Apatinib对弥漫大B细胞淋巴瘤细胞株的杀伤作用及分子机制的探讨
发布时间：2018-08-31 10:06
【摘要】：研究背景:弥漫大B细胞淋巴瘤(Diffuse large B-cell lymphoma,DLBCL)是成人最常见的淋巴系统肿瘤,占非霍奇金淋巴瘤(non-Hodgkin lymphoma,NHL)的30-40%,是其最常见的一种亚型。DLBCL是一种异质性很大的侵袭性恶性肿瘤,包括在组织形态学、细胞遗传学、分子生物学及临床预后方面。近年来,基于含有利妥昔单抗(rituximab)的联合化疗 R-CHOP(rituximab,cyclophosphamide,doxorubicin,vincristine,prednisolone)方案,DLBCL患者的治疗效果显著提高,生存期也明显延长。然而在R-CHOP的治疗下,仍有近1/3的难治复发患者,且只有很少的患者得益于挽救治疗及自体造血干细胞移植(autologous stem cell transplantation,ASCT)。那么新的药物对于治疗DLBCL是迫切需要的,目前靶向药物一直是恶性肿瘤研究的热点。血管生成对肿瘤生长及转移至关重要,选择性的抑制血管生成已成为抗肿瘤治疗的策略,目前越来越多的靶向血管生成药物在临床上运用于肿瘤。Apatinib是一种靶向血管的抑制剂,为血管内皮生长因子受体-2(VEGFR-2)酪氨酸激酶抑制剂;Apatinb能强效抑制肿瘤血管生成,主要作用机制是竞争性结合该受体胞内酪氨酸ATP结合位点,高度选择性地抑制VEGFR2酪氨酸激酶活性,阻断VEGFR2与内皮生长因子(VEGF)结合后的信号传导。VEGF与VEGFR2结合后可以激活多种信号通路,包括PI3K/Akt、Ras通路等,而Ras通路与人类绝大多数肿瘤密切相关,也是VEGF/VEGFR2激活后重要的信号途径。Apatinib作用于VEGFR2后,可以抑制VEGF/VEGFR2的激活,从而抑制其激活Ras信号通路,发挥抗肿瘤效应。研究目的:探讨Apatinib能否抑制弥漫大B细胞淋巴瘤细胞增殖并诱导其凋亡,并进一步研究其杀伤作用与Ras信号通路的关系,为开拓弥漫大B细胞淋巴瘤治疗新方法提供实验依据。研究方法:1、用 CCK-8 法检测不同浓度的(2.5、5、10、20、40μmol/L)Apatinib 作用48h后对DLBCL细胞株的增殖抑制作用。2、用Annexin-V/PI双染法流式细胞术检测不同浓度的(0、10、20、30、40μmol/L)Apatinb处理48h后对DLBCL细胞株凋亡的影响。3、利用蛋白免疫印迹法检测Apatinib作用oci-ly1和细胞SU-DHL-2 48h后pVEGFR2蛋白及Ras信号通路中Ras、c-Raf、pMEK1/2和pErK1/2的表达变化。4、统计学分析采用SPSS19.0软件,由于均是小样本数据的统计,两组独立样本均数的比较采用非参数检验(Mann-WhitheyU检验);多组间均数比较采用多个独立样本的非参数检验(Kruskal-WallisH检验)。计量资料用Mean±SD表示,所有的检验分析均是双侧检验,检验水准为α=0.05。研究结果:1.CCK8 结果显示:Apatinib 对 GCB 型 DLBCL 细胞株 oci-ly1、SU-DHL-4(S4)及ABC细胞株oci-ly3、SU-DHL-2(S2)具有明显的增殖抑制作用,并呈剂量依赖性。Apatinib 作用 oci-ly1、SU-DHL-4(S4)oci-ly3、SU-DHL-2(S2)细胞 48h 后,其 IC50 分别为(19.30±0.07)μmol/L、(18.15±0.15)μmol/L、(18.15±0.15)μmol/L、(15.29±0.13)μmol/L 和(12.34±0.27)μmol/L,经多个独立样本的非参数检验Kruskal-Wallis H检验分析结果表明与对照组比较差异均具有统计学意义(χ2=16.460,P=0.006;χ2=16.248,P=0.006;χ2=16.460,P=0.006 和χ2=16.648,P=0.005)。经两个独立样本非参数检验Mann-Whitney U分析,ABC型细胞株oci-ly3的增殖抑制率分别明显高于两种GCB型细胞株oci-ly1和SU-DHL-4(S4)(P=0.047和P=0.047);同样,另一株ABC型细胞株SU-DHL-2的增殖抑制率分别高于两种 GCB 型细胞株 oci-ly1 和 SU-DHL-4(S4)(P=0.047 和 P=0.047)。2.Annexin V/PI双染法流式细胞术检测Apatinib作用于GCB型DLBCL细胞株 oci-ly1、SU-DHL-4(S4)及 ABC 型细胞株 oci-ly3,SU-DHL-2(S2)48h 后的凋亡比例,发现 Apatinib 能够诱导 oci-ly1、SU-DHL-4(S4)、oci-ly3、SU-DHL-2(S2)细胞凋亡,呈浓度依赖性,经多个独立样本的非参数检验Kruskal-WallisH检验分析,与对照组比较差异均具有统计学意义(χ2=12.767,P=0.012;χ2=13.233,P=0.010;χ2=13.033,P=0.011 和χ2=12.933,P=0.012)。两种类型DLBCL细胞株的凋亡率的比较无统计学意义,经两个独立样本非参数检验Mann-Whitney U分析,ABC型细胞株oci-ly3的凋亡率与两种GCB型细胞株oci-ly1和SU-DHL-4(S4)比较均无明显差异(P=0.275和P=0.06);同样,另一株ABC型细胞株SU-DHL-2的凋亡率与GCB型细胞株oci-ly1和SU-DHL-4(S4)比较均无统计学意义(P=0.275和P=0.06)。3.蛋白质印迹法结果显示Apatinib可通过抑制VEGFR2的自磷酸化即抑制pVEGFR2蛋白的表达,进而抑制Ras(Ras、Raf、pMEK1/2、pErk1/2)信号通路。对蛋白条带进行灰度分析,经多个独立样本非参数检验Kruskal-Wallis H检验,Apatinib 组(20、40μmnol/L)中 pVEGFR2、Ras、c-Raf、pMEK1/2,pErk1/2 蛋白表达水平与对照组(0μmol/L)相比,差异均具有统计学意义(均χ2=7.2,均P=0.027)结论:1.不同浓度的Apatinib能抑制弥漫大B细胞淋巴瘤细胞oci-ly1、SU-DHL-4(S4)、oci-ly3、SU-DHL-4(S2)的增殖,并诱导其凋亡。且 Apatinib 对于ABC型的DLBCL细胞株的增殖抑制率效果更明显。2.Apatinib通过抑制VEGF与VEGFR2的结合,阻止VEGFR2发生自磷酸化成pVEGFR2而活化,从而抑制其激活的Ras信号通路,发挥杀伤弥漫大B细胞淋巴瘤细胞的作用。
