d-宁烯、丹参及姜黄素衍生物对ras基因产物膜结合和细胞间隙信息传导的影响

目的旨在寻找新型抗肿瘤药物，进一步研究ｄ宁烯、丹参及姜黄素衍生物的抗肿瘤机理。采用分子生物学方法及划痕标记染料示踪技术，研究了４种人实体瘤起源的细胞系的细胞间隙信息传导（ＧＪＩＣ）、Ｈｒａｓ癌基因表达以及ｒａｓ癌基因产物（Ｐ２１ｒａｓ蛋白）表达状态，并观察了４种天然产物对它们的影响。结果表明，细胞内染料传输功能的丧失与ｒａｓ基因突变率呈正相关；单萜化合物ｄ宁烯和酚类化合物丹参衍生物的抗肿瘤作用可能与抑制Ｐ２１ｒａｓ蛋白膜结合和增强细胞间隙信息传导功能有关。提示Ｒａｓ癌基因产物Ｐ２１ｒａｓ蛋白膜结合的抑制与细胞间隙信息传导功能的增强有直接关系     

乌骨藤中C21甾体苷诱导SACC-83和SACC-LM细胞凋亡的作用及其机制研究

目的 研究乌骨藤提取物C₂₁甾体苷对唾液腺腺样囊性癌（salivary adenoid cystic carcinomacv,SACC）低侵袭细胞株（salivary adenoid cystic carcinoma-83,SACC-83）和肺高转移细胞株（SACC-LM）增殖抑制和诱导凋亡的作用及其机制。方法 用不同浓度（5,10,20,40,60,80,100 μmol·L^-1） C₂₁甾体苷处理SACC-83和SACC-LM细胞48 h后,MTT法检测细胞活力,并计算药物的IC₂₀和IC₅₀;细胞克隆形成实验检测细胞增殖能力;流式细胞术检测SACC-83及SACC-LM细胞凋亡情况;实时荧光定量PCR和Western blot检测SACC-83和SACC-LM细胞Bcl-2、Bax、caspase 3的mRNA和蛋白的表达。结果 不同浓度的C₂₁甾体苷降低SACC-83和SACC-LM细胞活力,抑制细胞增殖,并且作用于SACC-83细胞C₂₁甾体苷的IC₂₀浓度为7.49 μmol·L^-1,IC₅₀浓度为38.34 μmol·L^-1;作用于SACC-LM细胞的C₂₁甾体苷IC₂₀浓度为9.30 μmol·L^-1,IC₅₀浓度为46.04 μmol·L^-1;细胞克隆集落形成明显减少。C₂₁甾体苷IC₂₀浓度分别促进SACC-83及SACC-LM细胞凋亡,且随着给药时间延长,凋亡率增加,具有显著性差异（P<0.05,P<0.01）。经7.49,9.30 μmol·L^-1 C₂₁甾体苷分别处理SACC-83及SACC-LM细胞后,Bcl-2的mRNA及蛋白水平显著降低（P<0.01）,而Bax、Caspase 3的mRNA和蛋白水平显著升高（P<0.05）。结论 乌骨藤C₂₁甾体苷抑制SACC-83及SACC-LM细胞增殖、促进凋亡,其作用机制可能与调控Bcl-2、Bax和Caspase 3表达有关。     

丹参柯巴基焦磷酸合酶基因的优化表达、纯化及抗体制备

用含丹参柯巴基焦磷酸合酶(Salvia miltiorrhiza copalyl diphosphate synthase，SmCPS)基因的重组质粒pET32CPS转化大肠杆菌BL21trxB (DE3)进行诱导表达，SDS-PAGE结果表明SmCPS基因在大肠杆菌中获得了表达；对影响可溶性表达的4个因素，即诱导温度、IPTG诱导浓度、诱导时宿主菌的密度(A₆₀₀)和诱导时间进行了优化。结果发现在宿主菌A₆₀₀达到1.0时加入0.4 mmol·L^-1 IPTG，在20 ℃诱导培养8 h表达的可溶性蛋白量最高，约占细胞总蛋白的35.6%。用Ni²⁺亲和色谱法纯化表达产物，纯化蛋白产率达到12.3 mg·L^-1。将纯化蛋白免疫制备兔源SmCPS抗血清，ELISA测定效价为1∶24 300，且经Western blotting鉴定该抗体可特异性识别SmCPS抗原，获得了效价高、免疫反应性好的SmCPS多克隆抗体，为进一步从蛋白水平研究SmCPS表达与丹参酮类有效成分生物合成相关性提供了物质基础。     

