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人髓性白血病细胞系K562购自上海富衡生物科技有限公司,RPMI1640培养基购自美国Gibco公司、胎牛血清购自民海公司,双抗(青、链霉素)、CCK-8购自北京Solarbio公司,CD235a抗体、Trizol裂解液购自美国Invitrogen公司,细胞凋亡检测试剂盒购自美国BD公司,cDNA反转录试剂盒购自美国Thermo Scientific公司,2×SYRB Green PCR Master Mix购自美国ABI公司,qPCR引物由武汉天一辉远生物科技公司合成,联苯胺购自天津光复,30%过氧化氢购自上海沪试,亚铁氰化钾购自上海中秦,冰乙酸购自天津富宇,氯化高铁血红素购自北京Solarbio公司,3-MA购自大连美仑公司。
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X射线生物辐照仪(X-RAD225,PXI公司)、流式细胞仪(Aminis,德国Merck Millipore公司)、酶标仪(Epoch,美国Biotech公司)、荧光正置显微镜(BX-53,德国Olympus公司)、荧光定量PCR仪(StepOnePlus,美国Applied Biosystems公司)。
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K562细胞常规培养使用体积分数为10%胎牛血清的RPMI1640培养基培养,于体积分数为5%CO2培养箱中37 ℃饱和湿润环境下培养。处于对数生长期的K562中加入氯化高铁血红素(40 μmol/L)诱导作为红系分化模型以进行后续实验。
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本实验采用细胞回转器来模拟微重力,其原理是样品在电机的带动下不停地绕水平轴匀速旋转,适当转速下的流体剪切力与自由落体相抵消,形成模拟微重力环境。回转器的半径是3 cm,采用转速30 r/min。实验采用X射线辐照仪进行辐照,剂量率为1.15 Gy/min。实验分为5组:空白模型组(Ctr组);1.0 Gy照射组(R组);微重力处理组(M组);1.0 Gy辐照+微重力联合处理组(R+M组);3-MA(PI3K抑制剂)+1.0 Gy辐照+微重力联合处理组(R+M+3-MA组)。Ctr组不经过任何照射和微重力处理;R组仅接受1.0 Gy照射处理;M组仅进行3 h微重力处理;R+M组先模拟微重力处理3 h后,接受1.0 Gy辐照。PI3K抑制剂3-MA的药物处理浓度为5 mmol/L。
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收集细胞,离心弃上清,加入新鲜培养基300 μL重悬,铺于96孔板中3孔,每孔100 μL,每孔再加入CCK8溶液30 μL,37 ℃继续孵育4 h,终止培养,用酶标仪检测各孔的490 nm吸光度,以空白孔(不接种细胞,其余操作一致)调零,细胞存活率%=实验组A均值/对照组A均值×100%。
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收集细胞后,离心后弃上清,重悬于250 μL PBS中,加入联苯胺溶液14 μL,30%过氧化氢1 μL,避光孵育2 min,后加入1 μL亚铁氰化钾,孵育10 min。重悬,弃上清,加入10 μL PBS重悬,于显微镜下拍照计数500个细胞,深色为含有血红蛋白细胞的阳性细胞,不着色的为阴性细胞,阳性细胞率%=阳性细胞数/细胞总数×100%。
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收集细胞,预冷的PBS洗两次,重悬于90 μL PBS中,加10 μL一定比例稀释好的抗体CD235a(1:500),避光孵育30 min。PBS洗细胞3次,重悬于100 μL PBS,用流式细胞仪收集数据,用IDEAS软件分析流式数据。
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收集细胞,预冷的PBS洗2次,重悬细胞于100 μL凋亡试剂盒中的1×染料结合缓冲液,再依次加入5 μL AnnexinV-FITC和5 μL碘化丙啶染液,混匀,室温避光孵育15 min,于流式细胞仪上进行检测。用IDEAS软件分析流式数据。
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各处理组细胞在24 h收集,离心后弃上清,将Trizol裂解液加入细胞。静置10 min。加入氯仿,震荡15 s,静置5 min。4 ℃,12 000 g离心10 min,弃去上清液,得到RNA沉淀。用无RNASe的水制备的75%乙醇溶液洗涤RNA沉淀,4 ℃,12 000 g离心5 min,弃上清,加入30~50 μl的无RNASe的水。Illumina HiSeQ-4000第二代测序平台用于测序。实验按照标准Protocol执行,总RNA提取后先进行质量评估,然后进行文库构建、文库质量检测和文库测序。
各样品Clean Data均在5.78 G以上,Q30碱基百分比能达到92.34%。HISAT2用于分别将各样品的Clean Reads与参考基因组进行序列对比。从比对结果统计来看,样品的Reads与参考基因组的比对效率在94.98%~96.05%之间,说明测序结果可信。使用FPKM(Fragments Per Kilobase of transcript per Million fragments mapped)作为基因表达水平的指标。使用DESeq2进行两种情况/组的差异表达分析,将Fold Change
$\geqslant 1.5$ 且FDR(False Discovery Rate)<0.05作为筛选差异表达基因的标准。 -
对GATA-1、EPOR、PIK3R2基因表达进行实时荧光定量PCR检测,引物列表见表1。取2 μg的总RNA用于每份cDNA反转录,cDNA合成按照逆转录试剂盒说明书进行。以获得的cDNA为模板,按照Qiagen SYRB Green荧光定量PCR试剂盒说明书进行实时荧光定量PCR。所有结果经Actin内参校正,用△△CT法计算表达量。
