视网膜母细胞瘤多药耐药相关蛋白的研究

目的 检测P-糖蛋白(P-Gp)、多药耐药基因相关蛋白(MRP)及肺耐药相关蛋白(LRP)在视网膜母细胞瘤(Rb)中的表达及临床意义;初步分析Rb患者临床病理指标与MRP间的关系;探讨Rb多药耐药现象的可能机制.方法 实验研究.应用免疫组织化学染色方法检测P-gp、MRP、LRP在75例Rb肿瘤标本中的表达情况.分析3种蛋白表达的相关性及其与患者年龄、性别、眼别、临床表现、组织病理分化程度等临床病理指标的相关性.各蛋白表达情况与一般临床特点、组织病理学特征的比较采用卡方检验,各蛋白间的相关性采用多元相关分析.结果 P-Gp、LRP、MRP蛋自在Rb中阳性表达率分别为64.0%、25.3%、36.0%.P-gp与LRP、P-gp与MRP、LRP与MRP的共表达阳性率分别为18.7%、32.0%、20.0%.P-gp、LRP、MRP在分化型Rb组织中的阳性表达率均高于杀分化型,且组间差异具有统计学意义(χ2=8.002,χ2=17.327,χ2=28.421;P<0.05).3种蛋白的表达均与年龄、性别、眼别无关(χ2=0.003～3.385,P>0.05).P-gp、LRP的表达分别与MRP的表达其有相关性(r=0.389,r=0.521;P<0.05).结论 Rb原发性多药耐药的形成是一个多因素、多步弱的复杂过程,与包括P-gp、LRP、MRP等在内的多项因素的参与有关.P-gp、LRP、MRP蛋白可以作为反映Rb耐药的分子基础,耐药相关标志的联合检测更有利于准确判断Rb的多药耐药状态,为Rb化学治疗提供科学的理论依据.     

HXO-Rb44视网膜母细胞瘤株多药耐药现象的基因研究

目的 研究视网膜母细胞瘤（retinoblastoma,Rb)细胞系HXO－Rb44细胞多药耐药基因（multidrug resistance gene,MDR1)和多药耐药相关蛋白（multidrug associated protein,MRP）基因的表达,探讨Rb多药耐药现象的发生机制。方法 用逆转聚合酶链反应检测HXO－Rb44细胞的MDR1和MRP基因的表达,并用免疫组织化学方法对其蛋白产物P－gp和P190进行检测。结果 HXO－Rb44系同时存在MDR1和MRP基因,并呈现P-gp和P190的过度表达（96％和97％）。结论 MDR1和MDRP基因的过度表达是Rb多药耐药现象发生的重要机制。     

多药耐药蛋白1和多药耐药相关蛋白5、7在糖尿病小鼠视网膜中表达的变化

目的 观察多药耐药蛋白1(muhidrug resistance 1,MDR1)、多药耐药相关蛋白(multidrug resistance-associated protein,MRP)5和MRP7在不同时期糖尿病小鼠视网膜中的表达变化.方法 通过腹腔注射链脲佐菌素诱导C57 BL/6小鼠建立糖尿病模型,伊文思蓝灌注检测血-视网膜内屏障变化.分别于糖尿病造模成功后的4周、12周和24周取小鼠视网膜,进行实时定量-PCR、免疫荧光、Western blotting等实验,检测各组小鼠视网膜中MDR1、MRP5和MRP7的表达变化.结果 伊文思蓝灌注结果显示在糖尿病12周就出现视网膜渗漏;实时定量-PCR结果发现与对照组相比,随糖尿病进展,MDR1、MRP5和MRP7在4周、12周和24周的表达均呈下降趋势(MDR1:P4周=0.028,P12周=0.003,P24周＜0.001;MRP5:P4周=0.045,P12周=0.009,P24周＜0.001;MRPy7:P4周=0.019,P12周＜0.001,P24周 =0.001),在糖尿病不同时期其变化也呈下降趋势(均为P＜0.05);免疫荧光观察到MDR1、MRP5和MRP7的荧光强度随糖尿病发展逐渐减弱;Western blotting检测可见在4周、12周、24周糖尿病小鼠视网膜中MDR1、MRP5和MRP7的表达均呈下降趋势.结论 在DR早期血-视网膜内屏障会受到破坏,视网膜中外排转运蛋白的表达也会受糖尿病的影响导致不同程度的下降.     

