靶向survivin的反义寡核苷酸联合三苯氧胺治疗乳腺癌的研究

目的研究联合应用靶向survivin的反义寡核苷酸(ASODN)和三苯氧胺(TAM)对MCF-7乳腺癌细胞的作用。方法将一条20mer的ASODN序列与TAM作用于MCF-7乳腺癌细胞,分别为TAM组(单独应用TAM)、ASODN组(单独应用ASODN)及TAM+ASOND联合组(联合应用TAM+ASODN).运用四甲基偶氮唑盐比色试验(MTT)、流式细胞仪(FCM)、原位杂交以及分光光度法分别检测各组MCF-7细胞的抑制率、细胞周期和凋亡率、survivin mRNA表达和caspase-3活性。结果 TAM+ASOND联合组对MCF-7细胞的抑制率〔(62.26±3.92)%〕明显高于TAM组〔(42.30±6.63)%〕及ASODN组〔(54.77±9.99)%〕,P0.05.TAM+ASOND联合组的细胞凋亡率为(28.08±4.32)%,明显高于TAM组〔(18.94±4.01)%〕及ASODN组〔(21.12±3.95)%〕,P0.01.TAM+ASODN联合组对MCF-7细胞的G0/G1期阻滞作用明显强于TAM组和ASODN组(P0.05,P0.01);TAM+ASODN联合组survivin mRNA表达阳性率为(13.38±3.45)%,明显低于TAM组〔(39.67±7.42)%〕或ASODN组〔(27.50±5.80)%〕,P0.01.TAM+ASOND联合组MCF-7细胞的caspase-3活性(0.93±0.13)高于TAM组(0.50±0.09)或ASODN组(0.64±0.08),P0.01.结论靶向survivin的反义寡核苷酸能够促进MCF-7乳腺癌细胞凋亡,增加其对TAM治疗的敏感性。     

槐耳清膏体外逆转人肝癌细胞HepG2/ADM多药耐药性

目的探讨槐耳清膏体外逆转人肝癌细胞多药耐药性的作用及可能的机制。方法MTT法检测槐耳清膏的细胞毒作用和处理前后耐药细胞对化疗药物的敏感性;流式细胞仪检测细胞内阿霉素浓度;RT-RCR检测MDR1基因的表达。结果槐耳清膏在0.01g/L以下剂量时对HepG2和HepG2/ADM耐药细胞株的抑制率均<10%,半数抑制率(IC_(50))分别为0.917 g/L和1.66 g/L,无细胞毒剂量即0.008 g/L的槐耳清膏能部分逆转HepG2/ADM细胞的耐药性,与之合用使耐药肝癌细胞对阿霉素、顺铂、丝裂霉素、氟尿嘧啶的相对逆转效率分别为79%、73.5%、97.4%和86.7%,HepG2/ADM细胞内阿霉素浓度明显增加,MDR1表达下降了33.7%。结论槐耳清膏具有体外逆转人肝癌细胞多药耐药性的作用,可能与下调MDR1表达、增加细胞内化疗药物积累有关。     

雷公藤内酯醇-聚乙烯亚胺-环糊精对乳腺癌多药耐药细胞MCF-7/Taxol逆转作用的研究

目的观察雷公藤内酯醇-聚乙烯亚胺-环糊精复合物(TP-PEI-Cy D)对乳腺癌细胞MCF-7/Taxol耐药性的逆转作用并探讨其可能的作用机制。方法 CCK-8法测紫杉醇和TPPEI-Cy D对MCF-7及MCF-7/Taxol的生长抑制率,计算出各自的IC50值、MCF-7/Taxol的耐药倍数以及TP-PEI-Cy D对MCF-7/Taxol的低毒浓度;根据所得IC50值选择低毒浓度TP-PEI-Cy D联合不同浓度的紫杉醇处理MCF-7/Taxol后,测得紫杉醇联合TP-PEI-Cy D后的IC50,并计算逆转倍数;选择低毒浓度TP-PEI-Cy D联合紫杉醇处理MCF-7/Taxol细胞后,应用流式细胞术测细胞凋亡情况;TP-PEI-Cy D和紫杉醇单独或联合分别作用于MCF-7/Taxol,用RT-PCR检测耐药基因多药耐药相关蛋白(MRP)、肺耐药蛋白(LRP)以及谷胱甘肽转移酶(GST-π)的表达水平。结果 TP-PEICy D对MCF-7/Taxol生长增殖有明显抑制作用,并提高MCF-7/Taxol对紫杉醇的敏感度,MCF-7/Taxol耐药性得到逆转(F=439.36,P0.01);紫杉醇联合TP-PEI-Cy D的细胞凋亡率明显高于紫杉醇单独用药(F=17.91,P0.05)。联合使用后MRP、LRP、GST-πmRNA表达水平降低(F=27.41、33.99、20.77,均P0.01)。结论 TP-PEI-Cy D对MCF-7/Taxol有增殖抑制和诱导凋亡作用,能有效逆转MCF-7/Taxo细胞的肿瘤耐药性,其机制可能与降低细胞耐药基因LRP、MRP以及GST-πmRNA表达水平相关。     

