辣椒素对家兔窦房结起搏细胞的电生理效应

目的:研究辣椒素对离体家兔窦房结起搏细胞的电生理效应及其作用机制。方法:应用经典玻璃微电极方法。结果:辣椒素(10 μmol/L)使窦房结起搏细胞的零相最大上升速度(V_(max))由(2.4±0.5)V/s降至(1.7±0.2)V/s(P<0.05);舒张期除极速度(VDD)由(91±34)mV/s降至(70±30)mV/s(P<0.01);起搏放电频率(RPF)由(186±14)beat/min降至(162±10)beat/min(P<0.01);最大舒张电位(MDP)绝对值由(49±3)mV降至(44±2)mV(P<0.01);动作电位幅度(APA)由(55±4)mV降至(49±4)mV(P<0.05)。复极化90％时间(APD_(90))则由(149±21)ms延长至(167±27)ms(P<0.01)。应用辣椒素受体阻断剂钌红(10 μmol/L)对辣椒素的上述电生理效应无影响。提高灌流液中钙离子浓度(5 mmol/L)以及应用L型钙通道开放剂Bay-K-8644(0.5μmol/L)均可抑制辣椒素对起搏细胞的电生理效应。β-肾上腺素能受体激动剂异丙肾上腺素(20 nmol/L)可逆转辣椒素所引起的除极化时间延长和MDP的降低。结论:辣椒素对家兔窦房结细胞有负性变时作用,这些效应可能与其抑制钙离子内流及/或钾离子外流有关,而非由辣椒素受体介导。     

2-03 氯丙烯对神经细胞NO含量和NOS活性的影响

目的探讨氯丙烯对神经细胞NO生成系统的影响,进一步研究氯丙烯的中毒机制.方法用NO、NOS检测试剂盒(均由南京建成生物工程研究所提供)研究氯丙烯染毒后(剂量依次为0.30、0.61、1.23mmol/L)的鸡胚脑神经细胞的NO含量、NOS活性,并与阴性对照组进行比较.结果随着氯丙烯浓度的增高,鸡胚脑神经细胞内NO含量明显增高,低、中、高3个剂量组依次为(4.63±0.37)、(4.65±0.19)、(5.95±0.223)μmo1/L.与对照组相比(3.17±0.89)μmol/L),均有显著增高(P＜0.01);细胞培养液中无明显改变(P＞0.05).细胞内,中、高剂量组NOS活性分别为(4.85±1.51)、(4.88±1.05)U.mg.protein-1,与对照组[(3.93±0.86)U.mg.protein-1]相比明显增高(P＜0.05);细胞外仅高剂量组差异有显著性[高剂量组为(19.96±3.32)U/ml,阴性对照组为(16.22±2.32)U/ml,P＜0.01].结论氯丙烯可引起神经细胞NO含量和NOS活性的增加,与我们以前报道的氯丙烯可引起细胞内Ca2+增高的结果相吻合.这可能由于细胞内Ca2+增高,激活cNOS,使其活性增强,导致NO生成增加.     

氟哌啶醇季铵盐衍生物抑制氯化钾所致大鼠主动脉平滑肌细胞内钙浓度增加

目的:动态观察氟哌啶醇季铵盐衍生物(F_3)对血管平滑肌细胞内钙浓度的影响。方法:利用激光共聚焦显微镜动态观察F_3(0.o1-10μmol/L)对由KCl(30mmol/L)诱导的大鼠血管平滑肌细胞钙荧光强度增加的作用。结果:KCl可诱发细胞内钙荧光强度迅速增强,F_3可以拮抗由KCl诱导的细胞内钙荧光强度增强作用,并呈量效依赖性和时间依赖性,终强度(KCl:67±24;F_3 0.o1μmol/L:57±13;0.1μmol/L:40±13;1μmol/L:29±9;10μmol/L:20±6)。在加入F_3后,钙荧光强度的变化最快时程是在给F_3后0-30s。结论:F_3拮抗血管收缩主要是由于阻断了Ca~(2 )通道。     

