該論文為《昆明理工大學(xué)》碩士論文，內(nèi)容就臍帶間充質(zhì)干細(xì)胞的生物學(xué)特性、分化潛能和機(jī)制做了詳細(xì)的概括和總結(jié)。并通過實(shí)驗(yàn)證明了黃芪在誘導(dǎo)干細(xì)胞分化為神經(jīng)細(xì)胞過程中的作用，并摸索出最佳誘導(dǎo)條件。論文對一些實(shí)驗(yàn)方法，如Western Blot 等有比較詳細(xì)的記錄。
 摘  要
中樞神經(jīng)系統(tǒng)疾病一直以來都是臨床治療上難點(diǎn)。隨著現(xiàn)代生物技術(shù)的發(fā)展，利用干細(xì)胞移植技術(shù)來治療神經(jīng)系統(tǒng)疾病成為可能的治療策略。人臍帶Wharton’s jelly源性間充質(zhì)干細(xì)胞（Wharton’s jelly mesenchymal stem cells,WJ-MSCs）是來源于分娩廢棄臍帶的一種成體干細(xì)胞，具有自我更新和多向分化潛能，且有獨(dú)特的免疫調(diào)節(jié)特性。與胚胎干細(xì)胞和骨髓間充質(zhì)干細(xì)胞相比，WJ-MSCs具有來源廣泛，在體外易于分離培養(yǎng)和擴(kuò)增，無倫理道德問題的爭議等優(yōu)勢，被認(rèn)為是組織工程中理想的種子細(xì)胞源。已有研究表明，WJ-MSCs具有分化為三個胚層來源細(xì)胞的潛能。近年來，國內(nèi)外學(xué)者通過不同的誘導(dǎo)方式，在體內(nèi)、外觀察到WJ-MSCs分化為神經(jīng)樣細(xì)胞。這無疑為神經(jīng)系統(tǒng)疾病的治療帶來了希望。
黃芪是一味扶正固本、補(bǔ)中益氣之要藥，在傳統(tǒng)中藥配方中被廣泛使用。據(jù)報(bào)道，黃芪對神經(jīng)組織具有一定的保護(hù)作用，能誘導(dǎo)WJ-MSCs分化為神經(jīng)樣細(xì)胞。本課題組已建立培養(yǎng)臍帶間充質(zhì)干細(xì)胞的技術(shù)平臺，并觀察基本的生物學(xué)特性?；谖覀兪橇⒆阌诜?wù)臨床的研究部門，需要實(shí)實(shí)在在的建立我們自身的實(shí)驗(yàn)數(shù)據(jù)。于此，本實(shí)驗(yàn)采用組織塊貼壁培養(yǎng)法獲取WJ-MSCs，用蛋白質(zhì)免疫印跡（Western blot）檢測NSE蛋白在不同濃度黃芪誘導(dǎo)WJ-MSCs分化過程中的表達(dá)以及時效關(guān)系。旨在探討黃芪誘導(dǎo)的適宜條件，以期為提高黃芪誘導(dǎo)效率奠定理論基礎(chǔ)。
WJ-MSCs的培養(yǎng)及蛋白提?。翰捎媒M織塊貼壁法分離培養(yǎng)原代WJ-MSCs，通過胰酶消化來純化、擴(kuò)增細(xì)胞。我們選用P3～P5的細(xì)胞進(jìn)行實(shí)驗(yàn)。在借鑒文獻(xiàn)的基礎(chǔ)上，設(shè)黃芪濃度為12.5mg/ml、25mg/ml、37.5mg/ml、50mg/ml、62.5mg/ml以及對照組，誘導(dǎo)24h后檢測NSE蛋白的表達(dá)。隨后，以50 g/L黃芪分別誘導(dǎo)WJ-MSCs6h、12h、24h、36h、48h后，檢測NSE蛋白的表達(dá)。
依照上述實(shí)驗(yàn)時間點(diǎn)，收集細(xì)胞，提取蛋白，用BCA法進(jìn)行蛋白定量，分裝，-20℃凍存?zhèn)溆谩?br /> 采用Western blot免疫印跡方法檢測NSE蛋白在黃芪誘導(dǎo)WJ-MSCs分化過程中的表達(dá)：兩組細(xì)胞蛋白中神經(jīng)元標(biāo)志物神經(jīng)元特異性烯醇化酶(NSE)的表達(dá)變化情況。
實(shí)驗(yàn)結(jié)果示，用組織塊貼壁培養(yǎng)法結(jié)合胰酶消化法獲取的WJ-MSCs為成纖維樣細(xì)胞，細(xì)胞增殖能力強(qiáng)，折光性好。經(jīng)不同濃度的黃芪誘導(dǎo)24h后，在12.5g/L誘導(dǎo)組中，NSE蛋白條帶亮度最高。提示在此濃度條件下，細(xì)胞中NSE蛋白表達(dá)量可能最高。50 g/L黃芪誘導(dǎo)不同時間后，在6hNSE蛋白條帶亮度最強(qiáng)，12h次之。
結(jié)論：NSE蛋白在? g/L黃芪誘導(dǎo)WJ-MSCs分化6h的表達(dá)量最大。