诺帝对人恶性胶质瘤细胞U87甲酰化肽受体功能的影响

探讨手性化合物诺帝对人恶性胶质瘤细胞甲酰化肽受体(formylpeptide receptor，FPR)功能的影响及其意义。以培养的人恶性胶质瘤细胞U87为研究对象，用FPR激动剂fMLF(n-formyl-methionyl-leucyl-phenylalanine)刺激细胞，以双室跨膜迁移模型检测细胞的迁移能力、计数法测定细胞的增殖能力、分光光度法测定细胞内钙流、RT-PCR方法测定血管内皮生长因子(vascular endothelial growth factor, VEGF)mRNA表达，以ELISA方法检测VEGF蛋白水平，并测定诺帝(25～100 μmol·L^-1)处理瘤细胞后上述功能指标的变化。50 μmol·L^-1诺帝能有效抑制fMLF诱导的细胞增殖、迁移(P<0.05)和细胞内钙流，100 μmol·L^-1诺帝能抑制VEGF mRNA表达，降低VEGF水平(P<0.05)。诺帝能抑制FPR介导的人恶性胶质瘤细胞增殖、迁移和VEGF产生，提示其具有抗肿瘤活性。     

蝙蝠葛酚性碱对离体大鼠心肌顿抑的保护作用

目的　探讨蝙蝠葛酚性碱(PAMD)对离体大鼠心肌顿抑的作用。方法　采用缺血10min后再灌注30min造成心肌顿抑模型(S) ,灌流液中加0.5mg·mL^-1 PAMD(P)组同样缺血再灌注。结果　灌注末S组LVSP ,+dp/dt_max和-dp/dt_max分别降至预灌末的49% ,53%和58% ,LVEDP则增至422% ;心肌钙含量为(514±142 ) μg·g^-1 (干重) ;出现可逆性心肌超微结构损伤。而P组再灌注末LVSP ,LVEDP ,+dp/dt_max和-dp/dt_max恢复为预灌末值的70% ,205% ,78%和79% ;心肌钙为(316±84) μg·g^-1 (干重) ;以上变化均有显著差异。结论　PAMD对离体大鼠顿抑心肌有保护作用     

5α-△9(11)-16α-甲基-3β,17α,21-三羟基-孕甾烯-3β,21-双醋酸酯-20酮的微生物转化

简单节杆菌A₁及耻垢杆菌MS₁两种菌混合培养转化地塞米松中间体5α-△⁹⁽¹¹⁾-16α-甲基-3μ,17μ,21-三羟基-孕甾烯-3μ,21-双醋酸酯-20酮(Ⅲ)可得到16α-甲基-△^1.4.0(11)-孕甾烯-17α,21-双羟基-3,20双酮(Ⅳ)(65%收率)及少量的16α-甲基-△^1.4孕甾烯-9α,11α-环氧-17α,21双羟基-3,20双酮(Ⅴ)。     

染料木素和槲皮素体外抗肝纤维化作用

目的　研究染料木素和槲皮素对体外肝维化的保护作用。方法　细胞增殖和胶原合成分别用结晶紫染色法和³H-脯氨酸参入法。原胶原mRNA水平用RT-PCR法测定。结果　染料木素(25 - 70 μmol·L^-1 )和槲皮素(6.25- 5 0 μmol·L^-1 )浓度依赖抑制PDGF促HSC-T6细胞增殖和TGFβ₁ 刺激的胶原合成及I型原胶原mRNA的表达。结论　染料木素和槲皮素抑制PDGF和TGFβ₁ 对星状细胞的作用可能对肝纤维化有保护作用     

