【摘要】：中樞神經(jīng)系統(tǒng)疾病一直以來(lái)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(lái)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(lái)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。在體內(nèi)、外適當(dāng)?shù)恼T導(dǎo)條件下,WJ-MSCs具有分化為神經(jīng)樣細(xì)胞的潛能。在誘導(dǎo)體系中,中國(guó)傳統(tǒng)中藥的應(yīng)用備受矚目。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。已有的研究表明,黃芪對(duì)神經(jīng)組織具有一定的保護(hù)作用,且可誘導(dǎo)WJ-MSCs分化為神經(jīng)樣細(xì)胞。本課題組已建立培養(yǎng)WJ-MSCs的技術(shù)平臺(tái),觀察基本的生物學(xué)特性,用免疫組化方法檢測(cè)到nestin和NF在黃芪誘導(dǎo)WJ-MSCs向神經(jīng)樣細(xì)胞分化的過(guò)程中有表達(dá),但其在mRNA水平的表達(dá)情況尚不清楚。目的：探討WJ-MSCs在黃芪誘導(dǎo)下分化為神經(jīng)樣細(xì)胞的潛能以及鈣離子阻斷劑的干預(yù)作用,觀察黃芪對(duì)WJ-MSCs表達(dá)NSE、nestin和NF-H的影響。方法：本研究采用組織塊貼壁培養(yǎng)法獲取WJ-MSCs,用胰酶消化傳代來(lái)純化細(xì)胞,取P3-P5代生長(zhǎng)狀態(tài)良好的細(xì)胞進(jìn)行誘導(dǎo)分化實(shí)驗(yàn)；用RT-PCR檢測(cè)黃芪在不同濃度(5ug/ml、50ug/ml、500ug/ml、5000ug/ml、25000ug/ml)和不同時(shí)間(3h、6h、24h、48h和72h)下,誘導(dǎo)NSE、nestin和NF-H基因的表達(dá)變化,并觀察鈣離子通道阻斷劑LY294002和氯化錳(MnCl2)對(duì)其表達(dá)的影響。另外,用蛋白質(zhì)免疫印跡(Western blot)檢測(cè)黃芪誘導(dǎo)WJ-MSCs分化過(guò)程中NSE蛋白表達(dá)的時(shí)效和量效關(guān)系。本研究對(duì)闡明黃芪的藥效以及WJ-MSCs的分化潛能具有重要意義,為建立WJ-MSCs分化為神經(jīng)樣細(xì)胞的誘導(dǎo)體系提供重要信息,以期為深入研究黃芪的誘導(dǎo)機(jī)制奠定理論基礎(chǔ)。結(jié)果：用組織塊貼壁培養(yǎng)法獲取的WJ-MSCs為長(zhǎng)梭形或成纖維細(xì)胞樣細(xì)胞,密集排列呈漩渦狀或束狀交織,細(xì)胞增殖能力強(qiáng),折光性好。RT-PCR檢測(cè)結(jié)果提示,NSE、nestin和NF-H基因在對(duì)照組和黃芪誘導(dǎo)組中均有表達(dá)。NSE基因在黃芪誘導(dǎo)WJ-MSCs分化48h和72h組中表達(dá)增加,有量效關(guān)系,與對(duì)照組比較,差異具有顯著性(P0.05)。NF-H基因在5ug/ml、50ug/ml和25000ug/ml黃芪誘導(dǎo)WJ-MSCs分化48h組中表達(dá)增加,與對(duì)照組比較,差異具有顯著性(P0.05)。在72h組中,NF-H基因在5ug/ml、50ug/ml黃芪誘導(dǎo)WJ-MSCs分化為神經(jīng)樣細(xì)胞的過(guò)程中表達(dá)增加,與對(duì)照組比較,差異具有顯著性(P0.05)。Nestin基因在5000ug/ml、25000ug/ml黃芪誘導(dǎo)WJ-MSCs分化24h組中表達(dá)增加,與對(duì)照組比較,差異具有顯著性(P0.05)。給鈣離子通道阻斷劑(LY294002和Mncl2), nestin和NF-H基因分別在6h組、24h組中表達(dá)減弱,與50ug/ml黃芪誘導(dǎo)組相比,差異有顯著性(P0.05)。但對(duì)NSE基因的表達(dá)無(wú)明顯影響。用Western blot檢測(cè)到NSE蛋白在不同濃度黃芪(12.5ug/ml-62.5ug/ml)誘導(dǎo)WJ-MSCs分化24h,50ug/ml為最佳濃度。另外,在不同時(shí)間點(diǎn)(6h、12h、24h、36h和48h),NSE蛋白在50 ug/ml黃芪誘導(dǎo)WJ-MSCs 6h的表達(dá)量高。結(jié)論：1、NSE、nestin和NF-H基因在黃芪誘導(dǎo)WJ-MSCs前后均有表達(dá)。2、黃芪誘導(dǎo)WJ-MSCs分化過(guò)程中NSE、nestin和NF-H基因及NSE蛋白表達(dá)上調(diào),LY294002和MnCl2下調(diào)nestin和NF-H基因表達(dá)。提示黃芪具有誘導(dǎo)WJ-MSCs分化為神經(jīng)樣細(xì)胞的潛能,且與鈣離子通路有關(guān)系。
[Abstract]:With the development of modern biotechnology, stem cell transplantation has become a possible treatment strategy for nervous system diseases. Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) from human umbilical cord are derived from childbirth waste. Adult stem cells abandoning umbilical cord have the potential of self-renewal and multi-differentiation, and have unique immunoregulatory properties. WJ-MSCs have the potential to differentiate into nerve-like cells in vivo and in vitro under appropriate induction conditions. In the induction system, the application of traditional Chinese medicine has attracted much attention. Astragalus membranaceus is widely used in traditional Chinese medicine formulas.Previous studies have shown that Astragalus membranaceus has a protective effect on nerve tissue and can induce WJ-MSCs to differentiate into neuron-like cells.Our team has established a technical platform for culturing WJ-MSCs to observe the basic biological characteristics and detected