【作者】 杜华平；

【导师】 金洁；

【作者基本信息】 浙江大学 ， 内科学， 2010， 博士

【摘要】 第一部分BBD9对淋巴瘤细胞的生长抑制和诱导凋亡作用目的：研究BBD9对淋巴瘤细胞生长的影响及诱导凋亡作用。方法：MTT法检测BBD9对多种淋巴瘤细胞株的生长抑制作用,瑞氏染色观察细胞形态学变化,AV-PI染色流式细胞术和DNA ladder方法检测细胞凋亡。结果：BBD9能显著抑制四种淋巴瘤细胞株(Raji, L428, namalwa, Jurkat)细胞的生长。在0-4μg/ml BBD9浓度范围内,随着浓度的升高,各种细胞株的细胞存活率逐步降低。同时,BBD9对四种细胞株的生长抑制作用也呈时间依赖性,同一浓度下,随着药物作用时间的延长,细胞存活率也降低。BBD9作用于Raji,L428,namalwa, Jurkat细胞24h的IC5o分别是：1.5μg／ml、1.89μg／ml、1.83μg／ml、1.59μg／ml。BBD9对2例病人原代细胞也呈现剂量依赖性的生长抑制。其IC50分别为2.09gg／ml,2.43 gg／ml。BBD9虽然对4例正常入骨髓单个核细胞也有抑制作用,但其IC50为3.46μg／ml,抑制作用弱于对淋巴瘤细胞株细胞的作用。细胞形态学观察可见,BBD9处理Raji细胞和L428细胞24h后出现了典型的凋亡细胞形态：核碎裂,核浓缩、凋亡小体。DNA片段检测发现经BBD9处理的Raji、L428细胞出现了典型"DNA梯形条带”,而且随着药物浓度的增加,“DNA梯形条带”也越明显。应用AV-PI染色法经流式细胞仪检测表明,BBD9可以诱导淋巴瘤细胞凋亡。0、1、2、3μg／ml BBD9处理Raji细胞24h后,早期凋亡细胞比例分别为2.87±0.35%、9.36±2.10%、11.69±0.89%、46.96±3.58%；而在L428细胞,相应浓度作用后早期凋亡细胞的比例分别为4.45±0.97%、9.02±0.64%、17.62±2.33%、35.87±4.44%。Western blot检测表明,BBD9能诱导Raji细胞和L428细胞的caspase-3,-8,-9及PARP剪切激活。结论：(1)BBD9能抑制多种淋巴瘤细胞株细胞生长。(2)BBD9能抑制原代淋巴瘤细胞生长,对正常骨髓单个核细胞抑制作用弱。(3)BBD9能诱导淋巴瘤细胞凋亡。第二部分BBD9对淋巴瘤细胞的各种信号通路的影响目的：观察线粒体凋亡通路上各种蛋白的改变,观察PTEN/PI3K/Akt信号传导通路中参与信号传导的各种蛋白的改变,观察NF-κB信号传导通路中参与信号传导的各种蛋白的改变,进一步阐明BBD9抑制淋巴瘤细胞生长的分子机制。方法：western blot法检测BCL-2家族蛋白和IAP家族蛋白表达水平,流式细胞术检测细胞周期。western blot法检测细胞周期调控蛋白,检测PI3K／Akt和NF-KB信号通路上各种蛋白的表达水平。Real-time PCR检测PTEN mRNA及miR-17-5p,miR-20a的表达。免疫荧光检测NF-κB在细胞核、细胞浆中的信号表达。结果：BBD9处理Raji细胞后,抑凋亡蛋白Mcl-1明显下调,BCL-2轻度下调,促凋亡蛋白Bak、Bax和Bim、pBad表达量则有明显升高。BBD9下调IAP家族蛋白,如XIAP、CIAP1和Survivin等的表达。细胞周期分析表明,BBD9使淋巴瘤细胞周期阻滞在G2／M期。2gg／ml BBD9处理Raji细胞0、4、8、12、24 h后,处于G2／M期的细胞分别为8.41±3.62%、11.51±2.44%、17.36±6.11%、23.09±2.40%和25.21±9.04%。细胞周期调控蛋白cyclinA、cyclinB1和CDK1在BBD9处理的Raji细胞出现下调。0-3μg／ml BBD9处理Raji细胞24小时后,总Akt无明显变化,而磷酸化Akt呈下降趋势；BBD9还能下调PI3K蛋白,而PTEN呈现明显上调趋势。1、2、3μg／ml BBD9处理Raji细胞24h后,PTEN mRNA拷贝数比对照组分别增加3.76、7.13和19.75倍。而对PTEN mRNA起负调控作用的microRNA-miR-17-5p、miR-20a出现下调。Western blot检测表明,经1～3μg／ml BBD9处理Raji细胞后,胞浆IκB升高,但pIκB减少,胞核内NF-κB表达呈下调趋势。免疫荧光方法检测结果表明,在BBD9处理前,胞核所在位置红色荧光较强,胞浆所在位置红色荧光较弱,提示胞核内NF-κB表达较高,胞浆内NF-κB表达相对较低。经2μg／ml BBD9处理24h后,胞核内的红色信号减弱,即核内NF-κB表达减少,说明BBD9抑制了NF-κB进入核内。结论：(1)BBD9上调促凋亡蛋白Bak、Bax、pBad和Bim,下调抑凋亡蛋白Mcl-1；同时能下调XIAP, CIAP1和Survivin等IAP家族蛋白；通过下调cyclinA、cyclinB1和CDKl使细胞周期阻滞于G2／M期；通过上述途径诱导细胞凋亡,从而抑制淋巴瘤细胞生长。(2)BBD9可抑制PI3K／Akt信号通路,该通路受阻是BBD9诱导淋巴瘤细胞凋亡的一个重要机制。(3)BBD9可下调miR-17-5p,miR-20a,上调mRNA水平,使PTEN蛋白表达上调,来抑制PI3K／Akt信号通路。(4)BBD9可通过抑制IκB磷酸化,阻止NF-KB与IKB解离而进入核内。NF-κB通路受阻可能是BBD9诱导淋巴瘤细胞凋亡的另一个重要机制。总结：BBD9具有抑制淋巴瘤细胞株及原代细胞生长的作用,并在一定范围内呈时间和剂量依赖性。这种生长抑制作用是通过诱导细胞凋亡来起作用的。在BBD9诱导的淋巴瘤细胞凋亡过程中,促凋亡蛋白Bak、Bax、pBad和Bim上调,而抑凋亡蛋白Mcl-1则下调,并能下调XIAP, CIAP1和Survivin等IAP家族蛋白。BBD9还能通过下调cyclinA、cyclinB1和CDK1使细胞周期阻滞于G2／M期。BBD9在mRNA水平、microRNA水平调控PTEN,从而上调PTEN蛋白的表达,进而抑制PI3K／Akt信号通路。该通路受阻是BBD9诱导淋巴瘤细胞凋亡的一个重要机制。此外,BBD9还可以通过胞浆下调磷酸化IκB,抑制NF-κB进入核内来抑制NF-κB信号通路。该通路受阻可能是BBD9诱导淋巴瘤细胞凋亡的另一个重要机制。更多还原

【Abstract】 Section 1 BBD9 inhibited cell growth and induced apoptosis In lymphoma cellsObjective:To study the effects of BBD9 on lymphoma cells.Methods:Cell growth was measured by MTT assay in lymphoma cell lines and primary cells from patients with lymphocytic leukemia. Cell morphology was observed by Wright-Giemsa stain. AV-PI staining and DNA ladder was used for detecting apoptosis.Results:The cell viabilities of four lymphoma cell lines, including Raji, L428, Namalwa and Jurkat, were significantly inhibited by BBD9 at the concentration of 0-4μg/ml. The cell survival rate was decreased with