摘要
目的：本课题主要探讨HL60细胞增殖分化过程中HOXA6mRNA的表达及HOXA6蛋白的分泌情况,用三氧化二砷(ATO)和／或全反式维甲酸(all-trans-retinoic acid,ATRA)进行干预,在基因水平探讨ATO和／或ATRA对HL60细胞增殖分化过程中HOXA6mRNA的表达及HOXA6蛋白的分泌的影响。方法：1.HL60细胞株(购自维康生物有限公司)2.实验分组。实验分4组：(1)空白对照组：HL60细胞对数生长期收集细胞并调节细胞数至1×105/ml,加以等量的RPMI 1640。(2) ATRA组：在培养体系分组中加入12ulATRA,终浓度1×10-6mol/l。(3)(3)ATO组：在培养体系分组中加入ATO,终浓度为1×10-6mol/l。(4) ATRA+ATO组：加入ATRA、ATO,终浓度同上。3.采用肿瘤细胞体外培养技术,以ATO和(或)ATRA持续干扰人类HL60细胞,观正常组、ATRA组、ATO组、ATRA+ATO组的HL60细胞第1天,2天和3天的分化情况。4.采用瑞氏姬姆萨染色法鉴定HL60细胞。5.第1天、第2天、第3天分别提取各组细胞总RNA,用1%甲醛变性琼脂糖凝胶电泳检测RNA分子的完整性。6.通过随机引物将总RNA逆转录为cDNA,采用实时荧光定量逆转录多聚酶链反应(fluorogenic quantitive reverse transcription polymerize chain reaction, FQ-RT-PCR)技术检测HL60细胞增殖分化过程中各组HOXA6基因的表达。7.随机抽取逆转录的HOXA6基因进行电泳分别获得HOXA6基因在1天、2天、3天电泳图。将HOXA6基因cDNA和ACTB基因阳性产物cDNA梯度稀释制作各自的标准曲线,根据各个样本循环指数增长期的起始点即循环域值(cycle threshold,Ct)和标准曲线斜率计算各自样本起始cDNA模板量倍数值,以ACTB基因作为内参照进行标化。8.PCR统计方法：结果用DNA相对拷贝数和RNA表达相对量(2-△Ct)表示HOXA6基因相对内参基因的表达量,采用均数加减标准差x±s)表示HOXA6基因的变化情况。进行方差齐性检验,REPEATED MEASURE方差分析,由统计学软件SPSS10.0完成。9.第1天、第2天、第3天分别裂解细胞提取各组细胞总蛋白。10.蛋白免疫印迹法(Western-blot):分别检测各组细胞中的HOXA6蛋白的表达：用BCA试剂盒测定样品总蛋白浓度、聚丙烯酰胺凝胶电泳(SDS-PAGE)、考马斯亮蓝染色观察蛋白条带、电转移(electrotransfer)、封闭PVDF膜、免疫反应(加入一抗和二抗)、化学发光显示目的条带。11.图像分析：将Western-blot结果传送至计算机,用QUANTITY ONE图像分析软件系统对蛋白条带进行分析,得到积分光密度值,用内参做校正,积分光密度值比值,代表蛋白相对表达量。12.Western-blot统计方法：分析各组细胞中HOXA6蛋白表达的差异,所有数据用x±s,采用REPEATED MEASURE方差分析,P<0.05有统计学意义,P<0.01为显著差异。结果：1.HL60细胞的形态学鉴定,瑞氏姬姆萨染色法证明所培养的细胞是HL60细胞,并可见药物处理组明显的粒系分化细胞。2.1%甲醛变性琼脂糖凝胶电泳显示RNA电泳图的5s,18s,28s三条带型整齐,无明显拖尾及弥散,说明RNA结构完整,无明显降解。3.逆转录PCR扩增三组均有目的基因HOXA6和内参照ACTB基因的cDNA的产物,与DNA分子Marker相比示扩增产物大小分别为：H0XA6为126bp,ACTB基因为111bp,与预定理论值大小符合。4.不同浓度ATRA处理细胞24h后,除5×10-6mol/l外其它浓度均会明显抑制细胞增殖,并随浓度的增加抑制作用也越显著。不同浓度ATO处理细胞后,除10-7mol/l外其它浓度均会明显抑制细胞增殖,并且浓度越大抑制作用出现的越早,效果越明显。不同浓度ATRA、ATO联合处理细胞后,均会明显抑制细胞增殖,并且浓度越大抑制作用出现的越早,效果越明显。5.空白对照组HOXA6mRNA和HOXA6蛋白在HL60细胞中的表达呈逐渐增高的趋势。6.ATRA组HOXA6mRNA和HOXA6蛋白表达增强,均高于正常组,其中第2天表达最强烈,第3天减弱。7.随时间推移,ATO组和ATO+ATRA组HOXA6mRNA和HOXA6蛋白的表达在第1天最强烈(高于正常组),第2天和第3天表达下调(低于正常组)。8.与正常对照组比较,HOXA6mRNA和HOXA6蛋白受ATRA (1×10-6mol/L)上调,而受ATO和ATO+ATRA先上调,然后下调。结论：1.1×10-6mol/L的ATRA、ATO及ATRA+ATO在诱导时间内能促进HL60细胞向成熟细胞分化。2.HOXA6基因、HOXA6蛋白在人髓性白血病细胞HL60细胞增殖过程中有表达,随着时间的推移表达增加,提示ATRA治疗白血病的机制可能与调节HOX基因的表达有关。3.ATRA (1×10-6mol/L)在诱导时间内能上调HOXA6基因和HOXA6蛋白的表达,提示ATRA治疗白血病的机制可能与调节HOX基因的表达有关。4.ATO (1×10-6mol/L)在HL60细胞分化过程中能使HOXA6基因和HOXA6蛋白表达下调,提示ATO治疗白血病的机制可能是通过调控HOXA6基因表达下调促进细胞分化成熟。
bjective:This topic mainly discusses expression on homeobox gene (HOX) HOXA6 and its protein in the proliferation or differentiation of HL60,and discusses its expression in following the intervention of use all-trans retinoic acid (ATRA) and/or arsenic trioxide(ATO),and explore HOX gene mediated pathogenesis of leukemia in gene level.Methods:1.HL60 cell line was purchased from Wellcome Bio Co.,Ltd..2.Experimental groups. Were divided into 4 groups:(1)control group (Normal):no drug, on behalf of the same amount by adding RPMI 1640 medium basic