银杏叶提取物对三氯乙烯诱导的人角质形成细胞NO合成的影响

背景与目的:观察银杏叶提取物(ginkgo biloba extract,GBE)对三氯乙烯(trichloroethylene,TCE)诱导的人角质形成细胞(kenatinocyte,KC)一氧化氮(NO)合成和一氧化氮合酶(NOS)基因表达及活性的影响,为探讨TCE职业性皮炎的机制及保护因子提供理论依据。材料与方法:分离的KC用无血清培养基进行原代培养至80%以上融合时,加入不同浓度的GBE预适应2h后再用2.0mmol/L的TCE染毒4h,以不含染毒的培养基作为对照组。根据试剂盒的方法检测NO含量和NOS活力,同时用RT_PCR的方法检测细胞i NOS mRNA的表达情况。结果:2.0mmol/L的TCE处理组NO含量和i NOS活力分别为(41.22±5.45)μmol/L和(0.53±0.07)U/4×105cell,明显高于对照组(24.20±3.72)μmol/L和(0.31±0.03)U/4×105cell。而TCE对KC cNOS活力无影响。10、50和100mg/L GBE保护组未见NO含量的明显下降,150mg/L GBE则能够拮抗2.0mmol/L TCE所致的NO水平和i NOS活力的升高。RT_PCR结果显示GBE的预处理可以抑制TCE诱导的KC i NOS mRNA的过表达。结论:TCE通过诱导KCiNOS基因上调产生大量的NO,GBE对TCE诱导的KCi NOS活力的升高以及iNOS基因表达的上调有抑制作用,也进一步抑制了NO的过量生成,可能对TCE皮肤损害具有保护作用。     

喹乙醇诱导HepG2 细胞自噬的实验研究

目的： 研究喹乙醇对HepG2细胞自噬的影响。方法：分别用不同浓度 (0、200、400、800 μg/mL)喹乙醇染毒HepG2细胞24 h后,进行单丹磺酰尸胺 (monodansylcadaverine,MDC)染色,分别通过荧光显微镜和流式细胞仪检测细胞自噬发生情况。另外,分别用不同浓度喹乙醇 (0、200、400、800 μg/mL)染毒HepG2细胞24 h和400 μg/mL喹乙醇染毒HepG2细胞不同时间 (0、1、3、6、12、24 h)后,采用Western blot方法检测自噬相关蛋白LC3-Ⅱ和Beclin 1的表达。结果：随着喹乙醇染毒剂量增加,MDC阳性细胞的荧光强度及细胞内MDC荧光颗粒数目明显增加。流式结果显示,与对照组相比,200、400和800 μg/mL染毒组MDC阳性细胞百分比均显著增加 (P<0.05或P<0.01)。随着喹乙醇染毒浓度和时间的增加,LC3-Ⅱ和Beclin 1表达量均明显增加,其中400 μg/mL 喹乙醇染毒细胞24 h组与对照组相比,LC3-Ⅱ和Beclin 1的表达量均明显上调 (P<0.01)。结论：喹乙醇诱导了HepG2细胞的自噬反应。     

绿茶对微囊藻毒素LR诱导肝细胞凋亡、Bcl-2表达及骨髓嗜多染红细胞微核的影响

背景与目的:研究绿茶(green tea,GT)对微囊藻毒素LR(MC-LR)诱导肝细胞凋亡、Bcl-2蛋白表达及微核发生的影响以探讨毒性拮抗机制.材料与方法:雄性小鼠50只随机分为5组,分别为空白对照、MC-LR染毒组、GT高低剂量拮抗组和环磷酰胺对照组.实验第1 d起GT高、低剂量拮抗组小鼠每日分别给予12 g/L和2 g/L两种浓度的GT自由饮用,连续18 d.自第6 d开始,染毒小鼠每日给予MC-LR 10 μg/kg腹腔注射1次,空白对照给予DMSO腹腔注射,连续13 d.环磷酰胺对照组以50 mg/kg剂量间隔24 h两次给药后6 h取材.小鼠处死后采用免疫组化和计数法对肝细胞凋亡、Bcl-2蛋白表达以及骨髓嗜多染红细胞(PCEs)微核发生率进行检测和分析.结果:(1)MC-LR染毒明显诱导小鼠肝细胞凋亡增加.高剂量GT处理后明显抑制MC-LR染毒所致小鼠肝细胞凋亡的发生(P＜0.05);(2)单纯MC-LR染毒肝细胞Bcl-2表达未见明显变化,GT各剂量组小鼠肝脏Bcl-2的表达明显增加,与MC-LR染毒组相比差异具有统计学意义(P＜0.01).(3)GT拮抗组小鼠骨髓嗜多染红细胞微核率(PCEs-MN)与MC-LR染毒对照和空白对照相比,其差异均无统计学意义(P＞0.05).结论:GT能上调抑癌基因Bcl-2的表达,抑制细胞凋亡.MC-LR染毒及GT拮抗对微核发生均未有显著影响.     