硒化壳聚糖促进体外培养皮肤成纤维细胞增殖

目的研究硒化壳聚糖对体外培养的皮肤成纤维细胞增殖的影响。方法用不同剂量（25、50、100、200、400mg/L）硒化壳聚糖作用于成纤维细胞,光镜下观察药物对细胞形态的影响;MTT法、3H-TdR掺入法、细胞生长动力学研究用于检测药物对细胞增殖影响,LDH漏出率检测药物对细胞增殖及损伤的影响,并以等体积细胞培养液处理为对照组。结果各药物浓度组处理细胞后,细胞形态未见明显改变,均可使细胞吸光度值增加,细胞倍增时间缩短,促进表皮细胞对3H-TdR的掺入（P〈0.05）,降低LDH漏出率（P〈0.05）。结论硒化壳聚糖对体外培养皮肤成纤维细胞增殖具有促进作用。     

吡格列酮对脂多糖引起大鼠大脑皮质神经元损伤的抑制作用

目的探讨吡格列酮对脂多糖(LPS)引起的神经元损伤的抑制作用,并探讨其信号传导机制。方法取培养7d的大鼠大脑皮质神经元细胞,除正常对照组外,其余各组先给予相应的处理30min～1h后,再加LPS10mg.L-1处理4～24h。MTT法测定细胞存活率;Hoechst33258核染色观察细胞凋亡的形态学改变;免疫荧光染色法测定磷酸化c-Jun氨基端激酶1(JNK1)细胞内定位;Western蛋白印迹法检测活性胱天蛋白酶3(caspase3)蛋白表达和磷酸化JNK1水平;Griess法测定细胞培养上清液中一氧化氮(NO)含量。结果与对照组相比,LPS可使体外培养神经元细胞存活率明显下降,(100.0±10.9)%vs(72.3±2.1)%;凋亡细胞百分率明显增加,(11.5±4.2)%vs(39.5±8.2)%;活性caspase3蛋白表达和磷酸化JNK1水平增加;细胞培养上清液中NO含量明显增加。吡格列酮0.01,0.1和1μmo.lL-1可明显对抗LPS引起的培养神经元细胞存活率下降,并呈一定浓度依赖性。吡格列酮0.1和1μmol.L-1也可明显对抗LPS引起的培养神经元凋亡细胞百分率、活性caspase3蛋白表达、磷酸化JNK1水平和NO含量增加。LPS+吡格列酮(1μmo.lL-1)组,细胞存活率为(97.8±9.7)%,凋亡细胞百分率为(20.6±5.0)%,NO含量为(6.8±1.3)μmol.L-1。过氧化物酶体增殖物激活受体γ(PPARγ)的特异性阻断剂GW9662不能去除吡格列酮对LPS引起的神经元细胞损伤的抑制作用,在LPS+吡格列酮(1μmol.L-1)+GW9662(10μmo.lL-1)组,细胞存活率为(90.7±6.9)%,凋亡细胞百分率为(23.4±4.1)%,NO含量为(5.8±0.7)μmol.L-1。GW966210μmol.L-1对LPS引起的细胞存活率降低没有影响。与LPS组相比,JNK特异性阻断剂SP6001255μmol.L-1可明显对抗LPS引起的神经元损伤,细胞存活率明显增加,(72.3±2.1)%vs(109.8±11.8)%;凋亡细胞百分率降低,(39.5±8.2)%vs(19.1±4.8)%;NO含量降低,(21.1±5.0)μmol.L-1vs(4.0±1.3)μmol.L-1。结论吡格列酮能明显抑制LPS引起的皮质神经元损伤,这种作用可能与抑制JNK信号传导通路有关,与PPARγ激活无关。     

肝硬化患者一氧化氮与丙氨酸转氨酶和自发性腹膜炎相互关系的研究

目的:观察肝硬化患者血浆一氧化氮(NO)和丙氨酸转氨酶(ALT)的变化,并分析其相关性;以及腹水一氧化氮(NO)与自发性腹膜炎的相关性。方法:取肝硬化患者血浆和腹水,用解放军三医大临床微生物研究室NO检测试剂盒检测NO,全自动生化仪(日立,7060)检测ALT。结果:伴有自发性腹膜炎肝硬化患者与单纯腹水型血浆NO分别为(21.10±11.23)μmol/L、(19.56±8.69)μmol/L;腹水NO分别为(22.45±15.51)μmol/L、(18.72±8.11)μmol/L;ALT各为(268.41±165.02)u/L、(246.82±133.51)u/L。与正常对照组比较,两组NO水平升高均较显著,P<0.01,且与ALT呈正相关。结论:肝硬化患者内源性NO水平随病情进展而升高,并与ALT水平有一定的相关性。腹水中的NO浓度能较好的反映血中的NO浓度,但与自发性腹膜炎程度无明确相关性;故血清或腹水中NO水平不能为腹膜炎的发生提供有价值的线索。     

