【作者】 陈公琰；

【导师】 郝希山；

【作者基本信息】 天津医科大学 ， 肿瘤学， 2006， 博士

【摘要】 目的：建立人肺腺癌耐药细胞模型Anip973／NVB，检测其生物学特性并从基因和蛋白质水平上探讨其诺维本的耐药机制。方法：本研究应用人肺腺癌细胞系Anip973，采用诺维本逐步增加剂量法，诱导建立人肺腺癌耐药细胞模型Anip973／NVB，检测其耐药细胞系和亲代细胞系的生物学特性；用基因芯片技术检测两细胞系间基因表达的差异；通过RT-PCR法验证部分基因芯片结果及检测传统耐药相关基因的表达；用2-DE技术初步分析其耐药细胞系与亲代细胞系间蛋白质表达的差异，探讨其诺维本的耐药机制。结果：1、本研究经过24个月建立了人肺腺癌耐药细胞系Anip973／NVB。2、通过Anip973／NVB生物学特性分析发现：（1）光镜及电子显微镜观察，Anip973／NVB细胞与Anip973细胞比较细胞形态不规则，呈长伪足的多角形及体积增大；线粒体数量增多，粗面内质网脱颗粒．（2）与亲代细胞相比Anip973／NVB细胞生长曲线的倍增时间无明显差异；（3）细胞周期分析发现：耐药细胞系Anip973／NVB的G₀-G₁期细胞明显增多，为62．90％，S期细胞明显减少，为28．32％，而亲代细胞系Anip973的上述各周期的百分比分别为53．73％、38．25％，两种细胞系的G₂-M期细胞无明显变化；（4）经MTT法测定Anip973／NVB细胞比较亲代细胞的NVB半数致死浓度(IC₅₀)增大至21．81倍，该耐药细胞系同时对顺铂、紫杉醇、异环磷酰胺、表阿霉素、氟尿嘧啶、达卡巴嗪、足叶乙甙等药物也产生了交叉耐药，耐药倍数分别为11．89、11．68、6．12、3．95、2．86、2．64、2．15，而对伊立替康、吉西他滨相对敏感，耐药倍数分别为1．38、1．27(均P＜0．05=（5）高效液相色谱法测定发现，耐药细胞中的药物浓度明显低于亲代细胞。3、基因芯片结果表明，在检测的21571对基因中，Anip973／NVB和Anip973细胞均有差异表达的基因143对，其中在Anip973／NVB细胞中表达上调的55对，下调的88对。在这143对基因中，已知功能的基因有77条，明确与肿瘤相关的基因21对，在这些基因中ANXA1、ABCC3、TIMP-3和部分细胞粘连相关基因的表达发生了变化，这可能是导致诺维本耐药的主要机制；4、通过RT-PCR检测经典的耐药相关基因，结果显示：MRP3及Bcl-2基因在耐药细胞系中表达增高，而未发现MDR、MRP1、ABCG2、GSTp1基因在这两种细胞系中的差异表达。5、通过双向凝胶电泳（2-DE）技术，筛选两种细胞系表达差异的蛋白，获得了分辨率较高，重复性较好的2-DE图谱。在亲代细胞系中检测到的平均蛋白质点为806个，平均匹配率为77％；在耐药细胞系中检测到的平均蛋白质点为711个，平均匹配率为78．33％。筛选出在两种细胞系中表达差异的蛋白质37个，其中在耐药细胞中上调的蛋白16个，下调的蛋白21个。查询数据库初步鉴定了17个蛋白。结论：1、初步证明了Anip973／NVB细胞系是一个明确的多药耐药细胞模型，具有耐药细胞的基本生物学特性。2、基因芯片结果表明，Anip973／NVB的耐药机制可能是ANXA1、ABCC3基因表达增强导致细胞内药物浓度的降低；细胞浸润转移相关基因表达的下调，使肿瘤细胞分化趋向成熟，逃避了化疗药物的攻击；3、RT-PCR证明ABCC3（MRP3）在Anip973／NVB细胞系中表达增高，与本实验基因芯片结果符合，表明MRP3的过表达可能是Anip973／NVB细胞系耐药的主要机制之一；Bcl-2的过表达也参与了该细胞系的耐药；4、两种细胞系部分差异蛋白鉴定结果表明，趋化素样因子超家族2、NME2蛋白、细胞色素P450、金属基质蛋白酶抑制剂-4、层粘连蛋白受体、钙结合蛋白等蛋白质在两种细胞系中表达存在差异，说明诺维本在对人肺腺癌细胞的分化增殖等方面产生了影响，并提示这些蛋白质可能参与了Anip973／NVB细胞系的耐药。更多还原

