R-dl-型维拉帕米调低KBv200肿瘤细胞对长春新碱和多柔比星的多药抗药性

目的:研究R-型维拉帕米对多药耐药的降低作用及其急性动物毒性,并与消旋维拉帕米的相应结果作比较。方法:细胞毒性的测定用MTT法;细胞内多柔比星积累的测定用萤光分光光度计法。急性毒性试验用BALB/c小鼠腹腔注射法。结果:R-型维拉帕米部分调低KBv200细胞对长春新碱和多柔比星的耐药性,其调低效应与作用浓度和作用时间有关。1.25μmol·L~(-1)的R-型维拉帕米与长春新碱对细胞作用24h,能够显著增加KBv200细胞对长春新碱的敏感性。在增敏和增加细胞内多柔比星累积方面,R-型维拉帕米与消旋维拉帕米效果一样,但R-型维拉帕米的急性动物毒性明显低于消旋维拉帕米。结论:R-型维拉帕米1.25μmol·L~(-1)提高耐药肿瘤细胞对长春新碱和多柔比星的敏感性,增加对长春新碱敏感性所需的药物作用时间可缩短至24h。     

多药耐药基因（mdr—1）RT／PCR检测方法的影响因素

多药抗药性是肿瘤化疗失败的主要原因，在多药耐药基因（mdr-1）检测方法中，PCR具有简便、灵敏、特异性强等特点，我室建立了检测mdr-1mRNA的逆转录／多聚酶链反应（RT／PCR）方法，并用KB耐药细胞对其进行了验证，检测灵敏度达8×104个细胞．在实验过程中摸索了RNA提取及反转录PCR过程的技术关键，为临床mdr-1检测提供了一种实用、稳定的手段。     

蛋白激酶C与耐药性KB细胞的多药抗药性

目的 :研究蛋白激酶C(PKC)在肿瘤细胞多药抗药性的作用及机制。方法 :测定KB细胞及其耐药株的PKC活性及PKC调节剂对细胞药敏、药物代谢和耐药膜糖蛋白表达及其磷酸化的影响。结果 :耐药株胞质和胞膜的PKC活性分别是KB细胞的 6 6倍和 5 2倍 ,膜糖蛋白阳性率为 4 7 5 % ,明显高于KB细胞的 2 8% ;而且膜糖蛋白的磷酸化明显增强。耐药株对罗丹明 12 3的外排显著加快 ,蓄积明显减少。PKC激活剂佛波乙酯使耐药株的药物外排增加了 60 % ,蓄积减少了 70 % ;PKC抑制剂Stau rosprin使耐药株药物外排后潴留增加了 2 5倍 ,蓄积增加了 14倍 ,抗药性被逆转了 14倍 ,但未影响耐药膜糖蛋白的表达。结论 :PKC活性增高与耐药株抗药性密切相关 ;而且抗药性是通过膜糖蛋白磷酸化增强 ,增加药物外排 ,减少药物蓄积而实现的     

牛精子蛋白激酶C抑制剂逆转KB/VCR的作用及其机理

目的研究牛精子蛋白激酶C抑制剂(PKCI)对肿瘤细胞多药耐药性的逆转作用及机理.方法选用KB细胞及其耐长春新碱的耐药株(KB/VCR)测定其蛋白激酶C活性及PKCI的影响,用MTT法测药敏,用流式细胞仪测定细胞对罗丹明123的蓄积及外排.结果耐药细胞细胞膜的蛋白激酶C活性是亲本细胞的2.9倍,耐药性是亲本细胞的172倍.经PKCI作用后,罗丹明123的蓄积量增加了6.8倍,外排明显延缓,外排后潴留增加了4.3倍,耐药性被逆转了10倍.PKCI对亲本株的蛋白激酶C活性和耐药性影响不大.结论PKCI可明显地逆转耐药细胞的多药耐药性,比PKC的相对特异性抑制剂H7作用略强.     