高血压患者血尿酸检测的临床意义分析

目的探讨血尿酸检测在高血压患者中的临床意义。方法选择2008年1月至2010年12月本院住院的高血压患者130例作为研究对象,空腹12h,清晨进行血液标本采集,采用酶学法用日立全自动化生化分析仪7600测定血尿酸浓度,观察高UA血症发生率,比较高UA与UA正常患者生化指标及预后差异。结果 130例高血压患者UA平均值为(471.22±80.34.39)μmol/L,高UA血症患者70例(53.84%),UA(531.69±87.34)μmol/L,UA正常患者60例(46.16%),(364.34±56.22)μmol/L。高UA血症组TC(6.23±1.30)mmol/L、TG(3.65±0.49)mmol/L、Cr(131.12±16.69)μmol/L水平高于正常UA血症组(4.24±0.68)mmol/L、(2.39±0.37)mmol/L、(102.53±13.56)μmol/L(P<0.05);随访(3.11±1.23)年,发现高UA血症组冠心病70.00%、脑血管意外41.43%、病死率17.71%高于正常UA血症组50.00%、26.67%、5.00%(P<0.05)。结论高尿酸血症是动脉硬化的危险因素之一,增高心脑血管事件发生率,对预后有不良影响,对高血压患者进行常规的UA监测有利于采取早期干预措施。     

单纯性肾病综合征患儿血中NO与ET的测定

目的:了解小儿肾病综合征发病过程中血浆中NO,ET浓度的变化特点。方法:采用分光光度法及放射免疫分析法检测了27例单纯性肾病综合征患儿及20例健康对照儿童血浆中NO,ET浓度。结果:血浆NO,NOS,ET在肾病患儿,正常对照组分别为(44.90±9.74μmol/L,13.84±3.44 U/mL,194.41±46.42 pg/ml);(80.46±13.43μmol/L,32.20±3.80U/mL,79.02±13.37pg/ml)。肾病患儿NO,NOS值显著降低(P<0.01),ET值显著增高。NO与NOS呈正相关(r=0.8798),NO与ET呈负相关(r=-0.6965)。结论:血浆NO,ET的浓度变化在肾病综合征患儿的病理生理过程中起着一定作用。     

甘氨酸-锌和SAC对人外周淋巴细胞DNA合成的激发作用

观察了不同浓度Zn-G络合物及其与SAC合用对培养外周全血淋巴细胞DNA合成的影响。结果表明,Zn-G(9～4.5)×10~(-5)mol/L时,[~3H]-TdR掺入淋巴细胞DNA的cpm和SI均显著增高(分别为P<0.025及P<0.001),而高浓度3.6×10~(-4)mol/L对淋巴细胞DNA的掺入出现明显抑制作用(P<0.05),阐明合适浓度Zn-G有明显激活淋巴细胞DNA的合成作用。单用SAC(10Dmg/L,400mg/L)培养时,[~3H]-TdR掺入B淋巴细胞DNA的两项参数均随浓度而增高,尤以SAC(100mg/L)与9×10~(-5)mol/L Zn-G合用时,比单用SAC激活B淋巴细胞DNA合成更强(3.78±0.33>2.23±1.27SI,P<0.05).本研究表明Zn-G参与SAC刺激活化B细胞增殖反应,具有明显的协同效应。     

