本文選題：生物反應(yīng)器　切入點(diǎn)：胎盤(pán)間充質(zhì)干細(xì)胞　出處：《中國(guó)醫(yī)科大學(xué)》2008年碩士論文

【摘要】： 目的 近年來(lái)研究發(fā)現(xiàn),間充質(zhì)干細(xì)胞(mesenchymal stem cells,MSCs)不僅支持造血系統(tǒng),還可以向中胚層和外胚層來(lái)源的組織分化,在特定的培養(yǎng)條件下可向骨、成骨、脂肪細(xì)胞、神經(jīng)細(xì)胞及肌細(xì)胞分化,且MSCs的免疫原性較低,在異基因移植中,用不相關(guān)供者的MSCs做滋養(yǎng)層支持造血干細(xì)胞(haemopoietic stemcell,HSC)體外擴(kuò)增并不引起異體淋巴細(xì)胞反應(yīng)。因而,MSCs在臨床上具有廣泛的應(yīng)用前景。目前,在實(shí)驗(yàn)室里,MSCs的體外擴(kuò)增主要采用小規(guī)模的靜態(tài)培養(yǎng)方式,靜態(tài)培養(yǎng)有其固有的局限性:培養(yǎng)環(huán)境不均一、培養(yǎng)參數(shù)不能及時(shí)監(jiān)測(cè)和調(diào)控以及不利于大規(guī)模的培養(yǎng)等。攪拌式生物反應(yīng)器培養(yǎng)技術(shù)可以為細(xì)胞的體外擴(kuò)增提供均衡的營(yíng)養(yǎng)環(huán)境,也可以在培養(yǎng)過(guò)程中對(duì)細(xì)胞代謝和培養(yǎng)液進(jìn)行監(jiān)控。人胎盤(pán)來(lái)源的間充質(zhì)干細(xì)胞(human placenta-derived mesenchymal stem cells,hPDMSCs)不僅來(lái)源廣泛,且對(duì)其研究不會(huì)涉及倫理道德和法律問(wèn)題,因此成為人類(lèi)MSCs新的來(lái)源。本實(shí)驗(yàn)應(yīng)用攪拌式生物反應(yīng)器體外擴(kuò)增hPDMSCs,通過(guò)檢測(cè)細(xì)胞體外擴(kuò)增速率、細(xì)胞表面標(biāo)記物、細(xì)胞周期、細(xì)胞代謝及培養(yǎng)液的PH和滲透壓的變化,探討體外大量擴(kuò)增干細(xì)胞的方法。 方法 1、分離培養(yǎng)hPDMSCs:取足月剖宮產(chǎn)胎兒胎盤(pán)子體面組織,PBS沖洗去除血跡,采用組織微塊貼壁培養(yǎng)法分離培養(yǎng)hPDMSCs。 2、Cytodex-3型微載體及攪拌式生物反應(yīng)器的預(yù)處理:以10mg/ml稱(chēng)取適量的cytodex-3微載體,PBS浸泡過(guò)夜、清洗、高壓滅菌、0.2%明膠包被后備用。無(wú)水乙醇浸泡攪拌式生物反應(yīng)器過(guò)夜,三蒸水反復(fù)沖洗,高壓滅菌后備用。 3、攪拌式生物反應(yīng)器體外擴(kuò)增hPDMSCs:分別將1×1O~7 cells接種于微載體上制備成細(xì)胞懸液,在培養(yǎng)瓶?jī)?nèi)預(yù)培養(yǎng)24h后,將該懸液轉(zhuǎn)移至攪拌式生物反應(yīng)器內(nèi)繼續(xù)培養(yǎng),逐漸調(diào)整攪拌式生物反應(yīng)器至適當(dāng)?shù)霓D(zhuǎn)數(shù),同時(shí)設(shè)置靜態(tài)培養(yǎng)的培養(yǎng)瓶為對(duì)照組。每2d半量換液一次。 4、hPDMSCs的生長(zhǎng)與代謝變化的檢測(cè):每日分別從培養(yǎng)瓶中和生物反應(yīng)器內(nèi)取樣,檢測(cè)細(xì)胞的擴(kuò)增速率和培養(yǎng)液的PH、乳酸含量、葡萄糖含量、滲透壓。 5、流式細(xì)胞儀檢測(cè)細(xì)胞表面標(biāo)記物與細(xì)胞周期:取應(yīng)用攪拌式生物反應(yīng)器培養(yǎng)前、后的hPDMSCs,流式細(xì)胞儀檢測(cè)CD13、CD14、CD29、CD31、CD44、CD45、CD73、CD90、CD105、CD166、HLA-DR、HLA-ABC的表達(dá)情況及細(xì)胞周期的變化并進(jìn)行比較。 結(jié)果 1、胎盤(pán)中提取的MSC形態(tài):呈梭形或成纖維狀,傳至20代時(shí)仍可穩(wěn)定生長(zhǎng)。 