【作者】 王彦；

【导师】 马梁明；

【作者基本信息】 山西医科大学 ， 内科学， 2015， 硕士

【摘要】 目的:费城染色体阳性(Ph+)急性淋巴细胞白血病(acute lymphoblastic leukemia,ALL)是起源于淋巴祖细胞并且在第22对染色体长臂和第9对染色体长臂相互易位后产生了BCR-ABL融合的致癌基因的肿瘤性疾病。Ph(+)ALL属于危险度分层较高的急性白血病,它的特点是缓解率较Ph(-)ALL低,缓解后复发时间短,预后不好。患者随着长期的化疗疾病缓解率可能会降低,缓解时间逐渐缩短,患者的身体长期无法耐受化疗所带来的副作用,比如感染、免疫力低下等。伴随着酪氨酸激酶抑制剂第一代产品针对BCR-ABL融合致癌基因靶向治疗药物的出现,治疗慢性髓细胞白血病(chronic myelocytic leukemia,CML)上取得了惊人的效果,使CML这个疾病从难以治疗的疾病变为存活期较长的疾病之一,之后该药物也用于治疗Ph+ALL上,同样大大提高了该疾病的缓解率,然而在临床用药的过程中逐渐出现了耐药情况,于是酪氨酸激酶抑制剂第二代产品也出现,但对于一些基因的突变效果不令人满意。异基因造血干细胞移植可能只有少数患者可以接受移植。因此,寻找有效一种特效的细胞毒药物十分迫切。近年来国外研究发现JQ1(溴结构域蛋白抑制剂)可抑制慢性髓细胞白血病(CML)BCR-ABLT315I基因突变细胞的增殖,而研究发现CML慢性期转变为急性期是由于MSI2过度表达激活了MSI2-Numb-Notch1的通路。Ph+ALL也具有与CML相似BCR-ABL融合基因。那么JQ1是否对Ph+ALL细胞有抑制作用,JQ1能否通过下调MSI2-Numb-Notch1通路达到抗肿瘤的作用,该研究国内外未见报道。本研究通过体外实验的方法观察JQ1对人Ph+ALL细胞株SUP-B15细胞的凋亡诱导作用及对Notch1通路上MSI2、Notch1、Hes1基因的影响,探讨JQ1治疗Ph(+)ALL中可能的抗肿瘤机制,为临床医生在Ph(+)ALL的治疗提供新的治疗方向。方法:1.不同浓度单药JQ1作用SUP-B15细胞株24h、48h、72h,实验采用四甲基偶氮唑蓝法试验(MTT法)检测的细胞生长抑制情况。2.不同浓度单药JQ1作用SUP-B15细胞48h,实验采用流式细胞术检测细胞周期的改变。3.不同浓度单药JQ1作用SUP-B15细胞株,实验采用实时荧光定量聚合酶链反应(RT-PCR)法检测MSI2、Notch1、Hes1、Bcr-Ablm RNA的表达水平。结果:1.实验采用四甲基偶氮唑蓝(MTT)法检测结果的说明:不同浓度单药的JQ1(1μmol/L、2μmol/L、4μmol/L)和SUP-B15细胞一起培养24h、48h、72h后,SUP-B15细胞生长有明显的抑制现象。在0-4μmol/L的浓度范围内,JQ1实验组的细胞生长抑制率会随着JQ1药物浓度的升高和作用时间延长而升高,呈现剂量-时间依赖性。与对照组比较,在相同时间下,差异有统计学意义(P<0.05)。2.实验采用流式细胞术检测SUP-B15细胞周期的结果说明:与对照组相比,不同浓度单药的JQ1(1μmol/L、2μmol/L、4μmol/L)作用SUP-B15细胞48h后,可以诱导细胞阻滞在S期,呈剂量依赖性。与对照组比较,在相同时间下,差异有统计学意义(P<0.05)。3.实验采用实时荧光定量聚合酶链反应(RT-PCR)法检测的结果说明:不同浓度单药的JQ1(1μmol/L、2μmol/L、4μmol/L)作用SUP-B15细胞48h后,随着JQ1浓度增加,MSI2、Notch1、Hes1、Bcr-Ablm RNA的表达水平逐渐减少,呈剂量依赖性,在相同时间段,与对照组比较,差异有统计学意义(P<0.05)。结论:1.JQ1可以明显抑制SUP-B15细胞的生长,可以有效诱导SUP-B15细胞的凋亡。2.JQ1可以使SUP-B15细胞在细胞的S期被阻滞,呈剂量依赖性,从而影响细胞的生长。3.JQ1可以使Notch1信号通路中关键基因MSI2、Notch1、Hes1的转录水平逐渐减少并呈剂量依赖性,说明JQ1对Ph+ALL细胞株SUP-B15细胞生长抑制可能是通过下调Notch1信号通路来完成的,该机制是JQ1抗肿瘤机制之一。更多还原