微囊藻毒素-LR对秀丽线虫精子形成的毒性作用

目的： 探讨微囊藻毒素-LR(MC-LR)对模式生物秀丽隐杆线虫精子形成的毒性作用。 方法：L4期him-5雄虫分别暴露于0.1、1、10和100 μg/L的MC-LR中,染毒48 h。通过测定精细胞大小和体外活化率分别观察MC-LR对精子竞争力和活化能力的影响,并利用实时荧光定量PCR测定相关基因表达水平,用杂交试验验证MC-LR对雄虫生育力的影响。 结果：暴露48 h后,100 μg/L 组秀丽隐杆线虫精细胞与对照组相比周长和面积均显著减小(P均<0.01);10和100 μg/L染毒组精细胞体外活化率显著下降(P均<0.01);spe-10和spe-15基因在染毒组出现了表达水平下调的现象;各染毒组后代数目与对照组相比差异均无统计意义(P均＞0.05)。 结论：MC-LR对秀丽隐杆线虫精子形成过程具有一定的毒性作用。     

丙酮酸乙酯对氯气染毒大鼠肺组织炎症的抑制作用

目的： 研究丙酮酸乙酯 (ethyl pyruvate,EP)对氯气导致的肺组织炎症反应的抑制作用及其机制。方法：60只SD雄性大鼠分为2批,每批随机分为阴性对照组、阳性对照组和EP处理组,每组各10只,阴性对照组以空气为对照。阳性对照组和EP处理组给予2 536 mg/m3的氯气动态染毒,染毒时间为20 min,染毒后立即分别腹腔注射生理盐水或EP (40 mg/kg)。染毒后6 h 采集肺泡灌洗液,检测TNF-α、IL-1β以及IL-8含量,取肺组织检测核因子NF-κB核转位。于染毒后24 h取肺组织检测高迁移率族蛋白B1(high mobility group box 1,HMGB1)的表达并检测ⅡA型分泌型磷脂酶A2 (type-ⅡA secretary phospholipase A2,sPLA2-ⅡA) 活性。结果：与阴性对照组比较,氯气染毒6 h后肺泡灌洗液中TNF-α、IL-1β、IL-8含量显著增加 (P<0.05),NF-κB核转位显著升高 (P<0.05),染毒后24 h肺组织中HMGB1表达以及sPLA2-ⅡA活性显著升高 (P<0.05)。与阳性对照组比较,给予EP处理后上述指标均显著降低 (P<0.05)。结论：氯气中毒后启动了早期和晚期炎症反应,引起肺组织释放大量炎症因子,导致炎症反应发生。EP处理可有效抑制肺组织炎症反应。     

人乳腺癌细胞过量产生NO对ADM和5-FU敏感性的调节作用

目的:研究内源性一氧化氮(NO)对于调节肿瘤细胞对化疗药物敏感性的影响.方法:应用IL-1β处理培养的MCF-7细胞检测NO的产生情况,并用蛋白质印迹法检测诱导型一氧化氮合酶(iNOS)蛋白的表达.设立实验组和对照组,采用MTT法检测MCF-7细胞在一氧化氮合酶(NOS)抑制剂NG甲基-L-精氨酸(L-NMMA)和NO合成原料L-精氨酸(L-Arg)作用下,对多柔比星(ADM)和氟尿嘧啶(5-FU)的药物敏感性.结果:内源性NO的产生量与IL-1β间存在着剂量依赖关系.蛋白质印迹法分析结果显示,在IL-1β诱导作用下,细胞大量表达iNOS蛋白.同时无论L-Arg和L-NMMA存在与否,iNOS蛋白都无差异.当ADM浓度为0.5和1μmol/L时,实验组细胞生存率有较明显的下降,P<0.05.L-NMMA的加入显著提高了实验组细胞的生存率,P<0..5;L-Arg的加入,在一定程度上提高了化疗药的敏感性,P<0.05.当L-Arg和L_NMMA与IL-1β同时存在,肿瘤细胞的生存率不会有明显下降,P<0.05.结论:在细胞因子IL-1β诱导下,MCF-7产生的内源性NO能提高肿瘤细胞的化学敏感性.     

