【作者】 罗沛华；

【导师】 程翼宇； 杨波；

【作者基本信息】 浙江大学 ， 药物分析学， 2009， 博士

【摘要】 研究目的:维甲酸(RA)是一组视黄醇(即维生素A)的活性分子衍生物,是目前临床上应用最为成功的分化诱导剂,可通过与维甲酸受体特别是维甲酸受体alpha(RARtα)结合,引起细胞周期蛋白依赖性激酶活化激酶(CAK)与RARα的解离,导致RARα的低磷酸化,从而达到诱导人急性髓性白血病细胞分化或凋亡的作用。在这一分子过程的基础上,本文进一步探讨CAK-RARα通路在维甲酸诱导的骨肉瘤细胞、造血干细胞粒性分化和急性T淋巴细胞凋亡的调控作用。研究方法:1)选用人骨肉瘤细胞U2OS和MG63为研究对象,以全反式维甲酸(ATRA)处理或采用慢病毒转染法导入低磷酸化的RARα,来评价ATRA诱导的RARα低磷酸化与诱导骨肉瘤细胞分化之间的相关性;采用台盼兰拒染法检测活细胞数量,并采用血细胞计数板计数来观察细胞增殖情况,描绘细胞生长曲线;细胞经碘化丙锭(PI)染色后采用流式细胞术分析细胞周期的变化情况;应用RT-PCR和Western blot检测骨肉瘤细胞分化标记基因转录水平和蛋白表达水平;采用Western blot检测细胞周期相关蛋白CDK7、p27、p21的表达;结合基因芯片技术检测RA及外源性低磷酸化RARα作用下人骨肉瘤细胞相关基因的变化。2)选用人造血干细胞(HSC)、髓性前体细胞(CMP)和髓性/粒性前体细胞(GMP)经RA作用或是采用慢病毒转染法导入低磷酸化的RARα,观察低磷酸化RARα与上述细胞分化之间的关系;应用台盼兰拒染法结合血细胞计数板计数,观察药物对细胞增殖情况的影响;吉姆萨染色观察药物作用后细胞形态学改变;采用RT-PCR和Western Blot检测ATRA诱导HSC,CMP,GMP和人造血干细胞CD34+细胞分化的相关基因转录和蛋白表达情况;应用细胞免疫荧光检测MAT1和CAK-RARα通路相关蛋白在分化各阶段的表达;结合免疫沉淀检测分化各个阶段血细胞CAK-RARα通路蛋白之间相互作用的关系。3)选用人急性T淋巴Molt3细胞为研究对象,RA作用,采用台盼兰拒染法结合血细胞计数板计数,观察其对细胞增殖情况的影响;采用PI染色,流式细胞术分析细胞凋亡;应用Western blot技术检测细胞凋亡的相关蛋白caspase-3、PARP、BCL-2、BAX的表达;应用RT-PCR和Westernblot技术检测RARα和p21的基因转录和蛋白表达。研究结果:1)通过台盼兰拒染法和血细胞计数板计数,显示ATRA(5μM)可显著抑制U2OS和MG63细胞增殖。流式细胞术分析细胞周期情况,显示ATRA(5μM)能使人骨肉瘤细胞的细胞周期阻滞在G1/G0期。同时,通过Western blot技术检测相关周期蛋白,发现ATRA(5μM)能降低U2OS和MG63细胞中CDK7和p21的表达,引起P27的上调。该浓度的ATRA也能诱导人骨肉瘤细胞分化标记物OPN的基因转录和蛋白表达增加,显示ATRA可诱导骨肉瘤细胞分化。通过基因芯片比较ATRA诱导分化细胞与空白对照细胞的基因表达谱,推测ATRA诱导骨肉瘤分化可能与RARα、RARβ、BMP、WNT和FGF蛋白有关。2)通过台盼兰拒染法和血细胞计数板计数及吉姆萨染色显示ATRA(1μM)能抑制分化各个阶段造血干细胞HSC、CMP和GMP细胞的增殖,增加三者的粒性分化。通过免疫荧光和Western blot技术确定了CAK-RARα通路上pRb、MAT1、CDK7和RAR RARαα在分化各个阶段造血干细胞HSC、CMP和GMP细胞中的表达情况,从中显示MAT1的表达随着粒性分化的成熟而降解,在HSC中表达最高、CMP次之,GMP最低。ATRA(1μM)诱导CMP细胞粒性分化过程中,CDK7和RARα的表达增加,P21的表达下降,同时pRb,CDK7及RARα的磷酸化水平都发生下调。结合免疫沉淀技术还可见ATRA(1μM)促进的CMP和GMP细胞粒性分化时,可使CAK-RARα复合体的解离,CMP细胞在粒性分化过程中还伴随着RARα和CDK7表达的下调,GMP细胞则伴随着MAT1的降解。3)通过慢病毒转染法,血细胞计数板计数,RT-PCR和Western Blot显示外源性低磷酸化的RARα能显著抑制人骨肉瘤细胞增殖,并能增加RA诱导分化的能力。结合基因芯片技术,比较了导入外源性低磷酸化RARα所上调的基因与全反式维甲酸作用所上调的基因。导入外源性低磷酸化RARα的U2OS细胞较空白对照细胞共有4个维甲酸受体相关通路基因显著上调。在成骨细胞分化相关通路基因中,U2OS细胞导入外源性RARαS77A后有18个基因显著上调,这一结果与全反式维甲酸诱导分化的人骨肉瘤细胞相似。对细胞因子类差异基因进行生物信息学分析表明,导入外源性RARαS77A后有20个基因显著上调。4)通过慢病毒转染法,血细胞计数板计数及吉姆萨染色显示显示外源性低磷酸化的RARα能显著抑制人CD34+细胞增殖,并能增加ATRA诱导分化的能力。通过RT-PCR可见外源性低磷酸化的RARα增加ATRA诱导的CD34+细胞中p21mRNA的水平。5)通过血细胞计数板计数可见ATRA(5μM)显著抑制Molt3细胞增殖。通过台盼兰拒染法和流式细胞术可见ATRA(5μM)处理Molt3细胞在抑制细胞增殖的同时引起了细胞的死亡。通过Western Blot可见ATRA(5μM)可使caspase 3发生裂解,激活的Caspase 3促进PARP发生水解,产生裂解片段。同时ATRA(5μM)可以抑制Molt3细胞中BCL-2的表达,促进BAX的表达。通过RT-PCR和Western Blot显示P21可以被配体结合的RARα所激活从而引起细胞凋亡。结论:通过对ATRA在体外诱导人骨肉瘤细胞和正常造血干细胞分化过程中CAK-RARα通路作用的系统研究,我们得出以下结论:1) ATRA 5μM可诱导骨肉瘤细胞分化,在这一过程中CAK和p21的蛋白水平下降,使细胞阻滞在G1/G0期,并使之最终走向分化。2) ATRA 1μM可诱导正常造血干细胞粒性分化,在这一过程中CDK7和RARα的表达增加,MAT1和P21的表达下降,同时pRb,CDK7及RARα的磷酸化水平都发生下调。3)基因芯片研究结果提示ATRA诱导骨肉瘤细胞分化可能与RARα、RARβ、BMP、WNT和FGF通路有关。4) RARαS77A可以在一定程度上产生与ATRA相似的作用,能显著抑制人骨肉瘤细胞和人CD34+细胞增殖,并能增加ATRA诱导分化的能力。5)筛选出的FGF8,BMP8A及WNT8B可作进一步研究,为细胞分化的机制研究以及新的肿瘤分化诱导剂研究提供实验依据。6) RARαS77A有助于诱导造血干细胞体外粒性分化。这一研究将为临床上体外扩增造血干细胞,诱导其粒性分化,进而治疗白血病提供实验依据。更多还原