槐耳清膏诱导人乳腺癌细胞MCF-7和MDA-MB-231凋亡的研究

本实验研究了槐耳清膏对人乳腺癌MCF-7及MDAMB-231细胞的生长抑制、凋亡诱导作用及其对凋亡相关基因p53,caspase-3表达水平的影响,为探索槐耳清膏诱导肿瘤细胞凋亡的机制提供线索,现报告如下.     

槐耳清膏诱导人直肠癌HR8348细胞凋亡的实验研究

目的　探讨槐耳清膏在体外对人直肠癌HR83 48细胞的生长抑制作用和凋亡诱导作用及其作用机理。方法　将处于对数生长期的HR83 48细胞用含槐耳清膏的培养基培养 3 6h ,用四氮唑蓝 (MTT )比色法检测吸光度 (OD)值 ,计算抑制率 ;用甲基绿 派若宁染色法及TUNEL法检测细胞凋亡指数 ;用免疫组织化学方法检测bcl 2、bcl xl、bax、bak及 p5 3基因表达的情况。 结果　槐耳清膏对HR83 48细胞的抑制率随浓度增加而上升 ,浓度为 4.0mg/ml时抑制率最大 (71.1% ) ,与 5 FU组 (浓度为 10 μg/ml)相比差异无显著性意义 (P>0 .0 5 )。甲基绿 派若宁染色法和TUNEL法均可见到典型的细胞凋亡、凋亡小体或出泡现象 ,凋亡指数随槐耳清膏浓度的增加而增加 ,当浓度达 4.0mg/ml时 ,凋亡指数迅速上升 ,甲基绿 派若宁法为 0 .162 0± 0 .0 12 8,TUNEL法为 0 .2 612± 0 .0 15 8,均大于 5 FU组的凋亡指数(0 .0 780± 0 .0 0 95和 0 .10 2 8± 0 .0 13 1) ,P<0 .0 5。槐耳清膏组bcl 2、bcl xl、bak、p5 3蛋白表达较空白对照组明显增强(P<0 .0 5 ) ,而bax变化不明显 (P>0 .0 5 )。槐耳清膏组bak/bcl 2和bak/bcl xl比值显著大于空白对照组。结论　槐耳清膏在体外对人直肠癌HR83 48细胞具有显著的抑制作用和凋亡诱导作用。槐耳清膏     

c-Src在TAM治疗耐药的人乳腺癌MCF-7细胞株中的表达及其意义

目的:探讨c-Src在乳腺癌三苯氧胺(TAM)中耐药的表达及意义.方法:用Western blot法检测TAM治疗耐药与敏感的两种人乳腺癌MCF-7细胞中c-Src的表达及其磷酸化水平;用c-Src阻滞剂PP2处理两种细胞后,采用酶联免疫吸附试验(ELISA)及吖啶橙/溴化乙锭(AO/EB)双荧光染色法检测细胞凋亡情况.结果:与TAM敏感MCF-7细胞比较,TAM耐药MCF-7细胞c-Src的表达量及其磷酸化水平均明显增高;PP2处理后,TAM敏感MCF-7细胞的富集指数(EF)及细胞凋亡率高于TAM耐药MCF-7细胞(均P＜0.05).结论:c-Src在TAM治疗耐药的人乳腺癌MCF-7细胞株中处于高表达及高活性状态,并可能参与乳腺癌内分泌治疗耐药的机制.     

葡萄籽多酚对人乳腺癌细胞MCF-7/ADR在裸鼠体内的多药耐药逆转作用

目的　研究葡萄籽多酚 (GSP)对人乳腺癌耐阿霉素细胞MCF 7/ADR的体内多药耐药逆转作用。方法　采用人乳腺癌裸鼠移植瘤模型研究GSP对肿瘤多药耐药的体内逆转作用 ;采用流式细胞术测定不同用药P 糖蛋白 (Pgp)表达变化及肿瘤细胞凋亡率变化。 结果　体内裸鼠抑瘤实验显示GSP有一定抑瘤作用 ,抑制率为 18 35 % ,联合应用阿霉素 (ADR)可显著抑制肿瘤生长 ,2 0mg/kgGSP可有效逆转MCF 7/ADR细胞的耐药性 ,抑瘤率为 5 4 6 4 %。流式细胞术结果显示应用GSP后肿瘤细胞Pgp表达显著降低 ( 32 0 3± 2 0 9) ,与对照组 ( 5 5 13± 2 12 )相比差异有显著意义 (P <0 0 5 )。平均凋亡率为 15 12 %± 1 0 4 % ,显著高于对照组 9 0 7%± 0 4 3% (P <0 0 5 )。结论　GSP能在体内逆转MCF 7/ADR细胞的耐药性 ,其机制可能是通过抑制Pgp表达 ,并可能通过诱导肿瘤细胞凋亡来起作用     