2、人胎盤(pán)組織來(lái)源的細(xì)胞細(xì)胞表面標(biāo)記特征:表達(dá)CD13、CD29、CD44、CD73、CD90、CD105、CD166、HLA-ABC,不表達(dá)CD14、CD31、CD45、HLA-DR。 3、hPDMCs的生長(zhǎng)與代謝的變化 (1)hPDMCs在cytodex-3型微載體上的貼附情況:對(duì)照組hPDMCs較為明顯的黏附在微載體的周?chē)?但僅看到少數(shù)微載體間的搭橋現(xiàn)象,且參與的微載體的個(gè)數(shù)較少;而在攪拌式生物反應(yīng)器中,微載體大都通過(guò)細(xì)胞基質(zhì)黏附在一起,呈現(xiàn)明顯的搭橋現(xiàn)象。 (2)hPDMCs的生長(zhǎng)曲線(xiàn):對(duì)照組中的hPDMCs開(kāi)始以較為平緩的趨勢(shì)擴(kuò)增,而且細(xì)胞密度一直呈現(xiàn)升高的趨勢(shì)并在第4d達(dá)峰值,隨后,細(xì)胞的密度就出現(xiàn)較為明顯的下降趨勢(shì);攪拌式生物反應(yīng)器中的hPDMCs在剛接入的48h內(nèi),其擴(kuò)增趨勢(shì)較對(duì)照組略低,隨后細(xì)胞出現(xiàn)明顯擴(kuò)增的跡象。 (3)hPDMCs的擴(kuò)增倍數(shù):hPDMCs在攪拌式生物反應(yīng)器中每代可以擴(kuò)增10.55±1.62倍,明顯高于對(duì)照組的6.10±0.11倍的擴(kuò)增值(P＜0.05)。 (4)營(yíng)養(yǎng)物質(zhì)的消耗與代謝:攪拌式生物反應(yīng)器內(nèi)的葡萄糖消耗較對(duì)照組的高,但乳酸的生成卻較對(duì)照組的低。 (5)培養(yǎng)液的PH值的變化:反應(yīng)器內(nèi)的PH變化幅度較對(duì)照組的小。 (6)培養(yǎng)液的滲透壓的變化:反應(yīng)器和對(duì)照組內(nèi)培養(yǎng)液的滲透壓均維持在(280-320)mmol/kg之間。 4、hPDMCs表面標(biāo)記特征及細(xì)胞周期在生物反應(yīng)器培養(yǎng)前、后的變化:流式細(xì)胞儀檢測(cè)應(yīng)用攪拌式生物反應(yīng)器培養(yǎng)前、后的細(xì)胞表面標(biāo)記特征及細(xì)胞周期無(wú)明顯改變(P＞0.05)。 結(jié)論 1、胎盤(pán)中存在著豐富的MSCs,不僅來(lái)源廣泛,且對(duì)其研究不會(huì)涉及倫理道德和法律問(wèn)題,為人類(lèi)提供了新的干細(xì)胞種子來(lái)源。 2、攪拌式生物反應(yīng)器為hPDMSCs提供了一種理想的體外擴(kuò)增的三維培養(yǎng)體系,與靜態(tài)的二維培養(yǎng)體系相比較,在這個(gè)三維培養(yǎng)體系中,細(xì)胞營(yíng)養(yǎng)物質(zhì)相對(duì)均一,使細(xì)胞表現(xiàn)出良好的擴(kuò)增能力,同時(shí)擴(kuò)增后的細(xì)胞干細(xì)胞特性無(wú)明顯變化。因而,攪拌式生物反應(yīng)器培養(yǎng)技術(shù)為體外獲得高數(shù)量的hPDMSCs提供了一種安全、有效的培養(yǎng)方法,同時(shí)為生物反應(yīng)器的研發(fā)提供了生物學(xué)依據(jù)。
[Abstract]:Purpose


Human placenta - derived mesenchymal stem cells ( hPDMSCs ) can be used to amplify hPDMSCs in vitro .


method


( 1 ) separating and culturing hPDMSCs : taking the fetal placenta sub - body surface tissues in full - term cesarean section , washing and removing blood stains by PBS , and separating and culturing hPDMSCs by using tissue micro - block adherent culture method .


