本文關(guān)鍵詞：大鼠少突膠質(zhì)細胞的分離培養(yǎng)與定向誘導分化　出處：《河北醫(yī)科大學》2012年碩士論文　論文類型：學位論文

  更多相關(guān)文章： 少突膠質(zhì)細胞前體細胞 細胞培養(yǎng) 細胞分化 大鼠 純化 細胞增值

【摘要】：目的：少突膠質(zhì)細胞(Oligodendrocyte, OL)是中樞神經(jīng)系統(tǒng)的髓鞘形成細胞，在神經(jīng)元信息的快速傳導以及軸突的功能維持方面起著關(guān)鍵的作用。少突膠質(zhì)細胞起源于胚胎神經(jīng)管的神經(jīng)上皮細胞,前腦的少突膠質(zhì)細胞由內(nèi)側(cè)腦室周圍腦室下區(qū)的祖細胞分化而來,是一種終末細胞不具有分裂能力，其來源于少突膠質(zhì)細胞/Ⅱ型星形膠質(zhì)細胞（oligodendrocyte/type2astrocyte, O2A）。在胚胎形成的晚期階段這些O2A/OPCs在中樞神經(jīng)系統(tǒng)內(nèi)增值并移行，最終分化成為生成髓鞘的少突膠質(zhì)細胞。目前認為在體外培養(yǎng)的少突膠質(zhì)細胞經(jīng)歷了幾個不同的階段，包括增值期的OPCs，其特征為前體細胞標記物如血小板來源生長因子（PDGFaR）的表達，形態(tài)上表現(xiàn)為兩級或三級的細胞；未成熟少突膠質(zhì)細胞，表達能被04抗體特異識別的多級形態(tài)的細胞；以及成熟的髓鞘生成少突膠質(zhì)細胞，其特征為表達髓鞘特異蛋白如髓鞘脂堿蛋。用更為簡單適用的方法分離和純化大量有生物活性的OPCs不僅有助于更好的了解少突膠質(zhì)細胞的功能、行為及軸突-神經(jīng)元的相互作用，更為髓鞘的修復(fù)研究提供了必不可少的工具。更為重要的是，可將體外培養(yǎng)增值的少突膠質(zhì)系細胞移植入脫髓鞘的中樞神經(jīng)系統(tǒng)，少突膠質(zhì)細胞前體通過移行進而產(chǎn)生大量成熟的少突膠質(zhì)細胞，因此O2A/OPCs移值治療脫髓鞘病已在嘗試中，這些實驗均需要大量不同階段的少突膠質(zhì)細胞。同時，少突膠質(zhì)系細胞的分離和培養(yǎng)會促進少突膠質(zhì)系細胞在體外的使用，如應(yīng)用于脫髓鞘病變的藥物研究等。 本實驗旨在研究少突膠質(zhì)細胞祖細胞（oligodendrocyte precursor cells,OPCs）培養(yǎng)及純化方法，并研究不同誘導條件對大鼠O2A/OPCs分化的影響。 方法： 1.混合膠質(zhì)細胞原代培養(yǎng)：取新生48h以內(nèi)的SD大鼠，冰埋麻醉，取出大腦并沿中線切開，去除嗅球、基底核、海馬、腦膜和血管組織，將皮層剪成1mm3的小組織塊，篩網(wǎng)過濾，移入離心管內(nèi)，加入含有DNase I儲備液(0.2mg/ml)和trypsin儲備液(0.25%)的HBSS中進行消化（組織培養(yǎng)箱37℃）,約15min后用DMEM20S停止胰酶消化，離心，種植于75mm玻璃培養(yǎng)瓶內(nèi),每2-3天換液，培養(yǎng)8-9天 2. OPCs分離、純化和增值：以振蕩分離法和差速貼壁法分離純化OPCs;加入促進增殖的細胞因子PDGFα和bFGF，獲得數(shù)量較多的OPCs，采用MTT法檢測OPCs的活力，通過A2B5鑒定，培養(yǎng)細胞純度在95%以上 3.定向誘導分化：更換含血清的培養(yǎng)基和不含血清的化學條件培養(yǎng)基，倒置相差顯微鏡觀察記錄每天細胞的形態(tài)變化,分化成熟的少突膠質(zhì)細胞和星形膠質(zhì)細胞進行免疫細胞化學測定 結(jié)果： 1.在接種后第7-8天，混合膠質(zhì)細胞鋪滿瓶底，少突膠質(zhì)細胞前體細胞位于星形膠質(zhì)細胞層之上； 2.少突膠質(zhì)細胞祖細胞胞體呈圓形，常有單極或雙極突起，形成克隆球，，A2B5標記陽性，免疫熒光鑒定細胞純度可達95％以上；3.生長因子PDGF和bFGF對少突膠質(zhì)細胞前體細胞的存活和增殖及其重要。OPCs具有雙向分化的能力，在不同誘導條件下可分化為星型膠質(zhì)細胞或少突膠質(zhì)細胞；結(jié)論:本實驗證實了培養(yǎng)新生大鼠皮層中的少突膠質(zhì)細胞前體細胞的方法簡單可靠。通過振蕩分離純化法及結(jié)合少突膠質(zhì)細胞定向培養(yǎng)基可以培養(yǎng)出高純度的少突膠質(zhì)細胞前體細胞。添加PDGF、bFGF可顯著提高細胞產(chǎn)量,并使細胞保持在未成熟階段。OPCs具有雙向分化的潛能，在不同化學成分的培養(yǎng)基中可定向誘導出高純度的少突膠質(zhì)細胞和星形膠質(zhì)細胞，這些細胞可用于藥物對于神經(jīng)膠質(zhì)細胞的影響等實驗研究中
[Abstract]:Objective: to oligodendrocytes (Oligodendrocyte, OL) is the myelin forming cells of the CNS, maintenance plays a key role in the rapid transmission of information and the function of neuronal axons. Neural epithelial cells of the oligodendrocyte originated from the embryonic neural tube, forebrain oligodendrocytes by medial periventricular brain the progenitor cells, is a kind of terminal cell does not have the ability to divide its source in oligodendrocytes / type II astrocytes (oligodendrocyte/type2astrocyte, O2A). In the late stage of embryonic development of these O2A/OPCs in the central nervous system and value migration, eventually become generation of myelin oligodendrocyte differentiation glial cells in vitro. The oligodendrocytes through several different stages, including proliferating OPCs, characterized by precursor cell markers such as platelet The source of growth factor (PDGFaR) expression, form two or three cells; immature oligodendrocytes, the expression can be multi form 04 antibodies to identify the cell; and mature myelinating oligodendrocytes, characterized by expression of myelin specific proteins such as myelin alkaline egg. A separation and purification method is simple and applicable to large biological activity of OPCs not only helps to better understand oligodendrocyte function, interaction behavior and neuron axon, more myelin repair research provides an essential tool. More importantly, the oligodendrocyte lineage cells after transplantation into the central nervous system demyelinating value-added of in vitro cultured oligodendrocyte precursor cells through the shift travel arising from a large number of mature oligodendrocytes, so O2A/OPCs transplant demyelinating disease treatment has been in the attempt, these A large number of oligodendrocytes in different stages are needed in the experiment. Meanwhile, the isolation and culture of oligodendroglial cells will promote the use of oligodendrocyte in vitro, such as drug research for demyelinating lesions.
The aim of this study is to investigate the culture and purification methods of oligodendrocyte precursor cells (OPCs), and to study the effect of different induction conditions on O2A/OPCs differentiation in rats.
Method錛

本文編號：1436127