人弥漫大B细胞淋巴瘤耐阿霉素和耐利妥昔单抗细胞株的构建及其耐药特性分析
发布时间：2018-08-13 12:32
【摘要】：目的:分别建立人弥漫大B细胞淋巴瘤(diffuse large B-cell lymphoma,DLBCL)阿霉素(Adriamycin,ADM)及利妥昔单抗(Rituximab,RTX)耐药细胞株,检测耐药株形态,增殖,药物敏感性的变化及多药耐药基因1的(multidrug resistance gene 1,MDR1)表达情况,探讨化疗药物与RTX之间是否存在交叉耐药现象。为DLBCL耐药研究奠定模型基础,对DLBCL继发耐药后的临床用药提供理论依据。方法:1.以体外低浓度梯度递增法诱导构建人耐阿霉素DLBCL细胞株Pfeiffer/ADM和耐RTX DLBCL细胞株Pfeiffer/R。2.显微镜下比较亲代及耐药细胞株的形态变化。3.运用细胞计数法,绘制亲代和耐药细胞株的生长曲线,计算倍增时间。4.阿霉素、依托泊苷(etoposide,VP-16)、长春新碱(vincristine,VCR)对Pfeiffer、Pfeiffer/ADM、Pfeiffer/R作用72h后,运用CCK-8法检测细胞增殖抑制情况,计算半数抑制浓度IC50。5.以健康人血清作为补体来源,1μg/mL,3μg/mL,10μg/mL,30μg/mL的RTX作用于Pfeiffer、Pfeiffer/ADM、Pfeiffer/R细胞株6h后,运用CCK-8法比较耐药细胞株及亲代细胞对RTX介导的补体依赖性细胞毒性(complement-dependent cytotoxicity,CDC)的差别。6.实时荧光定量PCR检测Pfeiffer、Pfeiffer/ADM、Pfeiffer/R细胞株中MDR1mRNA的表达情况。结果:1.成功构建耐ADM细胞株Pfeiffer/ADM及耐RTX细胞株Pfeiffer/R。2.显微镜下,Pfeiffer、Pfeiffer/ADM、Pfeiffer/R在细胞形态上无明显差别。3.Pfeiffer,Pfeiffer/ADM,Pfeiffer/R的倍增时间分别为(32.28±1.18)h,(31.41±2.03)h及(32.03±1.77)h,三细胞株体外增殖速度相似,差异无统计学意义(p0.05)。4.通过cck-8法测得adm,vp16,vcr对pfeiffer的ic50值分别为(0.038±0.0090)μg/ml,(0.26±0.045)μg/ml,(0.0093±0.0023)μg/ml,对pfeiffer/adm的ic50值分别为(0.48±0.049)μg/ml,(3.42±0.45)μg/ml,(0.16±0.0032)μg/ml,计算耐药指数分别为12.87,13.18,16.85,表明pfeiffer/adm具有多药耐药性。而adm,vp16,vcr对pfeiffer/r的ic50值分别为(0.038±0.046)μg/ml,(0.25±0.13)μg/ml,(0.0098±0.00014)μg/ml,与pfeiffer比较差异无统计学意义(p0.05),提示pfeiffer/r对化疗药物adm、vp-16和vcr依旧敏感。5.以健康人血清作为补体来源,1,3,10,30μg/ml的rtx作用于pfeiffer、pfeiffer/adm、pfeiffer/r细胞株6h后,对pfeiffer细胞的抑制率分别为28.58%,47.06%,69.00%,77.20%,对pfeiffer/adm为30.33%,47.21%,64.63%,72.53%,与pfeiffer细胞比较,差异无统计学意义(p0.05),提示pfeiffer/adm对rtx依旧敏感。pfeiffer/r细胞的抑制率分别为11.04%,24.12%,40.56%,53.58%,与pfeiffer细胞比较,差异具有统计学意义(p0.05),提示pfeiffer/r对rtx产生耐药。6.qrt-pcr检测发现,pfeiffer和pfeiffer/adm细胞的mdr1mrna的相对表达量分别为(1.00±0.10)和(82033.27±4187.24),差异具有统计学意义(p0.001)。然而pfeiffer与pfeiffer/r细胞的mdr1mrna的相对表达量分别为(1.03±0.33)和(3.00±1.32),两者差异无统计学意义(p0.05)。结论:运用低浓度梯度递增法可成功构建耐阿霉素人dlbcl细胞株pfeiffer/adm和耐rtx人dlbcl细胞株pfeiffer/r。pfeiffer/adm具有多药耐药性,其mdr1基因表达明显上调,对rtx介导的cdc效应仍敏感;pfeiffer/r对rtx介导的cdc效应产生耐药,但对化疗药物vp-16、vcr、adm仍敏感,其mdr1基因表达无明显变化。提示化疗药物vp-16、vcr、adm与rtx之间无交叉耐药。本研究所构建的耐药细胞株是进行下一步耐药机制或逆转耐药研究的理想模型。
[Abstract]:OBJECTIVE: To establish human diffuse large B-cell lymphoma (DLBCL) adriamycin (ADM) and rituximab (RTX) resistant cell lines, detect the morphology, proliferation, changes of drug sensitivity and expression of multidrug resistance gene 1 (MDR1), and explore the chemotherapeutic drugs. Whether there is cross-resistance between substances and RTX, which lays a foundation for the study of drug resistance of DLBCL and provides a theoretical basis for clinical drug use after secondary drug resistance of DLBCL. Methods: 1. To induce the construction of human doxorubicin-resistant DLBCL cell line Pfeiffer/ADM and RTX-resistant DLBCL cell line Pfeiffer/R.2 in vitro by low concentration gradient incremental method. The growth curves of parental and drug-resistant cell lines were drawn by cell counting method, and the doubling time was calculated. 