2 . Pre - treatment of Cytodex - 3 microcarriers and stirred bioreactor : Take a proper amount of cytodex - 3 microcarriers in 10 mg / ml , soak overnight in PBS overnight , clean , sterilize under high pressure , 0.2 % gelatin capsule was used for backup .


3 . In vitro amplification of hPDMSCs by a stirred bioreactor : 1 脳 1O - 7 cells were seeded onto the microcarriers to prepare the cell suspension . After the culture was pre - cultured for 24 h , the suspension was transferred to the stirred bioreactor to continue the culture , and the stirred bioreactor was gradually adjusted to the appropriate number of revolutions , while the culture flask with static culture was set as the control group .


4 . Detection of growth and metabolic changes of hPDMSCs : sampling from the culture flask and bioreactor , respectively , detecting the amplification rate of the cells and PH , lactic acid content , glucose content and osmotic pressure of the culture solution .


5 . The expression of CD13 , CD14 , CD29 , CD31 , CD44 , CD166 , CD73 , CD90 , CD105 , CD166 , HLA - DR , HLA - ABC and the changes of cell cycle were detected by flow cytometry .


Results


1 . MSC morphology extracted from placenta : fusiform or fibrous , and can be stably grown in 20 passages .


2 . Human placental tissue - derived cell surface labeling features : CD13 , CD29 , CD44 , CD73 , CD90 , CD105 , CD166 , HLA - ABC , CD14 , CD31 , CD45 , HLA - DR were not expressed .


3 . Changes of growth and metabolism of hPDGFs


( 1 ) The attachment of hPDGFs on the cytodex - 3 microcarriers : the control group of the microcarriers was obviously adhered to the periphery of the microcarriers , but only a small number of microcarriers were observed , and the number of the participating microcarriers was less ; and in the stirred bioreactor , the microcarriers were adhered together by the cell matrix , showing obvious bridging phenomenon .


( 2 ) The growth curve of hPDGFs showed that the growth curve of hPDGFs in the control group began to be expanded with a relatively gentle trend , and the density of cells had always increased and peaked at the 4th day , then the density of the cells appeared to decrease obviously . The amplification tendency of hPDLBs in the stirred bioreactor was slightly lower than that of the control group , and then there were some signs of obvious amplification of the cells .


( 3 ) The amplification factor of hPDMD1was 10.55 鹵 1.62times in the stirred bioreactor , which was significantly higher than that of the control group ( 6.10 鹵 0.11 ) ( P < 0.05 ) .


( 4 ) Consumption and metabolism of nutrient substances : glucose consumption in stirred bioreactor was higher than that of the control group , but the production of lactic acid was lower than that of the control group .


( 5 ) The PH value of the culture fluid varied : the pH change in the reactor was smaller than that of the control group .


( 6 ) The osmotic pressure of the culture fluid varied : the osmotic pressure of the culture solution in the reactor and the control group was maintained between ( 280 - 320 ) mmol / kg .


4 . The marked characteristics of the surface markers and the cell cycle before and after the bioreactor were cultured : flow cytometry showed that the cell surface markers and cell cycle did not change significantly ( P > 0.05 ) before and after the application of stirred bioreactor .


Conclusion


1 . There are abundant MSCs in the placenta , which is not only a wide source , but also does not involve ethics and legal issues . It provides a new source of stem cells for human beings .


2 . The stirred bioreactor provides an ideal three - dimensional culture system for hPDMSCs , and compared with the static two - dimensional culture system , the cell nutrient substances are relatively uniform in the three - dimensional culture system , so that the cells exhibit good amplification capability , and the characteristics of the expanded cell stem cells are not obviously changed .

【學(xué)位授予單位】：中國(guó)醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R329.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 白靈,樊瑜波,張明;離體培養(yǎng)細(xì)胞的力學(xué)實(shí)驗(yàn)方法[J];生物醫(yī)學(xué)工程學(xué)雜志;2002年02期

2 張毅,李長(zhǎng)東,江小霞,李荷蓮,唐佩弦,毛寧;Comparison of mesenchymal stem cells from human placenta and bone marrow[J];Chinese Medical Journal;2004年06期

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本文編號(hào)：1699102