初发及复发转移大肠癌患者外周血MDR-1基因表达的研究

目的：检测初发及复发转移大肠癌患外周血中多药耐药基因(MDR—1)的表达,探讨其与病理类型及化疗疗效的关系。方法：应用逆转录多聚酶链反应(RT—PcR)法对56例初发大肠癌及40例经术后辅助化疗后复发、转移大肠癌患的外周血进行检测,并与其病理类型及化疗疗效作对比研究。结果：56例初发大肠癌患外周血中MDR—1阳性表达率为27．2％,与病理类型无显相关(P＞0．05);但与肠系膜淋巴结转移密切相关,有淋巴结转移阳性表达率显高于无淋巴结转移(P＜0．05);40例化疗后复发、转移患外周血MDR—1阳性表达率为72．5％,与初发大肠癌患的MDR—1阳性表达宰相比差异有显性(P＜0．05),且MDR－1的表达与化疗疗效呈负相关,MDR—1阳性表达化疗疗效明显低于阴性表达(P＜0．05)。结论：大肠癌存在先天性和获得性多药耐药性;检测外周血MDR—1表达情况可以帮助预测化疗疗效,对制定临床化疗方案有指导意义。     

In vivo use of all-trans retinoic acid prior to induction chemotherapy improves complete remission rate and increases rhodamine 123 uptake in patients with de novo acute myeloid leukemia

All-trans retinoic acid (ATRA) is used in the treatment of acute promyelocytic leukemia. Because ATRA has effects (increase in apoptosis, suppression of bcl-2), it has also been used for the treatment of other French-American-British (FAB) subtypes of acute myelogenous leukemia (AML). To find out the in vivo and in vitro effects of ATRA in AML, we analyzed 37 patients with de novo AML. Twenty-seven patients received ATRA before remission-induction (RI) treatment (ATRA group). Results were compared to a control group (10 patients) that received induction without ATRA during the same time period. Bone marrow or peripheral blood samples were collected from all patients on d 0 and 4. The immunphenotype, myeloperoxidase (MPO), reaction and the efflux uptake of rhodamine 123 (Rh123) were analyzed on myeloblasts in these samples. In the myeloblasts from patients treated with ATRA, the uptake of Rh123 was increased significantly (p=0.026) from d 0 to d 4, and all other parameters remained unaltered. ATRA administration increased the complete remission (CR) rate (88%, 22/25 vs 55%, 5/9) significantly (p=0.042). Logistic regression analysis revealed that ATRA administration was the important factor in CR, among other potential factors including age, white blood count, bcl-2 expression, and the uptake and efflux of Rh123 (p=0.05). Estimated disease-free survival and overall survival were similar between these two groups (43% vs 37.5% and 51.2% vs 37.5%, respectively). In conclusion, ATRA treatment prior to RI treatment may improve the CR rate in patients with de novo AML, which seems to be related to its beneficial effect on multidrug resistance.     

BSO逆转人肺腺癌细胞株多药耐药性的实验研究

目的研究谷胱甘肽(GSH)合成酶抑制剂——BSO对人肺腺癌多药耐药细胞株A549DDP细胞内GSH含量影响;探讨BSO逆转多药耐药(MDR)作用机制及逆转效果。方法GSH还原酶循环法测定BSO对细胞内GSH含量的影响。MTT比色法测定经BSO预处理后顺氯氨铂(DDP)、阿霉素(ADM)对细胞50%抑制浓度(IC50)的影响。流式细胞仪检测BSO对MDR细胞内柔红霉素(DNR)荧光强度的影响。结果耐药细胞A549DDP细胞内GSH含量较人肺腺癌A549细胞内GSH含量明显增高。BSO在一定浓度范围内(50～200μmol·L-1)对A549细胞和耐药细胞A549DDP无明显细胞毒性作用(抑制率均小于10%)。BSO呈剂量依赖性非线性抑制细胞内GSH的合成,其对MDR细胞内GSH合成影响较为显著,而对A549细胞内GSH合成影响较小。BSO在一定浓度范围内能降低DDP、ADM对A549DDP细胞的IC50,而对A549细胞的IC50无明显影响。A549DDP细胞内DNR荧光强度较A549细胞显著降低,能不同程度提高A549DDP细胞内柔红霉素荧光强度,均较未处理组显著提高;与未经BSO处理的A549细胞比较,细胞内荧光强度轻度增高,但统计学上无显著性差异。结论BSO能有效逆转A549DDP细胞的MDR,其机制与降低MDR细胞内GSH含量有关。     