P-糖蛋白及多药耐药相关蛋白在眼眶横纹肌肉瘤中的表达及意义

目的　研究P 糖蛋白(P gp)、多药耐药相关蛋白 (MRP)在眼眶横纹肌肉瘤中的表达及临床意义。 方法　免疫组织化学法检测 42例眼眶横纹肌肉瘤和 10例正常眼眶组织中P gp、MRP的表达。 结果　P gp、MRP在眼眶横纹肌肉瘤组织中的表达阳性率分别为 38 10%和 54 76%,显著高于正常眼眶组织 (P<0 05 );P gp、MRP表达与年龄、性别、组织学类型、分期无关;复发组P gp和MRP的阳性表达率显著高于初发组(P<0 05);两蛋白间未见相关性。 结论　眼眶横纹肌肉瘤组织的多药耐药与P gp、MRP过表达有关,检测P gp、MRP的表达对判断预后有参考价值。     

葡萄膜黑色素瘤多药耐药基因的表达与患者预后因素的分析

目的 探讨葡萄膜黑色素瘤多药耐药基因的表达与患者预后的相关关系。 方法选择石蜡包埋葡萄膜黑色素瘤组织96例，通过脱色素免疫组织化学检测细胞周期素D1(cyclin D1)、上皮生长因子受体(epithelial growth factor receptor, EGFR)、转移抑制基因23(non-metastasis gene 23, nm 23)、P糖蛋白(P glucose protein,P-gp)、多药耐药相关蛋白(multidrug resistance relation protein,MRP)、肺耐药蛋白(lung-resistance-protein,LRP)基因表达。选择病例资料较完整者，进行随访，随访结果分类统计。 结果 96例葡萄膜黑色素瘤中类上皮细胞型21例、混合细胞型56 例和梭形细胞型19例，其中眼内期76例，眼外期20例。随着转移抑制基因nm 23表达的减低和Cyclin D1、EGFR表达的增高，耐药相关基因表达有增高趋势，其中MRP和LRP的表达与nm 23的表达呈明显负相关关系，与Cyclin D1、EGFR的表达呈明显正相关关系。随访结果显示，回访人数58例，生存超过 5年者26例，5年内死亡者19例。 结论 葡萄膜黑色素瘤多药耐药基因尤其LRP的表达与患者预后具有明显的相关性。 （中华眼底病杂志，2003，19：1-4）     

白藜芦醇对视网膜母细胞瘤细胞多药耐药性相关因子表达的影响

目的 观察白藜芦醇对视网膜母细胞瘤(RB)细胞多药耐药性(MDR)因子表达的影响.方法 体外培养RB细胞,取对数生长期细胞进行实验.实验分为实验组及对照组进行.采用浓度为6.25、12.50、25.00、50.00、100.00 μmol/L的白藜芦醇处理RB细胞,作用24、48 h后,噻唑蓝(MTT)比色法检测不同浓度组RB细胞的吸光度[A,旧称光密度(OD)]值,以此表示RB细胞活性.采用浓度为50.00 μmol/L的白藜芦醇处理RB细胞48 h,逆转录聚合酶链反应(RT-PCR)和蛋白质免疫印迹法(Western blot)分别检测MDR-1、环氧化酶(COX)-2、多药耐药相关蛋白(MRP)-1、谷胱苷肽转移酶(GST)-π的m RNA和蛋白表达.3种检测均以加入等体积0.5％二甲基亚砜细胞培养液为对照组.结果 MTT比色法检测结果显示,与对照组比较,6.25、12.50、25.00、50.00 μmol/L实验组A值呈剂量依赖性降低,差异有统计学意义(F=4.782,P＜0.05).50.00、100.00 μmol/L实验组A值间差异无统计学意义(F=6.351,P＞0.05).RT-PCR检测结果显示,实验组MDR-1、MRP1、COX-2、GST-π mRNA表达较对照组显著降低,差异均有统计学意义(t=9.170、5.758、4.152、4.638,P＜0.05).Western blot检测结果显示,实验组MDR-1、MRP1、COX-2、GST-π蛋白表达较对照组显著降低,差异均有统计学意义(t=3.848、5.955、4.541、3.514,P＜0.05).结论 白藜芦醇可降低RB细胞MDR因子的表达.     