the increasing concentration of BBD9. Furthermore, BBD9 inhibited cell growth in all four cell lines in a time dependent manner. When treated at the same concentration of BBD9, fewer cells would survive with increasing time. The 24h IC50 of BBD9 on Raji, L428, Namalwa and Jurkat were 1.5μg/ml、1.89μg/ml、1.83μg/ml、1.59μg/ml, respectively. Primary cells from two patients with lymphoma were treated with BBD9 for 24h, and also showed growth inhibition in a dose dependent manner. The IC50 for primary cells were 2.09μg/ml and 2.43μg/ml, respectively. The cell growth inhibition induced by BBD9 was also measured in mononuclear cells from normal bone marrow by MTT assay. The results showed that the cell growth of normal mononuclear cells was inhibited, in a dose dependant manner, by BBD9 at the concentration of 0-4μg/ml. However, normal mononuclear cells were less sensitive to BBD9 treatment than primary lymphoma cells were, and the 24h IC50 was 3.46μg/ml. After treated by BBD9 for 24h, Raji and L428 cells, observed by Wright-Giemsa stain, showed a typical apoptotic morphology, nuclear fragmentation and apoptotic body. Typical DNA ladders were also seen in Raji and L428 cells treated by BBD9 for 24h. With the concentration of BBD9 increasing, the DNA ladder became more significant. Further study revealed that BBD9 could induce apoptosis in lymphoma cells, as measured by AV-PI assay. After treated by 0,1, 2,3μg/ml BBD9 for 24h, the Raji cells in early apoptosis were 2.87±0.35%、9.36±2.10%、11.69±0.89%、46.96±3.58% respectively. Meanwhile, the L428 cells in early apoptosis were 4.45±0.97%.9.02±0.64%、17.62±2.33%、35.87±4.44%, respectively. Furthermore, caspase-3,-8,-9 and PARP were cleaved and activated, as measured by western blot, in Raji cells and L428 cells treated by BBD9.Conclusions:(1) BBD9 inhibited cell growth of four lymphoma cell lines(2) BBD9 inhibited cell growth of primary cells, but inhibited less in bone marrow normal mononuclear cells.(3) BBD9 induced apoptosis in lymphoma cells. Section 2 BBD9 affected some signaling pathway in lymphoma cellObjective:To reveal the alteration of proteins involved in mitochondria apoptosis; To reveal the expression of proteins involved in PI3K/Akt signaling pathway; To study the mechanism of PTEN protein upregulated in Raji cells after cells being treated by BBD9. To reveal the alteration of proteins involved in NF-κB signaling pathway, and to better understand the mechanism of BBD9 effecting on lymphoma cells.Methods:Western blot was used for detecting BCL-2 family proteins, IAP family proteins and PI staining was used for cell cycle analysis; Western blot was also used for assaying the expression of proteins involved in cell cycle regulating proteins, assaying the expression of proteins involved in PI3K/Akt and NF-κB signaling pathway. Real-time PCR was messured to detect the mRNA of PTEN and expression of the mir-17-5p and mir-20a. Immunofluorescence detection was messured to detect the expression of the NF-κB and IκB in cytosplasm and intranuclear.Results:After treated by BBD9, the expression of Raji cells’pro-apoptotic protein, Bak, Bax pBad and Bim were up-regulated and anti-apoptotic protein, Mcl-1 were down-regulated. BBD9 also inhibited the expression of IAP family proteins, such as XIAP, CIAP1 and Survivin. In addition, the results of cell cycle analysis showed that BBD9 induced cell cycle arrest in G2/M phase. After being treated by 2μg/ml BBD9 for Oh,4h,8h,12h,24h, the Raji cells arrested in G2/M phase were 8.41±3.62%. 