本文關(guān)鍵詞： 骨髓間充質(zhì)干細(xì)胞 分化 光感受器細(xì)胞　出處：《福建醫(yī)科大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的:探索骨髓間充質(zhì)干細(xì)胞(BMSCs)在體外定向分化成為視網(wǎng)膜光感受器細(xì)胞所需的微環(huán)境,為臨床上治療視網(wǎng)膜變性疾病提供新思路。 方法:無(wú)菌條件下自5位健康成年自愿骨髓捐贈(zèng)者髂前上棘采集骨髓,采用淋巴細(xì)胞分離液密度梯度離心法分離純化獲得BMSCs進(jìn)行原代培養(yǎng)及傳代培養(yǎng),培養(yǎng)傳代至第3代的BMSCs行流式細(xì)胞術(shù)鑒定,將第3代的BMSCs以含10%FBS的DMEM-LG培養(yǎng)基中加入堿性成纖維細(xì)胞生長(zhǎng)因子(bFGF)、表皮生長(zhǎng)因子(EGF)及腦源性神經(jīng)營(yíng)養(yǎng)因子(BDNF)三種因子首先進(jìn)行第一階段向神經(jīng)前體細(xì)胞誘導(dǎo)分化,對(duì)照組不用任何因子誘導(dǎo),只用含10%FBS的DMEM-LG培養(yǎng)基培養(yǎng)。應(yīng)用免疫細(xì)胞化學(xué)檢測(cè)細(xì)胞誘導(dǎo)后巢蛋白及微管相關(guān)蛋白-2的表達(dá)情況,連續(xù)檢測(cè)不同誘導(dǎo)時(shí)間巢蛋白的陽(yáng)性表達(dá)率,當(dāng)誘導(dǎo)至巢蛋白陽(yáng)性表達(dá)率達(dá)到最高時(shí)更換誘導(dǎo)因子,向培養(yǎng)基中加入色素上皮衍生因子(PEDF)及�；撬�(Taurine)進(jìn)行第二階段誘導(dǎo)2w,用免疫細(xì)胞化學(xué)及RT-PCR方法檢測(cè)誘導(dǎo)后細(xì)胞視紫紅質(zhì)的表達(dá)情況。 結(jié)果:實(shí)驗(yàn)組誘導(dǎo)BMSCs第3d開(kāi)始免疫細(xì)胞化學(xué)能檢測(cè)到巢蛋白表達(dá),第12d巢蛋白陽(yáng)性表達(dá)率達(dá)到最高,達(dá)(90.9±2.6)%, 14d時(shí)巢蛋白陽(yáng)性率減低為(75.5±3.7)%。微管相關(guān)蛋白-2在誘導(dǎo)第6d檢測(cè)到陽(yáng)性表達(dá)。第12d誘導(dǎo)因子更換為PEDF及Taurine繼續(xù)誘導(dǎo)2w后,免疫細(xì)胞化學(xué)方法檢測(cè)到有視紫紅質(zhì)表達(dá),第2w視紫紅質(zhì)陽(yáng)性率為(20.7±3.8)%,對(duì)照組均未檢測(cè)到視紫紅質(zhì)表達(dá)。在誘導(dǎo)第2w后采用RT-PCR方法檢測(cè)到誘導(dǎo)細(xì)胞有視紫紅質(zhì)表達(dá);對(duì)照組未見(jiàn)視紫紅質(zhì)表達(dá)。 結(jié)論:體外采用分階段誘導(dǎo)BMSCs,第一階段應(yīng)用因子bFGF、EGF及BDNF進(jìn)行向神經(jīng)前體細(xì)胞誘導(dǎo)分化,BMSCs能夠成功向神經(jīng)前體細(xì)胞分化,呈現(xiàn)神經(jīng)元細(xì)胞樣形態(tài),表達(dá)神經(jīng)前體細(xì)胞標(biāo)志物巢蛋白及神經(jīng)元細(xì)胞標(biāo)志物微管相關(guān)蛋白。第二階段應(yīng)用因子PEDF和Taurine能在體外誘導(dǎo)BMSCs表達(dá)光感受器細(xì)胞標(biāo)志物視紫紅質(zhì),結(jié)果顯示BMSCs在體外特定微環(huán)境的作用下能夠向表達(dá)光感受器細(xì)胞特異性標(biāo)志物的細(xì)胞方向分化,表明分化的細(xì)胞在某種程度上具有光感受器細(xì)胞類似特征。這為臨床上治療視網(wǎng)膜變性疾病提供新思路。
[Abstract]:Objective: to explore the microenvironment of bone marrow mesenchymal stem cells (BMSCs) to differentiate into retinal photoreceptor cells in vitro, and to provide a new idea for the clinical treatment of retinal degeneration. Methods: bone marrow was collected from the anterior superior iliac spine of 5 healthy adult voluntary bone marrow donors under aseptic condition. BMSCs was isolated and purified by density gradient centrifugation of lymphocytes. The primary culture and passage culture were carried out. The BMSCs from the culture to the third generation was identified by flow cytometry. The third passage of BMSCs was added to the DMEM-LG medium containing 10s with basic fibroblast growth factor (bFGF). Epidermal growth factor (EGF) and brain-derived neurotrophic factor (BDNF) were firstly induced into neural precursor cells in the first stage, but no factors were used in the control group. The expression of nestin and microtubule-associated protein -2 was detected by immunocytochemistry in DMEM-LG medium containing 10s. The positive expression rate of nestin was continuously detected at different induction time, and the induction factor was replaced when the positive expression rate of nestin reached the highest level. The pigment epithelium-derived factor (PEDF) and taurine Taurine were added to the culture medium for 2 weeks. The expression of rhodopsin was detected by immunocytochemistry and RT-PCR. Results: the expression of nestin was detected by immunocytochemistry on the 3rd day after induction of BMSCs in the experimental group, and the positive rate of nestin was the highest (90.9 鹵2.6%) on the 12th day. The positive rate of nestin decreased to 75.5 鹵3.7 at 14 days. The positive expression of microtubule-associated protein -2 was detected on the 6th day after induction, and the induction factor was replaced by PEDF and Taurine for 2 weeks on the 12th day. The expression of rhodopsin was detected by immunocytochemistry. The positive rate of rhodopsin was 20.7 鹵3.8% at the 2nd week. The expression of rhodopsin was not detected in the control group. After 2 weeks of induction, the expression of rhodopsin was detected by RT-PCR assay. No rhodopsin expression was found in the control group. Conclusion: BMSCs were induced in vitro by stepwise induction, and differentiation into neural progenitor cells was induced by bFGFEGF and BDNF in the first stage. BMSCs can successfully differentiate into neural precursor cells and present neuronal cell-like morphology. Expression of nestin, a neural precursor marker, and microtubule-associated protein, a neuronal marker. In the second stage, factors PEDF and Taurine could induce BMSCs to express photoreceptor cell labeling in vitro. The volunteers, rhodopsin. The results showed that BMSCs could differentiate into cells expressing specific markers of photoreceptor cells under specific microenvironment in vitro. It is suggested that differentiated cells have similar characteristics of photoreceptor cells to some extent, which provides a new idea for the clinical treatment of retinal degeneration.
【學(xué)位授予單位】：福建醫(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)】：R774.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 Claudia Lange;Helge Bruns;Dietrich Kluth;Axel R Zander;Henning C Fiegel;;Hepatocytic differentiation of mesenchymal stem cells in cocultures with fetal liver cells[J];World Journal of Gastroenterology;2006年15期

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本文編號(hào)：1464456