Alcohol exposure alters mouse lung inflammation in response to inhaled dust

Alcohol exposure is associated with increased lung infections and decreased mucociliary clearance. Occupational workers exposed to dusts from concentrated animal feeding operations (CAFOs) are at risk for developing chronic inflammatory lung diseases. Agricultural worker co-exposure to alcohol and organic dust has been established, although little research has been conducted on the combination effects of alcohol and organic dusts on the lung. Previously, we have shown in a mouse model that exposure to hog dust extract (HDE) collected from a CAFO results in the activation of protein kinase C (PKC), elevated lavage fluid cytokines/chemokines including interleukin-6 (IL-6), and the development of significant lung pathology. Because alcohol blocks airway epithelial cell release of IL-6 in vitro, we hypothesized that alcohol exposure would alter mouse lung inflammatory responses to HDE. To test this hypothesis, C57BL/6 mice were fed 20% alcohol or water ad libitum for 6 weeks and treated with 12.5% HDE by intranasal inhalation method daily during the final three weeks. Bronchoalveolar lavage fluid (BALF), tracheas and lungs were collected. HDE stimulated a 2-4 fold increase in lung and tracheal PKCε (epsilon) activity in mice, but no such increase in PKCε activity was observed in dust-exposed mice fed alcohol. Similarly, alcohol-fed mice demonstrated significantly less IL-6 in lung lavage in response to dust than that observed in control mice instilled with HDE. TNFα levels were also inhibited in the alcohol and HDE-exposed mouse lung tissue as compared to the HDE only exposed group. HDE-induced lung inflammatory aggregates clearly present in the tissue from HDE only exposed animals were not visually detectable in the HDE/alcohol co-exposure group. Statistically significant weight reductions and 20% mortality were also observed in the mice co-exposed to HDE and alcohol. These data suggest that alcohol exposure depresses the ability of the lung to activate PKCε-dependent inflammatory pathways to environmental dust exposure. These data also define alcohol as an important co-exposure agent to consider in the study of inhalation injury responses.     

蛋白激酶C信号通路对温石棉介导的肺成纤维细胞细胞周期调控蛋白表达的影响

目的 研究细胞周期调控蛋白在温石棉所致成纤维细胞（HEPF）增殖中的表达和蛋白激酶C（PKC）对细胞周期调控蛋白的影响。方法 利用体外细胞培养技术,用流式细胞仪测定温石棉介导的HEPF的细胞周期调控蛋白的表达,同时设立阳性对照（标准SiO2)、阳性对照（标准TiO2)、正常对照（未经粉尘处理的免肺泡巨噬细胞）和空白对照。结果 温石棉细胞周期蛋白（cyclin)Cyclin D1、CyclinE、增殖细胞核抗原（PCNA）和CyclinA表达阳性率分别为19．0％、22．8％、79．7％和65．1％,均较各对照组低,差异有显著性（P＜0．05）,且CyclinD1和CyclinE表达阳性率低于PCNA和CyclinA,差异有显著性（P＜0．01)而 CyclinB1和p34cdc2激酶的阳性表达率较空白对照和正常对照略高。当PKC信号通路被阻断后,PCNA在温石棉组表达的阳性率明显降低,差异有显著性（P＜0．05）,p34cdc2激酶的阳性表达率改变不明显。结论 这几种蛋白可能均参与了温石棉所致HEPF增殖,其中PCNA表达的改变可能与上游PKC信号通路有关。     

Prenatal alcohol exposure alters expression of neurogenesis-related genes in an ex vivo cell culture model

