本文關(guān)鍵詞：骨髓間充質(zhì)干細(xì)胞體外定向分化為神經(jīng)樣細(xì)胞的研究　出處：《廣州醫(yī)學(xué)院》2010年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 骨髓間充質(zhì)干細(xì)胞 生物學(xué)特性 誘導(dǎo)分化

【摘要】：脊髓損傷(spinal cord injury, SCI)后所引起的神經(jīng)源性膀胱癥狀嚴(yán)重影響患者的生活質(zhì)量,但目前尚無(wú)有效的治療手段,并且多數(shù)研究集中在尿流動(dòng)力學(xué)及神經(jīng)功能損傷等方面。隨著近年來(lái)干細(xì)胞研究的突飛猛進(jìn),干細(xì)胞移植用于治療血液系統(tǒng)惡性腫瘤、神經(jīng)系統(tǒng)自身免疫疾病并取得有效成果,甚至已經(jīng)應(yīng)用于臨床,這使通過(guò)干細(xì)胞聯(lián)合組織工程移植增加脊髓神經(jīng)數(shù)量、減少膠質(zhì)瘢痕和空洞的形成成為可能,采用干細(xì)胞接種至組織工程進(jìn)行移植可望成為一種有效治療SCI后神經(jīng)源性膀胱的新方法。然而,在組織工程構(gòu)建中選擇合適的種子細(xì)胞成為治療的重要環(huán)節(jié)。全能干細(xì)胞和多能干細(xì)胞具有高度自我更新能力和多向分化的潛能,是組織工程中重要的靶細(xì)胞。胚胎干細(xì)胞移植存在倫理道德和免疫排斥等問(wèn)題,神經(jīng)干細(xì)胞、嗅鞘細(xì)胞及雪旺細(xì)胞的來(lái)源均有限,且取材不便,很難應(yīng)用于臨床。BMSCs在體外具有強(qiáng)大的增殖能力,而且可以在不同的理化環(huán)境和細(xì)胞因子的誘導(dǎo)下向骨、脂肪、腱和肌肉等組織分化,還可以跨胚胎分化為神經(jīng)組織,展示出良好的研究和應(yīng)前用景。 實(shí)驗(yàn)?zāi)康? 通過(guò)對(duì)SD大鼠BMSCs的體外分離純化和培養(yǎng)擴(kuò)增,采用免疫熒光染色法對(duì)BMSCs進(jìn)行形態(tài)學(xué)觀察及表型鑒定,并利用其生物學(xué)特性進(jìn)行Nestin免疫細(xì)胞化學(xué)分析、誘導(dǎo)分化及分化鑒定,探討B(tài)MSCs定向分化為神經(jīng)樣細(xì)胞的潛能,為利用BMSCs治療SCI所引起的神經(jīng)源性膀胱提供一種理想的種子細(xì)胞和一定的理論依據(jù)。 實(shí)驗(yàn)方法: 1.采用全骨髓貼壁分離法體外提取、分離及純化大鼠BMSCs,并進(jìn)行傳代擴(kuò)增; 2.繪制BMSCs的生長(zhǎng)曲線,并進(jìn)行免疫熒光染色觀察其形態(tài)結(jié)構(gòu); 3.流式細(xì)胞分析儀檢測(cè)細(xì)胞表面抗原CD29、CD34、CD105的陽(yáng)性表達(dá)率; 4.在成骨細(xì)胞誘導(dǎo)劑和神經(jīng)細(xì)胞誘導(dǎo)劑條件下定向誘導(dǎo)BMSCs向成骨細(xì)胞和神經(jīng)樣細(xì)胞分化,并分別行茜素紅、Nestin免疫細(xì)胞化學(xué)染色鑒定; 5.將誘導(dǎo)形成的神經(jīng)球樣細(xì)胞團(tuán)進(jìn)一步培養(yǎng)分化,并進(jìn)行MAP-2、GFAP免疫細(xì)胞化學(xué)染色鑒定分化形成的神經(jīng)元、星形膠質(zhì)細(xì)胞結(jié)構(gòu)。 實(shí)驗(yàn)結(jié)果: 1.全骨髓貼壁分離法獲得的BMSCs在接種后24～36小時(shí)貼壁,梭形,核質(zhì)比大,呈漩渦狀生長(zhǎng),增殖能力強(qiáng); 2.細(xì)胞生長(zhǎng)經(jīng)過(guò)潛伏適應(yīng)期、對(duì)數(shù)增長(zhǎng)期、平臺(tái)期階段;免疫熒光染色后在熒光顯微鏡下可見(jiàn)全部細(xì)胞均呈綠色熒光,細(xì)胞為梭形或多邊形,細(xì)胞核未染熒光,保持了良好的形態(tài); 3.流式細(xì)胞分析儀顯示P3～P5代細(xì)胞表面抗原CD29、CD105表達(dá)陽(yáng)性,而CD34表達(dá)陰性; 4.在兩種不同誘導(dǎo)劑誘導(dǎo)下BMSCs能分化形成成骨細(xì)胞和神經(jīng)樣細(xì)胞并表達(dá)相應(yīng)的特異性抗原; 5.進(jìn)一步培養(yǎng)分化所形成的細(xì)胞能表達(dá)神經(jīng)元標(biāo)志物MAP-2或神經(jīng)膠質(zhì)細(xì)胞標(biāo)志物GFAP。 結(jié)論: 1.全骨髓貼壁分離法可分離出較純的BMSCs,經(jīng)培養(yǎng)擴(kuò)增后能獲取大量的BMSCs,細(xì)胞呈貼壁生長(zhǎng),增殖能力強(qiáng); 2.BMSCs具有多向分化的潛能,在體外可誘導(dǎo)分化為成骨細(xì)胞及神經(jīng)樣細(xì)胞; 3.分化形成的神經(jīng)球樣細(xì)胞能進(jìn)一步分化為神經(jīng)元和神經(jīng)膠質(zhì)細(xì)胞,是干細(xì)胞移植治療的理想種子細(xì)胞,具有極大的應(yīng)用前景。
[Abstract]:Spinal cord injury (spinal cord, injury, SCI) of neurogenic bladder symptoms caused by the serious influence the quality of life of patients, but there is no effective treatment, and most studies concentrated flow dynamics and nerve function injury in urine. Along with the rapid development of stem cell research, stem cell transplantation for the treatment of blood system malignant tumor, autoimmune diseases of the nervous system and achieved effective results, even has been used in clinical, which made by stem cells and tissue engineering transplantation increases the number of spinal nerves, the reduction of glial scar formation is possible, the stem cells were inoculated to tissue engineering transplant may be a new method for neurogenic bladder effectively after the treatment of SCI. However, in the construction of tissue engineering in the selection of suitable seed cells has become an important part of treatment. All stem cells and pluripotent stem Cells have self-renewal and multilineage differentiation potential, is an important target cells in tissue engineering. Embryonic stem cells exist ethical issues and immune rejection, neural stem cells, with limited sources of olfactory ensheathing cells and Schwann cells, and the sampling is inconvenient, it is difficult to apply to clinical.BMSCs has strong proliferation ability in vitro, but also fat to the bone, induced by different physical and chemical environment of cytokines, tendon and muscle tissue differentiation, can also cross the embryonic differentiation into nerve tissue, show good prospect of research and application.
Objective:
Purified and cultured by SD amplification of rat BMSCs in vitro, morphology and phenotype of BMSCs by immunofluorescence staining, and analyzed by Nestin immunocytochemistry using its biological characteristics, differentiation and differentiation, to explore the differentiation of BMSCs into neuron like cell potential, provides an ideal seed cell and a theoretical basis of neurogenic bladder caused by the use of BMSCs for the treatment of SCI.
Experimental methods:
1. the rat BMSCs was isolated and purified by the method of full bone marrow adherence separation in vitro.
2. the growth curve of BMSCs was plotted and the morphological structure was observed by immunofluorescence staining.
3. flow cytometry was used to detect the positive expression of cell surface antigen CD29, CD34 and CD105.
4., BMSCs was induced to differentiate into osteoblasts and neuron like cells under the condition of osteoblast inducers and nerve cell inducers. They were identified by alizarin red and Nestin immunocytochemical staining.
5., the induced neuron like cell clusters were further cultured and differentiated, and MAP-2 and GFAP immunocytochemical staining were used to identify the differentiated neurons and astrocytes.
Experimental results:
1. the BMSCs obtained by the full bone marrow adherence separation method was adhered to the wall 24~36 hours after inoculation, spindle shape, large nuclear substance ratio, whirlpool like growth and strong proliferation ability.
2., cell growth is in latent adaptation period, logarithmic growth stage and plateau stage. After immunofluorescence staining, all cells in the fluorescence microscope showed green fluorescence. The cells were spindle shaped or polygonal, and the nuclei were not fluorescent, and maintained a good morphology.
The 3. flow cytometry showed that the cell surface antigen of P3 to P5 was CD29, and the expression of CD105 was positive, but the expression of CD34 was negative.
4. under the induction of two different inducers, BMSCs can differentiate into osteoblasts and nerve like cells and express corresponding specific antigens.
5. the cells formed by further differentiation can express the neuron marker MAP-2 or the glial cell marker GFAP.
Conclusion:
1. the total bone marrow adherent separation method can separate the pure BMSCs. After culture amplification, a large number of BMSCs can be obtained. The cells are adhered to the wall, and the proliferation ability is strong.
2.BMSCs has the potential of multidifferentiation and can be induced to differentiate into osteoblasts and nerve like cells in vitro.
3., differentiated neuron like cells can be further differentiated into neurons and glial cells. They are ideal seed cells for stem cell transplantation, and have great potential for application.

