低剂量X线照射对骨髓间充质干细胞生物学影响

发布时间：2018-06-21 10:09

  本文选题：骨髓间充质干细胞 + 细胞凋亡　； 参考：《兰州大学》2010年硕士论文

【摘要】： 目的：建立人骨髓间充质干细胞在体外培养、扩增、鉴定的实验方法；观察不同剂量的X线照射对骨髓间充质干细胞的细胞凋亡、细胞周期及survivin基因表达量的影响。 方法：通过采集健康成人志愿者的髂后上棘骨髓,以密度为1.073g/ml的Percol分离液分离单个核细胞,在含有15%FBS的DMEM-LG培养体系中贴壁培养,消化传代扩增,在显微镜下观察细胞形态特点。流式细胞术检测骨髓间充质干细胞表面抗原；实验分组：A组OcGy组,B组5cGy组,C组10cGy组,D组20cGy组。P4代骨髓间充质干细胞,以总剂量为0、5、10、20cGy的X线下直接照射,照射剂量率0.1Gy/min,照射距离100cm。用MTT方法测量照射后1-7d的吸光度,然后根据吸光度绘制生长曲线；分别于照射后24,48和72h收集骨髓间充质干细胞,采用流式细胞仪检测细胞周期情况；采用膜连蛋白V (AnnexinV)和碘化丙啶(PI)双标法检测细胞凋亡率；用RT-PCR检测survivin表达的量；通过单个核细胞凝胶电泳检测DNA损伤情况。 结果：倒置显微镜下观察发现,骨髓间充质干细胞为均一的、梭形的成纤维样细胞,在体外可以持续培养,并能大规模扩增。流式细胞术结果显示：BM-MSC高表达CD44、CD13(93.37%),而造血细胞标志CD117、CD45、CD38及CD3呈阴性表达。我们从MTT结果发现,骨髓间充质干细胞在受到X线照射后,B组、C组和D组细胞增殖未受抑制,增殖力和增殖时间明显增加。细胞周期结果显示：5cGy、10cGy和20cGy的X线照射BM-MSC后,G0/G1期百分率逐渐减少,与同一时间对照组相比,G0/G1期细胞在48h和72h明显减低,其中10cGy照射组72h降低最低；与对照组S期相比,C组和D组在24h和48h S期细胞增加,P0.05,但B组无明显变化。与对照组同一时间比较,A组G2/M期无明显增加, C组和D组的24h、48h G2/M增加明显,P0.05,而72h又有下降,20cGy照射组72h降至最低,P0.05；细胞凋亡结果显示：与对照组相比,B组、C组和D组细胞凋亡率24h短暂性增高,P0.05,48h和72h又有下降趋势,72h降至最低,尤其是10cGy和20cGy组,P0.05；不同剂量的X线照射BM-MSC后,B组、C组和D组在24h和48h的survivin表达量都逐渐的增加,然而72h又有下降的趋势,但仍高于A组对照组P0.05；骨髓间充质survivin表达量在48h达到高峰。如彗星图显示：5cGy,10cGy、20cGy照射BM-MSC 24h后,骨髓间充质干细胞都有不同程度的损伤,A组彗星率(2%),B组彗星率(14%),C组彗星率(33%),D组彗星率(39%),并表现与剂量有一定的依赖性趋势。 结论：我们从生长曲线发现低剂量X线照射BM-MSC后细胞的增殖未被抑制,而表现为生长速度和生长时间明显高于对照组；细胞凋亡和细胞周期结果提示：照射可促进BM-MSC由G0/G1期向S期转化,S期细胞数增高；细胞凋亡率早期(24h)呈一过性增加,随后凋亡率随时间而降低,说明低剂量X线照射后BM-MSC由静止期迅速进入增殖期,S期百分率明显增多,而细胞凋亡在低剂量照射后早期有一过性增多,之后迅速下降,这可能是低剂量的X线照射对BM-MSC产生兴奋性效应的机制。低剂量X线照射促进MSCs的survivin的表达,48h达到高峰,提示survivin在骨髓间充质干细胞抵抗低剂量X线诱导的细胞凋亡中起到重要作用；低剂量X线照射后可引起BM-MSC细胞的DNA损伤,并表现与剂量有一定的依赖性趋势。
[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 .
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

【参考文献】

相关期刊论文 前10条

1 杨岩,王娟,陈晓,康丽花;成人骨髓间充质干细胞的体外培养及生物学性质[J];吉林大学学报(医学版);2005年02期

2 路艳蒙,傅文玉,朴英杰,乔东访,安连兵;人骨髓间充质干细胞的超微结构[J];电子显微学报;2002年04期

3 袁德晓;潘燕;赵镁嘉;陈红红;邵春林;;DNA损伤修复能力对辐射适应性反应的影响[J];核技术;2009年05期

4 杜日昌;谭丽珊;陈玉英;陈智慧;;环氧化酶-2和survivin在子宫内膜癌中的表达及临床意义[J];实用医学杂志;2007年11期

5 周雅丽;白海;孙延庆;王存邦;赵强;张玲;何晓霞;;Survivin在骨髓间充质干细胞中的表达研究[J];西北国防医学杂志;2009年02期

6 刘树铮;辐射免疫学中的剂量效应关系[J];中华放射医学与防护杂志;2002年06期

7 呼莹,马丽,马冠杰,姜学英,赵春华;成人和胎儿骨髓间充质干细胞的比较研究[J];中华血液学杂志;2002年12期

8 ;Correlation between expression of gastrin, somatostatin and cell apoptosis regulation gene bcl-2/bax in large intestine carcinoma[J];World Journal of Gastroenterology;2005年05期

9 Kang-Beom Kwon;Eun-Kyung Kim;Jung-Gook Lim;Eun-Sil Jeong;Byung-Cheul Shin;Young-Se Jeon;Kang-San Kim;Eun-A Seo;Do-Gon Ryu;;Molecular mechanisms of apoptosis induced by Scorpio water extract in human hepatoma HepG2 cells[J];World Journal of Gastroenterology;2005年07期

10 ;Cell proliferation, apoptosis and the related regulators p27, p53 expression in hepatocellular carcinoma[J];World Journal of Gastroenterology;2005年13期

，

本文编号：2048234