Apatinib對(duì)彌漫大B細(xì)胞淋巴瘤細(xì)胞株的殺傷作用及分子機(jī)制的探討
發(fā)布時(shí)間：2018-08-31 10:06
【摘要】：研究背景:彌漫大B細(xì)胞淋巴瘤(Diffuse large B-cell lymphoma,DLBCL)是成人最常見(jiàn)的淋巴系統(tǒng)腫瘤,占非霍奇金淋巴瘤(non-Hodgkin lymphoma,NHL)的30-40%,是其最常見(jiàn)的一種亞型。DLBCL是一種異質(zhì)性很大的侵襲性惡性腫瘤,包括在組織形態(tài)學(xué)、細(xì)胞遺傳學(xué)、分子生物學(xué)及臨床預(yù)后方面。近年來(lái),基于含有利妥昔單抗(rituximab)的聯(lián)合化療 R-CHOP(rituximab,cyclophosphamide,doxorubicin,vincristine,prednisolone)方案,DLBCL患者的治療效果顯著提高,生存期也明顯延長(zhǎng)。然而在R-CHOP的治療下,仍有近1/3的難治復(fù)發(fā)患者,且只有很少的患者得益于挽救治療及自體造血干細(xì)胞移植(autologous stem cell transplantation,ASCT)。那么新的藥物對(duì)于治療DLBCL是迫切需要的,目前靶向藥物一直是惡性腫瘤研究的熱點(diǎn)。血管生成對(duì)腫瘤生長(zhǎng)及轉(zhuǎn)移至關(guān)重要,選擇性的抑制血管生成已成為抗腫瘤治療的策略,目前越來(lái)越多的靶向血管生成藥物在臨床上運(yùn)用于腫瘤。Apatinib是一種靶向血管的抑制劑,為血管內(nèi)皮生長(zhǎng)因子受體-2(VEGFR-2)酪氨酸激酶抑制劑;Apatinb能強(qiáng)效抑制腫瘤血管生成,主要作用機(jī)制是競(jìng)爭(zhēng)性結(jié)合該受體胞內(nèi)酪氨酸ATP結(jié)合位點(diǎn),高度選擇性地抑制VEGFR2酪氨酸激酶活性,阻斷VEGFR2與內(nèi)皮生長(zhǎng)因子(VEGF)結(jié)合后的信號(hào)傳導(dǎo)。VEGF與VEGFR2結(jié)合后可以激活多種信號(hào)通路,包括PI3K/Akt、Ras通路等,而Ras通路與人類(lèi)絕大多數(shù)腫瘤密切相關(guān),也是VEGF/VEGFR2激活后重要的信號(hào)途徑。Apatinib作用于VEGFR2后,可以抑制VEGF/VEGFR2的激活,從而抑制其激活Ras信號(hào)通路,發(fā)揮抗腫瘤效應(yīng)。研究目的:探討Apatinib能否抑制彌漫大B細(xì)胞淋巴瘤細(xì)胞增殖并誘導(dǎo)其凋亡,并進(jìn)一步研究其殺傷作用與Ras信號(hào)通路的關(guān)系,為開(kāi)拓彌漫大B細(xì)胞淋巴瘤治療新方法提供實(shí)驗(yàn)依據(jù)。研究方法:1、用 CCK-8 法檢測(cè)不同濃度的(2.5、5、10、20、40μmol/L)Apatinib 作用48h后對(duì)DLBCL細(xì)胞株的增殖抑制作用。2、用Annexin-V/PI雙染法流式細(xì)胞術(shù)檢測(cè)不同濃度的(0、10、20、30、40μmol/L)Apatinb處理48h后對(duì)DLBCL細(xì)胞株凋亡的影響。3、利用蛋白免疫印跡法檢測(cè)Apatinib作用oci-ly1和細(xì)胞SU-DHL-2 48h后pVEGFR2蛋白及Ras信號(hào)通路中Ras、c-Raf、pMEK1/2和pErK1/2的表達(dá)變化。4、統(tǒng)計(jì)學(xué)分析采用SPSS19.0軟件,由于均是小樣本數(shù)據(jù)的統(tǒng)計(jì),兩組獨(dú)立樣本均數(shù)的比較采用非參數(shù)檢驗(yàn)(Mann-WhitheyU檢驗(yàn));多組間均數(shù)比較采用多個(gè)獨(dú)立樣本的非參數(shù)檢驗(yàn)(Kruskal-WallisH檢驗(yàn))。計(jì)量資料用Mean±SD表示,所有的檢驗(yàn)分析均是雙側(cè)檢驗(yàn),檢驗(yàn)水準(zhǔn)為α=0.05。研究結(jié)果:1.CCK8 結(jié)果顯示:Apatinib 對(duì) GCB 型 DLBCL 細(xì)胞株 oci-ly1、SU-DHL-4(S4)及ABC細(xì)胞株oci-ly3、SU-DHL-2(S2)具有明顯的增殖抑制作用,并呈劑量依賴(lài)性。