本文選題：骨髓間充質(zhì)干細(xì)胞 + 細(xì)胞凋亡��； 參考：《蘭州大學(xué)》2010年碩士論文

【摘要】： 目的：建立人骨髓間充質(zhì)干細(xì)胞在體外培養(yǎng)、擴(kuò)增、鑒定的實(shí)驗(yàn)方法；觀察不同劑量的X線照射對骨髓間充質(zhì)干細(xì)胞的細(xì)胞凋亡、細(xì)胞周期及survivin基因表達(dá)量的影響。 方法：通過采集健康成人志愿者的髂后上棘骨髓,以密度為1.073g/ml的Percol分離液分離單個核細(xì)胞,在含有15%FBS的DMEM-LG培養(yǎng)體系中貼壁培養(yǎng),消化傳代擴(kuò)增,在顯微鏡下觀察細(xì)胞形態(tài)特點(diǎn)。流式細(xì)胞術(shù)檢測骨髓間充質(zhì)干細(xì)胞表面抗原；實(shí)驗(yàn)分組：A組OcGy組,B組5cGy組,C組10cGy組,D組20cGy組。P4代骨髓間充質(zhì)干細(xì)胞,以總劑量為0、5、10、20cGy的X線下直接照射,照射劑量率0.1Gy/min,照射距離100cm。用MTT方法測量照射后1-7d的吸光度,然后根據(jù)吸光度繪制生長曲線；分別于照射后24,48和72h收集骨髓間充質(zhì)干細(xì)胞,采用流式細(xì)胞儀檢測細(xì)胞周期情況；采用膜連蛋白V (AnnexinV)和碘化丙啶(PI)雙標(biāo)法檢測細(xì)胞凋亡率；用RT-PCR檢測survivin表達(dá)的量；通過單個核細(xì)胞凝膠電泳檢測DNA損傷情況。 結(jié)果：倒置顯微鏡下觀察發(fā)現(xiàn),骨髓間充質(zhì)干細(xì)胞為均一的、梭形的成纖維樣細(xì)胞,在體外可以持續(xù)培養(yǎng),并能大規(guī)模擴(kuò)增。流式細(xì)胞術(shù)結(jié)果顯示：BM-MSC高表達(dá)CD44、CD13(93.37%),而造血細(xì)胞標(biāo)志CD117、CD45、CD38及CD3呈陰性表達(dá)。我們從MTT結(jié)果發(fā)現(xiàn),骨髓間充質(zhì)干細(xì)胞在受到X線照射后,B組、C組和D組細(xì)胞增殖未受抑制,增殖力和增殖時間明顯增加。細(xì)胞周期結(jié)果顯示：5cGy、10cGy和20cGy的X線照射BM-MSC后,G0/G1期百分率逐漸減少,與同一時間對照組相比,G0/G1期細(xì)胞在48h和72h明顯減低,其中10cGy照射組72h降低最低；與對照組S期相比,C組和D組在24h和48h S期細(xì)胞增加,P0.05,但B組無明顯變化。與對照組同一時間比較,A組G2/M期無明顯增加, C組和D組的24h、48h G2/M增加明顯,P0.05,而72h又有下降,20cGy照射組72h降至最低,P0.05；細(xì)胞凋亡結(jié)果顯示：與對照組相比,B組、C組和D組細(xì)胞凋亡率24h短暫性增高,P0.05,48h和72h又有下降趨勢,72h降至最低,尤其是10cGy和20cGy組,P0.05；不同劑量的X線照射BM-MSC后,B組、C組和D組在24h和48h的survivin表達(dá)量都逐漸的增加,然而72h又有下降的趨勢,但仍高于A組對照組P0.05；骨髓間充質(zhì)survivin表達(dá)量在48h達(dá)到高峰。如彗星圖顯示：5cGy,10cGy、20cGy照射BM-MSC 24h后,骨髓間充質(zhì)干細(xì)胞都有不同程度的損傷,A組彗星率(2%),B組彗星率(14%),C組彗星率(33%),D組彗星率(39%),并表現(xiàn)與劑量有一定的依賴性趨勢。 結(jié)論：我們從生長曲線發(fā)現(xiàn)低劑量X線照射BM-MSC后細(xì)胞的增殖未被抑制,而表現(xiàn)為生長速度和生長時間明顯高于對照組；細(xì)胞凋亡和細(xì)胞周期結(jié)果提示：照射可促進(jìn)BM-MSC由G0/G1期向S期轉(zhuǎn)化,S期細(xì)胞數(shù)增高；細(xì)胞凋亡率早期(24h)呈一過性增加,隨后凋亡率隨時間而降低,說明低劑量X線照射后BM-MSC由靜止期迅速進(jìn)入增殖期,S期百分率明顯增多,而細(xì)胞凋亡在低劑量照射后早期有一過性增多,之后迅速下降,這可能是低劑量的X線照射對BM-MSC產(chǎn)生興奮性效應(yīng)的機(jī)制。低劑量X線照射促進(jìn)MSCs的survivin的表達(dá),48h達(dá)到高峰,提示survivin在骨髓間充質(zhì)干細(xì)胞抵抗低劑量X線誘導(dǎo)的細(xì)胞凋亡中起到重要作用；低劑量X線照射后可引起B(yǎng)M-MSC細(xì)胞的DNA損傷,并表現(xiàn)與劑量有一定的依賴性趨勢。
[Abstract]:Objective : To establish an experimental method for the culture , amplification and identification of human bone marrow mesenchymal stem cells in vitro ;
To observe the effects of different doses of X - ray irradiation on apoptosis , cell cycle and survivin gene expression in bone marrow mesenchymal stem cells .