多药耐药相关基因在眼眶腺样囊性癌中的表达

目的 了解P53、GST-π、P170和鼠抗人MDR相关蛋白（MRP）在眼眶腺样囊性癌（ACC）组织中的表达情况.方法 免疫组织化学法检测51例眼眶ACC标本和10例正常眼眶组织中P53、GST-π、P170和MRP的表达状况.结果 P53、GST-π和P170在眼眶ACC组织中表达阳性率分别为52.94%、100%和82.35%,与眼眶正常组织相比差异有统计学意义.P53和P170在实体型ACC中的表达率明显高于其在腺样-管状型中的表达率;P53在复发组中的表达率明显高于其在初发组中的表达率;各指标之间在表达上无相关性.结论 眼眶ACC的多药耐药性与P53、GST-π和P170的过表达有关,但作用机制不尽相同,各指标的检测对于判断预后有参考价值.     

lncRNA HIF1A-AS1对长春新碱耐药视网膜母细胞瘤细胞化疗敏感性的影响

目的：探讨长链非编码RNA-HIF1A-AS1(lncRNA HIF1A-AS1)调节缺氧诱导因子-1α(HIF-1α))表达对长春新碱(VCR)耐药视网膜母细胞瘤(RB)细胞化疗敏感性的影响。

方法：建立人RB VCR耐药细胞株SO-RB50/VCR，实时荧光定量PCR(RT-qPCR)检测SO-RB50与SO-RB50/VCR细胞lncRNA HIF1A-AS1表达； 在SO-RB50/VCR细胞中抑制lncRNA HIF1A-AS1表达或同时过表达HIF-1α，检测SO-RB50/VCR细胞对VCR的半数抑制浓度(IC₅₀)及细胞增殖、凋亡情况； Western blot检测HIF-1α、多药耐药相关蛋白(MRP)、P-糖蛋白(P-gp)蛋白表达。

结果：与SO-RB50细胞相比，SO-RB50/VCR细胞中lncRNA HIF1A-AS1与HIF-1α蛋白表达水平升高(P<0.05)； 在SO-RB50/VCR细胞中抑制lncRNA HIF1A-AS1表达后，细胞凋亡率显著升高(P<0.05)，细胞吸光度(OD₄₅₀)值显著降低，VCR对细胞的IC₅₀值及HIF-1α、MRP、P-gp蛋白表达水平显著降低(P<0.05)； 过表达HIF-1α可减弱下调lncRNA HIF1A-AS1表达对SO-RB50/VCR细胞耐药性的抑制作用。

结论：lncRNA HIF1A-AS1在SO-RB50/VCR细胞中高表达，抑制lncRNA HIF1A-AS1表达可通过下调HIF-1α表达，降低SO-RB50/VCR细胞对VCR的耐药性。     

Pax6基因在视网膜母细胞瘤中的表达及临床意义

目的:探讨Pax6基因在视网膜母细胞瘤(retinoblastoma,Rb)中的表达及临床意义。方法:选择我院2001-01/2012-12收存在眼科病理室的15例Rb组织切片设为观察组,再选取15例正常视网膜组织切片设为对照组。应用Western-Blot及RT-PCR(逆转录酶链反应)法分别对正常视网膜组织及Rb组织中的Pax6蛋白和Pax6 mRNA的表达进行检测,同时应用Western-Blot法对Pax6基因下游的BRN3b及MATH5促分化基因在蛋白水平的表达进行检测,最后进行组间比较,进而对Pax6基因在Rb中的表达及临床意义进行探讨。结果:观察组Pax6基因mRNA表达平均值为0.99±0.03,Pax6基因蛋白表达平均值为2.07±0.15,BRN3b蛋白表达平均值为0.195±0.016,MATH5蛋白表达平均值为0.190±0.031,均明显高于对照组,差异有统计学意义(P<0.05)。结论:异常表达的Pax6基因可能对Rb的出现起到促进作用。     