[Abstract]:Background: Diffuse large B-cell lymphoma (DLBCL) is the most common lymphatic malignancy in adults, accounting for 30-40% of non-Hodgkin lymphoma (NHL) and one of its most common subtypes. DLBCL is a highly heterogeneous aggressive malignancy, including histomorphology and cell genetics. In recent years, based on R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisolone) regimen with rituximab, DLBCL patients have significantly improved treatment outcomes and significantly prolonged survival. However, under R-CHOP treatment, there are still nearly one third of refractory cases. Only a few patients benefit from salvage therapy and autologous stem cell transplantation (ASCT). New drugs are urgently needed for the treatment of DLBCL. Targeted drugs have been the focus of cancer research. Inhibiting angiogenesis has become a strategy of anti-tumor therapy. Nowadays, more and more targeted angiogenesis drugs are used in clinic. Competitively binding to the receptor's intracellular tyrosine ATP binding site, highly selectively inhibits the activity of VEGF R2 tyrosine kinase, and blocks the signal transduction of the binding of VEGF R2 to endothelial growth factor (VEGF). The binding of VEGF and VEGF R2 can activate a variety of signaling pathways, including PI3K/Akt, Ras pathway, and Ras pathway is closely related to most human tumors. Apatinib can inhibit the activation of VEGF/VEGFR2, thereby inhibiting the activation of Ras signaling pathway and exerting anti-tumor effect. Objective: To investigate whether Apatinib can inhibit proliferation and induce apoptosis of diffuse large B-cell lymphoma cells, and to further study its killing effect. Methods: 1. CCK-8 assay was used to detect the inhibitory effect of different concentrations (2.5, 5, 10, 20, 40 micromol/L) of Apatinib on the proliferation of DLBCL cell lines 48 hours later. 2. Annexin-V/PI double staining was used to detect the inhibitory effect of Apatinib on the proliferation of DLBCL cell lines. 20,30,40 micromol/L) Apatinb treatment for 48 hours on the apoptosis of DLBCL cell lines. 3. Protein immunoblotting was used to detect the expression of Ras, c-Raf, pMEK1/2 and pErK1/2 in the pVEGF R2 protein and Ras signaling pathway after Apatinib treatment for oci-ly1 and SU-DHL-248 hours. 4. SPSS19.0 software was used for statistical analysis, because the data were small sample statistics. Results: 1. The results of CCK8 showed that: Apatinib: 1. Apatinibacted on oci-ly1, SU-DHL-4 (S4) oci-ly3, SU-DHL-4 (S4) and ABC DLBCL cell lines oci-ly1, SU-DHL-4 (S4) and ABC cell lines oci-ly3, SU-DHL-2 (S2) in a dose-dependent manner. Apatinibacted on oci-ly1, SU-DHL-4 (S4) oci-ly3, SU-DHL-2 (S2) cells 48 h later, the IC50 values were (19.30 [(0.07) micromol/L, (18.15 [(18.15) micromol/L, (18.15) 15) micromol/L, (18.15) 18.15) micromol/L, (18.15) 18.L, (15.29 +) The results of Kruskal-Wallis H test showed that there were significant differences between the two groups (_2 = 16.460, P = 0.006; _2 = 16.248, P = 0.006; _2 = 16.460, P = 0.006; P = 16.460, P = 0.006, P = 0.006, P = 0.006, P = 0.006 and 2 = 16.648, P = 0, P = 0.006 and 2 = 16.648, P = 0.005). The inhibitory rate of proliferation of ABC cell line oci-ly3 was significantly higher than that of two GCB cell lines oci-ly1 and SU-DHL-4 (S4) (P = 0.047 and P = 0.047), respectively; similarly, the inhibitory rate of proliferation of another ABC cell line SU-DHL-2 was higher than that of two GCB cell lines oci-ly1 and SU-DHL-4 (S4) (P = 0.047 and P = 0.047) by Annexin V/PI double staining. The percentage of apoptosis in GCB DLBCL cell lines oci-ly1, SU-DHL-4 (S4) and ABC cell lines oci-ly3, SU-DHL-2 (S2) was measured by SPECT. It was found that Apatinib could induce apoptosis of oci-ly1, SU-DHL-4 (S4), oci-ly3, SU-DHL-2 (S2) cells in a