training system. (2) ATRA group:Add the diluent of ATRA, the final concentration of 1×10-6mol/1. (3)ATO group:Add the diluent of ATO, the final concentration of 1×10-6mol/1. (4) ATRA+ As2O3 group:while adding ATRA and ATO, the final concentration of 1×10-6mol/1.3.By the tumor cells culture techniques in vitro,and HL-60 cells continued to interfere with ATRA, and (or)ATO,then the formation conditions of normal group, ATRA group,ATO group, ATRA+ATO group of HL60 cells in culture were observed on the first,second,and third day.4.Their growth and differentiation was determined by Wright Giemsa staining.5.All group were extracted from total cellular RNA on the first, second, and third day, and total RNA was electrophoresed in 1% formaldehyde denaturalized agarose gel in order to validate integrity of RNA.6.By random primer to total RNA reverse transcriptase to cDNA, real-time fluorescent quantitative reverse transcription polymerase chain reaction (fluorogenic quantitive reverse transcription polymerize chain reaction, FQ-RT-PCR) to detect cell proliferation and differentiation process of gene expression in each group HOXA6.7.Randomly selected reverse transcriptase of the HOXA6 gene electrophoresis respectively HOXA6 gene in 1 day,2 days,3 days electrophoresis.The HOXA6 gene cDNA and the ACTB gene positive product of the respective cDNA dilution standard curve production, according to the exponential growth cycle of each sample is the starting point of the cycle threshold (cycle threshold, Ct) and standard curve slope of the calculated volume of each sample of the starting cDNA template multiple values to ACTB gene were standardized as internal reference.8.Statistical Methods of PCR:Results relative DNA copy number and RNA expression of the relative ACTB gene amount of (2-△Ct) said that the relative expression of HOXA6 gene, using the mean plus or minus standard deviation(X±S) that HOXA6 gene changes.Homogeneity of variance test, REPEATED MEASURE analysis of variance, Completed by the statistical software SPSS 17.0.9.Each group were lysed extract total cellular protein on the first day, the second day and the third day.10.Western blot:cells were detected in the HOXA6 protein: determination of samples with the BCA kit total protein concentration of polyacrylamide gel electrophoresis(SDS-PAGE), Coomassie brilliant blue staining observed protein bands and electricity transfer (electrotransfer),closed PVDF membrane, immune response (by adding an anti and two anti-), chemiluminescence showed that the target band.11,Image Analysis: Western-blot results will be sent to the computer and use Quantity one image analysis software to analyze the protein bands, get integral optical density, after correction with the internal reference, the ratio of integral optical density to represent the relative protein expression.12.statistical methods of Western-blot:analysis of cells in each group HOXA6 protein expression differences, all data with x±s, using REPEATED MEASURE analysis of variance, P<0.05 was statistically significant, P<0.01 for the significant.