表 1 qRT-PCR引物列表
基因名称 引物序列(5'-3') Actin F:GGACTTCGAGCAAGAGATGG R:CTGTACGCCAACACAGTGCT GATA-1 F:CCAAGCTTCGTGGAACTCTC R:ATTGTCAGTAAACGGGCAGG EPOR F:GAGCATGCCCA GGATACCTA R:TCTGCTGCCAGCTTTGAGTA PIK3R2 F:CCTGGCACCTATGTGGAGTT R:CAGTTCTCCCCACCTGATGT -
数值表示为均数±标准差,采用成组设计资料的t检验,SPSS统计软件和GraphPad Prism8绘图软件。
Researches on the Synergistic Effect of X-ray Radiation and Simulated Microgravity on Erythroid Differentiation of K562 Cells and Its Mechanism
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摘要: 使用氯化高铁血红素(hemin)诱导K562细胞分化作为红系分化模型,并对其进行X射线辐照及地面模拟微重力效应处理,研究辐照、微重力及二者的复合效应对红系分化的影响及机制。X射线辐照及模拟微重力效应处理后的联苯胺染色阳性率及CD235a蛋白表达均下调,细胞增殖抑制、细胞凋亡率及坏死率均增高,红系相关转录因子EPOR及GATA-1基因表达下调,且X射线辐照及模拟微重力效应的联合作用效应强于两者单独作用。辐照及微重力联合处理影响细胞中PI3K复合物调控亚基PIK3R2基因表达,加入PI3K抑制剂3-MA后细胞凋亡率及坏死率增高、红系分化率降低。以上结果表明,X射线辐照及模拟微重力效应对红系分化具有协同抑制作用,其机制与红系相关转录因子EPOR、GATA-1及生长信号通路因子PI3K相关。Abstract: In this study, using K562 cells induced by hemin as erythroid differentiation models, the synergistic effects and mechanisms of radiation and simulated microgravity on erythroid differentiation were investigated. Results showed that the positive rates of benzidine staining and the expression of CD235a were down-regulated after treatment with X-ray radiation and simulated microgravity, meanwhile, cell proliferation was inhibited and the rates of apoptosis and necrosis were increased. The expressions of transcription factors related to erythroid differentiation EPOR and GATA-1 were down-regulated. The synergistic effects of X-ray irradiation and simulated microgravity were much stronger than those of the two alone. After irradiation and microgravity combined treatment, PIK3R2 gene expression was down-regulated. After adding PI3K inhibitor 3-MA, the apoptosis and necrosis rate of cells increased, and the erythroid differentiation rates decreased. These results indicate that X-ray irradiation and simulated microgravity treatment have synergistic inhibitory effects on erythroid differentiation, and the mechanism is related to erythroid related transcription factors EPOR, GATA-1 and damage repair pathway factor PI3K.
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Key words:
- X-ray /
- simulated microgravity effects /
- erythroid differentiation /
- damage repair
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表 1 qRT-PCR引物列表
基因名称 引物序列(5'-3') Actin F:GGACTTCGAGCAAGAGATGG R:CTGTACGCCAACACAGTGCT GATA-1 F:CCAAGCTTCGTGGAACTCTC R:ATTGTCAGTAAACGGGCAGG EPOR F:GAGCATGCCCA GGATACCTA R:TCTGCTGCCAGCTTTGAGTA PIK3R2 F:CCTGGCACCTATGTGGAGTT R:CAGTTCTCCCCACCTGATGT -
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