nestin and NF in Astragalus membranaceus-induced WJ-MSCs by immunohistochemistry. Objective: To investigate the potential of WJ-MSCs to differentiate into neuron-like cells induced by Astragalus membranaceus and the effect of calcium blockers on the expression of NSE, nestin and NF-H in WJ-MSCs. WJ-MSCs were obtained by adherent culture and purified by trypsin digestion and passage, and the cells of P3-P5 generation with good growth status were induced to differentiate. The expression of NSE, nestin and NF-H genes were detected by RT-PCR at different concentrations (5ug/ml, 50ug/ml, 500ug/ml, 5000ug/ml, 25000 ug/ml) and at different times (3h, 6h, 24h, 48h and 72h). The effects of calcium channel blocker LY294002 and manganese chloride (MnCl2) on the expression of NSE protein were observed. In addition, the expression of NSE protein in WJ-MSCs induced by Astragalus membranaceus was detected by Western blot. This study is of great significance to clarify the pharmacodynamics of Astragalus membranaceus and the differentiation potential of WJ-MSCs. Results: WJ-MSCs obtained by tissue-block adherent culture method were spindle-shaped or fibroblast-like cells, which were densely arranged in a vortex or bundle-like interweave, with strong cell proliferation and good refraction. The results showed that NSE, nestin and NF-H genes were expressed in both the control group and the astragalus-induced group. The expression of NSE gene was increased in the astragalus-induced WJ-MSCs differentiation 48 h and 72 h groups, with a dose-effect relationship. There was a significant difference between the two groups (P 0.05). The expression of NF-H gene was increased in the 5 ug/ml, 50 ug/ml and 25 000 ug/ml astragalus-induced WJ-MSCs differentiation 48 h group. In 72 h group, the expression of NF-H gene increased during the differentiation of WJ-MSCs into neuron-like cells induced by 5 ug/ml and 50 ug/ml Astragalus membranaceus, and there was a significant difference between the two groups (P 0.05). The expression of Nestin gene increased in 5 000 ug/ml and 25 000 ug/ml Astragalus membranaceus-induced WJ-MSCs differentiation 24 h group, compared with the control group. Compared with the 50 ug/ml Astragalus induction group, the expression of NSE gene was significantly lower in the calcium channel blockers (LY294002 and Mncl2), nestin and NF-H genes in the 6h and 24h groups, respectively, but not in the 50ug/ml Astragalus induction group (P 0.05). The expression of NSE protein in different concentrations of Astragalus membranaceus was detected by Western blot. In addition, at different time points (6h, 12h, 24h, 36h and 48h), the expression of NSE protein in WJ-MSCs induced by 50 ug/ml was high. Conclusion: 1, NSE, nestin and NF-H genes were expressed before and after induction of WJ-MSCs by Astragalus membranaceus. LY294002 and MnCl2 down-regulated nestin and NF-H gene expression due to the up-regulation of NSE protein expression, suggesting that Astragalus membranaceus has the potential to induce WJ-MSCs to differentiate into neuron-like cells and is related to calcium ion pathway.
【學(xué)位授予單位】：昆明理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R741

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本文編號(hào)：2194996