黑色素瘤分化相关基因-7/白细胞介素-24促进阿霉素杀伤肝癌细胞MHCC-97L机制的研究

目的 探讨黑色紊瘤分化相关基因-7/白细胞介素-24(MDA-7/IL-24)基因促进阿霉素(ADM)杀伤肝癌细胞,逆转肝癌细胞多药耐药(MDR)的机制.方法 以人肝癌细胞株MHCC-97L为实验对象,使用噻唑蓝(MTT)比色法和流式细胞仪比较Ad. MDA-7联合ADM处理组与ADM组、Ad. MDA-7组对肝癌细胞MHCC-97L和正常肝细胞L02的作用差异.观察MDA-7/IL-24对多药耐药的逆转作用.荧光定量聚合酶链反应(PCR)检测MDR-1、STAT-3、bcl-2、bax mRNA的变化.Western blot检测gp-170、STAT-3、bcl-2、bax蛋白的表达的变化.结果 MTT表明Ad. MDA-7对正常肝细胞LO2无生长抑制作用(P>0.05).LO2细胞Ad. MDA-7联合ADM组与ADM组细胞生存率差异无统计学意义(P>0.05).低浓度(100 VP/cell)的Ad. MDA-7联合正常肝细胞的IC50浓度的ADM(1.5 mg/L)使得细胞抑制率从ADM组的17.46%上升到79.50%,生长抑制逆转4.55倍(P<0.05).MDR-1mRNA相对表达量从(16.49±0.11)下降至(5.48±0.05).STAT-3 mRNA相对表达量从(13.17±0.08)上升至(21.57±0.11).bcl-2及BAX表达与其他实验组比较差异均有统计学意义(P<0.05).联合实验组P-170蛋白的表达量较其他组明显降低,而磷酸化STAT-3蛋白的表达量亦增加.结论 Ad. MDA-7具有逆转肝癌细胞MHCC-97L多药耐药的作用,其下调MDR-1 mRNA的表达的同时,并通过活化STAT-3信号通路的表达促进肝癌细胞凋亡.     

Ad-p53对乳腺癌阿霉素耐药细胞株多药耐药基因1/P-gp表达的影响

目的 观察腺病毒介导的p53基因(Ad-p53)逆转乳腺癌阿霉素耐药细胞株MCF-7/Adr多药耐药作用及其对MDR1 mRNA/P-gP表达的影响.方法 以人乳腺癌MCF-7细胞及其阿霉素耐药株MCF-7/Adr为实验对象,以50 MOI Ad-p53感染MCF-7/Adr细胞,CCK-8法观察Ad-p53对多药耐药的逆转作用,实时荧光逆转录-聚合酶链反应(RT-PCR)法检测MDR1 mRNA变化,免疫荧光组织化学及Western blot检测P-gP蛋白表达的变化.结果 50 MOI Ad-p53能使MCF-7/Adr细胞阿霉素IC50由(4.54±0.91)mg/L降到(0.26±0.11)mg/L,逆转耐药倍数为18.1倍(P《0.01);MDR1 mRNA相对表达量由1.32下降至0.85(P《0.01),P-gP蛋白表达量亦下降.结论 Ad-p53具有对人乳腺癌阿霉素耐药细胞株MCF-7/Adr多药耐药的逆转作用,其可下调MDR1 mR-NA/P-gP表达.     

索拉非尼联合表阿霉素对乳腺癌MCF-7细胞作用的研究

目的:探讨索拉非尼(Sorafenib)联合表阿霉素(Epirubicin)对乳腺癌MCF-7细胞的作用。方法:实验分为4组:空白对照组、索拉非尼单药组、表阿霉素单药组、索拉非尼联合表阿霉素组。根据索拉非尼及表阿霉素的IC50值设定联合给药浓度及时间。MTT法测定72h时间点索拉非尼及表阿霉素单用与联合对MCF-7细胞的增殖抑制率。结果:索拉非尼72h的IC50值为1.820μmol/l,表阿霉素72的IC50值为0.99μmol/l。在72h时,两药联合给药表现为协同或相加作用(P<0.05)。结论:索拉非尼和表阿霉素对乳腺癌细胞MCF-7细胞的增殖有抑制作用,联合给药表现出协同或相加作用。     