【Abstract】 Objective:With the Philadelphia chromosome(Ph+) acute lymphocytic leukemia(acute lymphoblastic leukemia, ALL) is a relatively special in the biology of acute leukemia, is originated from B department or T lymphoid progenitor cells and chromosome 22 pairs of chromosomes long arms and long arm to chromosome 9 reciprocal translocation after the BCR- ABL fusion gene of tumor diseases.Ph(+) ALL belong to high-risk leukemia and low response rate, alleviate the time is short, the prognosis is poor.The Ph(+) there are three kinds of the main treatment of ALL: standard chemotherapy, tyrosinase inhibitor,allogeneic hematopoietic stem cell transplantation.Response rate is low, long-term regular chemotherapy patients relieve time is short, long body can not tolerate.With the advent of tyrosine kinase inhibitors, BCR ABL fusion gene in targeted therapy of chronic myelogenous leukemia(chronic myelocytic leukemia, CML) made a great success, then for the treatment of Ph + ALL greatly improves the response rate of disease, but in the process of clinical medication gradually discovered the body resistance.Allogeneic hematopoietic stem cell transplantation as a suitable donor, less expensive, only a minority of patients can transplant.Therefore,to find effective cytotoxic drugs and potential molecular targets is very urgent.In recent years, study abroad JQ1(bromine structure domain protein inhibitors) inhibits chronic myelogenous leukemia(CML) BCR-ABLT315 I gene mutations in the cell proliferation, but the study found that CML chronic phase into acute stage was due to the activation of MSI2- Numb- Notch1 pathway.Ph + ALL also have similar to CML BCR- ABL fusion gene.So JQ1 whether has the inhibitory effect on the Ph+ ALL cells, JQ1 can cut MSI2- Numb- Notch1 pathway to reach the role ofanti-tumor, the study reported for the first at home and abroad.his study observed by the method of in vitro experiments JQ1 SUP- B15 Ph+ ALL cell lines to the person on cell apoptosis inducing effect and of Notch1 pathway MSI2, Notch1, Hes1 gene, discuss in JQ1Ph(+) ALL possible antitumor mechanism, for the treatment of Ph(+) ALL provide new treatments.Methods:1.The experiment adopted four methyl azo thiazole blue method test(determined by MTT method) to detect different concentrations single-agent JQ1 role SUP-B15 cell line proliferation inhibition.2.Experiments using flow cytometry to detect different concentrations single drug JQ1 to SUP-B15 cell cycle of change.3.Real-time fluorescent quantitative polymerase chain reaction(RT-PCR) method to detect different concentration single-agent JQ1 SUP-B15 cell lines MSI2, Notch1, Hes1,Bcr- Ablm RNA expression level decrease.Results:1.The four methyl azo thiazole blue(determined by MTT method to detect the results showed that different concentrations of single-agent JQ1(1μmol/L, 2μmol/L, 4μmol/L)and SUP- B15 cell culture together after 24 h, 48 h, 72 h, SUP- B15 inhibition of cell growth has obvious phenomenon.At 0-4 mol/L concentration range, JQ1 cell growth inhibition rate of the experimental group with the extension of elevated JQ1 drug concentration and action time, dose- time dependence.At the same time, compared with control group, with statistical significance2.Flow cytometry detection SUP- B15 cell cycle result shows: compared with control group, different concentrations of single-agent JQ1(1μmol/L, 2μmol/L, 4μmol/L), after 48 h SUP- B15 cells can induce cell block in S phase, a dose dependent, at the same time,compared with control group, with statistical significance(P < 0.05).3.Real-time fluorescent quantitative polymerase chain reaction(RT-PCR) method to detect the results showed that different concentrations of single drug JQ1(1μmol/L,2μmol/L, 4μmol/L) to SUP- B15 cells after 48 h, with the increase of concentration of JQ1,MSI2, Notch1, Hes1, Bcr- Ablm RNA expression level gradually reduce, in dose dependent, at the same time, compared with control group, the difference was statistically significant(P < 0.05).Conclusions:1.JQ1 can obviously inhibit the growth of SUP- B15 cells, can induce the apoptosis of SUP- B15 cells effectively.2.JQ1 can make SUP- B15 cell block S phase of the cell and a dose dependent, which affects the growth of cells.3.JQ1 may cause the Notch1 signaling pathway in key genes MSI2, Notch1, Hes1 transcription level gradually reduce and a dose dependent, prompt JQ1 to Ph+ ALL cell lines SUP-B15 cell growth inhibition was done by Notch1 signaling pathways, the mechanism is one of JQ1 antitumor mechanism.更多还原