烟用香精香料对人支气管上皮细胞的毒性研究

为了探讨烟用香精香料对人支气管上皮细胞的毒性作用,并为其安全性评价提供毒理学依据,将永生化的人支气管上皮细胞(BEP2D细胞)分为纯卷烟组和卷烟加香加料组、卷烟加香精和卷烟加香料组、卷烟加不同香精组、卷烟加不同香料组,用MTT法测定细胞存活率,用分子探针氢化乙锭(HE)的荧光产物溴乙锭(EB)相对定量细胞内活性氧(ROS)水平.结果表明加香加料致卷烟烟气冷凝物(CSC)的细胞存活率降低,细胞内ROS水平增高,对细胞的氧化损伤大;与香料比较,香精的细胞存活率低,对细胞的氧化损伤大;1#香精的细胞存活率较高,但对细胞的氧化损伤较大;1#香料的细胞存活率较低,但对细胞的氧化损伤较小,表现在细胞内ROS水平较低.由此町知CSC对BEP2D细胞有毒性,烟用香精香料的添加增加了烟气对BEP2D细胞的毒性作用,而在香精和香料中,香精对细胞毒性较大.     

烟用香精香料的细胞毒性研究

将某种烟用全香精和伞香料按其配方拆分为10个组分,用微量注射法将其添加至某基质卷烟,以人支气管上皮细胞(BEAS-2B)为靶细胞,用噻唑蓝(MTT)法分别测定全香精、全香料、香精香料拆分组分,及其应用于卷烟后烟气凝集物的细胞毒性.研究发现(1)全香料10个拆分组分在所试剂量范围内(最高浓度50 ml/L)未观察到细胞毒性;(2)伞香精的细胞毒性极显著高于全香料(P<0.01);(3)全香精10个拆分组分的细胞毒性均极显著低于全香精(P<0.01);(4)添加全香精、全香料及其拆分组分卷烟CSC的细胞毒性与对照烟均无显著差异(P>0.05).结果表明香精香料添加至卷烟对卷烟的安全性无明显影响.     

橙皮苷调控Bcl-2/Bax-Caspase3信号通路阻抑H2O2诱导的奶牛乳腺上皮细胞凋亡

【目的】探析橙皮苷对奶牛乳腺上皮细胞凋亡的阻抑效果及其分子作用机制,为其在促进奶牛乳腺器官生长发育和维持泌乳功能领域的应用提供理论依据。【方法】以1000μmol/L H₂O₂处理奶牛乳腺上皮细胞8 h构建细胞凋亡模型,再用120μg/mL橙皮苷处理24 h,然后采用CCK-8法测定奶牛乳腺上皮细胞活性,通过激光共聚焦显微镜、流式细胞仪和DNA凝胶电泳检测细胞凋亡情况,利用实时荧光定量PCR检测奶牛乳腺上皮细胞的Bcl-2、Bax和Caspase3基因表达水平,并以Western blotting检测Bcl-2、Bax和Caspase3蛋白的表达变化。【结果】与空白对照组(CK)相比,H₂O₂组奶牛乳腺上皮细胞活性极显著下降(P<0.01,下同),发生凋亡的细胞比例极显著升高,且DNA片段呈梯状条带;橙皮苷组奶牛乳腺上皮细胞活性显著升高(P<0.05,下同),且细胞凋亡程度低,发生凋亡的细胞数目较少。与H₂O₂组相比,H₂...     

谷氨酰胺对高温应激奶牛乳腺上皮细胞凋亡及Bcl-2、HSP70表达的影响

为探讨谷氨酰胺预处理抗高温应激损伤的作用,以体外培养的奶牛乳腺上皮细胞为模型,取对数生长期的奶牛乳腺上皮细胞,分为对照组、谷氨酰胺组、高温组、谷氨酰胺+高温组,采用MTT法检测细胞存活率、Annexin V/PI双染法检测细胞凋亡、荧光定量PCR检测Bcl-2基因和HSP70基因的表达水平、比色法分析Caspase-3活性。结果显示,奶牛乳腺上皮细胞经谷氨酰胺预处理后,HSP70 mRNA表达量极显著增加(P 0. 01),提高了热应激状态下细胞的热耐受性和存活力;与高温组相比,谷氨酰胺+高温处理组Bcl-2 mRNA表达量在热恢复0、6、12 h分别提高3. 23倍(P 0. 05)、2. 49倍(P 0. 05)、1. 89倍(P 0. 05),Caspase-3活性分别下降68. 04%(P 0. 01)、40. 89%(P 0. 05)、52. 06%(P 0. 01)。综上,谷氨酰胺预处理促进了奶牛乳腺上皮细胞Bcl-2和HSP70表达,缓解了高温应激引起的Caspase-3活性上升,抑制了热应激所致的奶牛乳腺上皮细胞凋亡。     