CCL5对MCF-7中细胞增殖能力及CD44~+/CD24~-亚群比例的影响

目的 观察外源性趋化因子5(CCL5)对人乳腺癌MCF-7细胞增殖及CD44+/CD24-亚群比例的影响,探讨其在乳腺癌进展过程中的作用.方法 噻唑蓝(MTT)比色法检测浓度为10、50、100、500 μg/L的外源性rhCCL5(recombinant human CCL5)作用0、24、48 h后MCF-7细胞增殖水平的变化,流式细胞仪检测CD44~+/CD24~-亚群比例的变化.结果 rhCCL5作用48 h后MCF-7细胞增殖水平高于对照组(P＜0.05),并呈浓度依赖趋势;且高于作用24、0 h后细胞的增殖水平(P＜0.05).50、500μg/L rhCCL5处理MCF-7细胞后升高了细胞株中CD44~+/CD24~-亚群的比例,分别为对照组的5.68和6.05倍(P＜0.05).结论 外源性CCL5可以明显升高MCF-7细胞的增殖水平以及CD44~+/CD24~-亚群的比例,这种作用可能是其诱导乳腺癌进展的机制之一.     

酸性HEPES-KH液对离体未成熟心肌的作用

目的 观察不同pH值HEPES-KH复灌液对离体未成熟心肌的影响.方法 建立Langendorff离体灌注模型,分为2组:缺血/再灌(I/R,n=8),用pH 7.4 HEPES-KH液灌流20 min,缺血60min,恢复灌注30min;酸性灌注组(E,n=8),用pH7.4HEPES-KH液灌流20min,缺血60min后,应用pH 6.8、7.1和7.4 HEPES-KH液顺次灌注5、5、20 min.以血流动力学指标、生化指标作为观察指标.结果 E组与I/R组比较,左心室功能恢复、三磷酸腺苷含量(ATP)(0.93±0.12比0.56±0.04,P＜0.01)、超氧化物歧化酶活性(183.47±9.72比120.17±6.21,P＜0.01)、心肌线粒体Ca2+-ATP酶活性(16.74±1.42比6.78±0.64,P＜0.01)和心肌线粒体合成ATP的能力(105.37±9.51比50.83±4.75,P＜0.01)明显增强,在心肌含水量(74.56±1.68比86.20±2.33,P＜0.01)、丙二醛含量(1. 97±0.17比2.88±0.32,P＜0.01)、肌酸激酶(64.56±4.69比88.48±5.86,P＜0.01)和乳酸脱氢酶漏出率漏出率(96.41±6.57比128.42±9.80,P＜0.01)、心肌细胞内Ca2+含量(2.25±0.28比4.48±0.74,P＜0.01)和心肌线粒体Ca2+含量(36.10±4.05比68.29±6.90,P＜0.01)明显减少.结论 复灌初期应用梯度酸性复灌液对离体未成熟心肌具有明显保护作用.

Abstract：

Objective To study the protective effects of different pH HEPES-KH reperfusate solutions on immature myodium. Methods The isolated Langendorff perfused model from immature rabbit hearts was established. The rabbits in ischemia/reperfusion (I/R) group were perfused with pH7.4HEPES-KH solutions preischemia and postischemia. In experimental (E) group, pH 6. 8, pH 7. 1 and pH 7. 4 HEPES-KH solutions were perfused for 5, 5 and 20 min postischemia, respectively. The hemodynamics and biochemistry were tested. Results The left ventricular function was significantly improved, adenosine triphosphate (ATP) content (0. 93 ±0. 12 vs 0. 56 ±0. 04,P ＜0. 01 ), superoxide dismutase activity ( 183.47 ±9. 72 vs 120. 17 ± 6. 21, P ＜ 0. 01 ), Ca2+ -ATPase activity of mitothondia ( 16. 74 ± 1.42 vs 6. 78 ± 0. 64, P ＜ 0. 01 ), ATP activity of mitochondria ( 105.37 ± 9. 51 vs 50. 83 ± 4. 75, P ＜ 0. 01 ) were significantly increased in E group as compared with those in I/R group. Myocardial water content (74. 56 ± 1.68 vs 86. 20 ±2. 33 ,P ＜0. 01 ), malondialdehyde content ( 1.9710. 17 vs 2. 88 ±0. 32,P ＜0. 01 ), dehydrogenase (64. 56 ± 4. 69 vs 88. 48 ± 5. 86, P ＜ 0. 01 ) and creatine kinase leakage (96. 41 ±6.57 vs 128.42 ±9.80,P＜0.01), myocardial cell Ca2+ content (2.25 ±0.28 vs 4.48 ±0.74,P＜0.01) and mitochondrial Ca2+ content (36. 10 ±4.05 vs 68.29 ±6.90,P＜0.01) in E group were reduced as compared with those in I/R group. Conclusion pH paradox might be one of important mechnisms for immature myocardial I/R injury, and acidic perfusate, at the beginning of reperfusion, might attenuate pH paradox and ameliorate functional recovery on isolated immature rabbit hearts.     

