摘要
目的①探讨sari mRNA、SARI蛋白在白血病细胞株中的表达情况及相关性;探讨SARI信号通路在白血病细胞中的可能相关基因及在白血病中的作用。②探讨IFN-β作用于白血病细胞后对细胞生长、细胞周期的影响及对SARI信号通路可能相关基因表达的影响。③分析探讨sari mRNA及SARI信号通路可能相关基因mRNA在白血病患者中的表达情况及临床意义。
方法①收集急慢性髓系、淋系白血病细胞株,采用逆转录聚合酶链反应检测白血病细胞株sari及AP-1、白血病相关基因(bcl-2、bax、c-myc、p53、cyclinD1、caspase3)mRNA的表达;采用Western蛋白免疫印迹法检测白血病细胞株SARI蛋白的表达。
②一定浓度的IFN-β作用于白血病细胞一定时间后,MTT法检测IFN-β对细胞生长的影响;流式细胞周期分析检测IFN-β对细胞生长周期的影响;逆转录聚合酶链反应检测IFN-β对细胞中sari及SARI信号通路可能相关基因mRNA的表达情况的影响。
③收集血病患者标本218例,依据MIC分型诊断标准分型,并分为初治组、复发组、缓解组;收集正常对照标本30例。采用逆转录聚合酶链反应检测白血病患者及正常对照标本sari及AP-1、cyclinD1、p53 mRNA的表达。
结果①sari mRNA在髓系白血病细胞株中的表达量明显高于在急性淋系白血病细胞株的表达量(p<0.05);在慢性髓系白血病细胞株中的表达量明显高于在急性髓系白血病细胞株中的表达量(p<0.05);在急性髓性白血病细胞株的野生型的表达量高于耐药株(p<0.05)。
il-8 mRNA在急性髓系白血病细胞株中的表达量明显高于在慢性髓系白血病细胞株的表达量(p<0.05);在急性淋系白血病细胞株中没有表达;在白血病细胞株野生型和耐药株之间表达量没有显著差异。
bcl-2 mRNA在急性髓系白血病细胞株中的表达量明显高于在慢性髓系白血病细胞株中的表达量(p<0.05);在急性淋系白血病细胞株中的表达量高于在慢性髓系白血病细胞株的表达量而低于在急性髓系白血病细胞株中的表达量(p<0.05);在急性髓系白血病细胞株的野生型中的表达量明显高于耐药株(p<0.05)。
p53 mRNA在HL60、H/A、KAR细胞株中表达缺失,而在K562、Jurakt、CEM白血病细胞株中表达阳性。cyclinD1 mRNA在NB4、H/A、K562中呈阳性表达,而在其他几个白血病细胞株中表达缺失。bax mRNA在急性髓性白血病各细胞株中的表达量没有统计学差异,在慢性髓性白血病细胞株K562和KAR中的表达具有显著的统计学差异(p<0.05),在急性淋系白血病细胞株Jurkat和CEM中的表达具有显著的统计学差异(p<0.05)。caspase3 mRNA在急性髓性白血病细胞野生株和耐药株之间表达量没有差异,在慢性粒细胞白血病细胞耐药株中的表达量高于野生株(p<0.05)。c-myc mRNA在髓系白血病细胞株和急性淋系白血病细胞株的表达量没有显著差别。
SARI蛋白在白血病细胞株的表达量与sari mRNA的表达具有正性相关性(p<0.05)。
在髓性白血病细胞株(HL60、NB4、H/A、K562、KAR)中,sari mRNA与il-8 mRNA、bcl-2 mRNA的表达经Pearson相关分析,具有负相关性(p<0.05);il-8 mRNA与bcl-2 mRNA的表达经Pearson相关分析,具有正相关性(p<0.05)。
②一定浓度的IFN-β作用于白血病细胞一定时间后,对白血病细胞的生长、细胞周期及SARI、SARI信号通路可能相关基因的表达没有显著影响。
③各型白血病患者(初治组、复发组、缓解组)sari mRNA的表达量均小于正常对照组(p<0.01)。
各型白血病患者初治组间sari mRNA的表达量不同,从收集到的各型白血病初治组结果分析,表达量水平为M3 各型白血病初治组、复发组/未缓解组的表达量均小于对应的缓解组, (p<0.01);各型白血病初治组与复发组/未缓解组的表达量比较,没有统计学差异。
慢性粒细胞白血病慢性期初治组、加速期组、急变期组、干细胞移植后组sari mRNA的表达量依次增加,且相邻的两组之间比较均具有统计学差异(p<0.01)。
在髓性白血病初治组中sari mRNA与il-8 mRNA具有负相关(p<0.05)。结论①SARI在各种白血病细胞株中均有表达,SARI蛋白的表达与SARI转录水平的调控有关。②在髓性白血病细胞株中,SARI的表达水平与细胞的分化程度有关,在髓性白血病细胞株中SARI与AP-1、bcl-2的表达呈负相关(p<0.05),这三种基因在白血病的发生发展过程中可能存在相互作用;SARI与急性髓性白血病细胞的耐药性可能有关。③IFN-β对白血病细胞的生长和凋亡以及“SARI-AP1-下游基因”信号通路没有显著的影响。④sari mRNA在各型白血病患者中的表达有一定的差异,但均小于在正常对照中的表达,且各型白血病初治组、复发组/未缓解组的表达量均小于相应的缓解组,SARI可作为白血病诊断、疗效监测和预后判断的生物学指标之一。
[Objective]①To explore the expression of sari mRNA and SARI protein in leukemia cell lines.To explore the possible SARI signal transduction pathway-related genes and the role of them in leukemia.②To explore the effect of IFN-βon the growth,cell cycle and the expression of the possible sari-signaling pathway-related genes in leukemia cells.③To study the expression of sari and the possible sari signaling pathway-related genes and their clinical significance in leukemia patients.
