【作者】 匡叶红；

【导师】 陈翔；

【作者基本信息】 中南大学 ， 皮肤病与性病学， 2009， 博士

【摘要】 目的肿瘤的侵袭转移和多药耐药(MDR)是肿瘤治疗过程中的两大难题,亟待解决。临床工作中常见的化疗后肿瘤的侵袭转移加快以及转移性肿瘤更容易发生MDR等现象提示肿瘤的浸润转移和MDR之间可能存在共同的分子机制。CD147是一种属于免疫球蛋白超家族的细胞膜表面高糖基化蛋白,在许多恶性肿瘤中表达增高。CD147可通过诱导肿瘤细胞周围基质成纤维细胞及肿瘤细胞本身产生多种基质金属蛋白酶(MMPs),在肿瘤的浸润和转移中发挥重要作用。同时有研究发现,CD147通过刺激透明质酸(HA)的产生进而影响细胞凋亡,调控多药耐药基因MDR1的表达参与肿瘤多药耐药,但具体分子机制尚不清楚。本研究首先检测CD147在人口腔上皮鳞癌细胞(SCC)KB及其长春新碱诱导的耐药细胞KB/V的表达情况,再通过小干扰RNA技术(siRNA)抑制KB/V细胞的CD147表达,建立稳定转染的细胞株(KB/VsiCD147)。随后观察KB,KB/V和KB/VsiCD147细胞对于临床多种化疗药物的敏感性,与此同时采用蛋白质组学技术,分离并鉴定KB,KB/V和KB/VsiCD147细胞差异表达的蛋白质,并对部分差异表达蛋白质的表达水平进行了验证和功能分析。旨在发现新的受CD147调控的耐药相关蛋白,为阐明SCC多药耐药发生及机理提供新线索。另一方面,为明确CD147对于耐药肿瘤凋亡途径的调控作用,我们探讨X-连锁凋亡抑制蛋白(XIAP)和多种MDR相关分子(MDR1,MRP1,LRP)在KB及KB/V的表达情况,以KB,KB/V和先前构建的稳定转染细胞株KB/VsiCD147为研究对象,探讨各组细胞凋亡情况,XIAP表达和CD147的相关性,并分析各组细胞对于临床常用抗凋亡药物5-Fu的敏感性,旨在揭示CD147对于耐药肿瘤细胞凋亡途径的影响作用及内在机制,为逆转肿瘤耐药提供创新性的理论依据。方法1.RT-PCR,Western Blot和流式细胞仪法检测KB,KB/V细胞中CD147的表达,分析CD147表达和KB/V细胞耐药的相关性。2.运用siRNA干扰技术抑制CD147的表达,鉴定转染有效性,建立稳定转染细胞株(KB/VsiCD147)。3.运用MTT办法观察KB,KB/V和KB/VsiCD147细胞对于临床化疗药物(长春新碱:VCR,全反式维甲酸:ATRA,紫杉醇:taxol,5-氟尿嘧啶:5-Fu)的敏感性。4.应用双向凝胶电泳技术对KB,KB/V和KB/VsiCD147三种细胞的总蛋白进行双向凝胶电泳,PDQuest软件对双向电泳图谱进行比较分析找出差异表达的蛋白质,采用MALDI-TOF-MS质谱技术对差异表达的蛋白质进行肽质量指纹图谱(PMF)测定,数据库搜索并鉴定全部差异表达的蛋白质。5.RT-PCR和western Blot技术验证部分差异蛋白质在各组细胞的表达情况,并进行初步的功能研究。6.RT-PCR法检测KB和KB/V细胞中MDR1,MRP1,LRP和XIAP的mRNA水平,分析各分子与KB/V细胞耐药的相关性。7.RT-PCR和western B10t法研究KB,KB/V和KB/VsiCD147细胞XIAP的表达。8.运用流式细胞仪及电镜技术研究KB,KB/V和KB/VsiCD147细胞凋亡率及形态改变。9.采用MTT方法研究KB,KB/V和KB/VsiCD147细胞对于临床诱导凋亡相关药物5-Fu的敏感性。结果1.与KB细胞比较,KB/V细胞中CD147表达增高,半定量分析约为2.41倍(P<0.05),有显著差异性。2.成功建立pSUPER/CD147siRNA稳定转染的细胞克隆(KB/VsiCD147-C1a,C1b,C2),抑制率分别为50.63%,80.54%和86.50%(P<0.01),选择抑制效果较好的细胞克隆C1b和C2用于开展后续的研究工作。3.与KB细胞相比较,KB/V细胞对临床化疗药物长春新碱(VCR),全反式维甲酸(ATRA),紫杉醇(Taxol),5-氟尿嘧啶(5-Fu)均显示不同程度的耐药性(耐药指数分别为109.43,11.59,10.41和96.87),KB/VsiCD147-C2细胞对上述药物敏感性增强(耐药指数分别变为25.02,3.64,5.46和16.51,P<0.05)。4.建立了重复性和分辨率均较好的KB,KB/V和KB/VsiCD147-C2三组细胞总蛋白质的双向凝胶电泳图谱。通过分析肽质量指纹图谱(PMF)结合数据库搜索,鉴定了全部21个差异表达的蛋白质。5.差异表达的蛋白质GRP75在KB,KB/V和KB/VsiCD147-C1b,C2中的表达水平与比较蛋白质组学研究的结果一致。差异表达的蛋白质CyPA在各组细胞的蛋白表达水平与比较蛋白质组学研究的结果一致,而其mRNA水平在各组细胞无显著性(P>0.05)。CyPA功能抑制剂环孢素A(CsA)可部分逆转KB/V细胞对VCR和5-Fu的耐药性(P<0.05)。6.耐药相关基因MDR1,MRP1,LRP mRNA水平在KB和KB/V细胞株的表达无显著性差异(P>0.05),XIAP mRNA水平在KB/V细胞的表达为KB细胞的3.33倍(P<0.05),有显著差异性。7.KB和KB/VsiCD147-C1b,C2细胞株的XIAP蛋白表达水平分别为KB/V细胞株的39.44%,36.89%和37.59%(P<0.01),提示XIAP分子的表达受到CD147的调控。8.KB和KB/VsiCD147-C1b,C2细胞株的凋亡率分别为3.48%,5.48%和12.24%,显著高于对照组KB/V细胞(0.07%,P<0.01),提示KB/V细胞的凋亡受到CD147分子的调控。9.KB/VsiCD147-C1b,C2细胞形态出现环状、凝块状的核染色质或星月状小体(凋亡小体)等一系列凋亡细胞特征。结论1.CD147与人口腔上皮鳞癌细胞的多药耐药相关,可能是通过上调GRP75,CyPA等耐药相关分子起作用,CD147是一种新的逆转肿瘤耐药的靶分子;2.CD147通过影响XIAP分子的表达进而抑制人口腔上皮鳞癌耐药细胞的凋亡,影响药物敏感性,CD147是一种新的调控多药耐药肿瘤细胞凋亡途径的靶分子。更多还原