聚酰胺-氨纳米微粒介导5-氟尿嘧啶联合小RNA-21抑制乳腺癌细胞生长的研究

目的 观察聚酰胺-氨(PAMAM)纳米微粒介导5-氟尿嘧啶(5-Fu)联合小RNA-21抑制乳腺癌细胞MCF-7生长的效果.方法 透析法制备5-Fu/PAMAM,孵育法同载miR-21抑制剂;透射电镜观察形貌;紫外分光光度计检测载药率和包封率;流式细胞仪检测转染效率及细胞凋亡;噻唑蓝(MTT)比色法检测细胞增殖;Transwell检测细胞侵袭能力变化.结果 5-Fu/PAMAM形貌规整,包封率为(66.21±4.11)%,载药率为(31.77±0.73)%,转染效率为(60.54±6.97)%;联合治疗组肿瘤细胞生长缓慢,在观察截止期时生存率(55.85±3.71)%;联合治疗组细胞凋亡率(18.32±2.42)%,与对照组比较明显增多(F=58.326,P<0.01);联合治疗组视野内细胞数目仅为(18.96±3.14)个,侵袭能力显著降低(F=16.409,P<0.01).结论 PAMAM可以实现5-Fu和miR-21的同载,并有效抑制乳腺癌细胞的体外生长.     

同种异体肝细胞移植促进大鼠肝纤维化逆转

目的 探讨肝细胞移植对大鼠肝纤维化的改善作用及其机制.方法 经腹腔注射四氯化碳制备的肝纤维化Wistar大鼠接受正常Wistar大鼠肝细胞移植(实验组),另以不接受肝细胞移植的肝纤维化大鼠(肝纤维化组)和接受肝细胞移植的正常Wistar大鼠(移植对照组)为对照.肝细胞移植于注射四氯化碳后21 d进行.分别于移植前及移植后第1、2、7和14天采集抗凝血(肝纤维化组同步采集),测定血浆总转化生长因子β1(TGF-β1)、活性TGF-β1、纤溶酶及α2-巨球蛋白(α2M)水平,并取其肝脏,进行肝脏的纤维成像和定量分析,检测肝脏星形细胞活性.结果 肝细胞移植后的第14天,实验组肝脏内成胶原纤维面积百分比为(3.69±0.44)%,低于肝纤维化组的(8.25±0.69)%(P＜0.01);实验组肝内α-SMA阳性信号面积百分比为(0.43±0.03)%,明显低于肝纤维化组同期的(2.79±0.40)%(P＜0.01).移植前实验组大鼠血浆中总TGF-β1和活性TGF-β1水平分别为(4543.2±307.2)μg/ml和(2380.7±150.8)μg/ml,移植后1 d,二者均显著下降,分别为(2109.9±425.2)μg/ml(P＜0.01)和(1758.9±429.2)μg/ml(P＜0.05).实验组肝细胞移植前血浆纤溶酶水平为(5.16±0.42)μg/ml,移植后下降至(2.96±0.11)μg/ml(P＜0.01).实验组肝细胞移植前血浆α2M水平为(152.4±27.6)mg/ml,移植后升高至(343.0±48.8)mg/ml,为移植前的2倍,而移植对照组的α2M水平移植后升高30余倍.结论 肝细胞移植能有效改善大鼠肝纤维化,其机制可能与肝内活性星形细胞减少及TGF-β1水平降低有关.     

乳腺癌中雌激素诱导的细胞增殖与有丝分裂素激活蛋白激酶活性的关系

目的探讨雌二醇与有丝分裂素激活蛋白激酶(MAPK)信号通路的关系以及MAPK在乳腺癌细胞系中的表达情况。方法分别用上皮生长因子(EGF)和不同浓度的雌二醇诱导人类乳腺癌细胞系MCF-7细胞,诱导不同时间后用Western blot方法检测磷酸化ERK1／2的表达;用抗雌激素物质ICI182780和MAPK抑制剂PD98059作用于雌二醇诱导的MCF-7细胞,检测磷酸化ERK1／2的表达;用流式细胞仪检测雌二醇对MCF-7细胞周期的影响。结果MAPK信号通路的诱导剂EGF可以明显诱导磷酸化MAPK的表达;雌二醇也可以诱导磷酸化MAPK的表达。PD98059可以完全抑制雌二醇诱导的磷酸化ERK1／2水平,而ICI182780只能减少雌二醇诱导的磷酸化ERK1／2的表达。随着作用时间的延长,雌二醇可以使MCF-7细胞G2／M期的细胞数增加。结论雌二醇、雌激素受体与MAPK信号途径关系较为密切,MAPK是乳腺癌中重要的调节信号。     