茶多酚对氧化应激所致奶牛乳腺上皮细胞损伤的保护作用

用过氧化氢(H2O2)建立体外奶牛乳腺上皮细胞的氧化应激损伤模型,研究不同质量浓度茶多酚对氧化应激所致奶牛乳腺上皮细胞损伤的影响。MTT法检测细胞存活率,比色法检测细胞培养液中丙二醛(MDA)含量、超氧化物歧化酶(SOD)和乳酸脱氢酶(LDH)活性,流式细胞术检测细胞凋亡情况,分光光度法检测天冬氨酸特异的半胱氨酸蛋白酶3(Caspase-3)的活性。结果显示:与H2O2损伤模型组相比,20～100μg.mL-1茶多酚处理组的奶牛乳腺上皮细胞存活率、SOD活性显著升高,而MDA含量、LDH与Caspase-3相对活性与细胞凋亡率均下降,其中以100μg.mL-1茶多酚处理组效果最显著,说明一定质量浓度的茶多酚可以缓解氧化应激所致的奶牛乳腺上皮细胞的损伤,提高细胞存活率,抑制乳腺细胞凋亡。结论:茶多酚对乳腺上皮细胞氧化应激损伤具有保护作用,其保护作用可能与降低MDA含量,增强SOD活性及抑制Caspase-3活性有关。     

谷氨酰胺对呕吐毒素诱导IPEC-J2细胞凋亡和炎症的影响

[目的]本试验以猪空肠上皮细胞(IPEC-J2)为模型,探讨谷氨酰胺(Gln)对呕吐毒素(DON)诱导的IPEC-J2细胞凋亡和炎症的影响。[方法]通过MTT方法测定细胞活力来选择适宜的Gln浓度,以不添加Gln和DON的细胞为空白对照组,试验组分别为0.75 mmol·L~(-1) Gln组,2.0μg·mL~(-1) DON组和2.0μg·mL~(-1) DON+0.75 mmol·L~(-1) Gln组,处理24 h后测定各组细胞凋亡率、活性氧自由基(ROS)及细胞凋亡和炎症相关基因和蛋白的表达水平。[结果]与对照组相比,DON处理24 h细胞凋亡比例和ROS含量(ROS荧光密度/细胞总数)显著升高(P0.01);与DON组相比,DON+Gln组显著降低细胞的凋亡比例和ROS含量(P0.01)。与对照组相比,DON组上调IPEC-J2细胞白细胞介素1β(IL-1β)、白细胞介素6(IL-6)、环氧合酶2(COX-2)及肿瘤坏死因子α(TNF-α)炎症相关基因和Caspase-3及Caspase-8凋亡相关基因的表达水平;与DON组相比,DON+Gln组下调IPEC-J2细胞IL-1β、COX-2、Caspase-3、Caspase-8、BAK和Bcl-2基因的表达水平。蛋白免疫荧光结果显示,与对照组相比,DON组上调IPEC-J2细胞Caspase-3,核转录因子κB(NF-κB)和磷酸化核转录因子κB(phospho-NF-κB)蛋白的表达,而添加Gln后(DON+Gln组)下调了相关蛋白的表达。[结论]Gln通过清除DON诱导的IPEC-J2细胞中过量的ROS及调节炎症和凋亡相关基因及蛋白的表达量来缓解由DON引起的肠道上皮细胞损伤。     

NEFA对奶牛子宫内膜上皮细胞凋亡因子基因表达的影响

分析NEFA对奶牛子宫内膜上皮细胞内细胞凋亡相关因子mRNA表达的影响.培养奶牛子宫内膜上皮细胞,用CCK-8法检测不同浓度NEFA刺激不同时间后的细胞活性,以筛选出合适的NEFA刺激浓度和时间,再采用qRT-PCR法检测NEFA刺激子宫内膜上皮细胞后细胞凋亡相关因子caspase-3、caspase-8、Bcl-2、...     