Prenatal alcohol exposure can lead to long-lasting changes in functional and genetic programs of the brain, which may underlie behavioral alterations seen in Fetal Alcohol Spectrum Disorder (FASD). Aberrant fetal programming during gestational alcohol exposure is a possible mechanism by which alcohol imparts teratogenic effects on the brain; however, current methods used to investigate the effects of alcohol on development often rely on either direct application of alcohol in vitro or acute high doses in vivo. In this study, we used our established moderate prenatal alcohol exposure (PAE) model, resulting in maternal blood alcohol content of approximately 20 mM, and subsequent ex vivo cell culture to assess expression of genes related to neurogenesis. Proliferating and differentiating neural progenitor cell culture conditions were established from telencephalic tissue derived from embryonic day (E) 15–17 tissue exposed to alcohol via maternal drinking throughout pregnancy. Gene expression analysis on mRNA derived in vitro was performed using a microarray, and quantitative PCR was conducted for genes to validate the microarray. Student's t tests were performed for statistical comparison of each exposure under each culture condition using a 95% confidence interval. Eleven percent of genes on the array had significantly altered mRNA expression in the prenatal alcohol-exposed neural progenitor culture under proliferating conditions. These include reduced expression of Adora2a, Cxcl1, Dlg4, Hes1, Nptx1, and Vegfa and increased expression of Fgf13, Ndn, and Sox3; bioinformatics analysis indicated that these genes are involved in cell growth and proliferation. Decreased levels of Dnmt1 and Dnmt3a were also found under proliferating conditions. Under differentiating conditions, 7.3% of genes had decreased mRNA expression; these include Cdk5rap3, Gdnf, Hey2, Heyl, Pard6b, and Ptn, which are associated with survival and differentiation as indicated by bioinformatics analysis. This study is the first to use chronic low to moderate PAE, to more accurately reflect maternal alcohol consumption, and subsequent neural progenitor cell culture to demonstrate that PAE throughout gestation alters expression of genes involved in neural development and embryonic neurogenesis.     

锰致PC12细胞凋亡作用及与p-Erk关系

目的以鼠嗜铬神经瘤细胞(PC12)为模型,筛选锰对神经细胞增殖抑制作用的时间及剂量,观察锰作用下PC12细胞的细胞形态学、生化指标改变和磷酸化的胞外信号调节激酶(p-Erk)的表达。方法用200、400、600、800μmol/L MnCl2的培养液,分别作用对数生长期PC12细胞1、2、3、4 d后,四甲基偶氮塞唑蓝(MTT)筛选锰的细胞毒性剂量;透射电镜观察细胞形态学变化;琼脂糖凝胶电泳检测MnCl2对PC12细胞基因组DNA的影响。免疫印迹法(western blot)检测p-Erk。结果 MTT显示200～800μmol/L MnCl2作用1、2、3、4 d对PC12有显著的抑制作用,呈剂量和时间依赖趋势,600μmol/L MnCl2作用4 d对PC12的抑制率50%;600μmol/L MnCl2作用4 d电镜可见细胞凋亡,同样条件下细胞DNA碎片化;Western blot显示600μmol/L MnCl2作用1、2、3、4 d可见p-Erk2逐渐降低,其中作用2 d时较对照降低75%(n=3,P<0.05),200、400、600μmol/L MnCl2分别作用4 d时,p-Erk亦逐渐降低,当400μmol/L MnCl2作用4 d时较对照明降低78%(n=3,P<0.01);使用Erk通路MEK1/2特异性阻制剂PD98059实验结果表明:锰可能通过MEK1/2磷酸化下游的Erk,下调p-Erk。结论锰对PC12细胞的毒性作用可能是通过关闭胞外信号调节激酶ErK通路诱导细胞凋亡。     