concentration-dependent manner. Kruskal-Wallis H test was performed on several independent samples. The apoptosis rate of ABC cell line oci-ly3 was not significantly different from that of two kinds of GCBL cell lines (_2 = 12.767, P = 0.012; _2 = 13.233, P = 0.010; _2 = 13.033, P = 0.011 and_2 = 12.933, P = 0.012). Similarly, the apoptosis rate of another ABC cell line, SU-DHL-2, was not significantly different from that of GCB cell lines, oci-ly1 and SU-DHL-4 (S4) (P = 0.275 and P = 0.06). The expression of pVEGFR2 protein was inhibited and Ras (Ras, Raf, pMEK1/2, pErk1/2) signaling pathway was inhibited. The protein bands were analyzed by gray scale and Kruskal-Wallis H test. The expression levels of pVEGFR2, Ras, c-Raf, pMEK1/2 and pErk1/2 protein in Apatinib group (20,40 micron nol/L) were significantly different from those in control group (0 micromol/L). Conclusion: 1. Different concentrations of Apatinib can inhibit the proliferation and induce apoptosis of diffuse large B-cell lymphoma cells oci-ly1, SU-DHL-4 (S4), oci-ly3, SU-DHL-4 (S2), and the inhibitory effect of Apatinib on the proliferation of ABC-type DLBCL cell lines is more obvious. 2. Apatinib inhibits the binding of VEGF and VEGFR2. Inhibiting the autophosphorylation of VEGF R2 into pVEGF R2 and activating the Ras signaling pathway can kill diffuse large B-cell lymphoma cells.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.1
本文编号：2214664
[Abstract]:Background: Diffuse large B-cell lymphoma (DLBCL) is the most common lymphatic malignancy in adults, accounting for 30-40% of non-Hodgkin lymphoma (NHL) and one of its most common subtypes. DLBCL is a highly heterogeneous aggressive malignancy, including histomorphology and cell genetics. In recent years, based on R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisolone) regimen with rituximab, DLBCL patients have significantly improved treatment outcomes and significantly prolonged survival. However, under R-CHOP treatment, there are still nearly one third of refractory cases. Only a few patients benefit from salvage therapy and autologous stem cell transplantation (ASCT). New drugs are urgently needed for the treatment of DLBCL. Targeted drugs have been the focus of cancer research. Inhibiting angiogenesis has become a strategy of anti-tumor therapy. Nowadays, more and more targeted angiogenesis drugs are used in clinic. Competitively binding to the receptor's intracellular tyrosine ATP binding site, highly selectively inhibits the activity of VEGF R2 tyrosine kinase, and blocks the signal transduction of the binding of VEGF R2 to endothelial growth factor (VEGF). The binding of VEGF and VEGF R2 can activate a variety of signaling pathways, including PI3K/Akt, Ras pathway, and Ras pathway is closely related to most human tumors. Apatinib can inhibit the activation of VEGF/VEGFR2, thereby inhibiting the activation of Ras signaling pathway and exerting anti-tumor effect. Objective: To investigate whether Apatinib can inhibit proliferation and induce apoptosis of diffuse large B-cell lymphoma cells, and to further study its killing effect. Methods: 1. CCK-8 assay was used to detect the inhibitory effect of different concentrations (2.5, 5, 10, 20, 40 micromol/L) of Apatinib on the proliferation of DLBCL cell lines 48 hours later. 