汉防己甲素联合紫杉醇逆转脑胶质瘤C6/MDR细胞耐药及其机制研究

目的评价汉防己甲素联合紫杉醇(paclitaxel,PTX)逆转耐药型脑胶质瘤(C6/MDR)细胞多药耐药的可行性,并探讨可能的耐药逆转机制。方法通过非耐药型脑胶质瘤C6和耐药型脑胶质瘤C6/MDR细胞系作对比,以体外细胞毒性为指标,采用MTT法分别考察PTX、汉防己甲素、PTX+汉防己甲素抑制C6和C6/MDR细胞增殖作用差异;以细胞摄取能力为指标,借助HPLC和流式细胞仪技术分别测定PTX和荧光探针罗丹明123(R123)在2种细胞内的含量,评价汉防己甲素促C6/MDR细胞摄取PTX和R123的能力;以细胞凋亡率为指标,采用Annexin V-PE/7-AAD一步染色法定量PTX、汉防己甲素、PTX+汉防己甲素干预后的2种细胞凋亡率;采用P-糖蛋白(P-gp)抗体结合试剂盒和Pgp-Glo TM分析系统分别考察汉防己甲素对C6/MDR细胞P-gp表达以及P-gp ATP酶活性的影响。结果汉防己甲素+PTX对C6/MDR细胞的半数抑制浓度(IC50,以PTX浓度计)为(5.88±0.43)nmol/L;在10μmol/L汉防己甲素的干预下,C6/MDR细胞内的PTX和R123累积量相比PTX组分别提高9.4倍和12.3倍,凋亡率相应提高了2.3倍;这种汉防己甲素介导的耐药逆转机制可能与降低C6/MDR细胞P-gp表达、刺激细胞内P-gp ATP酶活性有关。结论汉防己甲素联合紫杉醇在体外具有逆转C6/MDR细胞耐药的潜力。     

ABCB1(1199G>A)基因多态性对多西他赛转运影响的分子机制

目的：在体外研究ABCB1（1199G>A）基因多态性对多西他赛转运影响的分子机制。方法：将ABCB1（1199G>A）野生型和突变型基因分别导入HEK293细胞,研究2种重组细胞株对多西他赛的摄取以及跨膜转运的差异。结果：在细胞毒性分析中,ABCB1_1199A/mut细胞对多西他赛表现出更强的耐药性。多西他赛在2种重组细胞中的含量均显著性的低于对照组细胞,证实了多西他赛是由P-糖蛋白介导转运的,并且在ABCB1_1199A/mut细胞中跨膜转运更高。ABCB1_1199A/mut细胞介导转运多西他赛的P-糖蛋白的活性较ABCB1_1199G/wt细胞的更强。结论：ABCB1（1199G>A）基因多态性能够显著性影响P-糖蛋白转运多西他赛的能力,由ABCB1突变型基因编码的P-糖蛋白能够更有效的转运多西他赛。因此ABCB1（1199G>A）基因多态性可能会影响P-糖蛋白的活性,并对药物的分布和消除产生影响,从而影响药物的治疗作用。     

UGCG通过调控MDR1/P-gp参与人结肠癌奥沙利铂耐药的初步研究

目的 建立人结肠癌耐奥沙利铂（L-OHP）细胞株,检测该细胞株的多药耐药性并初步探讨其可能的耐药机制。方法 以人结肠癌细胞HCT116为对象,采用药物浓度梯度递增诱导法建立人结肠癌耐奥沙利铂细胞株HCT-116/L-OHP。CCK-8法检测L-OHP、顺铂（DDP）、5-氟尿嘧啶（5-Fu）对亲本细胞和耐药细胞株的半数抑制浓度（IC50）。使用UDP-葡萄糖神经酰胺糖基转移酶（UGCG）siRNA转染HCT-116/L-OHP细胞,实时荧光定量 PCR和Western blot检测干扰前后UGCG基因和多药耐药基因1（MDR1）mRNA及其编码的蛋白表达水平。结果 HCT-116/L-OHP对L-OHP的耐药指数为10.5,与DDP有一定程度的交叉耐药,耐药指数为4.61,但对5-Fu无交叉耐药。耐药细胞HCT-116/L-OHP中UGCG、MDR1 mRNA和UGCG、P-糖蛋白（P-gp, MDR1编码的蛋白）表达均增加,相比HCT-116细胞,差异具有统计学意义（P<0.05）。UGCG siRNA成功抑制HCT-116/L-OHP细胞中UGCG的表达,各干扰组MDR1 mRNA、P-gp表达减少,与对照组相比差异具有统计学意义（P<0.05）。结论 成功构建了人结肠癌耐药细胞株;UGCG基因通过调控MDR1/P-gp的表达参与人结肠癌奥沙利铂的耐药机制的形成。     