基于网络药理学和实验验证的丹参-红花药对治疗心肌缺血的作用机制探讨

目的 通过网络药理学和动物实验探究丹参-红花药对治疗心肌缺血的潜在作用和机制。方法 检索中药系统药理学数据库与分析平台（TCMSP）和文献手动补充确定丹参-红花的活性成分及治疗心肌缺血靶点。以“心肌缺血”及“急性心肌缺血”为关键词,在CTD、DisGeNET、GeneCards和OMIM数据库中检索疾病的潜在靶点。利用Venny 2.1.0将筛选得到的有效化学成分靶点与疾病靶点进行交集,确定丹参-红花药对治疗心肌缺血的作用靶点。将交集靶点信息上传至STRING v11.0,构建活性成分-靶点网络及蛋白质相互作用网络（PPI）。通过DAVID数据库对丹参-红花治疗心肌缺血的靶点进行基因本体（GO）功能和京都基因与基因组百科全书（KEGG）通路的富集分析。40只SD雄性大鼠,根据体质量随机分成4组,每组10只,即对照组、模型组、复方丹参滴丸（73 mg·kg^-1·d^-1）组、丹参-红花（生药量16 g·kg^-1·d^-1）组。对照组和模型组大鼠每天ig等量0.9%氯化钠溶液,给药组ig相应剂量的药物,每天1次,共7 d。于第6天,除对照组外,其余各组给药1 h后sc异丙肾上腺素（ISO,85 mg·kg^-1）,连续2 d造模。HE染色观察各组大鼠心肌组织病理变化;蛋白免疫印迹法检测各组大鼠心肌组织磷酸化哺乳动物雷帕霉素靶蛋白（p-mTOR）、磷酸化转录激活蛋白3（p-STAT3）、缺氧诱导因子1α（HIF-1α）、血管内皮生长因子A（VEGFA）、血小板衍生生长因子A（PDGFA）和碱性成纤维细胞生长因子（bFGF）蛋白表达水平。结果 网络药理学筛选得到丹参-红花药对治疗心肌缺血的活性成分91个,靶点246个。PPI网络结果显示主要靶点包括STAT3、MAPK1、JUN、RELA、MAPK3、AKT1、SRC、APP、TNF、PIK3CA、CXCL8、KNG1、IL6、VEGFA、MAPK14、HSP90AA1、MAPK8、EGFR、FOS和IL2等。主要靶点相关的通路涉及血管生成（如HIF-1信号通路和VEGF信号通路）、炎症反应（如NF-κB信号通路和TNF信号通路）和细胞凋亡（如细胞凋亡信号通路和p53信号通路）等。动物实验HE染色结果表明,对照组大鼠心肌细胞正常;模型组大鼠心肌纤维肿胀且有断裂,心肌细胞增宽,局部有明显水肿、渗出、炎性细胞浸润;复方丹参滴丸组与模型组大鼠比较,心肌纤维排列略微紊乱且较窄,病理改变程度有所减轻。丹参-红花组大鼠的心肌纤维排列整齐,细胞形态结构完整,心肌纤维肿胀轻,无毛细血管扩张,接近正常心肌形态。蛋白质免疫印迹检测结果表明,与对照组比较,模型组大鼠心肌组织中HIF-1α、VEGFA、PDGFA和bFGF的蛋白表达水平显著升高（P＜0.05、0.01）,p-mTOR和p-STAT3的蛋白表达水平显著下降（P＜0.05、0.01）。与模型组比较,丹参-红花显著上调了p-mTOR、p-STAT3、HIF-1α、VEGFA、PDGFA和bFGF的蛋白表达水平（P＜0.05、0.01）。结论 丹参-红花可能通过上调与血管生成密切相关的蛋白水平来促进急性心肌缺血模型大鼠缺血心肌组织的血管生成,从而改善其缺血性损伤。     

硫代秋水仙碱衍生物的合成和抗肿瘤活性

目的寻找高效低毒的秋水仙碱抗肿瘤衍生物。方法秋水仙碱首先被转化为硫代秋水仙碱，然后硫代秋水仙碱通过水解得到7-(N-脱乙酰基硫代秋水仙碱)，最后经胺的酰化得到目标化合物。用¹H NMR，IR，MS和HR-MS确证了这些衍生物的结构，MTT法评价了目标衍生物的细胞毒性；用对小鼠肝癌H₂₂和宫颈癌U₁₄的抑制率评价了衍生物的体内抗肿瘤活性。结果合成了12个硫代秋水仙碱新衍生物。结论尽管一些硫代秋水仙碱衍生物的体外细胞毒性强于秋水仙碱，然而小鼠体内抑瘤活性却较低。     

