【摘要】：周圍神經(jīng)損傷和脊髓損傷是骨科最常見的神經(jīng)損傷性疾病，具有發(fā)生率高、損傷程度重、修復效果差、致殘率高、治療費用高等特點�！爸亟ńY(jié)構連續(xù)性和加快神經(jīng)生長速度”是國際公認的神經(jīng)損傷修復的關鍵，但這些問題至今尚未得到有效解決。組織工程細胞移植技術為神經(jīng)再生及修復帶來了希望，但由于取材來源有限和引發(fā)免疫排斥反應等問題，其應用受到限制。 骨髓間充質(zhì)干細胞具有高度自我更新能力和多向分化潛能，可被誘導分化為神經(jīng)細胞，被認為是理想的組織工程種子細胞。目前常用的化學誘導劑誘導法，易對細胞造成毒性損傷，導致誘導后細胞存活時間縮短、功能下降，無法滿足移植需求。因此，亟需尋找一種安全、有效的誘導分化方法。 電刺激在神經(jīng)損傷修復中的應用日益增加。有研究表明，電刺激可誘導脂肪干細胞分化成成骨細胞，并可促進骨髓間充質(zhì)干細胞向心肌細胞分化，提示電刺激可干預干細胞的分化行為。但至今為止，有關電刺激對骨髓間充質(zhì)干細胞誘導分化為神經(jīng)細胞的研究未見報道。因此，研究其有效性和可行的實施方法，對于構建組織工程種子細胞、促進神經(jīng)損傷修復具有重要意義。 本課題擬采用電刺激這一物理方法誘導骨髓間充質(zhì)干細胞分化，在篩選并確定適宜電刺激參數(shù)的基礎上，從細胞形態(tài)、細胞增殖能力、細胞凋亡情況、細胞分化等多個方面進行系統(tǒng)研究，為構建組織工程種子細胞、促進神經(jīng)損傷修復提供新的思路。具體內(nèi)容如下： 第一部分大鼠骨髓間充質(zhì)干細胞的分離、培養(yǎng)及鑒定 目的：分離培養(yǎng)并純化大鼠骨髓間充質(zhì)干細胞，采用形態(tài)學觀察、流式細胞技術和誘導分化成骨實驗進行骨髓間充質(zhì)干細胞的鑒定。 方法：應用全骨髓貼壁分離培養(yǎng)法培養(yǎng)純化SD大鼠骨髓間充質(zhì)干細胞，倒置顯微鏡觀察細胞形態(tài)學變化；CCK-8法檢測不同代數(shù)細胞的增殖能力；流式細胞技術檢測CD29、CD90、CD45表達情況；使用成骨誘導培養(yǎng)基誘導P3代BMSCs成骨分化，明確其分化能力。 結(jié)果：應用全骨髓貼壁分離培養(yǎng)法，成功培養(yǎng)出生長狀態(tài)較好的BMSCs，，CCK-8法檢測顯示細胞增殖能力良好。培養(yǎng)的細胞經(jīng)流式細胞檢測，大部分細胞呈現(xiàn)CD29、CD90陽性，CD45陰性，證明本實驗所培養(yǎng)的細胞為骨髓間充質(zhì)干細胞。進一步計數(shù)統(tǒng)計發(fā)現(xiàn)，CD29和CD90雙陽性、CD45陰性的細胞數(shù)占總培養(yǎng)細胞數(shù)的96.89%，說明本實驗獲得的細胞純度較高。采用常規(guī)化學法誘導培養(yǎng)細胞成骨分化實驗結(jié)果顯示，誘導后的細胞呈茜素紅、ALP染色陽性，證明所培養(yǎng)的細胞可被誘導分化為成骨細胞，具有良好的分化潛能。 結(jié)論：通過細胞增殖能力檢測、流式細胞鑒定以及成骨誘導分化實驗證實，本實驗所采用的全骨髓貼壁分離培養(yǎng)法，可穩(wěn)定獲得活性良好、具有自我更新能力和分化潛能的骨髓間充質(zhì)干細胞，可為后續(xù)研究提供充足的細胞來源。 第二部分電刺激對于骨髓間充質(zhì)干細胞誘導分化過程的作用研究 目的：采用電刺激誘導骨髓間充質(zhì)干細胞向神經(jīng)細胞分化，探索誘導分化的最適電刺激參數(shù)，初步闡明電刺激對骨髓間充質(zhì)干細胞誘導分化為神經(jīng)細胞的作用，為應用種子細胞移植修復神經(jīng)損傷提供新方法。 方法：（1）應用直流電刺激，選取不同刺激時間（A：15min/d、B：30min/d、C：1h/d、D：2h/d、E：空白對照）和不同電壓（A：1V、B：3V、C：5V、D：10V、E：空白對照），對骨髓間充質(zhì)干細胞進行干預，CCK-8法檢測細胞增殖情況變化，流式細胞技術檢測電刺激對細胞凋亡的影響，篩選最適宜的電刺激參數(shù)。（2）采用篩選出的適宜電刺激參數(shù)進行誘導分化研究。分別設立電刺激誘導組、化學誘導組、復合誘導組和空白對照組，MAP-2免疫熒光染色鑒定細胞誘導分化情況，并計算誘導分化比率。 結(jié)果：(1)刺激時間篩選實驗結(jié)果表明，當電壓設定為3V時，采用刺激時間為30min/d（B組）的參數(shù)刺激后，細胞形態(tài)及數(shù)量均明顯優(yōu)于其余各組；細胞增殖能力較其余各組明顯提高，且差異具有統(tǒng)計學意義；細胞凋亡檢測發(fā)現(xiàn)該參數(shù)的電刺激未明顯增加細胞凋亡。刺激電壓篩選實驗結(jié)果表明，當刺激時間為30min/d，采用刺激電壓為3V（B組）的參數(shù)刺激后，細胞形態(tài)及數(shù)量優(yōu)于其他各組，細胞增殖能力明顯提高，且差異具有統(tǒng)計學意義；細胞凋亡檢測表明該參數(shù)的電刺激未明顯增加細胞凋亡。從而確定了3V、30min/d為適宜電刺激參數(shù)。(2)應用常規(guī)化學誘導法作為陽性對照，對BMSCs進行化學誘導，通過誘導率和免疫熒光染色結(jié)果的比較，發(fā)現(xiàn)電刺激誘導組與化學誘導組，均可誘導BMSCs分化為神經(jīng)元，統(tǒng)計學無顯著差異（P＞0.05）；而復合誘導組的誘導率，優(yōu)于電刺激誘導組和化學誘導組，且具有統(tǒng)計學差異（P＜0.05）。但復合誘導組和化學誘導組，在細胞誘導成功后，出現(xiàn)細胞破裂、崩解及細胞殘片，而電刺激組未見此現(xiàn)象。 結(jié)論：通過對不同刺激時間和不同電壓參數(shù)進行篩選，確定3V，30min/d，連續(xù)刺激2d為適宜電刺激參數(shù)，應用于骨髓間充質(zhì)干細胞誘導分化為神經(jīng)細胞實驗，研究結(jié)果表明，電刺激不僅可達到與化學誘導劑相同的誘導分化效果，而且毒性及損傷作用小，可更加安全有效的對骨髓間充質(zhì)干細胞發(fā)揮誘導分化作用。本實驗為應用電刺激干預干細胞提供實驗依據(jù)，為組織工程種子細胞的構建提供了新的思路。
[Abstract]:Peripheral nerve injury and spinal cord injury are the most common nerve injury diseases in the department of orthopedics. It has the characteristics of high incidence, heavy injury degree, poor repair effect, high residual rate, high treatment cost and the like. "Reconstruction of structural continuity and speed of nerve growth" is the key to the internationally recognized repair of nerve injury, but these problems have not yet been effectively resolved. Tissue engineering cell transplantation technology has brought hope for nerve regeneration and repair, but its application is limited due to the limited source of material and immune rejection. The bone marrow mesenchymal stem cells have high self-renewal ability and multi-directional differentiation potential, can be induced to differentiate into nerve cells, and are considered to be ideal tissue engineering seeds The present general