泛素连接酶RING2对苯并[a]芘染毒人支气管上皮细胞周期和P53蛋白表达的影响

目的:探讨泛素连接酶RING2对苯并[a]芘(BaP)染毒人支气管上皮16HBE细胞周期和P53蛋白表达的影响。方法:以16HBE未处理组为阴性对照组,二甲基亚砜(DMSO)组为溶剂对照组,MOCK组为序列对照组。在使用RNA干扰技术降低16HBE细胞泛素连接酶RING2基因表达前后,分别采用不同浓度BaP(1、2、4、8、16、32 μmol/L)染毒24 h;或16 μmol/L BaP染毒不同时间(1、2、4、8、12、24 h)。用流式细胞术检测干扰前后两组细胞周期分布情况,用Western-blot法检测干扰前后两组细胞P53蛋白表达水平。结果:流式细胞术检测结果显示,与阴性对照组比较,16HBE细胞染毒后各浓度和各时点组S期细胞所占的比例均增加(P<0.05),而16HBE(siRNA-RING2)各浓度和各时点组S期细胞所占的比例均下降(P<0.05)。协方差分析显示分组因素(是否进行RNAi)和染毒浓度都对S期细胞比例有影响(P均<0.01),16HBE(siRNA-RING2)细胞组的修正均数(17.09%)比16HBE细胞组(31.55%)明显降低(P<0.01)。分组因素和染毒时间都对S期细胞比例有影响(P均<0.01),16HBE(siRNA-RING2)细胞组的修正均数(13.07%)比16HBE细胞组(28.04%)明显下降(P<0.01)。Western-blot结果显示,与阴性对照组比较,16HBE 细胞染毒后各浓度和各时点组P53的表达水平均增加(P<0.05),而16HBE(siRNA-RING2)细胞除16 μmol/L染毒8 h组外,其余各组P53的表达水平均降低(P<0.05)。协方差分析显示分组因素和染毒浓度都对P53的表达水平有影响,P值分别为0.026和0.028。16HBE(siRNA-RING2)细胞组的修正均数(0.989)比16HBE细胞组(1.375)明显下降(P<0.05);分组因素 和染毒时间都对P53的表达水平有影响,P值分别为0.007和0.035。16HBE(siRNA-RING2)细胞组的修正均数(0.857)比16HBE细胞组(1.541)明显下降(P<0.05)。结论:RING2参与的组蛋白泛素化修饰可能通过影响 P53表达和细胞周期S期的变化来发挥对DNA损伤修复的调控。     

原花青素对脂多糖诱导BV2小胶质细胞炎症介质分泌的影响

目的:探讨原花青素对脂多糖(LPS)激活小鼠小胶质细胞(BV2)炎症介质分泌的影响。方法:以LPS激活BV2细胞构建神经炎症模型。分别采用0.1、0.5、1.0、5.0 μg/mL原花青素预处理后,1.0 μg/mL的LPS刺激BV2细胞24 h。采用MTT法检测细胞存活率;Griess法检测BV2细胞培养液上清中一氧化氮(NO)水平;ELISA法检测肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)和白细胞介素-6(IL-6)的分泌水平。结果:在实验剂量范围内,原花青素及LPS对BV2细胞活性均无显著影响(P>0.05)。但1.0 μg/mL LPS可引起BV2细胞各炎症因子NO、TNF-α、IL-1β和IL-6水平明显升高(P<0.05)。与LPS组相比,原花青素(0.1、0.5、1.0、5.0 μg/mL)预处理能使激活的BV2细胞培养液上清中NO释放量减少(P<0.05),TNF-α、IL-1β和IL-6含量降低(P<0.05),抑制作用呈剂量-效应关系。结论:在体外实验中,原花青素对LPS诱导小胶质细胞所致的炎症反应具有保护作用。     

微囊藻毒素LR对大鼠卵巢颗粒细胞氧化损伤和凋亡的影响

目的：研究微囊藻毒素LR(MC-LR)对大鼠卵巢颗粒细胞的氧化损伤和致凋亡作用。方法：分别以10、20、30μg/mL的MC-LR对体外培养的大鼠卵巢颗粒细胞染毒48h,测定细胞内活性氧(ROS)含量、细胞培养液超氧化物歧化酶(T-SOD)活性、丙二醛(MDA)含量、卵巢颗粒细胞凋亡率和凋亡相关基因Bax、Bcl-2mRNA的表达水平。结果：与对照组比较,随着MC-LR剂量升高,染毒组细胞内ROS升高(rs=0.925,P<0.01);细胞培养液中T-SOD活性降低(rs= -0.865,P<0.05);MDA含量升高(rs=0.811,P<0.01)。卵巢颗粒细胞凋亡率随MC-LR剂量的升高而升高(P<0.05);凋亡相关基因BaxmRNA的表达量随MC-LR剂量的升高而升高(rs=0.972, P<0.01),但Bcl-2mRNA的表达量随MC-LR剂量的升高而降低(rs= -0.972,P<0.01)。结论：MC-LR对大鼠卵巢颗粒细胞具有氧化损伤作用,并能引起卵巢颗粒细胞凋亡和凋亡相关基因Bax、Bcl-2mRNA表达的改变。     