蛋白激酶抑制剂对青石棉诱导肺成纤维细胞的细胞周期调控蛋白表达的影响

目的 探讨青石棉致人胚肺成纤维细胞（ＨＥＰＦ）增殖的细胞周期变化的机制。方法 用流式细胞技术检测青石棉诱导ＨＥＰＦ的细胞周期调控蛋白表达的改变,观察酷氨酸蛋白激酶（ＴＰＫ）和蛋白激酶Ｃ（ＰＫＣ）的抑制剂对其表达的影响。结果 青石棉可诱导ＨＥＰＦ的Ｃｙｃｌｉｎ Ｄ１、Ｃｙｃｌｉｎ Ｅ、ＰＣＮＡ、Ｃｙｃｌｉｎ Ａ、Ｃｙｃｌｉｎ Ｂ１和Ｐ３４ｃｄｃ２激酶等蛋白的阳性表达率发生改变。在青石棉组,ＰＫＣ抑制剂使表达的阳性率较未加入ＰＫＣ抑制剂组明显降低,对Ｐ３４ｃｄｃ２激酶影响不大（Ｐ〉０．０５）;而ＴＰＫ抑制剂使ＰＣＮＡ表达的阳性率增高;Ｐ３４ｃｄｃ２激酶表达的阳性率降低（Ｐ〈０．０１）。结论 这几种蛋白可能均了青石棉致ＨＥＰＦ增殖,ＰＣＮＡ（和Ｐ３４ｃｄｃ２激酶）表达的改变可能与上游的ＰＫＣ（和ＴＰＫ）信号通路有关。     

Omega-3 free fatty acids attenuate insulin-promoted breast cancer cell proliferation

High insulin levels in obese people are considered as a risk factor to induce breast carcinogenesis. And consumption of fish oils which mainly contain omega-3 fatty acids is associated with a reduced risk of breast cancer. However, whether omega-3 free fatty acids (FFAs) modulate insulin signaling pathway to prevent breast cancer is poorly understood. The current study tested the hypothesis that omega-3 FFAs attenuate insulin-induced breast cancer cell proliferation and regulate insulin signaling pathway. We show here that omega-3 FFAs attenuate MCF-7 cell proliferation and Akt and Erk1/2 phosphorylation levels stimulated by insulin. Knockdown Shp2 by siRNA resulted in significantly elevated omega-3 FFAs–activated Akt phosphorylation but failed to change insulin-stimulated Akt and Erk1/2 phosphorylation. And viable cell number was not affected by either downregulation of Shp2 expression or Erk1/2 inhibitor U0126 treatment. These observations indicated that omega-3 FFAs attenuate insulin-promoted breast cancer cell proliferation and insulin-activated Akt phosphorylation.     

MDR1及MDR3基因与卵巢癌细胞顺铂耐药的关系

目的:探讨MDR1(Multidrug resistance gene-1)及MDR3(Multidrug resistance gene-3)基因与卵巢癌细胞顺铂耐药的关系。方法:两种质粒一起瞬时转染A2780/DDP细胞,空载体组及未转染组作为对照。Annexin V-FITC/PI双标流式细胞术、MTT法、RT-PCR、western blot分别检测A2780/DDP细胞早晚期凋亡、细胞增殖活性、MDR1和MDR3 mRNA表达及caspase-3蛋白水平。结果:两种质粒(pSuppressorNeo MDR1 siRNA,MDR3 siRNA)均未能逆转A2780/DDP细胞顺铂耐药:①流式显示转染MDR1 siRNA组、MDR3 siRNA组和pSuppressorNeo空载体组后相比较未转染A2780/DDP细胞凋亡率无显著改变(P>0.05);②在一定药物浓度范围内,转染MDR1和MDR3小分子干扰RNA均未能增强顺铂对A2780/DDP细胞的细胞毒作用;③与空载体组和未转染细胞相比,A2780/DDP细胞的MDR3及MDR1 mRNA表达均低下(P>0.05);④Caspase-3蛋白表达未见增加。结论:顺铂所致的卵巢癌细胞多药耐药与MDR1及MDR3基因过度表达均无关,卵巢癌细胞顺铂耐药为非P-gp     

Malondialdehyde-acetaldehyde adducts decrease bronchial epithelial wound repair.