Effects of WNK3 kinase on regulation of large-conductance calcium-activated potassium channels and its mechanisms

Objective To investigate the effects of WNK3 kinase on the regulation of large-conductance calcium-activated potassium channels (Maxi K channels) on African green monkey kidney fibroblast-like cells (Cos-7 cells) and its mechanisms. Methods (1) Cos-7 cells were transfected with 0, 0.6, 1.2, 1.8 μg WNK3 plasmid+0.5 μg Maxi K plasmid. The total protein expression of Maxi K channel and the phosphorylation of mitogen-activated protein kinase (MAPK) extracellular regulated kinase-1 and-2 (ERK1/2) were detected by Western blotting. (2) Cos-7 cells were divided into the control group (2.5 μg Maxi K plasmid) and the experimental group (2.5 μg WNK3 plasmid+2.5 μg Maxi K plasmid). Cell surface biotinylation was used to investigate the cell surface protein expression of Maxi K channel in Cos-7 cells. Immunoprecipitation and Western blotting were used to detect the ubiquitination of Maxi K channel protein. (3) WNK3 kinase was knocked down by WNK3 siRNA. The lysosomal degradation pathway was blocked by the proton pump inhibitor (Baf-A1). Cos-7 cells were divided into Maxi K+negative control siRNA group, Maxi K+WNK3 siRNA group and Maxi K+WNK3 siRNA+Baf-A1 group. The protein expression of Maxi K channel protein was detected by Western blotting. Results (1) Compared with those in 0 μg WNK3 plasmid groups, in 0.6, 1.2, 1.8 μg WNK3 plasmid groups the total protein expression of the Maxi K channel increased and the phosphorylation level of MAPK ERK1/2 reduced on a dose-dependent manner (all P＜0.01). (2) Compared with those in the control group, the total protein expression and cell surface membrane protein expression of the Maxi K channel increased in the experimental group (P＜0.01), while the ubiquitination of the Maxi K channel protein reduced (P＜0.01). (3) Compared with the Maxi K+negative control siRNA group, the expression of Maxi K protein reduced in the Maxi K+WNK3 siRNA group (P＜0.01), but did not change in the Maxi K+WNK3 siRNA+Baf-A1 group (P＞0.05). The expression of Maxi K protein in Maxi K+WNK3 siRNA+Baf-A1 group was higher than that in Maxi K+WNK3 siRNA group (P＜0.01). Conclusions WNK3 kinase inhibits the lysosomal degradation pathway of Maxi K channel protein by reducing the ubiquitination of Maxi K channel, and promotes the expression of Maxi K channel protein in cells and on cell membrane. These effects may be achieved by suppressing MAPK ERK1/2 signal transduction pathway.     

趋化因子2对乳腺癌细胞MCF-7趋化活性及趋化因子5mRNA表达的影响

目的 观察趋化因子2(CCL2)对MCF-7表达趋化因子5(CCL5)mRNA的影响,并观察CCL2对MCF-7趋化活性的影响.方法 使用不同浓度的CCL2作用于MCF-7细胞,通过实时荧光定量聚合酶链反应(RTFQ-PCR)测定不同时间CCL5 mRNA的表达,并使用趋化小室法检测CCL2作用下MCF-7趋化活性的改变.结果 当外源性CCL2浓度为200μg/L时,MCF-7中CCL5 mRNA相对表达量最大(15.22±2.32)(P＜0.01),随着作用时间的延长,CCL5 mRNA表达量增加;MCF-7的趋化活性与CCL2浓度呈正相关,当CCL2浓度为300μg/L时,穿膜细胞数最多(88.00±11.53)(P＜0.01);MCF-7的趋化活性与CCL2作用时间呈正相关,当CCL2作用时间为30 h时,穿膜细胞数最多(81.00±9.54)(P＜0.05).结论 加入外源性CCL2,MCF-7中CCL2 mRNA表达量增加,MCF-7趋化活性增强.

Abstract：

Objective To observe the effects of chemokine 2 (CCL2) on the expression of chemokine 5 ( CCL5) mRNA and chemotactic activity of MCF-7 cells. Methods MCF-7 cells were treated with different concentrations of CCL2, the expression of CCL5 mRNA was detected by using real-time fluorescence quantitative polymerase chain reaction ( RTFQ-PCR), and the chemotactic activity of MCF-7 was measured by using chemotaxis chamber method. Results When the concentration of exogenous CCL2 was 200 μg/L, the MCF-7 cells expressed the highest CCL5 mRNA (15. 22 ± 2. 3, P ＜0. 01). With the prolongation of CCL2 action time, the expression levels of CCL5 mRNA were increased. There was a positive correlation between the chemotactic activity of MCF-7 cells and the concentration of CCL2. When the concentration of exogenous CCL2 was 300 μg/L, the number of penetrating cells was the greatest (88.00 ±11. 53, P ＜0. 01). With the prolongation of CCL2 action time, the chemotactic activity of MCF-7 cells was enhanced. When the action time was 30 h, the number of penetrating cells was the greatest (81.00 ±9. 54, P ＜ 0.05 ). Conclusion Exogenous CCL2 could increase the expression of CCL5 mRNA and the chemotactic activity of MCF-7 cells.     