微囊藻毒素-LR对PC12细胞氧化性损伤作用的实验研究

目的:探讨微囊藻毒素-LR(microcystins-LR,MC-LR)对PC12细胞的毒性作用及其机制。方法:用MC-LR对PC12细胞进行染毒,以MTT法测定其对细胞的毒性作用;同时通过测定细胞培养液和细胞内的氧化应激指标初步判断MC-LR对细胞的氧化损伤作用。结果:MC-LR染毒12、24、48 h,对PC12细胞的半数抑制浓度(IC_(50))分别是89.06、47.24、33.13μg/L。MC-LR染毒24h后,细胞培养液中乳酸脱氢酶(LDH)活性升高。当染毒浓度大于等于10μg/L时,细胞中生成丙二醛(MDA)和超氧化物歧化酶(SOD)的含量随着染毒浓度的增加逐渐升高(P0.05);过氧化氢酶(CAT)含量的变化和SOD具有一致性。而谷胱甘肽过氧化物酶(GSH-Px)含量与对照组相比未见显著差异(P0.05)。结论:MC-LR可以抑制PC12细胞的增殖能力和引起细胞膜损伤,并发生氧化损伤作用。     

Nitric oxide inhibits autophagy and promotes apoptosis in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the second most common cause of cancer‐related mortality worldwide. The expression of nitric oxide synthase (NOS) and the inhibition of autophagy have been linked to cancer cell death. However, the involvement of serum nitric oxide (NO), the expression of NOS and autophagy have not been investigated in HCC. In the present study, we first established that the NO level was significantly higher in hepatitis B virus‐related HCC than in the liver cirrhosis control (53.60 ± 19.74 vs 8.09 ± 4.17 μmol/L, t = 15.13, P < 0.0001). Using immunohistochemistry, we found that the source of NO was at least partially attributed to the expression of inducible NOS and endothelial NOS but not neuronal NOS in the liver tissue. Furthermore, in human liver cancer cells, NO‐induced apoptosis and inhibited autophagy. Pharmacological inhibition of autophagy also induced apoptosis, whereas the induction of autophagy could ameliorate NO‐induced apoptosis. We also found that NO regulates the switch between apoptosis and autophagy by disrupting the Beclin 1/Vps34 association and by increasing the Bcl‐2/Beclin 1 interaction. Overall, the present findings suggest that increased NOS/NO promotes apoptosis through the inhibition of autophagy in liver cancer cells, which may provide a novel strategy for the treatment of HCC.     

Tumor cells suppress cytokine-induced nitric-oxide (no) production in cerebral endothelial cells

Nitric oxide (NO) produced by endothelial cells (EC) has been shown to exert cytotoxic activity on tumor cells. In order to analyze events involved in brain metastasis, the modulation of NO production in rat-brain-derived EC was investigated. NO release was increased by tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-1β, lipopolysaccharide or forskolin in EC2I9 cells, a rat-brain-microvessel-derived EC line. Dexamethasone decreased NO release by cytokine-activated EC219 cells. Tumor cells (DHD/K12/PROb, a rat coloncarcinoma cell line) were highly adherent to EC2I9 cells, and adhesion was not modified by TNF-α plus IFN-γ, or by dexamethasone. Addition of tumor cells or tumor-cell-conditioned medium significantly inhibited NO release induced by any of the stimuli examined, but only if added during the initial phase of endothelial-cell activation. Tumor-derived suppression of NO release was also observed in primary cultures of cerebral EC. NO synthase (NOS) activity in cytosol extracts of the cerebral EC line was Ca²⁺-independent and required both NADPH and tetrahydrobiopterin. NOS activity was increased by TNF-α and IFN-γ, and significantly reduced by tumor-cell-conditioned medium. These results suggest that rat colon-carcinoma cells may have developed a protective mechanism involving the release of (a) soluble factor(s) which inhibit(s) NO production by cerebral EC.     