Most people who abuse alcohol are cigarette smokers. Previously, we have shown that malondialdehyde, an inflammation product of lipid peroxidation, and acetaldehyde, a component of both ethanol metabolism and cigarette smoke, form protein adducts that stimulate protein kinase C (PKC) activation in bronchial epithelial cells. We have also shown that PKC can regulate bronchial epithelial cell wound repair. We hypothesize that bovine serum albumin adducted with malondialdehyde and acetaldehyde (BSA–MAA) decreases bronchial epithelial cell wound repair via binding to scavenger receptors on bronchial epithelial cells. To test this, confluent monolayers of bovine bronchial epithelial cells were grown in serum-free media prior to wounding the cells. Bronchial epithelial cell wound closure was inhibited in a dose-dependent manner (up to 60%) in the presence of BSA-MAA than in media treated cells (Laboratory of Human Carcinogenesis [LHC]-9-Roswell Park Memorial Institute [RPMI]). The specific scavenger receptor ligand, fucoidan, also stimulated PKC activation and decreased wound repair. Pretreatment with fucoidan blocked malondialdehyde–acetaldehyde binding to bronchial epithelial cells. When bronchial epithelial cells were preincubated with a PKC alpha inhibitor, Gö 6976, the inhibition of wound closure by fucoidan and BSA–MAA was blocked. Western blot demonstrated the presence of several scavenger receptors on bronchial epithelial cell membranes, including SRA, SRBI, SRBII, and CD36. Scavenger receptor–mediated activation of PKC alpha may function to reduce wound healing under conditions of alcohol and cigarette smoke exposure where malondialdehyde–acetaldehyde adducts may be present.     

Dietary copper affects azoxymethane-induced intestinal tumor formation and protein kinase C isozyme protein and mRNA expression in colon of rats

Previous studies have show that changes in protein kinase C (PKC) isoform expression may be related to increased susceptibility of copper-deficient rats to aberrant crypt formation. The purpose of this study was to determine whether dietary copper would affect azoxymethane-induced intestinal tumor formation and PKC isozyme expression in normal colonic mucosa and tumor samples. Eighty weanling Fischer-344 rats were randomly assigned to diets that contained either 0.8 or 5.3 microg Cu/g diet. After 24 and 31 d of diet consumption, 30 rats/diet were administered azoxymethane (15 mg/kg i.p.) and 10 rats/diet were administered saline. Rats continued to consume their respective diets for an additional 38 wk. Rats injected with azoxymethane and fed the low copper diet had a significantly (P < 0.0001) greater small intestinal and total tumor incidence compared with rats fed adequate dietary copper. However, dietary copper did not affect colon tumor incidence. Low dietary copper significantly (P < 0.004) decreased PKC alpha protein expression in normal but not in tumor tissue. In contrast, low dietary copper did not affect PKC delta or zeta protein expression in either the normal or tumor tissue. PKC alpha and delta protein and mRNA expression were lower in tumor tissue than in normal tissue. These results along with previous observations suggest that dietary copper-mediated changes in PKC alpha, delta and zeta protein expression are not as important for colon tumor promotion/progression as they are for tumor initiation.     

Increased translocation of cardiac protein kinase C beta2 accompanies mild cardiac hypertrophy in rats fed saturated fat

Signal transduction through protein kinase C (PKC) beta2 may modulate cardiac hypertrophy in pressure-overloaded rat myocardium. Because PKC beta2 can be activated by fatty acids and diacylglycerol, we hypothesized that altering the level and type of dietary fat might modulate cardiac PKC activation and stimulate hypertrophy in otherwise normal rat myocardium. Male Sprague-Dawley rats (n = 32) were randomly assigned to either a low fat [10% total energy intake (TEI)] or high fat diet (40% TEI) based on corn or coconut oil as a source of saturated or unsaturated fat. After 40 d of isoenergetic diet consumption, the heart/body weight ratio was slightly greater in rats fed saturated fat diets compared with those fed unsaturated fat (P = 0.05). Increased activation of PKC beta2, as evidenced by greater membrane translocation, was also observed in all rats fed saturated fat diets (P < 0.01). PKC alpha, beta1 and epsilon did not change. These results suggest that dietary fat type can alter PKC beta2 activation in the heart, and exert a mild hypertrophic effect on the heart.     