Results:1.Cell morphology identification, Wright's Giemsa staining demonstrated that the cultured cells are granulocytes.2.1% formaldehyde denaturing agarose gel electrophoresis showed that RNA electrophoresis of 5s, 18s,28s 3 band type and tidy, no significant tailing and dispersion, indicating structural integrity of RNA, no obvious degradation.3.RT-PCR amplification of the three groups were within the target gene HOXA6 and reference ACTB cDNA gene product, compared with the DNA molecule Marker said the size of PCR products were:HOXA6 to 126 base pairs (bp), ACTB gene 111bp, consistent with the intended size of the theoretical value.4.Different concentrations of ATRA-treated cells after 24h,in addition to 5×10-6mol/l outside the other concentrations are significantly inhibit cell proliferation. After the cells were treated with different concentrations of ATO, in addition to 10-7mol/l outside the other concentrations are significantly inhibited cell proliferation,and the greater the concentration of inhibition occurs earlier, the effect became. Different concentrations of ATRA,ATO combined treatment cells were markedly inhibited cell proliferation,and the greater the concentration of inhibition occurs earlier, the effect became.5.In normal group,the expression of HOXA6 gene and HOXA6 protein is more and more stronger when the time goes by.6.In ATRA group, the expression of HOXA6 gene and protein is always stronger than normal group in all three days, on the second day, the expression is the strongest.7.In ATO group and ATRA+ ATO group,the expression of HOXA6 gene and HOXA6 protein were were upregulated dramatically on the first day(higher than normal group) and downregulated on the secondly two days(lower than normal group). 8.Compared with normal group, HOXA6 gene and HOXA6 protein were upregulated by ATRA,and downregulated by ATO and ATO+ATRA.
Conclusion:1.1 X 10-6mol/l ATRA, ATO and ATRA+ATO can promote HL-60 cells to mature.2. HOXA6 gene and HOXA6 protein were expressed in human myeloid leukemia cells HL60 and upregulated when the cells' proliferation.which means HOXA6 can enhance the proliferation of cell and this gene may be cause leukemia.3 ATRA can upregulate the expression of HOXA6 gene and protein in the three observed days,so the mechanisms of treatment of leukemia by ATRA may be associated with the regulation of the expression of HOX genes.4.In the differentiation of HL60,ATO downregulated the expression of HOXA6 gene and protein,so the mechanisms of treatment of leukemia by ATO may be associated with the downregulation of the expression of HOXA6 genes.
引文
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