【Abstract】 Objective To establish a human lung adenocarcinoma multidrug-resistant cell lineAnip973/NVB and to study its biologic characteristics and mechanism of drugresistance.Methods The human lung adenocarcinoma multidrug-resistant cell line（A973/NVB） was established by inducing human lung adenocarcinoma cell lineAnip973 with step-wise increasing concentrations of NVB,and the biologiccharacteristics were observed.cDNA microarray technique was used to detect genedifference of the two cell lines.The results of microarray were validated and theclassics drug resistance related genes were detected by RT-PCR.2-DE techniquewas used to analyze the difference of proteins of the two cell lines.Results 1.The human lung adenocarcinoma multidrug-resistant cell lineAnip973/NVB was established successfully for 24 months.2.Its biologiccharacteristics were:（1） The cell morphous of A973/NVB cells were more irregularthan A973 cells,and there was significant difference in ultramicrostructure whileobserved by the light microscope and electron microscope.（2） The rate of cellproliferation of Anip973/NVB was no significantly different with that of Anip973cell line.（3） Cell cycle distribution of Anip973/NVB had changed compared withparental cells from flow cytometry.The percentage of cells in G₀-G₁ phase increasedto 62.90%,while the percentage of cells in S phase decreased to 28.32%.Thepercentage of Anip973 in G₀-G₁ and S phase were 53.73 %,38.25 % respectively,and there was no changes in G₂-M phase of the two cell lines.（4） The IC₅₀ of NVB inAnip973/NVB cells was 21.18 times than that of Anip973 cells by MTT.In addition, the Anip973/NVB showed various cross-resistance to CDDP,isofosfamide,pharmorubicin,5-fluorouracil,dacarbazine and etoposide,and the resistance indexeswere 11.89,11.68,6.12,3.95,2.86,2.64 and 2.15,respectively.The ceils remainedsensitive to irinotecan and gemcitabine and the sensitive resistance indexes were 1.38and 1.27 （p＜0.05）.（5）Medicine concentration in drug resistance cells was obviouslylower than that in parental cells by high efficiency liquid chromatography.3.The resultsof gene arrays showed that 143 genes of 21571 genes were differentially expressedbetween Anip973 and Anip973/NVB cell lines,of them,55 genes showed＞2 timesup-regulation,88 genes showed＜0.5 times down-regulation and the function ofseventy-seven genes had been identified.21 genes among identified genes had beenidentified function related to cancer,and,of 21 genes,the changes of ANXA1,ABCC3,TIMP-3 and cytoadherence related genes might be the major mechanism ofdrug resistance of NVB.4.The results by RT-PCR showed MRP3 and Bcl-2 wereoverexpressed in Anip973/NVB,and there were no significant expression of MDR,MRP1,ABCG2,GSTπ1 in two cell lines.5.Well-resolved,reproducible 2-DEpatterns of control and NVB-treated Anip973 were obtained.In Anip973/NVB group,average spots of 3 gels were 806 with an average matching rate of 77%,in Anip973group,average spots of 3 gels were 711 with an average matching rate of 78.33%.37significantly expressed protein spots were detected,among which,16 wereup-regulated and 21 were down-regulated in the drug-resistance group.17 spotswere identified initially with the data bank.Conclusions 1.A973/NVB is a reliable multi-drug resistant cell line of humanlung adenocarcinoma which has the basic biologic characteristics.2.Up regulationof ANXA1 and ABCC3 result in depression of intra-cellular drug concentration andchange of intra-cellular drug distribution,which may be the major mechanism ofdrug resistance of NVB.Down regulation of cell infiltration and metastasis relatedgenes result in better of cell differentiation and elusion of attack fromchemotherapeutics.3.Overexpression of ABCC3（MRP3） may be one of the major mechanism of MDR,that is consistent with the results of gene arrays.Overexpression of Bcl-2 inhibits apoptosis of drug resistance cells which is one ofthe drug resistance cause.4.Difference of chemokine-like factor superfamily 2,NME2 protein,Cytochrome P450,TIMP-4,laminin receptor,Calumenin,and so on,in the two cells shows NVB affects lung adenocarcinoma cell differentiation andproliferation,and the proteins may play a role in NVB resistance.更多还原