Role of Protein Kinase C in TBT-Induced Inhibition of Lytic Function and MAPK Activation in Human Natural Killer Cells

Human natural killer (NK) cells are lymphocytes that destroy tumor and virally infected cells. Previous studies have shown that exposure of NK cells to tributyltin (TBT) greatly diminishes their ability to destroy tumor cells (lytic function) while activating mitogen-activated protein kinases (MAPK) (p44/42, p38, and JNK) in NK cells. The signaling pathway that regulates NK lytic function appears to include activation of protein kinase C (PKC) as well as MAPK activity. TBT-induced activation of MAPKs would trigger a portion of the NK lytic signaling pathway, which would then leave the NK cell unable to trigger this pathway in response to a subsequent encounter with a target cell. In the present study we evaluated the involvement of PKC in inhibition of NK lysis of tumor cells and activation of MAPKs caused by TBT exposure. TBT caused a 2–3-fold activation of PKC at concentrations ranging from 50 to 300 nM (16–98 ng/ml), indicating that activation of PKC occurs in response to TBT exposure. This would then leave the NK cell unable to respond to targets. Treatment with the PKC inhibitor, bisindolylmaleimide I, caused an 85% decrease in the ability of NK cells to lyse tumor cells, validating the involvement of PKC in the lytic signaling pathway. The role of PKC in the activation of MAPKs by TBT was also investigated using bisindolylmaleimide I. The results indicated that, in NK cells where PKC activation was blocked, there was no activation of the MAPK, p44/42 in response to TBT. However, TBT-induced activation of the MAPKs, p38 and JNK did not require PKC activation. These results indicate the pivotal role of PKC in the TBT-induced loss of NK lytic function including activation of p44/42 by TBT in NK cells.     

苯乙基异硫氰酸盐对小鼠胚胎干细胞分化为神经细胞过程中基因表达影响的研究

目的探讨苯乙基异硫氰酸盐(PEITC)对小鼠胚胎干细胞(mESCs)体外定向诱导分化为神经样细胞中PKCα及其相关发育基因(pax-6、netrin-1)表达的影响。方法mESCs经RA法诱导后,第7天用RT-PCR法检测nestin、glutaminase、Brn-3基因等神经细胞特异性标志物。不同浓度PEITC作用条件下,分别取诱导后1、3、5、7及14天后的细胞,用RT-PCR方法测定细胞的PKCα及其相关发育基因pax-6和netrin-1的mRNA表达情况。结果诱导后第7天,分化的神经样细胞中nestin、glutaminase、Brn-3基因均高表达。正常诱导1天后,PKCα、pax-6和netrin-1基因的mRNA表达急剧下降,3、5、7天逐渐升高,至14天恢复至正常水平。PEITC作用下,PKCα、pax-6、netrin-1的mRNA在诱导后细胞中各阶段的表达水平与正常诱导条件下相比均下降,且随着作用浓度和作用时间的增加,下降趋势明显。结论PEITC对mESCs定向分化为神经样细胞过程中相关发育基因(pax-6、netrin-1)的表达的干扰作用可能与抑制PKCα的表达有关。     

Chronic prenatal ethanol exposure alters expression of central and peripheral insulin signaling molecules in adult guinea pig offspring

Maternal ethanol consumption during pregnancy can produce a range of teratogenic outcomes in offspring. The mechanism of ethanol teratogenicity is multi-faceted, but may involve alterations in insulin and insulin-like growth factor (IGF) signaling pathways. These pathways are not only important for metabolism, but are also critically involved in neuronal survival and plasticity, and they can be altered by chronic prenatal ethanol exposure (CPEE). The objective of this study was to test the hypothesis that CPEE alters expression of insulin and IGF signaling molecules in the prefrontal cortex and liver of adult guinea pig offspring. Pregnant Dunkin-Hartley-strain guinea pigs received ethanol (4 g/kg maternal body weight/day) or isocaloric-sucrose/pair-feeding (nutritional control) throughout gestation. Fasting blood glucose concentration was measured in male and female offspring at postnatal day 150–200, followed by euthanasia, collection of prefrontal cortex and liver, and RNA extraction. IGF-1, IGF-1 receptor (IGF-1R), IGF-2, IGF-2 receptor (IGF-2R), insulin receptor substrate (IRS)-1, IRS-2, and insulin receptor (INSR) mRNA expression levels were measured in tissues using quantitative real-time PCR. The mean maternal blood ethanol concentration was 281 ± 15 mg/dL at 1 h after the second divided dose of ethanol on GD 57. CPEE resulted in increased liver weight in adult offspring, but produced no difference in fasting blood glucose concentration compared with nutritional control. In the liver, CPEE decreased mRNA expression of IGF-1, IGF-1R, and IGF-2, and increased IRS-2 mRNA expression in male offspring only compared with nutritional control. Female CPEE offspring had decreased INSR hepatic mRNA expression compared with male CPEE offspring. In the prefrontal cortex, IRS-2 mRNA expression was increased in CPEE offspring compared with nutritional control. The data demonstrate that CPEE alters both central and peripheral expression of insulin and IGF signaling molecules at the mRNA level, which may be related to metabolic dysregulation in adult offspring. Furthermore, altered insulin and IGF signaling may be a mechanism of ethanol neurobehavioral teratogenicity.     