水飞蓟宾对口腔上皮KB细胞株增殖和侵袭的影响

目的:探讨水飞蓟宾对人口腔上皮样癌细胞KB细胞株增殖和侵袭的影响。方法:用不同浓度的水飞蓟宾和JNK抑制剂sp600125作用于体外培养的KB细胞株,应用CCK-8检测细胞24 h,48 h和72 h存活率;平板克隆形成实验检测KB细胞的增殖情况,Transwell检测KB细胞的侵袭能力,蛋白免疫印迹实验分析p-JNK,VEGF和MMP-9 蛋白的表达水平。结果:CCK-8结果显示水飞蓟宾对KB细胞株的增殖具有明显的抑制作用,且呈时间剂量依赖性;平板克隆实验表明,水飞蓟宾显著抑制KB细胞的集落形成;侵袭试验结果表明,水飞蓟宾能够以剂量依赖的方式抑制KB细胞的侵袭作用;蛋白免疫印迹试验显示水飞蓟宾处理后,JNK的磷酸化水平、VEGF和MMP-9的表达水平显著下调。 JNK抑制剂sp600125作用于KB细胞后,VEGF和MMP-9蛋白的表达水平明显下调,且集落的形成和侵袭均受到了明显的抑制。结论:水飞蓟宾具有明显的抑制KB细胞增殖和侵袭的作用;其作用机制可能是通过抑制JNK通路,进而下调VEGF和MMP-9的表达。     

吸烟个体血清在具核梭杆菌侵入 KB 细胞及分泌 MMP-1中的作用

目的：探讨慢性牙周炎患者中吸烟个体血清在具核梭杆菌（Fn）侵入 KB 细胞及在侵入过程中对 KB 细胞分泌基质金属蛋白酶-1（MMP-1）的作用。方法：提取慢性牙周炎吸烟个体10例（Y 组）、慢性牙周炎非吸烟个体10例（N 组）、牙周健康非吸烟个体5例（H 组）的血清。分别将各组血清200、400、800μl 添加至 Fn 感染 KB 细胞模型,作用24 h 后计数细菌菌落。用人酶联免疫吸附测定试剂盒检测各组上清液中 MMP-1的浓度。用单因素方差分析进行数据分析。结果：3组800μl血清引起 Fn 侵入 KB 细胞的侵入率（％）分别为12．59±1．27、8．03±0．075和7．99±0．14（P ＜0．05）,KB 细胞分泌 MMP-1的浓度（μg/L）分别为400．04±21．02、252．57±18．89、262．47±35．29（P ＜0．05）。200μl 和400μl 血清组与 N 组、H 组比较, Fn 侵入率及 KB 细胞 MMP-1分泌量无统计学差异（P ＞0．05）。结论：较高剂量吸烟个体血清有利于 Fn 侵入 KB 细胞并促进KB 细胞分泌 MMP-1。     

羟乙基淀粉130/0.4对内毒素诱导的大鼠血浆炎症因子表达和单个核细胞核转录因子活性的影响

目的 观察羟乙基淀粉130/0.4(HES 130/0.4)对内毒素腹腔注射大鼠炎症因子表达的影响,并通过其对单个核细胞核因子-、κB(NF-κB)和激活蛋白-1(AP-1)活性的影响探讨相关分子机制.方法 36只雄性Sprague-Dawley大鼠随机分为6组(n=6),对照组静脉输注生理盐水30 ml/kg,LPS组腹腔注射脂多糖(LPS)5 mg/kg后静脉输注生理盐水30 ml/kg,LPS HES组按照HES 130/0.4的剂量不同分为3个亚组,分别腹腔注射LPS 5 mg/kg,接着分别静脉输注HES 130/0.4 7.5、15、30 ml/kg,HES组静脉输注HES 130/0.4 30 ml/kg.在腹腔注射LPS 3 h后以ELISA法检测血浆肿瘤坏死因子-α(TNF-α)、白细胞介素-10(IL-10)、干扰素-γ(IFN-γ)浓度,用凝胶电迁移法检测NF-κB和AP-1的水平.结果 腹腔注射LPS后大鼠血浆TNF-α、IL-10和IFN-γ的浓度较对照组明显升高,且NF-κB和AP-1的活性明显增加(P＜0.05);HES 130/0.4静脉输注可降低血浆TNF-α和IFN-γ的浓度以及NF-κB和AP-1的活性(P＜0.05),而增加IL-10的浓度(P＜0.05),其中15 ml/kg剂量组对TNF-α、IFN-γ和NF-κB的作用最强(P＜0.01),而7.5 ml/kg剂量组对IL-10和AP-1的作用最强(P＜0.01).结论 HES 130/0.4可降低内毒素注射后促炎因子的表达,增强抗炎因子的表达,可能与其抑制转录因子NF-κB和AP-1的活性有关.     