【Abstract】 BackgroundMulti-drug resistance(MDR) is a major obstacle to the effective treatment of malignant tumors and its complex mechanisms remain to be elucidated.Most tumors with high metastatic potential exhibit the MDR phenotype and vice versa.A functional linkage between drug resistance and metastasis has been proposed in tumor cells.Studies reported that switched-on genes in metastatic cancer cells affected their sensitivity to chemotherapeutic drugs.There is growing evidence that the human cell-surface molecule CD147,an integral plasma membrane glycoprotein belonging to the Ig superfamily,participates in tumor metastasis and MDR.CD147 is highly expressed on the surface of various tumor cells.It stimulates the production of multiple matrix metalloproteinases(MMPs) by tumor cells and fibroblasts,serving as a key regulator of tumor cell invasiveness and metastasis.In addition,studies have discovered that CD147 expression was upregulated in MDR cancer cells and CD147 stimulated the production of hyaluronan(HA) in mammary carcinoma cells and induced MDR in a hyaluronan-dependent manner.Thus CD147 represents a potent target for both tumor metastatic behavior and drug resistance.Methods and ResultsWe studied the expression of CD147 in sensitive human oral squamous carcinoma(SCC) KB and MDR derivative KB/V cells. Reverse transcription-PCR,western blotting and flow cytometric analysis revealed that KB/V cells expressed CD147 at significantly higher levels than their parental KB cells(2.41-fold).Using stable RNA interference, we succeeded in establishing three CD147 knock-down KB/V cell lines (KB/VsiCD147-Cla,Clb,and C2).Compared to the control,CD147 expression was downregulated by 50.63%,80.54%,and 86.50%in KB/VsiCD147-Cla,Clb,and C2,respectively.MTT colorimetric assay showed an increase in the chemosensitivity to vincristine(VCR),all transretinoic acid(ATRA),taxol,and 5-fluorouracil(5-Fu) of KB/VsiCD147-C2 cells.We performed a comparative proteomics study of KB,KB/V,and KB/VsiCD147-C2 cells.The 2-DE pattern of each cell line was highly reproducible and well-resolved,and approximately 1000 protein spots were visualized.Using image analysis,we compared the 2-D PAGE patterns of