(xì)胞生長狀態(tài)較未加入腦活素前好,細(xì)胞數(shù)量增多,且具有統(tǒng)計(jì)學(xué)意義(P0.05),但細(xì)胞形態(tài)改變不明顯,細(xì)胞收縮,出現(xiàn)三角形樣細(xì)胞,但未見明顯細(xì)胞突起。 (2)法舒地爾體外誘導(dǎo)BMSCs增殖分化 法舒地爾誘導(dǎo)組誘導(dǎo)6小時(shí)后細(xì)胞形態(tài)逐漸發(fā)生變化,出現(xiàn)雙極形、多極形和錐形細(xì)胞,呈神經(jīng)元樣細(xì)胞。誘導(dǎo)24小時(shí)后,可見較多神經(jīng)元樣細(xì)胞,突觸逐漸形成并增多,細(xì)胞突起相互交織呈現(xiàn)網(wǎng)絡(luò)狀連接。誘導(dǎo)48小時(shí)后,細(xì)胞突觸發(fā)生斷裂溶解,網(wǎng)絡(luò)狀連接消失,細(xì)胞數(shù)量減少。 3免疫組織化學(xué)染色結(jié)果 對(duì)照組Nestin、NSE、NF、GFAP抗體均未見陽性染色。腦活素組中免疫組化可見少數(shù)Nestin、NSE、NF、GFAP染色陽性細(xì)胞,與空白對(duì)照組相比,無顯著性差異(P0.05)。 法舒地爾誘導(dǎo)實(shí)驗(yàn)組：誘導(dǎo)6小時(shí)后Nestin、NSE、NF、GFAP抗體染色呈陽性反應(yīng),與腦活素組及空白對(duì)照組相比,有顯著性差異(P0.05)。誘導(dǎo)24小時(shí)后Nestin、NF、GFAP抗體染色陽性率高于6小時(shí),有顯著性差異(P0.05)；NSE陽性率低于6小時(shí),無顯著性差異(P0.05)。誘導(dǎo)48小時(shí)后,Nestin、NF、GFAP陽性率高于24小時(shí),有顯著性差異(P0.05)；NSE陽性率低于24小時(shí),無顯著性差異(P0.05)。 Nestin、NF與GFAP:隨誘導(dǎo)時(shí)間延長陽性率逐漸增高,各組差異均有顯著性(P0.01)。 NSE：陽性細(xì)胞率6小時(shí)48小時(shí)24小時(shí),差異無顯著性(P0.05)。 結(jié)論： 通過貼壁篩選法體外分離、培養(yǎng),傳代后可獲得純度較高的骨髓間充質(zhì)干細(xì)胞。腦活素誘導(dǎo)BMSCs后細(xì)胞形態(tài)改變不明顯,胞體收縮,出現(xiàn)三角形樣細(xì)胞,但未見明顯細(xì)胞突起；法舒地爾在體外可誘導(dǎo)BMSCs分化為神經(jīng)元樣細(xì)胞,免疫組化顯示誘導(dǎo)細(xì)胞表達(dá)Nestin、NSE、NF、GFAP等神經(jīng)細(xì)胞特異性標(biāo)記物。為進(jìn)一步研究BMSCs應(yīng)用于神經(jīng)系統(tǒng)損傷和神經(jīng)退行性疾病的細(xì)胞治療提供了良好的實(shí)驗(yàn)基礎(chǔ)。
[Abstract]:Purpose: To study the isolation, culture and amplification of bone marrow mesenchymal stem cells (BMSCs) in rats to keep the characteristics of stem cells--self-renewal and multi-orientation To explore the potential and condition of the proliferation and differentiation of mesenchymal stem cells into neuron-like cells in vitro, and to provide a good theoretical basis for the treatment of nervous system injury and neurodegenerative diseases. Basis for inspection Methods: The bone marrow of the left and right SD rats and the bone marrow of the tibia were selected to be isolated and cultured. The bone marrow BMSCs and the differential apposed of the bone marrow of the primary rat were obtained by using the full-bone marrow direct attachment method. Purification of cells by digestion control method. The growth of BMSCs was detected by MTT assay. Best serum concentration and growth curve. The growth cycle of BMSCs and surface antigen CD34, CD44, CD29, CD90, and C were detected by flow cytometry. Expression of D45 and CD31. After the BMSCs were pre-induced with bFGF, the bone marrow mesenchymal stem cells were induced by using the brain activin and the method, and the morphological changes of the cells were observed with an inverted phase-contrast microscope. Nerve cells such as neurofilament protein (NF), neuron-specific enolase (NSE), glial fibrillary acidic protein (GFAP), and the like specificity The expression of the marker. 1. Isolation, culture and identification of BMSCs in rats (1) BMSCs with higher purity can be obtained by the method of apposition, and the primary culture 3 The cells were in the shape of a spindle after the day. The cells were in a group after 7 days. cluster, colony-like growth. (2) The concentration of 10% serum was the best concentration of BMSCs. (3) From the growth curve of the passage cells, the incubation period of the cells was 1-2 days, and the third-fourth day was the