11.51±2.44%、17.36±6.11%、23.09±2.40%、25.21±9.04%, respectively. Cell cycle regulators cyclin A, cyclin B1 and CDK1 were all down-regulated as measured by western blot assay, in Raji cells treated by BBD9 for 24h.After inhibited with 0-3μg/ml BBD9 for 24h, the expression of total Akt of Raji cell was not change, the pAkt and PI3K were decreased while PTEN was obiviously increased. Compared with the untreated control group, the expressions of PTEN mRNA were 3.76,7.13 and 19.75 folds. Furthermore, microRNA-miR-17-5p, miR-20a were significantly down regulated in Raji cells treated with BBD9.After inhibited with 0-3μg/ml BBD9 for 24h, the expression of cytoplasmic IκB of Raji cell increased, while the pIκB was decreased, meanwhile the expression of intranuclear NF-κB the was downregulated. Immunofluorescence detection revealed that the untreated control group, the red fluorescent light in the location of nuclear was bright, and weak in the cytoplasm which means the expression of NF-κB in nuclear was low. After treated with 2μg/ml BBD9 for 24h, the red fluorescent light in the blue fluorescent light location, which indicate the location of nuclear, was weak, that means the expression fo NF-κB in nuclear was downregulated and BBD9 inhibited the NF-κB transfer to nuclear.Conclusions:(1) BBD9 up-regulated pro-apoptotic protein such as Bak, Bax, pBad and Bim and down-regulated anti-apoptotic protein Mcl-1; and inhibited XIAP, CIAP1 and Survivin proteins; BBD9 also induced cell cycle arrested in G2/M phase through down-regulation of cyclin A, cyclin B1 and CDK1. By these signal pathways, BBD9 induce apoptosis of lymphoma cells so that inhibited the proliferation of lymphoma cells.(2) BBD9 inhibited PI3K/Akt signaling pathway which maybe the important mechanism that BBD9 induced apoptosis of lymphoma cells.(3) BBD9 downregulated miR-17-5p, miR-20a, and upregulated PTEN mRNA so as to upreglated the expression of PTEN protein, which inhibiter PI3K/Akt signaling pathway. (4) BBD9 inhibited pIκB, inhibited NF-κB transfer to nuclear so as to inhibit NF-κB signaling pathway, which maybe another important mechanism that BBD9 induced apoptosis of lymphoma cells.Summary:BBD9 inhibited cell growth in lymphoma cell lines and primary cells from patients through inducing apoptosis, which was time-and dose-depedent to some extent. BBD9 may induce Pro-apoptotic protein such as Bak, Bax pBad and Bim up-regulated and anti-apoptotic protein Mcl-1 down-regulated. IAP family proteins, include XIAP, CIAP1 and Survivin was downregulated by BBD9. Furthermore, BBD9 also can induce cell cycle arrested in G2/M phase through down-regulation of cyclin A cyclin B1 and CDK1. In addition, BBD9 increased the expression of mRNA of PTEN, and increase the expression of PTEN protein so as to inhibited PI3K/Akt signal pathway. BBD9 inhibited PI3K/Akt signal pathway, which maybe an important mechanism that BBD9 induced apoptosis of lymphoma cells. BBD9 also inhibited pIκB, inhibited NF-κB transfer to nuclear so as to inhibit NF-κB signaling pathway, which maybe another important mechanism that BBD9 induced apoptosis of lymphoma cells.更多还原