2',4'-二羟基-3'-甲基-3-甲氧基查耳酮上调p21抑制人肝癌HepG2细胞的生长

目的 探讨2'',4''-二羟基-3''-甲基-3-甲氧基查耳酮(C20)对人肝癌HepG2细胞的体外抗肿瘤作用及其潜在的作用机制。方法 通过CCK-8法、集落形成实验、5-乙炔基-2''-脱氧尿苷(EdU)染色法检测C20对人肝癌HepG2细胞增殖的影响;通过彗星实验检测C20(10 μmol·L^-1)对HepG2细胞DNA损伤的影响;通过流式细胞术检测C20(5、10 μmol·L^-1)对HepG2细胞周期阻滞的影响;通过Hoechst染色和流式细胞术检测C20(5、10 μmol·L^-1)对HepG2细胞凋亡的影响。借助Western blotting法检测C20(5、10 μmol·L^-1)处理对HepG2细胞中与凋亡、DNA损伤、细胞周期阻滞相关蛋白表达水平的调控作用。结果 与对照组比较,C20显著抑制HepG2细胞的活力(P<0.001),给药48 h的半数抑制浓度(IC₅₀)为7.937 μmol·L^-1;5 μmol·L^-1 C20能够显著抑制HepG2细胞的集落形成能力(P<0.01);EdU染色结果显示5、10 μmol·L^-1的C20能够抑制人肝癌HepG2细胞的增殖能力;5、10 μmol·L^-1的C20显著诱导HepG2细胞G₂/M期阻滞(P<0.001);5、10 μmol·L^-1的C20显著促进HepG2细胞凋亡(P<0.001),并显著上调Caspas-3、Caspase-9以及PARP的剪切水平(P<0.01);10 μmol·L^-1的C20能够诱导HepG2细胞发生DNA损伤,并且5、10 μmol·L^-1的C20显著上调γH2AX、p21的蛋白水平(P<0.01)。结论 C20能够造成HepG2细胞发生DNA损伤,上调p21蛋白水平,导致细胞G₂/M期阻滞,并进一步诱发凋亡,发挥体外抗肝癌作用。     

Inhibition of Farnesyl Protein Transferase, H-ras Oncogene Expression and P21ras Membrane Association by Natural Products in Human Solid Tumor Cell Lines

Ras proteins must be isoprenylated at a conserved cysteine residue near the carboxyl terminus (Cys-186 in mammalian Ras P²¹ proteins) in order to extend their biological activity. Previous studies indicated that an intermediate in mevalonate pathway, most likely farnesyl pyrophosphate, is the donor of this isoprenyl group, that using inhibitors of the mevalonate pathway could block the transforming properties of ras oncogene. Unfortunately, mevalonate is the precursor of various end products essential to mammalian cells, such as dolichols, ubiquinones, heme A, and cholesterol. In this study, we partially purified farnesyl protein transferase (FPTase) capable of catalyzing the farnesylation of unprocessed Ras P²¹ proteins in vitro from porcine kidney epithelial-like LLC-PK1 cells, human lung adenocarcinoma A549 cells and human pancreatic cancer MIA PaCa-2 cells. We observed the effects of the monoterpene compound, d-limonene (1); turmeric derivatives, TD-I (2) and TD-II (3); polyphenol compound, gallotannin; salviol derivative, SMD; and retinoid acid derivative, RAD on FPTase activity. We found that turmeric derivatives and gallotannin had a strong inhibition on FPTase besides d-limonene, while gallotannin was the strongest among synthetic and natural compounds tested. Saliviol and retinoid acid derivatives had no influence on FPTase activity. Our results suggest that compounds containing polyphenol hydroxyl may be a new source of FPTase inhibitors. The experiment also showed that availability of an in vitro FPTase assay could be useful in screening for potential inhibitors of ras oncogene function that will not interfere with other aspects of the mevalonate pathway.

Concomitantly, we also studied gap junction intercellular communication (GJIC), H-ras oncogene expression and ras oncogene product (P21^ras protein) expression in four human solid     

Inhibition of tumor growth by S-3-1, a synthetic intermediate of salvianolic acid A