食管癌及癌旁组织中p53、MDR基因表达的临床意义

目的　探讨食管癌和癌旁组织p5 3、MDR基因的表达及其临床意义。方法　采用免疫组化ABC法及逆转录酶 -聚合酶链反应的方法 ,对 5 0例食管癌和 3 7例癌旁组织中p5 3、MDR基因的表达情况进行检测。结果　p5 3基因蛋白在食管癌中表达为 68%(3 4 5 0 ) ,癌旁组织中表达 5 9% (2 2 3 7) ;其中在正常组织中占 5 4% (13 2 4) ,在非典型增生中占 69% (9 13 )。MDR基因在食管癌高、中、低分化中的表达水平有极显著意义 ,癌与正常组织有极显著意义。在不同年龄、性别、肿瘤大小上p5 3、MDR基因的表达无显著意义。结论　p5 3、MDR基因在食管癌中均呈高水平表达 ,癌旁组织中MDR有表达 ,p5 3基因突变早于组织形态学改变     

子宫内膜癌MDR基因的表达与临床意义

目的　探讨子宫内膜癌多药耐药基因的表达与临床病理学特征的关系。方法　采用免疫组织化学ABC法检测 50例子宫内膜癌组织与 2 6例正常或各型增生子宫内膜组织MDR基因的表达。结果　正常或各型增生子宫内膜存在MDR基因的表达 ,子宫内膜癌MDR基因的阳性表达率为 48% ,其中组织学分级G1 、G2 级MDR的表达显著高于G3级 ,手术分期Ⅰ期MDR基因的表达显著低于Ⅱ～Ⅳ期 ,MDR的表达与肌层浸润的深度无关。结论　子宫内膜癌抗药性可能与MDR基因的表达有关 ,其化疗时应选择敏感药物以增强化疗疗效     

MRP及GST-π在胰腺癌中的表达及临床意义

目的 :了解胰腺癌中多药耐药相关蛋白及谷胱甘肽 S 转移酶π 2种多药耐药基因产物的表达与其临床意义。方法 :采用免疫组化的方法检测胰腺癌中上述 2种蛋白的表达。结果 :多药耐药相关蛋白、谷胱甘肽 S 转移酶π在胰腺癌中的阳性率分别为 81%和 5 9% ,并且多药耐药相关蛋白与谷胱甘肽 S 转移酶π的表达具有相关性 ,二者的表达与临床分期、病理分型、病理分级及淋巴结转移无明显相关 ,同时有 2种蛋白阳性者的生存期短于相应阴性者。结论 :多药耐药相关蛋白及谷胱甘肽 S 转移酶π在胰腺癌的多药耐药现象中占有重要地位     

转染肿瘤坏死因子及反向多药耐药基因对乳腺癌细胞耐药基因表达的影响

目的构建反向pEGFP-MDR1重组表达载体和pDsRed2-TNF-α重组表达载体,在乳腺癌耐药细胞株MCF-7/ADR中进行表达,并观察它们对多药耐药(MDR)基因的影响。方法应用RT-PCR和DNA重组技术构建反向绿色荧光蛋白pEGFP-MDR1融合蛋白表达载体和红色荧光蛋白pDsRed2-TNF-α融合蛋白表达载体,分别和同时导入乳腺癌耐药细胞株MCF-7/ADR中进行表达,应用荧光显微镜直接观察融合蛋白的表达情况,RT-PCR和免疫细胞化学染色法分别检测转染前后MDR1-mRNA和P糖蛋白表达情况。结果转染了反向pEGFP-MDR1载体的细胞可见到绿色荧光,转染了pDsRed2-TNF-α载体的细胞可见到红色荧光,而同时转染了反向pEGFP-MDR1载体和pDsRed2-TNF-α载体的细胞可见到红色及绿色荧光。转染后的细胞在mRNA水平及蛋白水平明显降低了耐药细胞MCF-7/ADR的MDR1基因的表达。结论反向pEGFP-MDR1融合蛋白和pDsRed2-TNF-α融合蛋白能在乳腺癌耐药细胞MCF-7/ADR中分别及同时获得表达,并能抑制MDR1基因在MCF-7/ADR细胞中的表达,为进一步研究基因治疗联同免疫治疗逆转乳腺癌耐药建立了很好的基础。     