cell growth latent period. The cell cycle analysis showed that the cells in the G0/ G1 phase were 90.99%, the cells in the non-proliferation state, the S-phase cells were 5.46%, and the cells in the G2/ M0 stage were 0.54%. G0/ G1 The proportion of the whole cell population was 90.99%, and the high differentiation potential of BMSCs was also described. (5) Flow cytometry was used to detect the cells. Surface markers: expression of CD90, CD29, C D44, not expressing CD34, CD45, CD 31.2. Morphological changes (1) brain activity after induction of BMSCs in vitro In vitro, the proliferation and differentiation of BMSCs were pre-induced by 10 ng/ ml bFGF for 24 hours, and the cell morphology was not changed before the pre-induction. and had statistical significance (P0.05), but the cells The morphological changes were not clear, the cells were contracted, triangular-like cells were present, but no significant cell projections were found. (2) in-vitro induction of the method The proliferation and differentiation of BMSCs induced a gradual change in the morphology of the cells after 6 hours of induction, and the two-pole, multipolar, in that form of a neuron-like cell, in the form of a neuron-like cell, more neuron-like cells are visible after 24 h of induction. The synapses gradually form and increase, fine. The cells of the cells were in the form of a network-like connection. After 48 hours of induction, the cells of the cells were broken. in that control group, Nestin, NSE, NF and GFAP were all of the control group. No positive staining was found. The positive cells of Nestin, NSE, NF and GFAP were found in the brain activin group, and there was no significant difference compared with the blank control group (P0.05). The results showed that in the experimental group, the staining of Nestin, NSE, NF and GFAP was positive in 6 hours, and there was a significant difference (P0.05). The positive rate of Nestin, NF and GFAP was higher than 6 hours after 24 hours, and there was a significant difference (P0.05). The positive rate of NSE was lower than 6 hours without significant difference (P0.05). After 48 hours, the positive rate of Nestin, NF and GFAP was higher than 24 hours, and there was a significant difference (P0.05). The positive rate of E was lower than 24 hours without significant difference (P0.05)., NF The positive rate of GFAP and GFAP was increased with the time of induction, and there was a significant difference in each group (P0.001). 1). NSE: The rate of positive cells was 6 h, 48 h and 24 h, and the difference was not significant (P0.05). Conclusion: The bone marrow mesenchymal stem cells with high purity can be obtained by in vitro isolation, culture and passage of the adherent screening method. After Cs, the morphological changes of the cells were not obvious, and the cells were collected. In vitro, BMSCs can be induced to differentiate into neuron-like cells.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 項(xiàng)鵬,夏文杰,張麗蓉,陳振光,張秀明,李艷,李樹濃;堿性成纖維生長因子等誘導(dǎo)間質(zhì)干細(xì)胞分化為神經(jīng)元樣細(xì)胞的研究[J];中華神經(jīng)科雜志;2002年03期

2 劉愛軍,項(xiàng)平,黃錦桃,李海標(biāo);堿性成纖維細(xì)胞生長因子對(duì)大鼠骨髓間質(zhì)干細(xì)胞增殖的影響[J];中山大學(xué)學(xué)報(bào)(醫(yī)學(xué)科學(xué)版);2004年06期

，

本文編號(hào)：2374716