【Abstract】 Objective:Retinoic Acid(RA),the most potent biologically active metabolite of vitamin A,suppresses proliferation by inducing differentiation and apoptosis in a variety of cell types through modifying expression of RA-target genes.The biological effects of RA on cell differentiation of malignant cells are mediated by retinoid receptors,notably retinoic acid receptor alpha(RARα).The recent finding that CAK- RARαdiassociation, me’nage a’ troisl(MAT1) degradation and RARαhypophosphorylation in the presence of RA,coordinate cell cycle G1 exit and transition into differentiation in myeloid leukemia.We further study CAK- RARαsignaling events on osteosarcoma cell,human hematopetic stem cell differentiation and acute T lymphoma leukemia cell apoptosis based on this.We first test the inhibition and mecenism of RA on osteosarcoma cells,as well as comfirm the hypothesis that RA-induced loss of RARαphosphorylation mediates differentiation of osteosarcoma cells.We then investigated whether CAK-RARαsignaling coordinates CAK-dependent cell division and transition into differentiation in normal primitive hematopoietic cells.At last we investigated the effects and mechanism of ATRA in mediating antiproliferative responses in T lymphoma Molt3 cells.Methods:1) For proliferation assay,human osteosarcoma cells U2OS and MG63 treated with 5μM ATRA or trandusec with lentiviral- RARαS77A,cell growth curve was performed by counting cells each day using typen blue staining.For cell cycle analysis,cells were stained with PI and analyzed by flow cytometry.Osteoblastic differentiation marker OPN expression was detected by RT-PCR and Western blot.The expressions of CAK-RARαsignaling factors CDK7,p27 and p21 were shown by Western blot.Microarray was used to compare the gene profile of ATRA-treated or lentiviral- RARαS77A transduced U2OS cell.2) For proliferation assay,human hematopoietic stem cell(HSC),common myeloid progenitor cell(CMP) and granucytic myeloid progenitor cell(GMP) treated with 1μM ATRA or trandused with lentiviral- RARαS77A,cell growth curve was performed by counting cells each day using trypan blue staining.For cell differentiation analysis,cells were stained with giemsa and the morphology was dectect under microscope.The expressions of CAK-RARαsignaling factors CDK7,MAT1,RARαand pRb during RA-induced myeloid differentiation in HSC,CMP and GMP cells were shown by immuonfluorence, RT-PCR and Western blot.The interaction of CAK-RARαsignaling factors was detected by immunonprecitation.3) For proliferation assay,human acute T lymphoblastic leukemia cell treated with 5μM ATRA,cell growth curve was performed by counting ceils each day using trypan blue staining.For cell apoptosis assay,cells were stained with PI and analyzed by flow cytometry.The expression of apoptosis related protein caspase-3,PARP,Bcl-2 and BAX were detected by Western blot.The mRNA and protein expressions of CAK-RARαsignaling factors RARαand p21 were shown by RT-PCR and Western blot.Results:1) Here we report that ATRA(5μM) suppresses proliferation in both U2OS and MG63 osteosarcoma cells by inducing G1/G0 phase arrest through trypan blue staining and FACS.By using RT-PCR and Western