 關(guān)鍵詞：臍帶；間充質(zhì)干細(xì)胞；黃芪；NSE；免疫印跡

ABSTRACT
                                   
      Diseases of the central nervous system has been a clinical difficult. With the development of modern biotechnology, the stem cell transplantation to treat nervous system diseases become possible treatment strategies. Umbilical cord mesenchymal stem cells derived from umbilical cord delivery abandoned. Is a kind of adult stem cells. Embryonic stem cells and bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells in vitro has extensive sources, easy isolation and culture and proliferation, no ethical issues and other advantages, is considered to be the ideal source of tissue engineering seed cells. In addition, umbilical cord mesenchymal stem cells also have the differentiation potential, in appropriate culture conditions in vitro, can be induced to differentiate into various tissue cells. In recent years, scholars at home and abroad through different ways, in vitro to complete the umbilical cord mesenchymal stem cells differentiate into neuronlike cells. In this process, the application of traditional Chinese medicine China attention, has been reported to use traditional Chinese medicine Astragalus mongholicus induced differentiation of umbilical cord mesenchymal stem cells into neuron like cells. This is the clinical treatment of diseases of the nervous system brings hope. But at the same time, the problem of Astragalus membranaceus induced differentiation is not clear, this for clinical cell transplantation in the treatment of some limitations.
      Based on this, the technique and Western blot method for primary cell culture, the umbilical cord mesenchymal stem cells differentiation in Astragalus, Astragalus induced concentration and suitable for induction time problems are investigated and discussed. In order to lay a theoretical foundation for improving the efficiency of Astragalus membranaceus induced differentiation. The experiment is divided into two parts.
     1.Isolation and culture of primary human umbilical cord mesenchymal stem cells by tissue adherent method, through the passage to purified cells. P3-P5 as a substitute for good growth state of cells as experimental products, in accordance with the Astragalus concentrations were 12.5mg/ml, 25mg/ml, 37.5mg/ml, 50mg/ml, 62.5mg/ml and blank control group 6 group, the induction time was 24h, another group according to the induction time were 6h, 12h, 24h, 36h, 48h and blank control group 6 group, Astragalus membranaceus induced concentration was 50mg/ml. To observe the morphological changes of cells, induced group discovered in adherent cell shedding, but no obvious changes in cell morphology. Protein extraction, quantification by BCA protein concentration determination of cell protein, the protein -20 ℃ storage backup.
     2.The two groups were detected in neurons cell protein markers of neuron specific enolase by Western blot blotting method (NSE) expression changes. The experimental results showed the expression of NSE protein in display, the control group and the experimental group in the experimental group, difference in the concentration of Astragalus, induced by 24h, the concentration of 12.5mg/ml in the group of Astragalus, electrophoresis band maximum brightness, the concentration of 25mg/ml group. Show that this concentration condition, is the highest expression of NSE protein in cells. In the induction time difference group, control group and experimental group has band appeared in the experimental group, the induction time for the brightness of 6h bands was the strongest, followed by 12h, showed that the induction time in the 6-12h, the highest expression of NSE proteins in the cell rate. We believe in Astragalus membranaceus induced mesenchymal stem cells differentiate into neuron like cells in the process of Astragalus, the suitable concentration of 12.5-25mg/ml, the best time induced by 6-12h.
       Conclusion: the tissue culture method can conveniently obtain umbilical cord mesenchymal stem cells, and the cell growth, proliferation speed; in Astragalus membranaceus induced differentiation of mesenchymal stem cells, cell morphology did not change, Western blot, the experimental group and the control group. NSE protein expression, the band comparing the brightness of Astragalus, concentration of 12.5mg/ml and induced 6h two group with the highest brightness, it also shows that under these conditions, the NSE expression in cells of the maximum. Therefore, we believe that in Astragalus membranaceus induced differentiation of mesenchymal stem cells, the optimal concentration range of 12.5-25mg/ml, the best induction when the long range 6-12h.