罗格列酮治疗前后糖尿病并发冠心病患者纤溶系统的变化

目的观察罗格列酮治疗前后糖尿病(DM)并发冠心病(CHD)患者纤溶系统的变化。方法 2型糖尿病合并冠心病患者100例随机分为常规治疗组(A组)和罗格列酮治疗组(B组)检测2组治疗前后一氧化氮(NO)、组织型纤溶酶原激活物(t-PA)和纤溶酶原激活物抑制剂(PAI-1)的含量变化。结果 A组、B组治疗后NO、t-PA浓度[A组:(62.76±7.25)μmol/L、(32.75±9.36)μg/L;B组:(69.86±5.97)μmol/L、(40.62±10.56)μg/L]均显着高于治疗前[A组:(42.73±6.14)μmol/L、(20.83±8.13)μg/L;B组:(41.76±6.65)μmol/L、(19.93±7.87)μg/L](t=2.545,t=2.911,P均<0.05);A组、B组治疗后PAI-1浓度[(62.76±7.25)μg/L、(51.01±5.09)μg/L]均显着低于治疗前[(88.23±9.91)μg/L、(89.46±9.15)μg/L](t=2.445,t=2.411,P均<0.05);B组治疗后PAI-1(51.01±5.09)μg/L明显低于A组(62.61±7.63)μg/L(t=2.589,P<0.05)。结论罗格列酮具有抗动脉粥样硬化作用对糖尿病并发冠心病患者是一个较好的治疗方法。     

芦荟凝胶对人皮肤成纤维细胞的增殖作用及其机制研究

目的研究芦荟凝胶对人皮肤成纤维细胞增殖的影响及其作用机制。方法体外培养人皮肤成纤维细胞,MTT法测定芦荟凝胶作用后人皮肤成纤维细胞的增殖能力,用实时荧光定量PCR法检测Ⅰ、Ⅲ型胶原mRNA水平,用免疫蛋白印迹法检测Ⅰ、Ⅲ型胶原蛋白的表达。结果 MTT检测结果表明,芦荟凝胶对人皮肤成纤维细胞具有促增殖作用,且呈现时间依赖性和剂量依赖性。芦荟凝胶作用于人皮肤成纤维细胞72h后,Ⅰ、Ⅲ型胶原mRNA及蛋白的表达显著增加(P<0.05,P<0.01)。结论芦荟凝胶可通过促进人皮肤成纤维细胞增殖以及Ⅰ、Ⅲ型胶原mRNA和蛋白的表达加速创面愈合。     

Ex vivo evaluation of prolidase loaded chitosan nanoparticles for the enzyme replacement therapy.

Prolidase loaded chitosan nanoparticles were set up in order to suggest an innovative therapeutic approach for Prolidase Deficiency (PD), a rare autosomal inherited disorder of the connective tissue. The satisfactory drug loading efficiency (42.6+/-2.1%) as well as the suitable physical characteristics (mean diameter of 365.5+/-35.1nm and a positive zeta-potential of 17.94+/-0.12mV) was achieved. In order to verify the compatibility of the chitosan nanoparticles with cells, the influence of the nanoparticles on the growth and the viability (MTT assay) of cultured skin fibroblasts were determined: the nanoparticles showed a good biocompatibility up to 5mug of chitosan/10,000 fibroblasts. Uptake of chitosan nanoparticles by fibroblasts was verified by confocal microscopy using FITC-labelled chitosan nanoparticles. The ex vivo experiments were performed by incubating different amounts of prolidase loaded chitosan nanoparticles with skin human fibroblasts from PD patients for scheduled times. The restored prolidase activity was quantitatively monitored by a capillary electrophoretic method and confirmed by cells morphological observations. Standing from the nanoparticles internalization, the enzymatic activity was progressively restored reaching the best value (about 66%) after 5 days of co-incubation. Moreover, prolidase loaded chitosan nanoparticles permitted to restore prolidase activity in PD fibroblasts for a prolonged period of time (8 days).     

Calyculin A from Discodermia Calyx Is a Dual ActionToxin that Blocks Calcium Influx and Inhibits Protein Ser/Thr Phosphatases

Calyculin A (Caly A) is cell permeable toxin widely used in cell biology research as an inhibitor of type 1 and type 2A protein Ser/Thr phosphatases of the PPP family. Here we tested effects of low concentrations of Caly A on proliferation of human cancer and non-cancer cell lines. We found that long-term 0.3 nM Caly A prevented G1 to S phase cell cycle progression in human Hs-68 fibroblasts and ARPE19 epithelial cells, but not human breast cancer MDA-MB-468, MDA-MB-231 and MCF7 cells. These conditions produced no change in cyclin D1 levels or in the phosphorylation of endogenous proteins. However, acute application of 0.3 nM Caly A blocked serum-induced increase in intracellular calcium levels in Hs-68 fibroblasts, but not in MDA-MB-468 breast cancer cells. We propose that subnanomolar Caly A prevents cell cycle progression because it blocks calcium uptake by fibroblasts. This probably involves non-selective cation channels and cancer cell proliferation was not affected because calcium enters these cells by other channels. Our results suggest that calyculin A has dual actions and acts as a channel blocker, in addition to its well-established effects as a phosphatase inhibitor.     