blot,we found that the expression of mature osteoblastic marker OPN was induced,CAK-RARαsignaling factors CDK7 and p21 were downregulated,and p27 was upregulated in ATRA-treated U2OS cells.By using microarray,the regulator in RARαrelated pathway,osteoblastic differentiation pathway and cytokines were activated with ATRA treatment in U2OS cells.2) Here we report that ATRA(1μM) suppresses proliferation in HSC,CMP and GMP cells by inducing myeloid differentiation through trypan blue staining for growth curve and giemsa staining for morphology change.By using immunoflunrence and Western blot, we found that the expression of CAK- RARαsignaling factors CDK7 and RARαwere increased,MAT1 and p21 were downregulated,and meanwhile the phosphoralate level of CDK7,pRb and RARαwas reduced in ATRA-treated U2OS induced normal myeloid differentiation cells.By using microarray,the regulator in RARαrelated pathway, osteoblastic differentiation pathway and cytokines were activated with ATRA treatment in U2OS cells.By using immunonprecipetation,we found that CAK-RARαdiassociation,MAT1 degradation and RARαhypophosphorylation in the presence of ATRRA,coordinate with myeloid differentiation in hematopoietic stem cells.3) Here we report that lentiviral- RARαS77A suppresses proliferation in both U2OS osteosarcoma cells through trypan blue staining.By using RT-PCR and Western blot,we found that the expression of mature osteoblastic marker OPN was enhanced in RARαS77A transduced cell with ATRA treatment.By using microarray,the 4 regulators in RARαrelated pathway,18 factors in osteoblastic differentiation pathway and 20 cytokines were activated in RARαS77A transduced cell compared with blank cells.4) Here we report that lentiviral-RARαS77A suppresses proliferation in CD34+ cells through trypan blue staining.By giemsa staining,it was shown that RARαS77A enhanced ATRA-induced myeloid differentiation.By using RT-PCR,we found that the expression of p21 was upregulated in RARαS77A transduced cells.5) Here we report that ATRA(5μM) suppresses proliferation in acute T lymphoblastic leukemia Molt3 cells by inducing apoptosis through trypan blue staining and FACS.In our study,a decrease in Bcl-2 and increase in Bax protein expression was observed in Molt3 cells, following the increase of Bax/Bcl-2 ratio,we observed an activation of both caspase 3 and PARP that were cleaved after 24 h treatment with ATRA treatment by using Western blot.Meanwhile it was shown that RARαreduction is involved in upregulating p21 expression in mediating RA-induced Molt3 cell apoptosis.Conclusion:It was concluded that CAK- RARαevents play a pivotal role in ATRA-induced osteosarcoma cell and hematopoietic stem cell differentiation.This regulation is similar to ATRA-induced myeloid differentiation in leukemia cell. Transduced hypophosphoralate RARαcan not only inhibit osteosarcoma and CD34+ cell proliferation but also enhance ATRA-induced differentiation.At last RARαreduction is involved in upregulating p21 expression in mediating RA-induced Molt3 cell apoptosis.更多还原