Stearate Preferentially Induces Apoptosis in Human Breast Cancer Cells

Stearic acid (stearate) is an 18-carbon saturated fatty acid that has been shown to inhibit invasion and proliferation and induce apoptosis in various human cell types. The specificity of stearate-induced apoptosis for cancerous versus noncancerous breast cells has not been examined, and the mechanism underlying stearate-induced apoptosis is unknown. Morphological analysis, cell viability, and caspase-3 activity assays demonstrated that stearate activated apoptosis preferentially in cancerous breast cells in a time- and dose-dependent manner. Inhibition of de novo diacylgycerol synthesis or protein kinase C (PKC) blocked stearate-induced caspase-3 activity, indicating the involvement of a novel or classical PKC isozyme. To our knowledge this is the first study showing that stearate induces apoptosis preferentially in breast cancer cells and implicates protein kinase C in the signaling cascade. These results raise the possibility of dietary stearate having a beneficial role in the prevention or treatment of breast cancer.     

Restricted feed intake in lactating primiparous sows. II. Effects on subsequent litter sex ratio and embryonic gene expression

Expression of panels of candidate genes controlling myogenesis, angiogenesis and gender-specific imprinting of development were analysed in embryonic, placental and endometrial tissues recovered at Day 30 of gestation from a subset of primiparous sows that were either feed restricted (Restrict; n=17) or fed to appetite (Control; n=15) during the last week of the previous lactation. Embryos were also sex typed to investigate gender bias in response to treatments. Average embryonic weight was lower in the subset of Restrict compared with Control litters (1.38±0.07vs 1.59±0.08g, respectively) and the male:female sex ratio was higher (P<0.05) in embryos (litters) recovered from Restrict sows. Treatment affected (P≤0.05) the expression of embryonic and placental genes involved in insulin-like growth factor (IGF) 2 signalling, including IGF2, INSR and IGF2R. Embryonic expression of ESR1 was also affected by treatment (P<0.03) and sex×treatment interactions were observed for the expression of embryonic ESR1 (P<0.05) and placental ANGPT2 (P<0.03). At the molecular level, these results support the suggestion that changes in placental function are not the primary mechanism mediating detrimental effects of previous sow catabolism on early embryonic development in the feed-restricted lactational sow model. However, perturbations in the IGF2 system are implicated as mediators of these effects.     

Low-dose of ionizing radiation enhances cell proliferation via transient ERK1/2 and p38 activation in normal human lung fibroblasts

