【摘要】：間充質(zhì)干細胞（Mesenchymal stem cells，MSCs）在臨床治療方面有廣泛的應(yīng)用價值。然而，MSCs在體外擴增培養(yǎng)過程中由于多次傳代培養(yǎng)會進入復(fù)制性衰老，進而發(fā)生一些生物學(xué)性狀的改變，，影響臨床應(yīng)用與研究。已有研究證明干細胞微環(huán)境與干細胞的衰老有密切聯(lián)系，但是不同血清微環(huán)境對細胞衰老的影響尚不清楚。目前實驗室體外培養(yǎng)MSCs常用到胎牛血清（fetal bovine serum，F(xiàn)BS）或小牛血清（calf serum，CS），兩種血清對MSCs的衰老進程有何影響，至今未見相關(guān)報道。因此，本實驗擬在探究胎牛血清和小牛血清對MSCs衰老進程的影響。 首先采用組織塊貼壁培養(yǎng)法獲取人臍帶源MSCs，并對傳統(tǒng)的組織塊貼壁法進行了改良，將本應(yīng)丟棄的組織塊進行二次貼壁培養(yǎng)，以獲得更多的原代MSCs。首次組織塊貼壁培養(yǎng)第5-7天可見有細小梭形細胞從組織塊邊緣爬出，到10天左右，可見細胞克隆形成。將組織塊轉(zhuǎn)移到新的培養(yǎng)瓶中進行二次貼壁培養(yǎng)，2天后即有細胞爬出，5天即可形成細胞克隆。對二次貼壁培養(yǎng)得到的MSCs進行流式檢測，結(jié)果顯示，細胞高表達CD90、CD105，不表達CD34、CD45、HLA-DR，說明第二次組織塊貼壁培養(yǎng)得到的細胞也是間充質(zhì)干細胞。同時，我們將得到的細胞與胎盤來源MSCs進行了生物學(xué)特性的比較，發(fā)現(xiàn)兩種細胞沒有明顯的差異性。 對于分離的臍帶源MSCs，從第一代開始，分別采用胎牛血清和小牛血清進行體外長期培養(yǎng)，直至細胞衰老。兩組采用相同的接種密度，并在傳代培養(yǎng)時計算所得細胞數(shù)和群體倍增數(shù)，進而累加得到累計群體倍增數(shù)。選取處于相同累計群體倍增數(shù)的三個階段，對兩組細胞分別進行增殖、分化、凋亡以及衰老相關(guān)指標(biāo)的檢測。結(jié)果發(fā)現(xiàn)，隨著傳代次數(shù)的增加，MSCs的增殖分化能力下降，凋亡率升高，同時，衰老相關(guān)的β-半乳糖苷酶活性增強，細胞內(nèi)ROS水平升高，p53、p21、p16等基因表達量增高。結(jié)果還顯示胎牛血清組MSCs的增殖和分化能力要優(yōu)于小牛血清組，而細胞的凋亡比率、細胞內(nèi)ROS水平以及p53等衰老相關(guān)基因的表達水平要低于小牛血清組。說明胎牛血清能夠?qū)SCs的生長提供一個更好的微環(huán)境，有助于保持其生物學(xué)特性，延緩衰老，而小牛血清則會加速細胞的衰老進程。
[Abstract]:Mesenchymal stem cell (Mesenchymal stem cells,MSCs) has been widely used in clinical treatment. However, in the process of MSCs amplification in vitro, the repeated passage culture will enter into replicative senescence, and then some biological characters will change, which will affect the clinical application and research. It has been proved that stem cell microenvironment is closely related to stem cell senescence, but the effect of different serum microenvironment on cell senescence is not clear. At present, fetal bovine serum (fetal bovine serum,FBS (FBS) or calf serum (calf serum,CS) are commonly used in laboratory culture of MSCs in vitro. There are no reports on the effects of the two serums on the aging process of MSCs. Therefore, the effect of fetal bovine serum and calf serum on the aging process of MSCs was studied. Firstly, the human umbilical cord derived MSCs, was obtained by using tissue mass adherent culture method, and the traditional tissue block adherent method was improved to obtain more primary MSCs. by secondary adherent culture of tissue mass that should have been discarded. On the 5-7 days after the first tissue mass adherent culture, there were small fusiform cells crawling out from the edge of the tissue mass, and about 10 days later, the cell clone formation was observed. The tissue mass was transferred to a new culture bottle for secondary adherent culture. After 2 days, the cells crawled out, and the cell clone was formed in 5 days. The MSCs obtained from the secondary adherent culture was detected by flow cytometry. The results showed that the cells with high expression of CD90,CD105, did not express CD34,CD45,HLA-DR, indicating that the cells obtained from the second tissue mass adhesion culture were also mesenchymal stem cells. At the same time, we compared the biological characteristics of the obtained cells with placental MSCs, and found that there was no significant difference between the two kinds of cells. For isolated umbilical cord MSCs, fetal bovine serum and calf serum were used for long-term culture in vitro from the first generation, respectively, until cell senescence. The two groups used the same inoculation density and calculated the cell number and the population multiplication number during the passage and then accumulated the cumulative population multiplication number. The cell proliferation, differentiation, apoptosis and senescence were detected in three stages of the same cumulative population multiplication. The results showed that with the increase of passage times, the proliferation and differentiation ability of MSCs decreased and the apoptosis rate increased. At the same time, the activity of 尾 -galactosidase increased, the level of ROS increased, and the expression of p53, p21, p16 and other genes increased. The results also showed that the proliferation and differentiation ability of MSCs in fetal bovine serum group was better than that in calf serum group, while the apoptosis ratio, intracellular ROS level and the expression level of senescence related genes such as p53 in fetal bovine serum group were lower than those in calf serum group. The results showed that fetal bovine serum could provide a better microenvironment for the growth of MSCs, which could help to maintain its biological characteristics and delay senescence, while calf serum could accelerate the process of cell senescence.
【學(xué)位授予單位】：天津大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R329.2

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1 徐燕;李長虹;孟恒星;郝牧;邱錄貴;;人臍帶間充質(zhì)干細胞分離培養(yǎng)條件的優(yōu)化及其生物學(xué)特性[J];中國組織工程研究與臨床康復(fù);2009年32期

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