Salvianolic acid A (1) is one of the active components from Salvia miltiorrhiza, which was found to suppress the growth of mouse tumors. S-3-1 (a 2-allyl-3,4-dihydroxybenzaldehyde, 2) is a synthetic intermediate of a salvianolic acid A derivative with strong inhibitory effects on the growth of cancer cells in vitro. The inhibitory effects of 2 on tumor growth and its molecular targets were studied. 2 significantly suppressed the growth of mouse Lewis lung carcinoma, S180 sarcoma and H22 hepatic carcinoma in a dose-dependent manner. With a simple scrape-loading dye transfer method, 20 μg/ml of 2 was found to significantly enhance gap junction intercellular communication (GJIC) in human pancreatic adenocarcinoma PaCa Cells, human lung epithelial carcinoma W₁-38 cells and human lung adenocarcinoma A549 cells, but 2 had no marked effect on GJIC in human colon cancer CACO₂ cells. With Northern blot analysis, 2 was found to inhibit the expression of c-myc gene in A549 cells and have no marked effect on H-ras oncogene expression, and increase the cellular P53 mRNA contents, though it did not affect the expression of RB tumor suppressor gene. 2 also suppressed the P46 (JNK/SAPK) expression in A549 cells. Western blot analysis was applied to visualize the P21^ras protein. Results shows that 2 at concentrations ranging from 10 to 20 μg/ml decreases the contents of the membranous P21^ras and total P21^ras and increases the contents of cytosolic P21^ras protein in a time-dependent manner. However, 2 had no significant effects on farnesyl protein transferase activities at the concentrations that could efficiently decrease the membranous P21^ras content. This suggested that 2 might suppress tumor growth partly through enhancement of GJIC and reversion of the transformed phenotypes. The other mechanisms may be that 2 can suppress the overexpression of c-myc oncogene, inhibit the function of Ras oncoprotein, increase the expression of P53 tumor suppressor gene and interrupt P46-associated mitogen-activated pathway other than farnesylation of Ras protein.     

热疗上调细胞间隙连接通讯对胶质瘤侵袭性的影响及其机制

目的探讨热疗降低胶质瘤侵袭性的作用与细胞间隙连接通讯(gap junctional intercellular communication,GJIC)的关系。方法热疗处理C6胶质瘤细胞后,免疫组织化学法和Western blot法动态检测HSP70和Cx43的表达水平;划痕标记染料示踪技术检测胶质瘤GJIC功能变化;结晶紫染色法检测胶质瘤侵袭性的改变。结果 C6细胞经热疗后,HSP70表达增加,于30 min时含量最多。C6细胞内Cx43的表达水平也在热疗后明显增加,并于热疗后的120 min达高峰,后逐渐减少。热疗后GJIC功能的恢复与C6细胞内Cx43的表达相一致,且GJIC功能越强,胶质瘤侵袭性越低。结论胶质瘤细胞经热疗后HSP70表达增加,增加的HSP70可能是通过其分子伴侣作用提高Cx43的表达水平,进而上调GJIC功能而引起胶质瘤侵袭性的下降。     

Tumor promoters induce inhibition of gap junctional intercellular communication in mouse epidermal cells by affecting the localization of connexin43 and E-cadherin

The molecular and histological effects of tumor promoters on gap junctional intercellular communication (GJIC) were studied in three mouse epidermal cell types, representing different stages of tumor formation. GJIC was inhibited by most of the studied compounds (l-ethionine, d-limonene, o-anisidine, clofibrate, Aroclor 1260 and 1,1'-(2,2,2-trichloroethylidene)bis(4-chlorobenzene) (DDT)) except NaF and phenobarbital (PB). Whatever their effect on GJIC, most of the studied compounds increased the phosphorylation state of the gap junction protein expressed in these cells, connexin43 (Cx43), as shown by Western analysis. All agents with GJIC inhibiting capacity changed the intensity of the immunofluorescent staining of Cx43 on the membrane of the cells, whereas NaF and PB had no effect on Cx43 immunostaining. No association could be found between the type of change in Cx43 localization (changed membrane and/or cytosolic staining) and Cx43 phosphorylation or GJIC inhibition. Because the cell adhesion molecule E-cadherin also regulates GJIC, the effects of tumor promoters on E-cadherin protein and localization were studied. No quantitative change could be observed in E-cadherin protein content of cells treated with any of the selected agents. However, all agents which decreased GJIC, affected E-cadherin immunostaining of the membrane, while PB and NaF had no effect. These results show that an association exists between inhibition of GJIC and localization of both connexin43 and E-cadherin protein, but not with Cx43 phosphorylation.     