4. The inhibition of cell proliferation was detected by CCK-8 method after 72 hours of action of adriamycin, etoposide (VP-16), vincristine (VCR) on Pfeiffer, Pfeiffer/ADM and Pfeiffer/R. The inhibitory concentration IC50.5. The complement-dependent cytotoxicity (CDC) of the drug-resistant cell lines and their parental cells mediated by RTX was compared by CCK-8 assay after 6 hours of treatment of Pfeiffer, Pfeiffer/ADM, Pfeiffer/R cell lines with healthy human serum as complement source, 1 ug/mL, 3 ug/mL, 10 ug/mL, 30 ug/mL RTX. The expression of MDR1 mRNA in Pfeiffer, Pfeiffer/ADM and Pfeiffer/R cell lines was detected by fluorescence quantitative PCR. Results: 1. ADM-resistant cell lines Pfeiffer/ADM and RTX-resistant cell lines Pfeiffer/R.2 were successfully constructed. Under microscope, Pfeiffer, Pfeiffer/ADM, Pfeiffer/R had no significant difference in cell morphology. The proliferation rates of the three cell lines in vitro were similar, but there was no significant difference (p0.05). 4. the IC50 values of adm, VP16 and VCR to Pfeiffer were (0.038 + 0.0090) UG / ml, (0.26 + 0.045) UG / ml, (0.0093 + 0.0023) UG / ml, and (0.04 + 0.04) icfeiffer / ADM by cck-8, respectively. 9) UG / ml, (3.42 (+ 0.45) UG / ml, (0.16 (+ 0.0032) UG / ml, and the calculated drug resistance indices were 12.87, 13.18, 16.85, respectively, indicating that Pfeiffer / ADM had multidrug resistance, while the IC50 values of adm, vp16, VCR for Pfeiffer / r were (0.038 (+ 0.046) UG / ml, (0.25 (+ 0.13) UG / ml, (0.0098 (+ 0.00014) UG / ml, respectively, which had no significant difference with Pfeiffer (p0.05). Pfeiffer / R was still sensitive to adm, VP-16 and vcr. 5. the inhibition rates of Pfeiffer / R cells were 28.58%, 47.06%, 69.00%, 77.20%, 30.21%, 64.63% and 72.53% respectively, after 6 hours of Pfeiffer / adm, Pfeiffer / ADM and Pfeiffer / R cells were treated with 1,3,10,30 UG / ml RTX from healthy human serum as a complement source. The inhibition rates of Pfeiffer / R cells were 11.04%, 24.12%, 40.56%, 53.58% respectively. the difference was statistically significant compared with Pfeiffer cells (p0.05), suggesting that Pfeiffer / R had resistance to rtx. 6.qrt-pcr detection showed that Pfeiffer and Pfeiffer / ADM cells were still sensitive to MDR1 mrn. The relative expression of a was (1.00 0.10) and (82033.27 4187.24), respectively. however, the relative expression of MDR1 mRNA in Pfeiffer and Pfeiffer / R cells was (1.03 0.33) and (3.00 1.32), respectively. there was no significant difference between them (p0.05). conclusion: the low concentration gradient method can be successfully used to construct a drug-resistant strain. Human DLBCL cell lines pfeiffer/adm and rtx-resistant human DLBCL cell lines