芦荟大黄素对硬皮病成纤维细胞增殖的影响

目的探讨芦荟大黄素对系统性硬皮病患者皮肤成纤维细胞增殖的影响。方法以不同浓度的芦荟大黄素处理体外培养的成纤维细胞,显微镜下观察成纤维细胞生长状况并摄片;用四甲基偶氮唑盐（MTT）方法测定芦荟大黄素对成纤维细胞增殖的影响。结果荟大黄素对硬皮病成纤维细胞的增殖具有显著的抑制作用。结论芦荟大黄素可显著的抑制硬皮病成纤维细胞的生长,降低成纤维细胞的增殖指数并诱导其调亡。     

Involvement of reactive oxygen species in aclarubicin-induced death of human trisomic and diabetic fibroblasts.

We investigated the mode of cell death induced by aclarubicin in human trisomic and diabetic fibroblasts. The cells were incubated with aclarubicin for 2 h and then were cultured in drug-free medium for up to 96 h. Aclarubicin in trisomic and diabetic fibroblasts, compared with normal cells, induced lesser changes in the level of reactive oxygen species (ROS) and the content of intracellular calcium. The drug induced ROS-mediated apoptotic and necrotic pathways in all cell lines. The extent of apoptosis and necrosis was strongly dependent on the cell line, sensitivity to drug and post-treatment time. These results indicate that most resistant diabetic cells died prevalently by apoptosis. In the case of trisomic fibroblasts, the number of apoptotic cells decreased with post-incubation time. The role of reactive oxygen species in aclarubicin-induced cell death was confirmed by the diminution effects of antioxidants (N-acetylcysteine and pyrrolidine-dithiocarbamate) on drug-induced ROS formation, increase of intracellular calcium and the extent of apoptosis and necrosis in fibroblast cell lines.     

维拉帕米和糖基化对细胞内胆固醇含量的影响

用国产酶联试剂检测培养细胞内微量胆固醇的方法简便、快速、价廉、灵敏度高,以此观察了不同培养条件及糖基化低密度脂蛋白和维拉帕米对细胞内胆固醇含量的影响。     

转化生长因子β1对人脐静脉血管内皮细胞活性的影响

目的观察转化生长因子β1（TGF—β1）刺激增生性瘢痕成纤维细胞上清液对人脐静脉血管内皮细胞（HUVEC）增殖和趋化能力的影响。方法以增生性瘢痕成纤维细胞为实验组,正常皮肤为对照组,添加TGF—β1（10μg/L）为TGF—β1组,向TGF—β1组内添加丝氨酸-苏氨酸蛋白激酶（Akt）阻断剂Tricirlbine（5Ixmol／L）为Akt阻断剂组,二者皆未添加的瘢痕成纤维细胞为非干扰组,利用各组细胞上清液（条件培养基）培养HUVEC,使用四甲基偶氮唑盐（MTT）法观察各条件培养基对HUVEC增殖的影响,并利用细胞划痕实验和Transwell细胞迁移实验对HUVEC的趋化情况进行评估。结果对照组吸光度值为0．91±0．34,非干扰组1．75±0．15,TGF—B,组3．26±0．58,Akt阻断剂组1．14±0．81。非干扰组吸光度值与对照组和TGF-β1组比较,差异均有统计学意义（均P〈0．01）;而Akt阻断剂组成纤维细胞增殖吸光度值明显低于TGF—β1组（P〈0．01）。细胞划痕24h后,TGF—β1组促使HUVEC划痕的闭合率较非干扰组高（P〈0．01）;而Akt阻断剂组则明显减弱HUVEC划痕闭合的速度（P〈0．01）。Transwell细胞迁移实验中,TGF—β1组迁移的细胞较非干扰组多[TGF—β1组细胞迁移数目：（1718±15）个,非干扰组：（829±2）个,P〈0．01];而Akt阻断剂组阳性细胞迁移率又明显降低[Akt阻断剂组：（935±11）个,P〈0．01]。对照组血管内皮生长因子（VEGF）（65．2±0．3）ng／L,非干扰组（87．0±0．5）ng／L,TGF—β1组（132．7±0．4）ng／L,Akt阻断剂组（70．5±0．6）ng／L。非干扰组与对照组、TGF-β1组与非干扰组、Akt阻断剂组与TGF—β1组比较,差异均有统计学意义（均P〈0．01）。结论TGF—β1可以通过刺激增生性瘢痕成纤维细胞旁分泌VEGF等细胞因子对血管内皮细胞增殖、趋化等能力产生影响。     