反式—BPDE诱发人胎气管上皮细胞H—ras癌基因点突变的研究

用苯并[a]芘的代谢物反式-BPDE,作用于6例体外培养人胎气管上皮细胞,2至6周后,用PCR结合限制性内切酸酶切片段多态性分析法,检测H-ras癌基因12位点密码子的点突变情况。结果显示,经反式-BPDE处理的气管上皮细胞,都有H-ras癌基因第12位点密码子的点突变,其中部分细胞还同时伴有起始密码子上游24bp处的另一个CCGG序列的点突变,而这些细胞都未出现典型的转化细胞的形态特征的改变。提示癌基因的点突变早于细胞形态学的改变。     

The effect of the PQ1 anti-breast cancer agent on normal tissues

Gap junctions are intercellular channels connecting adjacent cells, allowing cells to transport small molecules. The loss of gap junctional intercellular communication (GJIC) is one of the important hallmarks of cancer. Restoration of GJIC is related to the reduction of tumorigenesis and increase in drug sensitivity. Previous reports have shown that PQ1, a quinoline derivative, increases GJIC in T47D breast cancer cells, and subsequently attenuates xenograft breast tumor growth. Combinational treatment of PQ1 and tamoxifen can lower the effective dose of tamoxifen in cancer cells. In this study, the effects of PQ1 were examined in normal C57BL/6J mice, evaluating the distribution, toxicity, and adverse effects. The distribution of PQ1 was quantified by high-performance liquid chromatography and mass spectrometry. The expressions of survivin, caspase-8, cleaved caspase-3, aryl hydrocarbon receptor (AhR), and gap junction protein, connexin 43 (Cx43), were assessed using western blot analysis. Our results showed that PQ1 was absorbed and distributed to vital organs within 1 h and the level of PQ1 decreased after 24 h. Furthermore, PQ1 increased the expression of survivin, but decreased the expression of caspase-8 and caspase-3 activity. Interestingly, the expression of AhR increased in the presence of PQ1, suggesting that PQ1 may be involved in the AhR-mediated response. Previously, PQ1 caused an increase in Cx43 expression in breast cancer cells; however, PQ1 induced a decrease in Cx43 in normal tissues. Hemotoxylin and eosin staining of the tissues showed no histological change between the treated and the untreated organs. Our studies indicate that the administration of PQ1 by an oral gavage can be achieved with low toxicity to normal vital organs.     

环磷酰胺诱发叙利亚地鼠胚胎细胞恶性转化的分子生物学特征

采用杂交，测序，ＲＴ－ＰＣＲ等方法对环磷酰胺（ＣＰ）转化的不同叙利亚地鼠胚胎（ＳＨＥ）细胞株癌基因和抑癌基因进行了分析，结果表明，ｃ－ｍｙｃ癌基因以扩增的方式被激活，在Ｈａ－ｒａｓ和Ｋｉ－ｒａｓ癌基因上均存在着突变．在对Ｈａ－ｒａｓ２０７ｂｐ的一段ｃＤＮＡ序列分析中发现，总共发生了３５处碱基对的替换，除两处是在密码子的第一位碱基外，其余均发生于第三位碱基上，但除第６７位密码子的第三位碱基由于Ｇ－Ｔ颠换导致所编码的甲硫氨酸被异亮氨酸取代外，其余碱基的替换均未导致氨基酸的改变．分析还表明，ＣＰ致Ｈａ－ｒａｓ癌基因的突变无位点专一性．结果还表明，ｐ５３抑癌基因也发生了突变，并且可能属于点突变性质．因此，ＣＰ引起的ＳＨＥ细胞恶性转化的分子机理可能是以点突变和基因扩增的方式引起ｃ－ｍｙｃ，Ｋｉ－ｒａｓ，Ｈａ－ｒａｓ癌基因的激活及ｐ５３抑癌基因的失活或表达改变     

Inhibitory effects of organochlorine pesticides on intercellular transfer of Lucifer Yellow in cultured bovine oviductal cells

