本文選題：葉酸 + 骨髓間充質(zhì)干細(xì)胞��； 參考：《山西醫(yī)科大學(xué)》2010年碩士論文

【摘要】： 目的：本實(shí)驗(yàn)旨在研究葉酸對(duì)骨髓基質(zhì)干細(xì)胞(Human Bone Marrow Stromal Cells, HBMSCs)誘導(dǎo)分化為神經(jīng)細(xì)胞的影響及其移植治療大鼠腦缺血模型神經(jīng)細(xì)胞的增值分化作用,探討HBMSCs的移植機(jī)制,主要包括以下兩個(gè)方面：①研究葉酸對(duì)HBMSCs向神經(jīng)細(xì)胞誘導(dǎo)的影響；探討HBMSCs在傳代培養(yǎng)和向神經(jīng)細(xì)胞分化過程中的分泌作用及其機(jī)制。②觀察葉酸誘導(dǎo)后HBMSCs經(jīng)尾靜脈注射治療大鼠腦缺血后七天原位神經(jīng)前體細(xì)胞的增殖、分化情況,評(píng)價(jià)HBMSCs治療價(jià)值并探討其移植入大鼠體內(nèi)作用機(jī)制。 方法：HBMSCs由正常成年人骨髓經(jīng)密度梯度離心加貼壁離心獲得,取第5代HBMSCs以1mM二巰基乙醇(BME)預(yù)誘導(dǎo)24h后,用羥基茴香醚(BHA)、2%二甲基亞砜(DMSO)、和3種不同濃度的葉酸(4mg/L、40 mg/L、400mg/L)誘導(dǎo)分化,采用免疫細(xì)胞化學(xué)法檢測神經(jīng)細(xì)胞特異性抗原標(biāo)志神經(jīng)元特異性烯醇化酶NSE、膠原纖維酸性蛋白GFAP的表達(dá),MTT(四唑鹽比色)法檢測不同時(shí)間段對(duì)HBMSCs增殖的影響。選取16只健康Wistar大鼠分為假手術(shù)組、缺血對(duì)照組、低濃度HBMSCs組、高濃度HBMSCs組,后三組采用接扎雙側(cè)頸總動(dòng)脈法建立大鼠大腦中動(dòng)脈缺血再灌注模型(MCAO),模型制作24h后將葉酸誘導(dǎo)后不同濃度HBMSCs經(jīng)尾靜脈注射入大鼠體內(nèi)。各組腹腔注射5-溴脫氧尿嘧啶(bromodeoxyuridine, BrdU)用以標(biāo)記處于增殖狀態(tài)的神經(jīng)前體細(xì)胞,應(yīng)用免疫組織化學(xué)染色檢測缺血后7d腦組織BrdU、BrdU+NSE、BrdU+GFAP陽性細(xì)胞數(shù),結(jié)果進(jìn)行統(tǒng)計(jì)學(xué)分析。 結(jié)果：葉酸誘導(dǎo)后3h細(xì)胞胞體成圓形,折光性強(qiáng),多級(jí)細(xì)長的突起,隨著時(shí)間的延長突起變的更多更長,并相互連接交織成網(wǎng)。誘導(dǎo)后8h細(xì)胞免疫化學(xué)顯示,對(duì)照組約70%細(xì)胞呈陽性染色,GFAP陽性為主,葉酸低劑量組陽性細(xì)胞數(shù)與對(duì)照組相近,葉酸中高劑量組陽性細(xì)胞數(shù)占90%,以NSE為主,統(tǒng)計(jì)學(xué)顯示,對(duì)照組,葉酸低劑量組與葉酸中高劑量組比較差異有顯著性(P0.01)。使用40 mg／L葉酸誘導(dǎo)HBMSCs后干預(yù)治療大鼠腦缺血,腦梗死后7d側(cè)腦室室管膜下區(qū)、海馬齒狀回區(qū)BrdU、BrdU+NSE、BrdU+GFAP陽性細(xì)胞數(shù)開始增多,且高濃度組的上述陽性細(xì)胞數(shù)明顯多于對(duì)照組(P0.05)和低濃度組(P0.05)。 結(jié)論：①不同劑量的葉酸均能促進(jìn)HBMSCs向神經(jīng)細(xì)胞增值分化,以40 mg／L中等劑量的葉酸誘導(dǎo)HBMSCs向神經(jīng)細(xì)胞增值分化最為理想。②葉酸誘導(dǎo)HBMSCs干預(yù)治療后,可以促進(jìn)腦梗死大鼠損傷原位內(nèi)源性神經(jīng)前體細(xì)胞的增殖、分化,具有潛在的治療價(jià)值。
[Abstract]:Objective: to study the effect of folic acid on the differentiation of bone marrow stromal cells (BMSCs) into neural cells induced by human Bone Marrow Stromal Cells, HBMSCs) and the effect of transplantation on the proliferation and differentiation of neural cells in rat model of cerebral ischemia, and to explore the mechanism of HBMSCs transplantation. The effects of folic acid on the induction of HBMSCs into nerve cells were studied in the following two aspects: 1. To investigate the secretory role of HBMSCs in the process of passage culture and differentiation into nerve cells and its mechanism .2 to observe the proliferation and differentiation of in situ neural precursor cells in rats treated with folic acid-induced HBMSCs via caudal vein for 7 days after cerebral ischemia. To evaluate the therapeutic value of HBMSCs and to explore the mechanism of its transplantation into rats. Methods BMSCs were obtained from normal adult bone marrow by density gradient centrifugation and adherent centrifugation. The fifth passage of HBMSCs was preinduced by 1mM dimercaptoethanol for 24 hours, then induced by 2% dimethyl sulfoxide DMSOL and 3 different