本文選題：細(xì)胞培養(yǎng) + 外周血纖維細(xì)胞�。� 參考：《第四軍醫(yī)大學(xué)》2012年碩士論文

【摘要】：外周血纖維細(xì)胞（peripheral blood fibrocytes）是近年來(lái)發(fā)現(xiàn)的一個(gè)多分化潛能細(xì)胞，它具有成纖維細(xì)胞的特性，并被最新研究證實(shí)能分化為肌成纖維細(xì)胞與血管周細(xì)胞。外周血纖維細(xì)胞能夠在組織損傷后在炎癥期迅速遷移至創(chuàng)面局部，進(jìn)而發(fā)揮它抗原呈遞、收縮創(chuàng)面、合成釋放多種細(xì)胞因子和細(xì)胞外基質(zhì)并促進(jìn)創(chuàng)面新生血管形成等等一系列作用，因此可以說(shuō)，PBFCs的定向遷移是其在創(chuàng)傷修復(fù)中發(fā)揮作用的重要基礎(chǔ)。而骨髓來(lái)源的PBFCs是受到哪些因子或細(xì)胞的趨化而定向遷移到創(chuàng)傷部位的，關(guān)于這一問(wèn)題目前尚無(wú)相關(guān)報(bào)道。此外，關(guān)于外周血纖維細(xì)胞的體外分離培養(yǎng)，目前常用的方法都需要免疫磁珠篩選來(lái)對(duì)PBFCs進(jìn)行純化，此法費(fèi)用高昂且獲得的目的細(xì)胞數(shù)量少，不利于臨床應(yīng)用研究的開展。 實(shí)驗(yàn)?zāi)康模?探索更為簡(jiǎn)便易行的外周血纖維細(xì)胞體外培養(yǎng)方法、了解體外培養(yǎng)的PBFCs生物學(xué)特征及功能，，研究體外共培養(yǎng)條件下人真皮微血管內(nèi)皮細(xì)胞對(duì)外周血纖維細(xì)胞有無(wú)趨化作用。 方法： 采用Ficoll密度梯度離心分離法分離成人外周血，所獲得的白細(xì)胞在一定條件要求下體外培養(yǎng)，觀察不同接種密度、換液時(shí)間、血清濃度等培養(yǎng)條件下對(duì)PBFCs生長(zhǎng)分化的影響，采用流式細(xì)胞技術(shù)、細(xì)胞免疫熒光染色等對(duì)體外培養(yǎng)的細(xì)胞進(jìn)行鑒定，在掃描電鏡下進(jìn)一步觀察其形態(tài)結(jié)構(gòu)，Transwell小室共培養(yǎng)初步探索人真皮微血管內(nèi)皮細(xì)胞對(duì)PBFCs有無(wú)趨化作用。 結(jié)果： 1．采用“密度梯度離心法”分離成人外周血得到的白膜層細(xì)胞中含有大量淋巴細(xì)胞、單核細(xì)胞和少量粒細(xì)胞，在適當(dāng)?shù)呐囵B(yǎng)條件下，這些細(xì)胞于兩周左右分化培養(yǎng)出血液來(lái)源的前體細(xì)胞——外周血纖維細(xì)胞。 2．多種因素均會(huì)對(duì)外周血纖維細(xì)胞的培養(yǎng)造成影響，經(jīng)過(guò)我們反復(fù)實(shí)驗(yàn)，認(rèn)為血液來(lái)源的取材、首次換液的時(shí)間、細(xì)胞的接種密度、血清濃度等均是細(xì)胞培養(yǎng)成敗與否的關(guān)鍵因素。我們認(rèn)為選取年齡在20至45歲的健康志愿者采血，以pH值為7.2~7.3，含有20%胎牛血清的DMEM培養(yǎng)基在六孔培養(yǎng)板上按5×106/cm2接種，4天后首次換液，之后每三天換液一次，是PBFCs培養(yǎng)的適宜條件，經(jīng)以上條件培養(yǎng)得到的PBFCs細(xì)胞純度可達(dá)80%以上，能夠滿足科研實(shí)驗(yàn)的一般要求。 3．免疫熒光染色鑒定的結(jié)果顯示培養(yǎng)12天時(shí)CD34和COLⅠ均為強(qiáng)陽(yáng)性表達(dá)，繼續(xù)培養(yǎng)至28天時(shí)，血液來(lái)源的細(xì)胞表面抗原CD34發(fā)生明顯丟失，免疫熒光染色幾乎不能顯色，相反COLⅠ持續(xù)表達(dá)陽(yáng)性，顯示PBFCs不斷向成纖維細(xì)胞分化的特性。 4．Transwell穿膜實(shí)驗(yàn)證實(shí)了人真皮微血管內(nèi)皮細(xì)胞對(duì)外周血纖維細(xì)胞的趨化作用，為進(jìn)一步研究血管新生過(guò)程中PBFCs的定向遷移及其機(jī)制打下基礎(chǔ)。 結(jié)論： 采用密度梯度離心法配合適當(dāng)?shù)呐囵B(yǎng)條件，成人外周血中存在的前體細(xì)胞經(jīng)體外分離、培養(yǎng)可分化為外周血纖維細(xì)胞，并保持其生物學(xué)特性，建立了較為可靠、穩(wěn)定的分離培養(yǎng)方法。初步掌握了體外培養(yǎng)過(guò)程中PBFCs的生物學(xué)特性和表性變化，在此基礎(chǔ)上，研究證實(shí)了人真皮微血管內(nèi)皮細(xì)胞對(duì)外周血纖維細(xì)胞的趨化作用。
[Abstract]:Peripheral blood fibroblasts ( PBFs ) are a multi - differentiation potential cell found in recent years , which has the characteristics of fibroblasts . It has been proved to be able to differentiate into myofibroblasts and perivascular cells .

Purpose of the experiment :

Objective To explore the biological characteristics and function of cultured human dermal vascular endothelial cells ( PBEC ) in vitro and to explore the effect of human dermal vascular endothelial cells on the presence or absence of human dermal vascular endothelial cells in vitro .

Method :

Using Ficoll density gradient centrifugation method to separate adult peripheral blood , the cultured white blood cells were cultured in vitro under certain conditions , and cultured in vitro under the conditions of different inoculation density , time of change , serum concentration and so on . The cultured cells were identified by flow cytometry and immunofluorescence staining .

Results :

1 . Large amounts of lymphocytes , monocytes and a small amount of leukocytes were isolated from peripheral blood of adult by " density gradient centrifugation " . These cells were differentiated into precursor cell _ peripheral blood cells from the blood source for two weeks under appropriate culture conditions .

2 . The culture of peripheral blood cells was affected by various factors . After repeated experiments , it was concluded that the blood - derived materials , the time of the first liquid exchange , the density of inoculated cells and the serum concentration were the key factors for the success or failure of cell culture .

3 . The results of immunofluorescence staining showed that CD34 and COL 鈪

本文編號(hào)：1772575