Methods : Single core cells were isolated from the iliac spine of healthy adult volunteers with density of 1.073 g / ml . The cells were cultured in DMEM - LG culture system containing 15 % FBS . The cell morphology was observed under the microscope . Flow cytometry was used to detect the surface antigen of bone marrow mesenchymal stem cells .
Experimental group : Group A OcGy group , group B 5cGy group , group C 10cGy group , group D 20cGy group . P4 bone marrow mesenchymal stem cells were irradiated directly under X - ray irradiation with total dose of 0 , 5 , 10 , 20cGy . The irradiation dose rate was 0.1Gy / min , the irradiation distance was 100cm . The absorbance of 1 - 7d after irradiation was measured by MTT method , and then the growth curve was drawn according to absorbance .
Bone marrow mesenchymal stem cells were collected 24 , 48 and 72 h after irradiation , and the cell cycle was detected by flow cytometry .
The apoptosis rate was determined by the double standard method of annexinV and propidium iodide ( PI ) .
The expression of survivin was detected by RT - PCR .
DNA damage was detected by single core cell gel electrophoresis .


The results showed that bone marrow mesenchymal stem cells were homogeneous and fusiform fibroblast - like cells were cultured in vitro . The results showed that BM - MSC expressed CD44 and CD13 ( 93.37 % ) .
Compared with control group , group C and D had no significant changes in S phase in 24 h and 48 h , but there was no significant change in group B . Compared with the control group , the G2 / M phase of group A was not significantly increased , 24h , 48h G2 / M increased significantly in group C and D , P 0.05 , while at 72 h decreased , and that of 20cGy irradiation group decreased to the lowest , P0.05 ;
The results showed that the apoptosis rate of cells in group B , group C and D was significantly higher than that in control group ( P0.05 , 48h and 72h ) , and decreased to the lowest in 72h , especially in the group of 10cGy and 20cGy , P0.05 ;
After irradiation of BM - MSC with different doses of X - ray , the expression of survivin was increased in group B , C and D at 24 h and 48 h , however , there was a tendency of decrease in 72 h , but it was still higher than that in group A ( P0.05 ) .
The expression of survivin in bone marrow reached its peak at 48h . The comet assay showed that the bone marrow mesenchymal stem cells had different degrees of injury after irradiation with 5 cGy , 10 cGy and 20cGy . The comet rate in group A ( 2 % ) , comet rate in group B ( 14 % ) , comet rate in group C ( 33 % ) , comet rate in group D ( 39 % ) showed a certain dependence tendency .


Conclusion : From the growth curve , we found that the proliferation of the cells after irradiation of BM - MSC with low dose X - ray was not inhibited , and the growth rate and the growth time were significantly higher than those in the control group .
Apoptosis and cell cycle results suggested that irradiation could promote the transformation of BM - MSC from G0 / G1 phase to S phase , and the number of S phase cells increased ;
The apoptosis rate of BM - MSC was increased with time after low dose X - ray irradiation . The apoptosis rate of BM - MSC increased rapidly after low - dose X - ray irradiation , and then declined rapidly after low - dose X - ray irradiation .
The DNA damage of BM - MSC cells can be caused by low dose X - ray irradiation .
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R329

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