线粒体ATP敏感性钾通道阻断剂对在体大鼠肺缺血后处理的作用及其机制

目的 探讨缺血后处理(IPO)对大鼠在体肺缺血-再灌注损伤(I/R)的保护作用及线粒体ATP敏感性钾通道(mitoKATP)在缺血后处理效应中的作用.方法 将Wistar大鼠35只随机分为5组:假手术组(Sham组)、缺血再灌注损伤组(I/R组)、缺血后处理组(IPO组)、缺血再灌注损伤+5-羟基葵酸盐组(I/R+5-HD组)、缺血后处理+5-羟基葵酸盐组(IPO+5-HD组).观察各组肺组织中丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性、湿/干比值(W/D)以及病理形态学改变.结果 I/R组与Sham组比较MDA含量增加[(5.07±1.60)nmol/mg prot比(1.43±0.41)nmol/mgprot,P＜0.01],SOD活性减低[(12.38±2.24)U/mg prot比(45.51±5.42)U/mg prot,P＜0.01],W/D比值增高(5.45±0.82比3.05±0.47,P＜0.01),肺组织形态及超微结构明显受损;IPO+5-HD组与IPO组比较MDA含量增加[(3.74±0.71)nmol/mg prot比(2.60±0.43)nmol/mg prot,P＜0.01],SOD活性减低[(22.91±2.71)U/mg prot比(28.74±2.03)U/mg prot,P＜0.01],W/D比值增高(4.64±0.79比3.89±0.60,P＜0.01),肺组织形态及超微结构明显受损;IPO组与I/R组比较,肺组织MDA含量减少[(2.60±0.43)nmol/mg prot比(5.07±1.60)nmol/mg prot,P＜0.01],SOD活性增高[(28.74±2.03)U/mg prot比(12.38±2.24)U/mg prot,P＜0.01],W/D比值减低(3.89±0.60比5.45±0.82,P＜0.01),肺组织病理形态学改变轻于I/R组;I/R+5-HD组与I/R组比较,肺组织MDA含量[(5.14±1.30)mol/mg prot比(5.07±1.60)mol/mg prot,P＞0.05)、SOD活性[(11.65±1.82)U/mg prot比(12.38±2.24)U/mg prot,P＞0.05]、W/D比变化(5.54±0.61比5.45±0.82),差异无统计学意义(P＞0.05),肺组织病理形态学改变无明显差异.IPO+5-HD组的各项指标介于IPO组和I/R组之间.结论 缺血后处理能减轻大鼠在体肺缺血再灌注损伤,mitoKATP参与了肺缺血后处理效应.

Abstract：

Objective To investigate the protective effect of ischemic postconditioning (IPO) on lung ischemic reperfusion (L/R) in rats in vivo and the mechanism of mitochondrial ATP-sensitive potassium channel (mitoKATP) blocker in the ischemic postconditioning. Methods Thirty five Wistar rats were randomly divided into 5 groups: sham group, I/R group, ischemic postconditioning (IPO) group, I/R +5-hydroxydecanoate (I/R + 5-HD) group, IPO + 5-HD group. The concentration of malondialdehyde (MDA) and activity of superoide dismutase (SOD) were determined in the lung homogenate, wet to dry weight ratio (W/D) was measured and pathological changes were also observed. Results The levels of MDA[(5.07±1.60) vs (1.43 ±0.41) nmol/mg prot,P＜0. 01]and W/D (5.45 ±0.82 vs 3.05 ±0. 47,P ＜0. 01 ) were increased significantly in I/R group as compared with sham group, while the activity of SOD[( 12. 38 ±2. 24) vs (45.51 ±5.42) U/mg prot,P ＜0. 01]was decreased, and the injury of lung tissues was significantly aggravated in IPO + 5-HD group as compared with IPO group[MDA: (3.74 ±0. 71 ) nmol/mg prot vs (2. 60 ± 0. 43 ) nmol/mg prot , P ＜ 0. 01]; W/D: 4. 64 ± 0. 79 vs 3. 89 ± 0. 60,P＜0.01; SOD:[(22.91 ±2.71) U/mg prot vs (28.74±2.03) U/mg prot,P＜0. 01]. The levels of MDA[(2.60±0.43) vs (5.07 ±1.60) nmol/mg prot,P＜0. 01]and W/D (3.89 ±0.60 vs 5.45 ±0. 82,P ＜0. 01 ) were decreased significantly in IPO group as compared with I/R group, the activity of SOD[(28.74±2.03) vs (12.38 ±2.24) U/mg prot,P＜0. 01]increased and lung tissue histological damage attenuated. The difference in MDA[(5.14 ± 1.30) vs (5.07 ± 1.60) nmol/mg prot, P ＞ 0. 05],W/D (5.54±0.61 vs5.45 ±0.82,P＞0.05) and SOD[(11.65 ±1.82) vs (12.38 ±2.24) U/mgprot,P ＞ 0. 05]levels had no statistical significance between I/R + 5-HD group and I/R group, and the injury of lung tissues had no significant difference too. Each index in IPO + 5-HD group was between IPO and I/R groups. Conclusion Ischemic postconditioning can attenuate the lung I/R injury, and mitoKATP plays a vital role in the protective procession of ischemic postconditioning on lung ischemic reperfusion.     