香附醋制前后对Caco-2细胞P-糖蛋白功能和表达的影响

目的:通过研究香附醋制前后对Caco-2细胞P-糖蛋白(P-gp)功能和表达的影响,初步阐明“香附醋制增效”理论的机制.方法:采用Caco-2细胞模型,细胞以2×105/mL的密度接种于Trenswell 6孔板,加入体积分数分别为5％,10％,20％的香附生品及醋制品含药血清培养24 h,流式细胞仪检测香附醋制前后对Caco-2细胞Pgp功能的影响;免疫组化法检测P-糖蛋白的表达.结果:香附生品低、中、高浓度可使细胞内地高辛累积增加8.25％,9.57％,16.84％,醋制品低、中、高浓度可使细胞内地高辛累积增加22.44％,29.12％,33.09％.二者均可使细胞内地高辛累积增加,并使P-gp表达的阳性率降低,对P-gp功能和表达均呈现出抑制作用,且醋制品作用更加明显.结论:香附醋制后可增加其抑制Caco-2细胞P-gp功能和表达的作用,从而促进P-gp底物的吸收.     

Absorption properties and effects of caffeic acid phenethyl ester and its p-nitro-derivative on P-glycoprotein in Caco-2 cells and rats

Context: Caffeic acid phenethyl ester (CAPE), isolated from honeybee propolis, has pharmacological applications. A synthesized CAPE derivative, p-nitro-caffeic acid phenethyl ester (CAPE-NO₂), showed similar activities with CAPE. The pharmacological activities of CAPE and CAPE-NO₂ are related to their absorption properties.

Objective: To understand the pharmacokinetic profiles of CAPE and CAPE-NO₂ in rats and investigate the absorption mechanisms and effects on P-glycoprotein in Caco-2 cells.

Materials and methods: The pharmacokinetic profiles of CAPE and CAPE-NO₂ were obtained after oral administration (10?mg/kg) to rats. Transport studies of CAPE and CAPE-NO₂ (5, 10, 20?μM) were performed in Caco-2 cell model. P-gp activities were assayed by rhodamine 123 cellular retention. Expression of P-gp was determined after the cells were administrated with CAPE and CAPE-NO₂ (5, 20?μM) for 48 and 72?h.

Results: The AUC_(0?t) of CAPE-NO₂ (3239.9?±?352?ng?×?h/mL) was two-time greater than CAPE (1659.6?±?152?ng?×?h/mL) in rats. The P_app values of CAPE and CAPE-NO₂ were (4.86?±?0.90)?×?10^?6?cm/s and (12.34?±?1.6)?×?10^?6?cm/s, respectively. The accumulation of rhodamine 123 was increased by 1.3- to 1.9-fold and 1.4- to 2.3-fold in CAPE and CAPE-NO₂ groups after 1?h administration, respectively. However, CAPE and CAPE-NO₂ increased the P-gp levels by 2.1- and 1.7-fold, respectively.

Conclusion: The absorption of CAPE-NO₂ can be enhanced in rats and Caco-2 cells compared with CAPE. The two compounds are potential inhibitors of P-gp. The increased P-gp levels generated by CAPE and CAPE-NO₂ played a role as a defense mechanism by limiting intracellular xenobiotic levels.     

新基因HA117编码蛋白在儿童急性白血病中的表达及其临床意义

目的探讨多药耐药新基因HA117编码蛋白在儿童急性白血病(acute leukemia,AL)患儿骨髓单个核细胞(bone marrow mononuclear cells,BMMNC)中的表达及其临床意义。方法将67例AL患儿分为初诊组、完全缓解组和未缓解组,10例ITP患儿为对照组。应用免疫组织化学法检测BMMNC中HA117编码蛋白及P-糖蛋白(P-gp)表达。结果HA117编码蛋白在AL患儿中阳性表达率从低至高为:完全缓解组、初诊组、未缓解组,对照组呈阴性表达;初诊组中强阳性(+++)表达者其首次缓解(CR1)率(16.67%)显著低于阴性表达组(94.74%)(P<0.01)及中度表达组(87.50%)(P<0.05);同一患儿中HA117编码蛋白表达与P-gp表达无相关性(κ=0.195,P>0.05),两者均表达者CR率(30.00%)显著低于HA117+/P-gp-、HA117-/P-gp+组(83.33%)(P<0.05)及HA117-/P-gp-组(94.44%)(P<0.01)。结论 HA117编码蛋白的过表达与临床化疗耐药密切相关,是儿童AL预后的不利因素,且其耐药机制可能是独立的非P-gp介导的;HA117编码蛋白与P-gp两者同时高表达提示预后不良。     

多药耐药基因及P—糖蛋白在正常涎腺组织的表达

目的 探讨多药耐药基因（mdrl)与P－糖蛋白（P－gp)在正常涎腺组织的表达与功能,方法 采用PCR和免疫组化法对15例正常涎腺组织进行mdrlm RNA及p-gp的检测,结果 mdrlmRNA阳性率为40％,IOD均值为0．245,P－gp阳性率为66．7％,定位于正常涎腺的分泌管,结论 P－gp可能参与了涎腺的正常生理功能,尤其是涎腺的分泌功能。     