Apatinib 作用 oci-ly1、SU-DHL-4(S4)oci-ly3、SU-DHL-2(S2)細(xì)胞 48h 后,其 IC50 分別為(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,經(jīng)多個(gè)獨(dú)立樣本的非參數(shù)檢驗(yàn)Kruskal-Wallis H檢驗(yàn)分析結(jié)果表明與對(duì)照組比較差異均具有統(tǒng)計(jì)學(xué)意義(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)。經(jīng)兩個(gè)獨(dú)立樣本非參數(shù)檢驗(yàn)Mann-Whitney U分析,ABC型細(xì)胞株oci-ly3的增殖抑制率分別明顯高于兩種GCB型細(xì)胞株oci-ly1和SU-DHL-4(S4)(P=0.047和P=0.047);同樣,另一株ABC型細(xì)胞株SU-DHL-2的增殖抑制率分別高于兩種 GCB 型細(xì)胞株 oci-ly1 和 SU-DHL-4(S4)(P=0.047 和 P=0.047)。2.Annexin V/PI雙染法流式細(xì)胞術(shù)檢測(cè)Apatinib作用于GCB型DLBCL細(xì)胞株 oci-ly1、SU-DHL-4(S4)及 ABC 型細(xì)胞株 oci-ly3,SU-DHL-2(S2)48h 后的凋亡比例,發(fā)現(xiàn) Apatinib 能夠誘導(dǎo) oci-ly1、SU-DHL-4(S4)、oci-ly3、SU-DHL-2(S2)細(xì)胞凋亡,呈濃度依賴(lài)性,經(jīng)多個(gè)獨(dú)立樣本的非參數(shù)檢驗(yàn)Kruskal-WallisH檢驗(yàn)分析,與對(duì)照組比較差異均具有統(tǒng)計(jì)學(xué)意義(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)。兩種類(lèi)型DLBCL細(xì)胞株的凋亡率的比較無(wú)統(tǒng)計(jì)學(xué)意義,經(jīng)兩個(gè)獨(dú)立樣本非參數(shù)檢驗(yàn)Mann-Whitney U分析,ABC型細(xì)胞株oci-ly3的凋亡率與兩種GCB型細(xì)胞株oci-ly1和SU-DHL-4(S4)比較均無(wú)明顯差異(P=0.275和P=0.06);同樣,另一株ABC型細(xì)胞株SU-DHL-2的凋亡率與GCB型細(xì)胞株oci-ly1和SU-DHL-4(S4)比較均無(wú)統(tǒng)計(jì)學(xué)意義(P=0.275和P=0.06)。3.蛋白質(zhì)印跡法結(jié)果顯示Apatinib可通過(guò)抑制VEGFR2的自磷酸化即抑制pVEGFR2蛋白的表達(dá),進(jìn)而抑制Ras(Ras、Raf、pMEK1/2、pErk1/2)信號(hào)通路。對(duì)蛋白條帶進(jìn)行灰度分析,經(jīng)多個(gè)獨(dú)立樣本非參數(shù)檢驗(yàn)Kruskal-Wallis H檢驗(yàn),Apatinib 組(20、40μmnol/L)中 pVEGFR2、Ras、c-Raf、pMEK1/2,pErk1/2 蛋白表達(dá)水平與對(duì)照組(0μmol/L)相比,差異均具有統(tǒng)計(jì)學(xué)意義(均χ2=7.2,均P=0.027)結(jié)論:1.不同濃度的Apatinib能抑制彌漫大B細(xì)胞淋巴瘤細(xì)胞oci-ly1、SU-DHL-4(S4)、oci-ly3、SU-DHL-4(S2)的增殖,并誘導(dǎo)其凋亡。且 Apatinib 對(duì)于ABC型的DLBCL細(xì)胞株的增殖抑制率效果更明顯。2.Apatinib通過(guò)抑制VEGF與VEGFR2的結(jié)合,阻止VEGFR2發(fā)生自磷酸化成pVEGFR2而活化,從而抑制其激活的Ras信號(hào)通路,發(fā)揮殺傷彌漫大B細(xì)胞淋巴瘤細(xì)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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R733.1
本文編號(hào)：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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R733.1
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