本文選題：睪丸支持細(xì)胞 + 骨髓間充質(zhì)干細(xì)胞　； 參考：《華東理工大學(xué)》2012年碩士論文

【摘要】：睪丸支持細(xì)胞(SCs)具有分泌多種細(xì)胞因子與營(yíng)養(yǎng)物質(zhì)的功能,能滋養(yǎng)或促進(jìn)共培養(yǎng)細(xì)胞的增殖或分化,且可形成局部免疫豁免的功能。骨髓間充質(zhì)干細(xì)胞(bmMSCs)作為源自于骨髓的間充質(zhì)干細(xì)胞,bmMSCs在不同的誘導(dǎo)條件下可以分化為多種跨胚層細(xì)胞,在組織工程領(lǐng)域具有廣泛的研究?jī)r(jià)值。本論文主要對(duì)SCs與bmMSCs分離鑒定、SCs促進(jìn)bmMSCs定向分化及分化的特性進(jìn)行初步研究,并建立了利用攪拌式生物反應(yīng)器實(shí)現(xiàn)bmMSCs在微載體上大量增殖的方法,并對(duì)擴(kuò)增后的bmMSCs進(jìn)行生理特性的研究。具體結(jié)果如下： (1)SCs被從10d大的小鼠睪丸中成功分離與鑒定。經(jīng)蘇木精伊紅染色與電子掃描顯微鏡鑒定為支持細(xì)胞,具有明顯的雙核仁特征；通過(guò)對(duì)培養(yǎng)基與轉(zhuǎn)瓶微載體培養(yǎng)體系的工藝優(yōu)化,成功實(shí)現(xiàn)SCs大量擴(kuò)增,支持細(xì)胞由初始2.25×105cells/ml的接種密度增殖至2.37×106cells/ml。 (2)利用密度梯度離心的方法從骨髓中成功分離出bmMSCs。體外培養(yǎng)時(shí)呈現(xiàn)典型的魚(yú)群或漩渦狀排列,經(jīng)流式細(xì)胞術(shù)檢測(cè)表面抗原CD29. CD90與CD34鑒定為bmMSCs。通過(guò)檢測(cè)不同血清濃度對(duì)bmMSCs體外增殖的影響,發(fā)現(xiàn)高濃度血清培養(yǎng)可能會(huì)引起bmMSCs分化,影響bmMSCs的生理特性,因此確定含1%FBS的培養(yǎng)基更適宜bmMSCs的體外培養(yǎng)。 (3)采用兩種細(xì)胞共培養(yǎng)與條件培養(yǎng)基(CM)的培養(yǎng)模式,進(jìn)行SCs促進(jìn)bmMSCs定向分化為軟骨細(xì)胞與成骨細(xì)胞的特性研究。通過(guò)免疫細(xì)胞化學(xué)、細(xì)胞化學(xué)、RT-PCR與、Vestern-blot等方法進(jìn)行檢測(cè),結(jié)果表明SCs能夠顯著促進(jìn)bmMSCs定向分化。 (4)建立1.5L攪拌式生物反應(yīng)器的bmMSCs體外大規(guī)模擴(kuò)增的微載體培養(yǎng)方法。在細(xì)胞與微載體接種密度分別為2.5×105cells/ml與4mg/ml,攪拌速率實(shí)時(shí)調(diào)控的條件下,利用Cytodex3微載體與低血清(1%)培養(yǎng)基以及1.5L攪拌式反應(yīng)器,成功地實(shí)現(xiàn)了bmMSCs體外大規(guī)模擴(kuò)增。通過(guò)每24h更換50%培養(yǎng)基的策略,bmMSCs的擴(kuò)增倍數(shù)達(dá)到了10.4倍,最大細(xì)胞密度達(dá)到2.6x106cells/ml。通過(guò)流式細(xì)胞術(shù)與定向分化檢測(cè),用胰酶從微載體上消化下的bmMSCs仍能保留其表型特征以及分化為軟骨與成骨細(xì)胞的潛能。
[Abstract]:Testicular Sertoli cells (SCS) can secrete many cytokines and nutrients, can nourish or promote the proliferation or differentiation of co-cultured cells, and form the function of local immunity. Bone marrow mesenchymal stem cells (BMSCs), as mesenchymal stem cells derived from bone marrow, can differentiate into many kinds of transdermal cells under different induction conditions. In this paper, we mainly studied the characteristics of promoting the directional differentiation and differentiation of BmMSCs by isolation and identification of SCs from bmMSCs, and established a method to realize the proliferation of bmMSCs on microcarriers by stirred bioreactor. The physiological characteristics of amplified bmMSCs were studied. The results are as follows: 1) the stem cells were isolated and identified successfully from the testis of 10 d old mice. Sertoli cells were identified by hematoxylin eosin staining and electron scanning microscope. Sertoli cells proliferated from the initial inoculation density of 2.25 脳 105cells/ml to 2.37 脳 106 cells / ml 路ml. 2) bmMSCs were isolated from bone marrow by density gradient centrifugation. In vitro culture showed a typical fish colony or whirlpool arrangement, and the surface antigen CD29 was detected by flow cytometry. CD90 and CD34 were identified as bmMSCs. By detecting the effects of different serum concentrations on the proliferation of bmMSCs in vitro, it was found that the high concentration of serum culture might induce the differentiation of bmMSCs and affect the physiological characteristics of bmMSCs. Therefore, the culture medium containing 1s was more suitable for the culture of bmMSCs in vitro. (3) the characteristics of SCs promoting the differentiation of bmMSCs into chondrocytes and osteoblasts were studied by using two culture modes of co-culture and conditioned medium (CMM). The results of immunocytochemistry RT-PCR and Vestern-blot showed that SCs could significantly promote the directional differentiation of bmMSCs. Under the condition that the cell and microcarrier inoculation densities were 2.5 脳 105cells/ml and 4 mg / ml, respectively, and the stirring rate was adjusted in real time, using Cytodex3 microcarrier and low serum 1 layer) medium and 1.5L stirred reactor, BmMSCs were successfully amplified in vitro on a large scale. By replacing 50% culture medium every 24 hours, the expansion times of BmMSCs reached 10.4 times, and the maximum cell density reached 2.6x106 cells / ml. By flow cytometry and directional differentiation detection, bmMSCs digested with trypsin from microcarriers could still retain their phenotypic characteristics and the potential to differentiate into cartilage and osteoblasts.
【學(xué)位授予單位】：華東理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329

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本文編號(hào)：1998732