The present study investigated the effects of dichlorodiphenyltrichloroethane (DDT), methoxychlor (MXC), and gamma-hexachlorocyclohexane (gammaHCH, lindane) on gap junctional intercellular communication (GJIC) in cultured bovine oviductal cells. GJIC was evaluated by microinjecting fluorescent dye Lucifer Yellow and observing the inhibition of the spreading of dye into adjacent cells. After incubation for 1 h at 37 degrees C, a dose-dependent inhibition of GJIC was observed over a concentration range of 16 to 128 microM DDT, MXC, or gammaHCH compared with nonexposed controls. A significant inhibition began at 32 microM DDT, MXC, or gammaHCH. After incubation for 5 h, a dose-dependent inhibition of GJIC was obtained in the concentration range from 8 to 64 microM of the pesticides. The first significant inhibitory effect on GJIC was caused by 8 microM DDT, 16 microM MXC, and 32 microM gammaHCH. The 128 microM concentration of the pesticides was toxic. At pesticide concentration of 64 microM, the decrease in dye-coupling observed was not due to lethal cell injury, as is indicated by the use of trypan blue dye exclusion. After removal of 64 microM DDT from the culture medium, intercellular communication was reestablished within 3 h. Measurement of cytosolic free Ca2+ concentration [Ca2+]i in fura-2/AM-loaded oviductal cells showed that the inhibition of GJIC by addition of DDT, MXC, or gammaHCH was not associated with a detectable increase in [Ca2+]i. Coincubation of the DDT with dibutyryl-cAMP prevented the 64 microM DDT-induced inhibition of intercellular communication in adherent oviduct cells. It is suggested that organochlorine pesticides can influence cells responsible for reproduction.     

Effects of cadmium on gap junctional intercellular communication in WB-F344 rat liver epithelial cells

Cadmium has been associated with a number of tumors but its role in tumor promotion has not been elucidated clearly or the results obtained from various studies have been conflicting. This study was designed to investigate the effects of cadmium on the gap junctional intercellular communication (GJIC), number of gap junctions per cell, and cell proliferation in WB-F344 rat liver epithelial cells from the viewpoint of tumor promotion. GJIC was monitored by counting the cells stained with Lucifer yellow CH dye, using the scrape-loading and dye-transfer method. The numbers of gap junctions per cell were visually quantitated after an indirect immunostaining for gap junction protein using an antibody to connexin 43. Cell proliferation was assayed by direct counting of the living cells using the trypan blue dye exclusion method. In the time course study, cells treated with 200 microM CdCl2 showed rapid and nearly complete inhibition of GJIC (approximately 14% of the control) and a decrease in the number of gap junctions per cell (approximately 21% of the control) at 30 min, and the decrease continued up to 4 h without any changes in the cell viability. Treatment with CdCl2 (7.4-200 microM) for 4 h resulted in the decrease of GJIC and gap junction numbers per cell in a dose-response pattern without changes in the cell viability. In the long-term (14 days) exposure studies at doses of 0.01-7.4 microM CdCl2, an increase in cell proliferation was observed at low doses of 0.03-2.5 microM CdCl2, with GJIC also decreasing. These data demonstrate that cadmium inhibits GJIC, reduces the number of gap junctions per cell, and induces cell proliferation while decreasing the function of the gap junction.     

双环醇拮抗DDT引起的细胞间隙连接通讯功能抑制研究

目的研究治肝炎药物双环醇对促癌剂滴滴涕(DDT)引起细胞间隙连接通讯(GJIC)功能抑制的拮抗作用及作用机制。方法划痕标记染料示踪技术直接观察DDT引起的大鼠肝上皮WB-F344细胞GJIC功能抑制并分析双环醇的作用。利用Western blot方法检测间隙连接蛋白43(Cx43)、磷酸化Cx43、E-cadherin及β-Catenin的表达和活性。细胞免疫荧光技术考察WB-F344细胞Cx43蛋白亚细胞定位、间隙连接的形成及E-cadherin和β-Catenin在细胞内的表达。结果 DDT能剂量依赖性的抑制WB-F344细胞GJIC功能,20μM浓度时小分子荧光染料Luciferyellow CH仅能从伤沿细胞向后传递1-2列细胞。双环醇能部分恢复DDT引起的GJIC功能抑制,且具有一定剂量依赖关系,其作用机制与抑制DDT引起的磷酸化Cx43蛋白表达量升高,进而恢复DDT损伤的间隙连接形成有关。DDT和双环醇对与Cx43蛋白功能密切相关的E-cadherin及β-Catenin的表达、活性及细胞内定位均无明显影响。结论双环醇能通过影响Cx43蛋白的磷酸化水平,部分恢复环境促癌剂DDT引起的WB-F344细胞间隙连接的形成,改善GJIC功能。对GJIC的功能抑制是多种促癌剂诱发肿瘤发生的重要原因,前期研究显示双环醇具预防二甲基亚硝胺/苯巴比妥诱发肝癌发生的作用,本文研究结果进一步提示,双环醇在预防杀虫剂DDT(一种主要的环境致癌物)诱发的肿瘤方面亦具有一定潜能。