paired KB/V and KB cells or KB/V and KB/VsiCD147-C2 cells,the average number of differential protein spots that showed more than 2-fold changes in expression were 93±5.84,98±7.91,respectively.We converged those spots detected in both paired cells and found that 21 proteins were differentially expressed.In KB/V cells, 15 spots were specifically upregulated while 6 spots were downregulated as compared to KB and KB/VsiCD147-C2 cells.We performed automated MALDI-TOF-MS and database searches on the 21 differentially expressed protein spots.All 21 proteins were identified on MALDI-TOF-MS maps and by database query.The enhanced expression of representative active proteins,GRP75 and CyPA,were confirmed by western blotting and RT-PCR analysis,the protein expression were in accordance with previous proteomics assessment,while no changes in CyPA mRNA were detectable.In addition,pretreatment of KB/V cells with a CyPA-binding immunosuppressive drug,cyclosporine A(CsA), enhanced the chemosensitivity to VCR and 5oFu of KB/V cells.We also aimed to reveal the anti-apoptotic effect of CD147 on the MDR phenotype of KB/V cells and its possible pathways.RT-PCR analysis revealed that KB/V cells expressed equal levels of MDR related genes MDR1,MRP1,and LRP,whereas significantly higher leve of X-linked inhibitor of apoptosis(XIAP) than its sensitive counterpart KB cells. Western blotting analysis showed that XIAP expression level in KB was 39.44%of that in KB/V cells.Down-regulation of CD147 by transfection with CD147 siRNA resulted in decreased XIAP expression by 36.89% and 37.59%in KB/VsiCD147-C1b and C2 cell lines.We analyzed apoptosis in KB,KB/V and KB/VsiCD147-C1b and C2 cells by flow cytometry.The average%apoptosis in KB,KB/VsiCD147-C1b and C2 was 3.48%,5.48%,and 12.24%,respectively,significantly higher than those of KB/V(0.07%).Electron microscopic observation comfirmed the morphology changes of apoptosis related to CD147 expression. Additionally,chemo-sensitivity to 5-Fu of KB/V was increased by CD147 silencing as measured by MTT colorimetric assay.ConclusionWe propose that CD147 is at least partly involved in the MDR of KB/V cells by upregulating GRP75 and CyPA,CD147 may represent a therapeutic target to overcome MDR by oral SCC.Our results also suggest that inhibition of CD147 and subsequent XIAP depletion may have an anti-tumor effect through enhancing the susceptibility of cancer cells to apoptosis.更多还原