[Methods]①Reverse polymerase chain reaction (RT-PCR) was used to detect the expression of sari and AP-1﹑leukemia-related genes(bcl-2﹑bax﹑c-myc﹑p53﹑cyclinD1﹑caspase3 )mRNA in leukemia cell lines.Western blot was used to detect the expression of SARI protein in leukemia cell lines.②MTT assay was used to explore the effect of IFN-βon the growth in leukemia cells;Flow cytometry was used to explore the effect of IFN-βon cell cycle;Reverse polymerase chain reaction (RT-PCR) was used to detect the effect of IFN-βon the expression of sari and the possible sari signaling pathway-related genes in leukemia cell lines.③Reverse polymerase chain reaction (RT-PCR) was used to detect the expression of sari and AP-1﹑cyclinD1 and p53 in 218 leukemia patients,30 normal controls.
[Results]①The expression of sari mRNA in myeloid leukemia cell lines was significantly higher than that in acute lymphocytic leukemia cell lines(p<0.05);The expression of sari mRNA in chronic myeloid leukemia cell lines was significantly higher than that in acute myeloid leukemia(p<0.05);The expression of sari mRNA in myeloid leukemia wild strains was higher than that in drug-resistant strains(p<0.05).
The expression of il-8 mRNA in acute myeloid leukemia cell lines was significantly higher than that in chronic myeloid leukemia cell lines(p<0.05);The expression of il-8 mRNA was absent in acute lymphocytic leukemia cell lines;there was no difference between drug-resistant strains and wild strains.
The expression of bcl-2 mRNA in acute myeloid leukemia cell lines was significantly higher than that in chronic myeloid leukemia cell lines(p<0.05);The expression of bcl-2 mRNA in acute lymphocytic leukemia cell lines was higher than that in chronic myeloid leukemia cell lines(p<0.05),lower than that in acute myeloid leukemia cell lines;The expression of bcl-2 mRNA in acute myeloid leukemia wild strains was higher than that in drug-resistant strains (p<0.05).
The expression of p53 mRNA was absent in leukemia cell lines HL60, H/A,KAR,was positive in K562, Jurakt and CEM.The expression of cyclinD1 mRNA was positive in NB4﹑H/A﹑K562,and was absent in several other leukemia cell lines.The expression of bax mRNA was no statistically significant difference among acute myeloid leukemia cell lines;The expression of bax mRNA was statistically significant difference between K562 and KAR(p<0.05), between lymphocytic leukemia cell lines Jurkat and CEM(p<0.05).The expression of caspase3 mRNA was no statistically significant difference between drug-resistant strains and wild strains of acute myeloid leukemia;The expression of caspase3 mRNA in chronic myeloid leukemia drug-resistant strains was higher than that in wild strains(p<0.05). The expression of c-myc mRNA in myeloid leukemia cell lines and acute lymphocytic leukemia cell lines showed no significant difference.
The expression of SARI protein was positively correlated to the expression of sari mRNA in leukemia cell lines (p<0.05).
The expression of sari mRNA was negatively correlated to the expression of il-8,bcl-2 mRNA in myeloid leukemia cell lines (p<0.05).The expression of il-8 mRNA was positively correlated to the expression of bcl-2 mRNA in myeloid leukemia cell lines (p<0.05).
②IFN-βhas no effect on the growth, cell cycle and the expression of sari and the possible sari signaling pathway-related genes in leukemia cells.
③The expression of sari mRNA in leukemia patients(newly diagnosed cases,relapsed cases,remission cases) was lower than that in normal control group(p<0.01).
The level of sari mRNA expression in newly diagnosed cases was:M3 The expression of sari mRNA in leukemia patients(newly diagnosed cases,relapsed cases)was lower than that in their corresponding remission cases (p<0.01);The expression of sari mRNA in newly diagnosed cases and relapsed cases was not statisticaly significant different.
The expression of sari mRNA in chronic myeloid leukemia was:stem cell transplantation cases>blast crisis>accelerated phase>newly diagnosed cases, and there was statistically significant difference between adjacent groups(p<0.01).
The expression of sari mRNA was negatively correlated to the expression of il-8 mRNA in the newly diagnosed cases in acute myeloid leukemia (p<0.05).
[Conclusions]①The expression of SARI was positive in leukemia cell lines, The expression of SARI protein was regulated by the transcription of sari.②The level of SARI expression is related with the degree of differentiation in myeloid leukemia cell lines,The expression of SARI was negatively correlated to AP-1,bcl-2 in myeloid leukemia(P <0.05),These three genes may have interactions in the pathogenesis and progress of leukemia;SARI may play roles in drug-resistance in acute myeloid leukemia cells.③IFN-βhas no effect on the growth,apoptosis and "SARI-AP1-downstream genes"in leukemia cell lines.④The expression of sari mRNA was some differences in different leukemia type patients, but was lower than the expression of normal control cases, and the expression of sari mRNA in newly diagnosed cases and relapsed cases was lower than their corresponding remission cases,SARI may be a indicator in the diagnosis,efficacy monitoring and prognosis judgement.
引文
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