This study shows the human cellular responses and the mechanism of low-dose ionizing radiation in CCD 18 Lu cells, which are derived from normal human lung fibroblasts. Cell proliferation and viability assay were measured for the cells following gamma-irradiation using trypan blue, BrdU incorporation, and Wst-1 assay. We also examined genotoxicity using a micronuclei formation assay. The activation of the MAPKs pathway was determined by Western blot analysis, and the siRNA system was used to inhibit the expression of ERK1/2 and p38. We found that 0.05 Gy of ionizing radiation stimulated cell proliferation and did not change Micronuclei frequencies. In addition, 0.05 Gy of ionizing radiation activated ERK1/2 and p38, but did not activate JNK1/2 in cells. A specific ERK1/2 inhibitor, U0126, decreased the phosphorylation of ERK1/2 proteins induced by 0.05 Gy of ionizing radiation, and a similar suppressive effect was observed with a p38 inhibitor, PD169316. Suppression of ERK1/2 and p38 phosphorylation with these inhibitors decreased cell proliferation, which was stimulated by 0.05 Gy of ionizing radiation. Furthermore, downregulation of ERK1/2 and p38 expression using siRNA blocked the cell proliferation that had been increased by 0.05 Gy of ionizing radiation. These results suggest that 0.05 Gy of ionizing radiation enhances cell proliferation through the activation of ERK1/2 and p38 in normal human lung fibroblasts.     

RNA interference against aldehyde dehydrogenase-2: development of tools for alcohol research

Liver alcohol dehydrogenase oxidizes ethanol to acetaldehyde, which is further oxidized to acetate by aldehyde dehydrogenase-2 (ALDH2*1). Individuals who carry a low-activity ALDH2 (ALDH2*2) display high blood acetaldehyde levels after ethanol consumption, which leads to dysphoric effects, such as facial flushing, nausea, dizziness, and headache (“Asian alcohol phenotype”), which result in an aversion to alcohol and protection against alcohol abuse and alcoholism. Mimicking this phenotype may reduce alcohol consumption in alcoholics. RNA interference (RNAi) is a cell process in which a short interfering RNA (siRNA) of 21-25 bp guides the degradation of a complementary target mRNA. Thus, siRNAs may be useful in mimicking the Asian phenotype by inhibiting ALDH2 gene expression. We determined the inhibitory effect of three chemically synthesized siRNAs targeted against rat ALDH2 mRNA in human embryonic kidney cells (HEK-293 cell lines) transfected with a plasmid carrying the rat ALDH2 cDNA. Two of the three siRNAs were active, yielding a 65-75% reduction of ALDH2 activity. Based on the most promising siRNA sequence, three short hairpin RNA (shRNA) genes driven by the human U6 RNA promoter were designed and cloned in a plasmid. After transfection of HEK-293 cells, one of the genes was shown to be active, yielding a 50% reduction of ALDH2 activity. This effect is consistent with a 50% reduction in ALDH2 mRNA, whereas neither β-actin mRNA nor the interferon-inducible transmembrane protein-1 mRNA levels were affected. This study describes chemically synthesized siRNAs and an endogenously synthesized shRNA, which reduce ALDH2 activity and constitute tools that should be of value for further alcohol research.     

沉默信息调节因子1小干扰RNA诱导前列腺癌细胞PC3细胞凋亡

摘要:目的　观察沉默信息调节因子1（SIRT1）小干扰RNA（siRNA）对前列腺癌细胞PC3细胞生长增殖、DNA合成、细胞凋亡和Bcl-2和Bax蛋白的表达变化,探讨SIRT1在前列腺癌发生中的可能机制。方法　体外培养PC3细胞,分空白对照组（mock组）,转染阴性对照组（scramble siRNA组）和SIRT1 siRNA转染组;Western blot检测PC3细胞中SIRT1的干涉效能;MTT法测定PC3细胞的增殖率;BrdU掺入法测定DNA合成;流式细胞术检测细胞凋亡;Western blot检测PC3细胞中细胞凋亡关键调控因子Bcl-2和Bax的蛋白表达。结果　与对照组比较,SIRT1 siRNA组SIRT1蛋白表达降低（P＜0.01）,PC3细胞的增殖和DNA合成明显受抑制（P＜0.01）,细胞凋亡比例增加（P＜0.01）,Bcl-2蛋白表达减少,Bax的表达增加。结论　下调SIRT1的表达抑制细胞增殖和DNA合成,诱导前列腺癌PC3细胞发生凋亡,其机制可能与改变细胞凋亡关键调控因子Bcl-2和Bax的蛋白表达相关。