Keywords: Umbilical cord mesenchymal stem cells；Astragalus membranaceus；
             Neuron specific enolase；Western blot

目  錄
摘  要 I
ABSTRACT III
目  錄 VI
插圖和附表清單 XI
英文縮寫表 XII
第一章 緒論 1
1.1 前言 1
1.2 干細(xì)胞研究概況 1
1.2.1 胚胎干細(xì)胞 1
1.2.2 臍帶間充質(zhì)干細(xì)胞 2
  1.2.2.1 間充質(zhì)干細(xì)胞生物學(xué)特性 2
  1.2.2.2 間充質(zhì)干細(xì)胞的鑒別 3
  1.2.2.3 間充質(zhì)干細(xì)胞分化潛能 3
  1.2.2.4 間充質(zhì)干細(xì)胞的免疫原性 4
1.3 UCMSC的誘導(dǎo)分化及其機(jī)制 4
1.3.1 UCMSC的誘導(dǎo)分化方法 4
1.3.2 UCMSC的誘導(dǎo)分化機(jī)制 5
  1.3.2.1 外源性調(diào)控 5
  1.3.2.2內(nèi)源性調(diào)控 6
1.4 中藥誘導(dǎo)MSC分化神經(jīng)樣細(xì)胞 8
1.4.1 MSC向神經(jīng)樣細(xì)胞分化的特點(diǎn) 8
  1.4.1.1 MSC誘導(dǎo)分化為神經(jīng)樣細(xì)胞 8
  1.4.1.2 MSC促進(jìn)其他細(xì)胞分化為神經(jīng)樣細(xì)胞 9
1.4.2 中藥誘導(dǎo)MSC分化為神經(jīng)樣細(xì)胞 9
  1.4.2.1中藥復(fù)方制劑誘導(dǎo)MSC分化為神經(jīng)樣細(xì)胞 9
  1.4.2.2單味中藥誘導(dǎo)MSC分化為神經(jīng)樣細(xì)胞 10
  1.4.2.3中藥有效成分誘導(dǎo)MSC分化為神經(jīng)樣細(xì)胞 11
1.5 黃芪藥理作用及誘導(dǎo)MSC分化為神經(jīng)樣細(xì)胞 13
1.5.1 黃芪藥理作用 13
  1.5.1.1對免疫系統(tǒng)的作用 13
  1.5.1.2抗腫瘤作用 14
  1.5.1.3神經(jīng)保護(hù)作用 14
1.5.2 黃芪誘導(dǎo)MSC分化為神經(jīng)樣細(xì)胞 14
第二章 黃芪誘導(dǎo)MSC及蛋白提取 16
2.1 引言 16
2.2 材料與方法 17
2.2.1 實(shí)驗(yàn)材料 17
2.2.2 實(shí)驗(yàn)藥品與試劑 17
2.2.3 實(shí)驗(yàn)儀器 18
2.2.4 臍帶MSCs的原代培養(yǎng) 19
2.2.5 臍帶MSCs的傳代 20
2.2.6 臍帶MSCs生長曲線 20
2.2.7 臍帶MSCs的凍存與復(fù)蘇 21
2.2.8 黃芪誘導(dǎo)臍帶MSC 21
2.2.9 細(xì)胞蛋白提取及定量 24
2.3 實(shí)驗(yàn)結(jié)果 26
2.3.1 臍帶MSCs分離、純化及形態(tài)學(xué)觀察 26
2.3.2 細(xì)胞生長曲線分析 29
2.3.3 黃芪誘導(dǎo)臍帶MSC 29
2.3.4 細(xì)胞蛋白定量標(biāo)準(zhǔn)曲線 29
2.3 實(shí)驗(yàn)結(jié)果 30
2.5 本章小結(jié) 32
第三章 黃芪誘導(dǎo)MSC中NSE的表達(dá)差異 33
3.1 引言 33
3.2 實(shí)驗(yàn)材料與儀器 33
3.2.1 實(shí)驗(yàn)試劑 33
3.2.2 實(shí)驗(yàn)儀器 34
3.2.3 實(shí)驗(yàn)準(zhǔn)備 34
3.3 實(shí)驗(yàn)方法 35
    3.3.1 SDS-PAGE電泳 35
3.3.2 轉(zhuǎn)膜 37
3.3.3 免疫反應(yīng) 37
3.3.4 化學(xué)發(fā)光顯影 37
3.5 討論 39
3.6 本章小結(jié) 40
致  謝 41
參 考 文 獻(xiàn) 42