Determination of the potency and subunit-selectivity of ribonucleotide reductase inhibitors with a recombinant-holoenzyme-based in vitro assay

Ribonucleotide reductase (RR) is an important therapeutic target for anticancer drugs. The structure of human RR features a 1:1 complex of two homodimeric subunits, hRRM1 and hRRM2. p53R2 is a newly identified homologue of hRRM2. We have devised a holoenzyme-based in vitro assay for the determination of the potency and subunit-selectivity of small-molecule inhibitors of RR. The assay was implemented using two forms of recombinant RR (hRRM2/hRRM1 and p53R2/hRRM1) and based on their [(3)H]CDP reduction activity. Hydroxyurea was used to standardize the assay. We found that the activities of hRRM2/hRRM1 and p53R2/hRRM1 were decreased by hydroxyurea in a dose-dependent manner. The -NH-OH segment of hydroxyurea was shown to be essential for inhibition. In the presence of Fe(III) and reductants, less inhibition of enzymatic activity by hydroxyurea was observed, especially for p53R2/hRRM1. The potency of four hydroxyurea analogues (Schiff bases of hydroxysemicarbazide, SB-HSC) decreased in the order SB-HSC 21 > SB-HSC 24 > SB-HSC 2 > hydroxyurea (HU) > SB-HSC 29. SB-HSC 2 and SB-HSC 24 inhibited p53R2/hRRM1 significantly more than hRRM2/hRRM1, whereas SB-HSC 21 and SB-HSC 29 showed low subunit-selectivity. Electron paramagnetic resonance (EPR) measurements showed that inhibition of RR was accompanied by reduction of its tyrosyl radical. The method was validated by comparison with data obtained using cell-based assays. We suggest that this novel recombinant-holoenzyme-based in vitro assay is a useful tool for the discovery of more potent and subunit-selective inhibitors of RR.     

Evaluation of anticancer activity of the alkaloid fraction of Alstonia scholaris (Sapthaparna) in vitro and in vivo

The anticancer effect of various doses of an alkaloid fraction of Sapthaparna, Alstonia scholaris (ASERS), was studied in vitro in cultured human neoplastic cell lines (HeLa, HepG(2), HL60, KB and MCF-7) and in Ehrlich ascites carcinoma bearing mice. Treatment of HeLa cells with 25 microg/mL ASERS resulted in a time dependent increase in the antineoplastic activity and the greatest activity was observed when the cells were exposed to ASERS for 24 h. However, exposure of cells to ASERS for 4 h resulted in 25% viable cells and hence this time interval was considered to be the optimum time for treatment and further studies were carried out using this time. Treatment of various cells with ASERS resulted in a concentration dependent decline in the viable cells and a nadir was reached at 200 microg/mL in all the cell lines studied. The IC50 was found to be 5.53, 25, 11.16, 10 and 29.76 microg/mL for HeLa, HePG2, HL60, KB and MCF-7 cells, respectively. Similarly, administration of ASERS, once daily for 9 consecutive days to the tumor bearing mice caused a dose dependent remission of the tumor up to 240 mg/kg body weight, where the greatest antitumor effect was observed. Since 240 mg/kg ASERS showed toxic manifestations, the next lower dose of 210 mg/kg was considered as the best effective dose, in which 20% of the animals survived up to 120 days post-tumor-cell inoculation as against no survivors in the saline treated control group. The ASERS treatment resulted in a dose dependent elevation in the median survival time (MST) and the average survival time (AST) up to 240 mg/kg ASERS and declined thereafter. The surviving animals were healthy and disease free. The effect of ASERS was better than cyclophosphamide, which was used as a positive control, where all the animals succumbed to death by 40 days and the MST and AST were 19.5 and 18.3 days, respectively. The effective dose of 210 mg of ASERS was 3/10 of the LD50 dose, which increased the MST and AST up to 54 and 49.5 days, respectively.     

核因子-κB与眼科疾病的研究进展

核因子-κB(nuclearfactor-kappaB,NF-κB)是一个广泛存在于细胞中具有多向调节作用的核转录因子,在细胞生长分化、增殖、凋亡、免疫应答、炎症反应等生理及病理过程中发挥重要的调节作用。NF-κB的活化是炎症反应的中心环节,NF-κB的过度活化与许多急慢性炎性相关疾病的发生发展密切相关。近年的研究显示NF-κB在眼科多种疾病的发病机制中占有重要的地位,尤其是眼表疾病、青光眼、葡萄膜病、白内障、眼底病等,针对NF-κB活性的抑制可能成为眼科新型药物的治疗靶点。本文将对NF-κB与眼科相关疾病的研究报道进行综述。