pfeiffer/r.pfeiffer/adm have multidrug resistance, and their MDR1 gene expression is significantly up-regulated, and they are still sensitive to rtx-mediated CDC effect; pfeiffer/r is resistant to rtx-mediated CDC effect, but still sensitive to chemotherapy drugs vp-16, VCR and adm, and their MDR1 gene expression is not significantly changed. There is no cross-resistance between chemotherapy drugs vp-16, vcr, ADM and rtx. The drug-resistant cell lines constructed in this study are ideal models for further study of drug resistance mechanism or reversal of drug resistance.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.1
本文编号：2181003
[Abstract]:OBJECTIVE: To establish human diffuse large B-cell lymphoma (DLBCL) adriamycin (ADM) and rituximab (RTX) resistant cell lines, detect the morphology, proliferation, changes of drug sensitivity and expression of multidrug resistance gene 1 (MDR1), and explore the chemotherapeutic drugs. Whether there is cross-resistance between substances and RTX, which lays a foundation for the study of drug resistance of DLBCL and provides a theoretical basis for clinical drug use after secondary drug resistance of DLBCL. Methods: 1. To induce the construction of human doxorubicin-resistant DLBCL cell line Pfeiffer/ADM and RTX-resistant DLBCL cell line Pfeiffer/R.2 in vitro by low concentration gradient incremental method. The growth curves of parental and drug-resistant cell lines were drawn by cell counting method, and the doubling time was calculated. 4. The inhibition of cell proliferation was detected by CCK-8 method after 72 hours of action of adriamycin, etoposide (VP-16), vincristine (VCR) on Pfeiffer, Pfeiffer/ADM and Pfeiffer/R. The inhibitory concentration IC50.5. The complement-dependent cytotoxicity (CDC) of the drug-resistant cell lines and their parental cells mediated by RTX was compared by CCK-8 assay after 6 hours of treatment of Pfeiffer, Pfeiffer/ADM, Pfeiffer/R cell lines with healthy human serum as complement source, 1 ug/mL, 3 ug/mL, 10 ug/mL, 30 ug/mL RTX. The expression of MDR1 mRNA in Pfeiffer, Pfeiffer/ADM and Pfeiffer/R cell lines was detected by fluorescence quantitative PCR. Results: 1. ADM-resistant cell lines Pfeiffer/ADM and RTX-resistant cell lines Pfeiffer/R.2 were successfully constructed. Under microscope, Pfeiffer, Pfeiffer/ADM, Pfeiffer/R had no significant difference in cell morphology. The proliferation rates of the three cell lines in vitro were similar, but there was no significant difference (p0.05). 4. the IC50 values of adm, VP16 and VCR to Pfeiffer were (0.038 + 0.0090) UG / ml, (0.26 + 0.045) UG / ml, (0.0093 + 0.0023) UG / ml, and (0.04 + 0.04) icfeiffer / ADM by cck-8, respectively. 