视网膜母细胞瘤血管生成拟态与临床病理学特征关系的研究

目的 探讨视网膜母细胞瘤(Rb)标本中是否存在血管生成拟态(VM),并分析VM与Rb临床病理特征及其与缺氧诱导因子-1αa(HIF-1α)、血管内皮生长因子(VEGF)蛋白表达的关系,进一步阐述VM的临床意义.方法 实验研究.收集60例Rb患者、10例正常人视网膜组织石蜡标本及相关临床病理资料,应用过碘酸-雪夫(PAS)与CD34双重染色以及免疫组织化学染色法检测60例Rb中是否存在VM和分布,观察与临床病理学特征的关系特点;检测HIF-1α、VEGF蛋白表达,分析Rb瘤组织中VM与HIF-1α、VEGF表达的关系;用CD34抗体进行肿瘤血管内皮染色,并计数肿瘤微血管密度(MVD).Rb和正常视网膜组织中的VM、HIF-1α、VEGF蛋白阳性表达的比较采用χ2检验;VM阳性组和VM阴性组的MVD值的比较采用q检验.结果 HE染色法观察到Rb中存在VM,其为Rb细胞构成的管腔样结构,无内皮细胞衬覆,腔内可见红细胞.CD34和PAS双重染色结合CD34、神经元特异性烯醇化酶(NSE)免疫组织化学染色进一步证明Rb中存在VM,60例Rb中VM阳性率为18.33%(11/60),其中随着R-E分级的增高,VM阳性表达率明显增加,差别具有统计学意义(χ2=8.861,P<0.05);分化型Rb中VM阳性率(4.34%)低于未分化型中VM阳性率(22.02%)(χ2=4.872,P<0.05);此外,HIF-1α、VEGF在Rb的VM形成过程中的阳性表达率依次为VM阳性组大于VM阴性组大于正常组;微血管密度计数高低与VM的表达有关,VM阴性组的MVD值(49.77±2.05)高于VM阳性组MVD值(36.53±1.15).结论 Rb中存在VM,且肿瘤恶性程度越高,形成VM能力越强.存在VM的Rb组织HIF-1α、VEGF高表达,提示HIF-1α、VEGF在VM的形成中可能起促进作用.     

Expression of multidrug resistance proteins in retinoblastoma

AIM: To elucidate the mechanism of multidrug resistance in retinoblastoma, and to acquire more insights into in vivo drug resistance. METHODS: Three anticancer drug resistant Y79 human RB cells were generated against vincristine, etoposide or carboplatin, which are used for conventional chemotherapy in RB. Primary cultures from enucleated eyes after chemotherapy (PCNC) were also prepared. Their chemosensitivity to chemotherapeutic agents (vincristine, etoposide and carboplatin) were measured using MTT assay. Western blot analysis was performed to evaluate the expression of p53, Bcl-2 and various multidrug resistant proteins in retinoblastoma cells. RESULTS: Following exposure to chemotherapeutic drugs, PCNC showed less sensitivity to drugs. No significant changes observed in the p53 expression, whereas Bcl-2 expression was found to be increased in the drug resistant cells as well as in PCNC. Increased expression of P-glycoprotein (P-gp) was observed in drug resistant Y79 cells; however there was no significant change in the expression of P-gp found between primary cultures of primarily enucleated eyes and PCNC. Multidrug resistance protein 1 (Mrp-1) expression was found to be elevated in the drug resistant Y79 cells as well as in PCNC. No significant change in the expression of lung resistance associated protein (Lrp) was observed in the drug resistant Y79 cells as well as in PCNC. CONCLUSION: Our results suggest that multidrug resistant proteins are intrinsically present in retinoblastoma which causes treatment failure in managing retinoblastoma with chemotherapy.     