槲皮素对缺血再灌注大鼠离体心脏的作用

目的 观察槲皮素(Quercetin)对缺血再灌注损伤(IRI)离体大鼠心脏的作用.方法 将32只SD大鼠随机分为4组:空白对照组(Control);给药对照组(Control+Que);缺血再灌注组(I/R);缺血再灌注给药组(I/R+Que),行Langendorff心脏灌注,给药组预防性给予槲皮素(5 μmol/L).监测各组心功能(±dp/dtmax),比较再灌注1 h心肌尼克酰胺腺嘌呤二核苷酸磷酸氧化酶(NOX2)、一氧化氮合酶(iNOS、eNOS)表达和超微结构的变化.结果 I/R组与Control组比较心功能显著降低(分别为18.91±3.38、-22.43±8.84和60.65±11.65、-56.62±8.49,P＜0.01),NOX2、iNOS、eNOS mRNA(分别为0.1590±0.0539、0.0897±0.0236、0.0154±0.0061和0.0247±0.0070、0.0377±0.0135、0.0091±0.0033,P＜0.05)和蛋白的表达均显著增加,心肌超微结构严重损伤;与I/R比较I/R+Que组(45.77±8.05,-42.10±8.71)显著增强心功能(P＜0.01),显著降低NOX2、iNOS、eNOS mRNA(分别为0.0864±0.0358、0.0445±0.0104、0.0085±0.0032,P＜0.05)和蛋白的表达,明显减轻心肌超微结构的损伤.结论 在离体水平预防性给予槲皮素能够显著减轻缺血再灌注对大鼠心肌造成的损伤,保护心脏.

Abstract：

Objective To observe the effect of quercetin (Que) on isolated rat hearts after ischemia-reperfusion injury (IRI). Methods Thirty-two SD rats were divided randomly into 4 groups with 8 in each group: ( 1 ) Control group, isolated hearts contiuosly peffused without ischemia; (2) Control + Que group: isolated hearts contiuosly perfused without ischemia but the adminstration of Que (5 μmol/L) 5 min after perfusion; (3) I/R group: isolated hearts perfused with 30 min global ischemia followed by reperfusion; (4) I/R + Que group: isolated hearts perfused with 30 min global ischemia followed by reperfusion and the adminstration of Que (5 μmol/L) 10 min before ischemia. Hemodynamic parameters ( ± dp/dtmax),myocardial ultrastructure, nicotinamide adenine dinucleotide phosphate (NADPH) oxidases 2 ( NOX2),inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) mRNA and protein expression after reperfusion were compared among the four groups. Results As compared with control group, hemodynamic parameters were greatly decreased after reperfusion ( 18.91 ± 3. 38, - 22. 43 ± 8. 84vs 60. 65 ± 11.65, - 56. 62 ± 8. 49 ,P ＜ 0. 01 ), myocardial ultrastructures were significantly destroyed and the expression levels of NOX2, iNOS, eNOS mRNA and protein were significantly increased after 60-min reperfusion (0. 1590 ±0.0539, 0.0897 ±0.0236, 0.0154 ±0.0061 vs 0.0247 ±0.0070, 0.0377 ±0. 0135, 0. 0091 ± 0. 0033, P ＜ 0. 05 ) in I/R group. As compared with I/R group, hemodynamic parameters were significantly recovered (45.77 ± 8.05, - 42. 10 ± 8. 71, P ＜ 0. 01 ), myocardial ultrastructures were well protected and the expression levels of NOX2, iNOS, eNOS were significantly decreased (0. 0864± 0. 0358, 0. 0445 ± 0. 0104, 0. 0085 ± 0. 0032, P ＜ 0. 05 ) in I/R + Que group, but there was no significant difference between control group and control + Que group ( P ＞ 0. 05 ). Conclusion Que can protect isolated perfused rat hearts from IRI by its antioxidative effect.