【學(xué)位授予單位】：廣州醫(yī)學(xué)院
【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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4 徐路堯;Rac1與骨髓間充質(zhì)干細(xì)胞向神經(jīng)樣細(xì)胞分化過(guò)程關(guān)系的研究[D];蘇州大學(xué);2008年

5 呂曉琳;bFGF對(duì)骨髓間充質(zhì)干細(xì)胞向視網(wǎng)膜神經(jīng)樣細(xì)胞增殖分化的影響[D];吉林大學(xué);2010年

6 馬躍東;VEGF基因轉(zhuǎn)染人臍帶間充質(zhì)干細(xì)胞向神經(jīng)樣細(xì)胞誘導(dǎo)分化的研究[D];遼寧醫(yī)學(xué)院;2011年

7 李鵬斌;骨髓基質(zhì)干細(xì)胞定向分化為神經(jīng)細(xì)胞及其功能特征的研究[D];華中科技大學(xué);2010年

8 于濱濱;微囊化共培養(yǎng)誘導(dǎo)骨髓間充質(zhì)干細(xì)胞體外定向分化新體系的研究[D];大連醫(yī)科大學(xué);2010年

9 宋國(guó)強(qiáng);IGF-1體外誘導(dǎo)人臍帶間充質(zhì)干細(xì)胞向神經(jīng)樣細(xì)胞分化[D];河北醫(yī)科大學(xué);2012年

10 于雁玲;人臍血間充質(zhì)干/祖細(xì)胞誘導(dǎo)為神經(jīng)樣細(xì)胞的實(shí)驗(yàn)研究[D];山西醫(yī)科大學(xué);2005年

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