【In Press】 Expression of multidrug resistance proteins in retinoblastoma

AIM: To elucidate the mechanism of multidrug resistance in retinoblastoma (RB), and to acquire more insights into in vivo drug resistance. METHODS: Three anticancer drug resistant Y79 human RBcells were generated against vincristine, etoposide or carboplatin, which are used for conventional chemotherapy in RB. Primary cultures from enucleated eyes after chemotherapy (PCNC) were also prepared. Their chemosensitivity to chemotherapeutic agents (vincristine, etoposide and carboplatin) were measured using MTT assay. Western blot analysis was performed to evaluate the expression of p53, Bcl-2 and various multidrug resistant proteins in RB cells. RESULTS: Following exposure to chemotherapeutic drugs, PCNC showed less sensitivity to drugs. No significant changes observed in the p53 expression, whereas Bcl-2 expression was found to be increased in the drug resistant cells as well as in PCNC. Increased expression of P-glycoprotein (P-gp) was observed in drug resistant Y79 cells; however there was no significant change in the expression of P-gp found between primary cultures of primarily enucleated eyes and PCNC. Multidrug resistance protein 1 (Mrp-1) expression was found to be elevated in the drug resistant Y79 cells as well as in PCNC. No significant change in the expression of lung resistance associated protein (Lrp) was observed in the drug resistant Y79 cells as well as in PCNC. CONCLUSION: Our results suggest that multidrug resistant proteins are intrinsically present in RB which causes treatment failure in managingRB with chemotherapy.     

人腺样囊性癌多药耐药细胞系的建立

目的:建立人腺样囊性癌耐药细胞系,为阐明腺样囊性癌的多药耐药机制及逆转尉药提供模型及研究依据.方法:以长春新碱(VCR)为诱导剂,通过浓度递增间断刺激法对人腺样囊性癌细胞系(ACC)进行体外诱导耐药,建立耐VCR的腺样囊性癌细胞系ACC/VCR.细胞计数法绘制细胞生长曲线,噻唑蓝(MTT)比色法检测细胞对化疗药物敏感性.结果:ACC经体外诱导后,ACC/VCR细胞对长春新碱(VCR)、阿霉素(ADM)和平阳霉素(PYM)的耐药性明显增强,具有交叉耐药,对环磷酰胺(CTX)、氟尿嘧啶(5-FU)和顺铂(DDP)耐药性无明显变化.耐药前后ACC细胞的生长曲线、群体倍增时间和光镜下的形态无明显改变.结论:VCR可诱导腺样囊性癌细胞产生多药耐药.     

Immunohistochemical detection of multidrug-resistant protein expression in retinoblastoma treated by primary enucleation

PURPOSE: To compare the expression of multidrug-resistant proteins in retinoblastoma tumors among eyes treated with primary enucleation. METHODS: A group of 18 patients with unilateral retinoblastoma with advanced intraocular disease was selected for the study. All patients had undergone primary enucleation. A histologic specimen from each patient was retrieved from the pathology archives and a tissue gene microarray was constructed (0.6 x 3-4 mm). Standard immunohistochemical techniques were used to study the tissue microarrays for the expression of the ATP-binding cassette (ABC) transporters: breast cancer resistance protein (BCRP; ABCG2), multidrug-resistant protein 1/P-glycoprotein (MDR1/Pgp; ABCB1), multidrug-resistant-associated protein 1 (MRP1; ABCC1), MRP2 (ABCC2), and MRP4 (ABCC4). RESULTS: Of the 18 specimens retrieved, 16 had adequate tissue for study. MRP1 was expressed in 8 (50%) of 16 tumors, and MRP2 was expressed in 5 (31%) of 16 tumors. MDR1/Pgp was found in 2 (12%) of 16 retinoblastomas. MRP4 and BCRP were not detected in any of the tumors studied. CONCLUSIONS: The results show that multiple ABC transporters were present in a cohort of sporadic patients with unilateral retinoblastoma who underwent primary enucleation. Studies are planned of the expression of ABC transporters in eyes treated by chemotherapy and/or radiation as a comparison with this group.     