concentrations of folate 4 mg / L 40 mg / L 4 mg 路L ~ (-1) 路L ~ (400) mg 路L ~ (-1). The immunocytochemistry method was used to detect the expression of neuron-specific enolase (NSEs) and the expression of collagen fibrillary acidic protein (GFAP) to detect the effect on the proliferation of HBMSCs in different time periods. Sixteen healthy Wistar rats were divided into sham operation group, ischemic control group, low concentration HBMSCs group and high concentration HBMSCs group. The model of middle cerebral artery ischemia-reperfusion was established by ligating bilateral common carotid artery in the latter three groups. 24 hours after folic acid was induced, different concentrations of HBMSCs were injected into rat caudal vein. 5-bromodeoxyuridine (BrdU) was injected intraperitoneally in each group to label the proliferating neural precursor cells. The number of BrdU BrdU NSE-BrdU GFAP positive cells in brain tissue 7 days after ischemia was detected by immunohistochemical staining. The results were statistically analyzed. Results: three hours after folic acid induction, the cell bodies became round, the refraction was strong, the multistage slender protrusions became more and longer with the prolongation of time, and the cells interlinked with each other to form a network. At 8 h after induction, 70% of the cells in the control group showed positive staining for GFAP, and the number of positive cells in the low dose group was similar to that in the control group. The number of positive cells in the middle and high dose group of folic acid accounted for 90%, mainly in NSE, and the statistics showed that the number of positive cells in the low dose group was similar to that in the control group. In the control group, there was a significant difference between the low dose folic acid group and the middle and high dose group (P 0.01). After 40 mg/L folic acid induced HBMSCs, the number of BrdU NSEN BrdU GFAP positive cells in the subependymal and hippocampal dentate gyrus began to increase 7 days after cerebral infarction in rats with cerebral ischemia. The number of positive cells in high concentration group was significantly higher than that in control group (P 0.05) and low concentration group (P 0.05). Conclusion different doses of folic acid can promote the proliferation and differentiation of HBMSCs into neural cells, and 40 mg/L folic acid at medium dose can induce the differentiation of HBMSCs into neuronal cells. The most ideal treatment is that folic acid can induce the differentiation of HBMSCs into neuronal cells after the intervention of HBMSCs. 2 folic acid. It can promote the proliferation and differentiation of in situ endogenous neural precursor cells in rats with cerebral infarction, and has potential therapeutic value.
【學(xué)位授予單位】：山西醫(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

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