2. Annexin-V/PI double staining was used to detect the inhibitory effect of Apatinib on the proliferation of DLBCL cell lines. 20,30,40 micromol/L) Apatinb treatment for 48 hours on the apoptosis of DLBCL cell lines. 3. Protein immunoblotting was used to detect the expression of Ras, c-Raf, pMEK1/2 and pErK1/2 in the pVEGF R2 protein and Ras signaling pathway after Apatinib treatment for oci-ly1 and SU-DHL-248 hours. 4. SPSS19.0 software was used for statistical analysis, because the data were small sample statistics. Results: 1. The results of CCK8 showed that: Apatinib: 1. Apatinibacted on oci-ly1, SU-DHL-4 (S4) oci-ly3, SU-DHL-4 (S4) and ABC DLBCL cell lines oci-ly1, SU-DHL-4 (S4) and ABC cell lines oci-ly3, SU-DHL-2 (S2) in a dose-dependent manner. Apatinibacted on oci-ly1, SU-DHL-4 (S4) oci-ly3, SU-DHL-2 (S2) cells 48 h later, the IC50 values were (19.30 [(0.07) micromol/L, (18.15 [(18.15) micromol/L, (18.15) 15) micromol/L, (18.15) 18.15) micromol/L, (18.15) 18.L, (15.29 +) The results of Kruskal-Wallis H test showed that there were significant differences between the two groups (_2 = 16.460, P = 0.006; _2 = 16.248, P = 0.006; _2 = 16.460, P = 0.006; P = 16.460, P = 0.006, P = 0.006, P = 0.006, P = 0.006 and 2 = 16.648, P = 0, P = 0.006 and 2 = 16.648, P = 0.005). The inhibitory rate of proliferation of ABC cell line oci-ly3 was significantly higher than that of two GCB cell lines oci-ly1 and SU-DHL-4 (S4) (P = 0.047 and P = 0.047), respectively; similarly, the inhibitory rate of proliferation of another ABC cell line SU-DHL-2 was higher than that of two GCB cell lines oci-ly1 and SU-DHL-4 (S4) (P = 0.047 and P = 0.047) by Annexin V/PI double staining. The percentage of apoptosis in GCB DLBCL cell lines oci-ly1, SU-DHL-4 (S4) and ABC cell lines oci-ly3, SU-DHL-2 (S2) was measured by SPECT. It was found that Apatinib could induce apoptosis of oci-ly1, SU-DHL-4 (S4), oci-ly3, SU-DHL-2 (S2) cells in a concentration-dependent manner. Kruskal-Wallis H test was performed on several independent samples. The apoptosis rate of ABC cell line oci-ly3 was not significantly different from that of two kinds of GCBL cell lines (_2 = 12.767, P = 0.012; _2 = 13.233, P = 0.010; _2 = 13.033, P = 0.011 and_2 = 12.933, P = 0.012). Similarly, the apoptosis rate of another ABC cell line, SU-DHL-2, was not significantly different from that of GCB cell lines, oci-ly1 and SU-DHL-4 (S4) (P = 0.275 and P = 0.06). The expression of pVEGFR2 protein was inhibited and Ras (Ras, Raf, pMEK1/2, pErk1/2) signaling pathway was inhibited. The protein bands were analyzed by gray scale and Kruskal-Wallis H test. The expression levels of pVEGFR2, Ras, c-Raf, pMEK1/2 and pErk1/2 protein in Apatinib group (20,40 micron nol/L) were significantly different from those in control group (0 micromol/L). Conclusion: 1. Different concentrations of Apatinib can inhibit the proliferation and induce apoptosis of diffuse large B-cell lymphoma cells oci-ly1, SU-DHL-4 (S4), oci-ly3, SU-DHL-4 (S2), and the inhibitory effect of Apatinib on the proliferation of ABC-type DLBCL cell lines is more obvious. 2. Apatinib inhibits the binding of VEGF and VEGFR2. Inhibiting the autophosphorylation of VEGF R2 into pVEGF R2 and activating the Ras signaling pathway can kill diffuse large B-cell lymphoma cells.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.1
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