Molecular evidence and functional expression of multidrug resistance associated protein (MRP) in rabbit corneal epithelial cells

Multidrug resistance associated protein (MRP) is a major family of efflux transporters involved in drug efflux leading to drug resistance. The objective of this study was to explore physical barriers for ocular drug absorption and to verify if the role of efflux transporters. MRP-2 is a major homologue of MRP family and found to express on the apical side of cell membrane. Cultured Rabbit Corneal Epithelial Cells (rCEC) were selected as an in vitro model for corneal epithelium. [14C]-erythromycin which is a proven substrate for MRP-2 was selected as a model drug for functional expression studies. MK-571, a known specific and potent inhibitor for MRP-2 was added to inhibit MRP mediated efflux. Membrane fraction of rCEC was used for western blot analysis. Polarized transport of [14C]-erythromycin was observed in rCEC and transport from B-->A was significantly high than from A-->B. Permeability's increased significantly from A-->B in the presence of MK-571 and ketoconozole. Uptake of [14C]-erythromycin in the presence of MK-571 was significantly higher than control in rCEC. RT-PCR analysis indicated a unique and distinct band at approximately 498 bp corresponding to MRP-2 in rCEC and MDCK11-MRP-2 cells. Immunoprecipitation followed by Western Blot analysis indicated a specific band at approximately 190 kDa in membrane fraction of rCEC and MDCK11-MRP-2 cells. For the first time we have demonstrated high expression of MRP-2 in rabbit corneal epithelium and its functional activity causing drug efflux. RT-PCR, immunoprecipitation followed by Western blot analysis further confirms the result.     

耐长春新碱视网膜母细胞瘤细胞株的建立

目的：研究视网膜母细胞瘤获得性多药耐药现象，探讨其产生机制。方法：利用长春新碱(vincristree，VCR)对人视网膜母细胞瘤细胞系HXO-RB₄₄细胞在体外进行浓度递增法逐步诱导实验，建立一株耐长春新碱视网膜母细胞瘤细胞亚株HXO—RB/VCR。并对其细胞生长，药物含量，药物敏感性与放射性敏感性进行检测。 结果：该耐药细胞株HXO—RB/VCR细胞对长春酰胺、丝裂霉素C、阿霉素、足叶乙甙、顺铂、卡铂等有交叉耐受，对卡氮芥、氟尿嘧啶无明显抗药性，而对氨甲喋呤的敏感性反而增加。对⁶⁰ Coγ射线有轻度耐受性。细胞内药物含量测定显示：耐药细胞内长春新碱浓度明显低于敏感细胞，钙通道阻断剂异博定可使细胞内VCR浓度增加，部分逆转其耐药性。 结论：VCR可诱导视网膜母细胞瘤产生多药耐药和轻度放射耐受，获得性多药耐药作用可被异博定逆转。 （中华眼底病杂志，1997，13：6-9）     

Multidrug resistance in ocular melanoma.

AIMS/BACKGROUND: Metastatic disease in patients with ocular melanoma is resistant to chemotherapy. One of the main mechanisms of modulating multidrug resistance is the expression of the multidrug resistance gene 1 (MDR1) product (p-glycoprotein) by tumour cells. The purpose of this study was to evaluate the frequency of expression of the MDR1 gene in ocular melanoma whose primary treatment was surgical excision or enucleation. METHODS: Twelve recent ocular melanomas were received fresh, snap frozen and cryostat sections of tumour were analysed for expression of MDR1 by immunohistochemistry using a well characterised monoclonal antibody to MDR1. Tumour explants were established in short term tissue culture from four tumours and cell blocks were examined by immunohistochemistry. RESULTS: MDR1 expression was present in five of 12 ocular melanomas. Upregulation of protein expression was found in four cell lines established in short term culture from tumour explants. A recurrent tumour, initially treated by local excision and radioactive plaque, showed overexpression of MDR1 mRNA. CONCLUSIONS: These results suggest that significant level of MDR1 may be intrinsically present in ocular melanomas before exposure to drugs involved in multidrug resistance, and indicate the possible importance of MDR1 in modulating chemoresistance in ocular melanoma. Chemosensitisation may be of potential value in planning adjuvant chemotherapy for patients with metastatic disease.