chemical inducers are easy to cause toxic damage to the cells, and the survival time of the cells after induction is shortened, the function is reduced, and the transplantation needs can not be met. Please, therefore, there is an urgent need to find a safe and effective method of inducing differentiation The application of electrical stimulation in the repair of nerve injury The results show that the electrical stimulation can induce the differentiation of the adipose-derived stem cells into the osteoblast and promote the differentiation of the bone marrow-derived mesenchymal stem cells into the cardiac muscle cells, suggesting that the electrical stimulation can interfere with the differentiation of the stem cells. The effect of electrical stimulation on the induction and differentiation of bone marrow-derived mesenchymal stem cells into nerve cells has not been studied. See the report. Therefore, to study its effectiveness and feasible implementation methods, for the construction of tissue engineering seed cells, it is of great importance to promote the repair of nerve injury The purpose of this study is to induce the differentiation of bone marrow-derived mesenchymal stem cells by the physical method of electrical stimulation. On the basis of screening and determining appropriate electric stimulation parameters, the cell morphology, cell proliferation ability, cell apoptosis, cell differentiation and so on are introduced. The study of the line system in order to construct the seed cells of tissue engineering and promote the repair of nerve injury To supply new ideas. The contents of the body are as follows: the first part of the rat bone marrow mesenchymal stem cells The purpose of isolation, culture and identification was to separate and culture and purify the rat bone marrow-derived mesenchymal stem cells. Identification of mesenchymal stem cells. Methods: The bone marrow mesenchymal stem cells of SD rats were cultured by full-bone marrow adherent culture method. The morphological changes of cells were observed by inverted microscope. The proliferation ability of different algebraic cells was detected by CCK-8 method. CD29 and CD were detected by flow cytometry. 90, CD45 expression; using osteogenic induction medium to induce P3 generation BMSCs to become The results showed that BMSCs and CCK-8 with better growth status were successfully cultured by the method of full-bone marrow adherent separation and culture. The cultured cells were detected by flow cytometry. Most of the cells exhibited CD29, CD90 positive and CD45 negative. The cultured cells were bone marrow mesenchymal stem cells. The results showed that the number of CD29 and CD90 positive and CD45-negative cells accounted for 96.89% of the total number of cultured cells. The results of induction of osteogenic differentiation of cultured cells by conventional chemical method showed that the induced cells showed a positive staining and the ALP staining was positive, and that the cultured cells could be induced to differentiate into the cells. Osteoclasts, with good differentiation potential. Conclusion: Through cell proliferation, flow cytometry and osteogenic differentiation, the whole bone marrow adherent culture method is used in this experiment, which can be stably obtained with good activity and self-renewal. the mesenchymal stem cells of the force and the differentiation potential, Adequate source of cells can be provided for subsequent studies. The second part of the electrical stimulation is for bone The Role of Mesenchymal Stem Cells in the Differentiation of Mesenchymal Stem