9) UG / ml, (3.42 (+ 0.45) UG / ml, (0.16 (+ 0.0032) UG / ml, and the calculated drug resistance indices were 12.87, 13.18, 16.85, respectively, indicating that Pfeiffer / ADM had multidrug resistance, while the IC50 values of adm, vp16, VCR for Pfeiffer / r were (0.038 (+ 0.046) UG / ml, (0.25 (+ 0.13) UG / ml, (0.0098 (+ 0.00014) UG / ml, respectively, which had no significant difference with Pfeiffer (p0.05). Pfeiffer / R was still sensitive to adm, VP-16 and vcr. 5. the inhibition rates of Pfeiffer / R cells were 28.58%, 47.06%, 69.00%, 77.20%, 30.21%, 64.63% and 72.53% respectively, after 6 hours of Pfeiffer / adm, Pfeiffer / ADM and Pfeiffer / R cells were treated with 1,3,10,30 UG / ml RTX from healthy human serum as a complement source. The inhibition rates of Pfeiffer / R cells were 11.04%, 24.12%, 40.56%, 53.58% respectively. the difference was statistically significant compared with Pfeiffer cells (p0.05), suggesting that Pfeiffer / R had resistance to rtx. 6.qrt-pcr detection showed that Pfeiffer and Pfeiffer / ADM cells were still sensitive to MDR1 mrn. The relative expression of a was (1.00 0.10) and (82033.27 4187.24), respectively. however, the relative expression of MDR1 mRNA in Pfeiffer and Pfeiffer / R cells was (1.03 0.33) and (3.00 1.32), respectively. there was no significant difference between them (p0.05). conclusion: the low concentration gradient method can be successfully used to construct a drug-resistant strain. Human DLBCL cell lines pfeiffer/adm and rtx-resistant human DLBCL cell lines pfeiffer/r.pfeiffer/adm have multidrug resistance, and their MDR1 gene expression is significantly up-regulated, and they are still sensitive to rtx-mediated CDC effect; pfeiffer/r is resistant to rtx-mediated CDC effect, but still sensitive to chemotherapy drugs vp-16, VCR and adm, and their MDR1 gene expression is not significantly changed. There is no cross-resistance between chemotherapy drugs vp-16, vcr, ADM and rtx. The drug-resistant cell lines constructed in this study are ideal models for further study of drug resistance mechanism or reversal of drug resistance.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.1
【参考文献】
相关期刊论文 前10条
1 盛佳钰;时百玲;陈红风;;三阴性乳腺癌MDA-MB-231顺铂耐药细胞株的建立及鉴定[J];肿瘤防治研究;2016年03期
2 潘梅芳;岑洪;谭晓虹;王明月;;应用基因表达谱芯片技术研究Rituximab的耐药机制[J];中国癌症防治杂志;2015年03期
3 王文廉;万里新;屈中玉;;利妥昔单抗耐药淋巴瘤细胞株构建及其表型分析[J];中华肿瘤防治杂志;2015年03期
4 向定朝;王存祖;陆晓峰;欧阳琦;;大剂量冲击法建立胶质瘤耐替莫唑胺细胞株及生物学鉴定[J];江苏大学学报(医学版);2013年01期
5 李小秋;李甘地;高子芬;周小鸽;朱雄增;;中国淋巴瘤亚型分布:国内多中心性病例10002例分析[J];诊断学理论与实践;2012年02期
6 王金华;赵万洲;陈小祥;赵一兵;曲军卫;彭素蓉;;人卵巢癌紫杉醇耐药细胞株OV1228/Taxol的建立[J];中华肿瘤防治杂志;2010年23期
7 张红雨;陈红涛;管忠震;黄岩;张星;林桐榆;;抗CD20单克隆抗体诱导B淋巴瘤细胞株凋亡的实验研究[J];中国实验血液学杂志;2009年04期
8 刘银星,范冬梅,熊冬生,许元富,邵晓枫,许元生,彭晖,杨铭,秦岚,朱祯平,杨纯正;单价抗CD20抗体诱导人B细胞淋巴瘤Raji细胞的凋亡[J];癌症;2003年12期
9 龚福生,汪相如,陈蓉明,谢云青,郑秋红;RT-PCR法定量检测恶性淋巴瘤多药耐药基因的表达[J];中国肿瘤;2003年01期
10 李志革,匡志鹏,于起涛,曾爱屏,韦劲松,宋向群;多药耐药基因及P-糖蛋白在恶性淋巴瘤组织中的表达[J];右江民族医学院学报;2002年06期
相关硕士学位论文 前1条
1 黄谷瑜;Ibrutinib联合Rituximab靶向治疗套细胞淋巴瘤的实验研究[D];广西医科大学;2015年
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