多药耐药基因介导的骨髓保护在肝癌小鼠化疗时的富集效应

目的 探讨多药耐药基因转染小鼠骨髓单个核细胞移植后,化疗时其在体内的表达、分布以及在骨髓和外周血的富集效应.方法 骨髓造血细胞转染外源性多药耐药基因后移植入荷瘤Balb/c小鼠,免疫组化和RT-PCR观察mdr1基因在骨髓造血细胞、外周血单个核细胞及重要脏器的表达和分布;监测外周血白细胞变化及肿瘤组织P-糖蛋白表达.结果 外源性多药耐药基因在重要脏器和肿瘤组织无表达;化疗前骨髓细胞和外周血的P-糖蛋白表达阳性率分别为(9.36±1.84)%、(8.52±1.26)%,化疗药物剂量递增时,P-糖蛋白表达阳性率增高;移植未转染组与移植转染组外周血白细胞计数差异有统计学意义(P＜0.01).结论 外源性多药耐药基因转染在骨髓造血细胞和外周血单个核细胞有持续较高表达,在重要脏器无表达;随化疗药物剂量递增时骨髓和外周血有明显的富集效应;多药耐药基因转染介导保护骨髓在超剂量化疗中获得显著效果.     

P-Glycoprotein Is Positively Correlated with p53 Protein Accumulation in Human Colorectal Cancers

To explore the relationship between mutant p53 and Pgp expression, we have examined the levels of both proteins in human colorectal adenocarcinomas. Serial frozen sections of 40 surgical samples were stained with an anti-Pgp (MRK16) and two different anti-p53 protein antibodies (Abs), PAb421 and Pabl80l. Nineteen (47.5%) of 40 samples examined were positive for Pgp, and 18 (45%) of 40 were positive for p53. The samples that stained positively with PAb421 also stained positively with Pahl80l. Pgp expression was detected in 13 (76.5%) of 17 samples that were positive for p53 using PAb421 and in 15 (83.3%) of 18 samples that were positive for p53 using Pabl80. Thus, we found that p53 and Pgp were co-expressed in a significant number of samples ( P < 0.002). There was no relationship between Pgp or p53 protein accumulation and histologic grade or stage. The present results demonstrate that Pgp expression is closely associated with p53 protein accumulation in human colorectal cancers. These data provide evidence to support the idea that mutant p53 activates the MDR1 gene in vivo .     

Selective modulation of P-glycoprotein's ATPase and anion efflux regulation activities with PKC α and PKC ɛ in Sf9 cells

The modulation of P-glycoprotein's (Pgp) ATPase activity and its ability to regulate swelling-activated ¹²⁵I efflux, by PKC α and PKC ɛ, was examined in insect cells. Recombinant baculovirus was used to express human Pgp in Sf9 cells and Pgp was also co-expressed with either PKC α or PKC ɛ. ATPase assays showed the enzyme activity of Pgp to be elevated during co-expression with the Ca²⁺ dependent isoform PKC α, but not with the Ca²⁺ independent variant PKC ɛ. Furthermore, neither isoform, when co-expressed with Pgp, altered the swelling-activated efflux of ¹²⁵I from Sf9 cells. However, in cells co-expressing Pgp/PKC (α or ɛ), pre-treatment with the phorbol ester TPA significantly reduced the swelling-activated ¹²⁵I efflux with both PKC isoforms. Our results suggest that phosphorylation with the Ca²⁺ independent variant PKC ɛ does not regulate the ATPase activity of Pgp and that stimulation of PKC with TPA alters the swelling-activated efflux of anions from insect cells expressing Pgp. Received: 8 March 2000 / Accepted: 5 June 2000