Correlation between P-glycoprotein (P-gp) expression in parathyroid and Tc-99m MIBI parathyroid image findings

The major factor to influence localization of parathyroid adenomas is tumor size. P-glycoprotein (P-gp) expression in parathyroid adenomas has been considered to be an another possible factor to influence localization of parathyroid adenomas because false-negative studies have been reported with large tumors and true-positives reported with very small tumors in previous studies. The aim of this study was to characterize Tc-99m MIBI uptake and retention by parathyroid adenomas and to correlate this with cell surface expression of P-gp. Sixteen patients with parathyroid adenoma (larger than 1.5 gm) underwent dual-phase (10min and 2hr) Tc-99m MIBI parathyroid image immediately before parathyroid exploration. Tissues were obtained from normal and abnormal parathyroid glands and from the thyroid gland. Immunohistochemistry (IHC) was obtained with monoclonal antibodies to identify P-gp expression in all tissues. All of the 16 parathyroid adenomas and 32 normal control specimens (16 normal parathyroid and 16 normal thyroid specimens) were submitted for P-gp detection by IHC. The dual-phase Tc-99m MIBI parathyroid image accurately localized 14 parathyroid adenomas, but not the remaining 2 adenomas. The 14 parathyroid adenomas with significant Tc-99m MIBI uptake in delayed 2hr images revealed negative P-gp expression, but the 2 adenomas without significant Tc-99m MIBI uptake, as well as normal parathyroid and normal thyroid specimens, revealed positive P-gp expression when evaluated by IHC. Not only the size of parathyroid adenomas, but also significant P-gp expression limited the sensitivity of dual-phase Tc-99m MIBI parathyroid image to localize parathyroid adenomas before operation.     

紫杉醇及塞来昔布对胃癌细胞株ＣＯＸ-２及Ｐ-ｇｐ表达的影响

目的：观察紫杉醇及环氧化酶-２（ＣＯＸ-２）选择性抑制剂塞来昔布（Ｃｅｌｅｃｏｘｉｂ）对人胃腺癌细胞株ＢＧＣ-８２３ ＣＯＸ-２及Ｐ-糖蛋白（Ｐ-ｇｐ）表达的影响,探讨ＣＯＸ-２在化疗药物诱发多药耐药（ＭＤＲ）产生机制中的作用。方法：采用ＭＴＴ法检测紫杉醇不同剂量、不同时间点及塞来昔布不同剂量对胃腺癌细胞株ＢＧＣ-８２３生长的影响,在此基础上采用Ｗｅｓｔｅｒｎｂｌｏｔ方法检测一定剂量范围内某一时间点的紫杉醇对ＢＧＣ-８２３ ＣＯＸ-２、Ｐ-ｇｐ表达的影响及联合塞来昔布后两种蛋白表达的变化。结果：紫杉醇对胃腺癌细胞株ＢＧＣ-８２３的生长为细胞毒作用,呈时间和剂量依赖性。在一定剂量范围内和某一时间点,随着紫杉醇剂量逐渐升高,ＢＧＣ-８２３的ＣＯＸ-２、Ｐ-ｇｐ表达均呈上升趋势;且这两种蛋白在联合塞来昔布应用后表达均呈下降趋势。结论：紫杉醇可诱导胃腺癌细胞株ＢＧＣ-８２３ ＣＯＸ-２及Ｐ-ｇｐ蛋白的表达,这种表达能被塞来昔布所抑制。ＣＯＸ-２表达的诱导在紫杉醇诱导的Ｐ-ｇｐ表达中起着重要作用。     

HIF-1α及其靶基因在宫颈癌组织中的表达

目的探讨缺氧诱导因子-1α及其调节的靶基因,在宫颈癌发生发展中的作用机制以及对宫颈癌多药耐药的影响。方法采用免疫组化SP染色法,检测缺氧诱导因子-1α（HIF-1α）、血管内皮生长因子（VEGF）、P糖蛋白（P-gp）在158例人宫颈上皮中的表达。结果NNC组、CIN组和ICC组中HIF-1α表达率分别为10.71%、37.50%、76.53%;VEGF表达率分别为14.29%、46.88%、70.41%;P-gp表达率分别为21.43%、37.50%、54.08%。HIF-1α和VEGF表达与宫颈病变程度呈正相关,三组间HIF-1α和VEGF表达比较有显著性差异（P均〈0.05）;P-gp表达亦与宫颈病变程度有关,NNC组与ICC组比较有显著性差异（P〈0.05）,NNC组与CIN组及CIN组与ICC组比较无显著性差异（P均〉0.05）。HIF-1α表达与VEGF表达显著相关（R=0.538,P=0.000）,而与P-gp无明显相关性（P〉0.05）。结论HIF-1α可能通过促进血管生成参与宫颈癌的发生、发展及转移,它不能独立调控宫颈癌多药耐药行为。HIF-1α和VEGF可以作为诊断宫颈癌的分子标志物。