Nitric oxide of human colorectal adenocarcinoma cell lines promotes tumour cell invasion

The present study investigates the role of nitric oxide and the involvement of nitric oxide synthase II isoform on the invasion of human colorectal adenocarcinoma cell lines HRT-18 and HT-29. HRT-18 cells, which constitutively express nitric oxide synthase II mRNA were three-fold more invasive in a Matrigel invasion assay than nitric oxide synthase II mRNA negative HT-29 cells. Treatment of HT-29 cells with the nitric oxide donor Deta NONOate (50 nM) as well as induction of nitric oxide synthase II mRNA and production of endogenous nitric oxide by inflammatory cytokines (IFN-gamma and IL-1alpha) increased the invasiveness of HT-29 cells by approximately 40% and 75%, respectively. In HT-29 cells nitric oxide synthase II mRNA was also induced in co-culture with human monocytes. The invasiveness of HRT-18 cells and stimulated HT-29 cells was partly inhibited by the nitric oxide synthase II inhibitor 1400 W. These results show that nitric oxide increases the invasion of human colorectal adenocarcinoma cell lines HRT-18 and HT-29, and the involvement of nitric oxide synthase II isoform in tumour cell invasion. Therefore, the production of nitric oxide and secretion of pro-inflammatory cytokines by tumour-associated macrophages, which in turn induce nitric oxide synthase II isoform in tumour cells, promotes tumour cell invasiveness.     

Evaluation of antitumor effects following tumor necrosis factor-α gene delivery using nanobubbles and ultrasound

The antitumor effects of tumor necrosis factor (TNF-α) were evaluated following transfection of TNF-α plasmid DNA into solid mouse tumors using the nanobubbles (NBs) and ultrasound (US) gene delivery system. Murine breast carcinoma (EMT6) cells expressing luciferase (1 × 10(6) cells) were injected intradermally into the flanks of 6-7-week-old male SCID mice on day 0. Ten microliters of TNF-α (5 μg/μL) or TNF-α mock plasmid DNA (5 μg/μL) with/without NBs (15 μL) and saline was injected intratumorally in a total volume of 30 μL, and tumors were exposed to US (frequency, 1 MHz; intensity, 3.0 W/cm(2); duty cycle, 20%; number of pulses, 200; and exposure time, 60 s) on days 2, 4, 7, and 9. Changes in tumor size were measured with an in vivo bioluminescent imaging system and a mechanical caliper. Changes in tumor vessel area were quantified using contrast-enhanced US imaging with Sonazoid and a high frequency US imaging system (40 MHz) and immunohistochemistry (CD31). At the mRNA level, expression of TNF-α, caspase-3, and p53 were quantified using real-time quantitative RT-PCR. At the protein level, expression of caspase-3 and p53 were confirmed by immunohistochemistry. We show that repeated TNF-α gene delivery using NBs and US can lead to the local production of TNF-α. This results in antitumor effects, including activation of p53-dependent apoptosis, decrease in tumor vessel density, and suppression of tumor size. In this study, we showed the effectiveness of using NBs and US for TNF-α gene delivery into tumor cells.     

小胶质细胞及其细胞因子对乳腺癌细胞的影响

目的:体外培养小胶质N9细胞并观察其条件培养液(conditioned medium,CM)对乳腺癌MCF-7细胞生存率的影响,以探讨小胶质细胞在脑转移癌中的作用机制.方法:混合培养N9细胞和MCF-7细胞,在倒置光学显微镜下观察细胞形态学的变化.采用脂多糖(lipopolysaccharide,LPS)、MCF-7细胞及其条件培养液MCF-7-CM刺激N9细胞获得N9-CM; ELISA法检测N9-CM中肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-17 (interleukin-17,IL-17)的表达,Griess体系检测一氧化氮(nitric oxide,NO)的生成量.将N9-CM按不同浓度(10％、25％、50％、75％和100％)作用于MCF-7细胞,MTT法检测Ng-CM对MCF-7细胞生存率的影响.结果:MCF-7细胞与N9细胞共培养,N9细胞由静止态变为激活态;LPS、MCF-7细胞及其上清液均可以诱导N9细胞产生TNF-α、IL-17和NO;N9-CM浓度达50％～100％时对MCF-7细胞生长有抑制作用,且作用强度呈时间-浓度相关性.结论:小胶质细胞可以被转移到CNS的乳腺癌细胞激活并发挥肿瘤抑制作用.其作用强度与小胶质细胞分泌NO及细胞因子TNF-α和IL-17的浓度和作用时间呈正相关性.