Cells: Induction of Mesenchymal Stem Cells from Mesenchymal Stem Cells to the Nerves by Electrostimulation The effects of electrical stimulation on the induction and differentiation of bone marrow-derived mesenchymal stem cells into nerve cells were discussed, and the effects of electrical stimulation on the induction and differentiation of bone marrow-derived mesenchymal stem cells into nerve cells were discussed. Methods: (1) DC stimulation was applied to select different stimulation time (A:15 min/ d, B:30 min/ d, C:1 h/ d, D: 2h/ d, E: blank control) and different voltage (A:1 V, B: 3 V, C:5 V, D:10 V, E: blank control), the bone marrow mesenchymal stem cells were intervened, the proliferation of the cells was detected by the CCK-8 method, and the electropuncture was detected by flow cytometry. The most appropriate parameters of the electrical stimulation were selected by the effect of the stimulation on the apoptosis of the cells. (2) The induced differentiation was induced by the appropriate electrical stimulation parameters. The electrical stimulation induction group, the chemical induction group, the compound induction group, the blank control group and the MAP-2 immunofluorescence staining were respectively established. Results: (1) The experimental results of the stimulation time showed that, when the voltage was set to 3 V, the cell morphology and number of the cells were significantly superior to the other groups after the stimulation time was 30 min/ d (group B). The colonizing ability was significantly higher than the other groups, and the difference was of statistical significance. It was found that the electrical stimulation of the parameters did not significantly increase the apoptosis of the cells. The experimental results of the stimulation of the voltage showed that the cell morphology and number were superior to that of the other cells after the stimulation time was 30 min/ day and the stimulation voltage was 3 V (group B). The cell proliferation ability of the group was significantly improved, and the difference was of statistical significance; fine The cell apoptosis test showed that the electrical stimulation of the parameter did not significantly increase the apoptosis of the cells. 3 V and 30 min/ d were determined to be suitable for electrical stimulation. (2) BMSCs were induced by chemical induction, induction rate and immunofluorescence staining, and BMSCs were induced by the comparison of induction rate and immunofluorescence staining. The induction rate of the compound induction group was superior to that of the electric stimulation induction group. And the combination induction group and the chemical induction group, after the cell induction was successful, appeared. The results showed that 3 V,30 min/ d and 2 d of continuous stimulation were suitable for stimulation of bone by screening different stimulation time and different voltage parameters. The induction and differentiation of the mesenchymal stem cells into the nerve cell experiments show that the electrical stimulation can not only achieve the same induction and differentiation effect as the chemical inducers, but also the toxicity and the damage effect are small, The invention can more safely and effectively induce differentiation of the bone marrow mesenchymal stem cells.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R329

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相關期刊論文 前10條

1 龍乾發(fā);劉衛(wèi)平;玉石;劉陽;韓蕊;王孝安;李娟;;體外誘導大鼠骨髓間充質(zhì)干細胞向GABA能神經(jīng)元分化[J];中華神經(jīng)外科疾病研究雜志;2009年06期

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本文編號：2502817