Lecithin/chitosan nanoparticles for transdermal delivery of melatonin

In this study, the potential of lecithin/chitosan nanoparticles (NPs) as colloidal nanosystem for transdermal melatonin delivery was investigated. Mean diameter and zeta-potential of NPs differing in lecithin type (Lipoid S45 and S100) and chitosan content ranged between 113.7 and 331.5?nm and 4.6 and 31.2?mV, respectively. Melatonin loadings were up to 7.2%. The potential of lecithin/chitosan NPs to enhance transdermal melatonin delivery was investigated by determining the drug flux across dermatomed porcine skin and its skin deposition. Lecithin/chitosan NPs provided 1.3-2.3-fold higher flux compared to melatonin solution. The highest flux, 9.0?±?0.21?μg/cm2/h, was observed for S45 lecithin/chitosan NPs with lecithin/chitosan weight ratio of 20:1. NP possible cytotoxicity in vitro was evaluated using human skin keratinocytes and fibroblasts. It was demonstrated that lecithin/chitosan NPs can be applied to skin cells at concentrations up to 200?μg/mL without inducing plasma membrane damage or cell viability decrease.     

Mibefradil-induced inhibition of proliferation of human peripheral blood mononuclear cells

To evaluate the role of intracellular calcium and particularly Ca2+-uptake in the initiation of lymphocyte mitogenesis, the effect of mibefradil, which blocks both L- and T-type calcium channels with a more selective blockade of T-type channels, on the proliferation of human peripheral blood mononuclear cells (PBMCs) is compared with the effect of nifedipine, which blocks only the L-type calcium channel. The rate of [3H]thymidine incorporation into control and concanavalin A-stimulated PBMCs in the presence or absence of the calcium channel blockers mibefradil or nifedipine (1, 10, or 50 microM), and of the intracellular calcium antagonist 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester (TMB-8; 1, 10, 25, or 50 microM) was assayed in the cells cultured for 3 days. The cellular cytotoxicity and the cell number in growing cultures also was determined in mibefradil- or nifedipine-treated control or stimulated cells. Restoration of the proliferative response in mibefradil- or nifedipine-treated cells was investigated by addition of exogenous interleukin-2. Interleukin-2-receptor expression in the cells was monitored by using anti-activated T-cell antigen (Tac) antibody, and the interleukin-2 production in the cell supernatants of the cultures was determined by an enzyme-amplified sensitive immunoassay. Mibefradil and nifedipine concentration-dependently reduced the cell number and the [3H]thymidine incorporation or the de novo DNA synthesis in control and concanavalin A-stimulated human PBMCs. Mibefradil exhibited a more pronounced inhibition of the proliferation of human PBMCs than did nifedipine. The inhibitory effect of mibefradil or nifedipine on DNA synthesis was dependent on the timing of treatment with the drugs. The inhibitory effect of mibefradil or nifedipine on the lymphoproliferative response was nearly abolished if the drugs were added 20 h after cell stimulation. A markedly reduced inhibitory effect was found when mibefradil or nifedipine was added 1-7 h after cell stimulation. However, regardless of time of addition, TMB-8 caused a persistent inhibition of the proliferation of human PBMCs. The inhibitory effect of mibefradil or nifedipine on the proliferation of human PBMCs is nearly abolished by addition of the calcium channel activator Bay K 8644. The proliferative response of mibefradil- or nifedipine-treated cells is restored by addition of exogenous interleukin-2. The normal expression of interleukin-2 receptors was preserved, whereas the interleukin-2 production was blocked in the presence of mibefradil or nifedipine. Our data show that mibefradil has a more pronounced inhibitory effect on the proliferation of human PBMCs than nifedipine and that this inhibitory effect on DNA synthesis is dependent on the timing of treatment with both drugs.