黄芪多糖对H_2O_2诱导的奶牛乳腺上皮细胞氧化损伤及凋亡的影响

[目的]本试验旨在研究黄芪多糖(APS)对H_2O_2诱导的奶牛乳腺上皮细胞凋亡、氧化损伤及相关基因、蛋白表达的影响。[方法]体外培养奶牛乳腺上皮细胞并用H_2O_2(600μmol·L~(-1),2 h)诱导建立乳腺上皮细胞氧化应激模型;试验分组:空白对照组、H_2O_2组、H_2O_2+APS组(8 mg·mL~(-1)APS)。[结果]与空白对照组相比,H_2O_2组细胞活力显著下降(P0.05);与H_2O_2组相比,H_2O_2+APS组的细胞凋亡率和细胞中的活性氧(ROS)含量均显著降低(P0.05),而超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)活性显著升高(P0.05),从而缓解H_2O_2诱导奶牛乳腺上皮的氧化损伤;此外,与H_2O_2组相比,H_2O_2+APS组凋亡基因Bax/Bcl-2 mRNA比值及Caspase-3 mRNA的表达量极显著降低(P0.01),Bax蛋白表达水平显著降低(P0.05),且APS降低了H_2O_2诱导细胞凋亡率。[结论]APS能够提高H_2O_2诱导的奶牛乳腺上皮细胞抗氧化酶活性,并减缓氧化损伤引起的奶牛乳腺上皮细胞凋亡。     

烟用香精香料的细胞毒性研究

将某种烟用全香精和全香料按其配方拆分为10个组分,用微量注射法将其添加至某基质卷烟,以人支气管上皮细胞（BEAS-2B）为靶细胞,用噻唑蓝（MTT）法分别测定全香精、全香料、香精香料拆分组分,及其应用于卷烟后烟气凝集物的细胞毒性。研究发现（1）全香料10个拆分组分在所试剂量范围内（最高浓度50mL/L）未观察到细胞毒性;（2）全香精的细胞毒性极显著高于全香料（p〈0.01）;（3）全香精10个拆分组分的细胞毒性均极显著低于全香精（p〈0.01）;（4）添加全香精、全香料及其拆分组分卷烟CSC的细胞毒性与对照烟均无显著差异（p〉0.05）。结果表明香精香料添加至卷烟对卷烟的安全性无明显影响。     

鸡IL-2基因cDNA输卵管组织特异性表达载体的构建与表达

将克隆并测序的鸡卵清蛋白基因5′端调控序列和鸡IL-2基因cDNA,以串联方式置于真核表达载体pcDNA3的CM V启动子下游,构建了鸡输卵管定位表达载体pcDNA3-OVP-IL 2。将真核表达载体pcDNA3-IL 2和构建的输卵管定位表达载体pcDNA3-OVP-IL 2分别转染鸡输卵管上皮细胞,激素诱导72 h后,用淋巴细胞转化试验检测细胞培养液中IL-2的表达水平及生物学活性。结果显示,转染细胞均可表达IL-2,且所表达的IL-2有促进T淋巴细胞转化和淋巴母细胞成熟的活性;转染pcDNA3-OVP-IL 2的输卵管上皮细胞表达产物在1∶256稀释水平下仍具有促进T淋巴细胞转化和淋巴母细胞成熟的活性。转染pcDNA3-IL 2载体的输卵管上皮细胞虽有IL-2表达,但水平较低。     

Cooperative regulation of cell polarity and growth by Drosophila tumor suppressors

Loss of cell polarity and tissue architecture are characteristics of malignant cancers derived from epithelial tissues. We provide evidence from Drosophila that a group of membrane-associated proteins act in concert to regulate both epithelial structure and cell proliferation. Scribble (Scrib) is a cell junction-localized protein required for polarization of embryonic and, as demonstrated here, imaginal disc and follicular epithelia. We show that the tumor suppressors lethal giant larvae (lgl) and discs-large (dlg) have identical effects on all three epithelia, and that scrib also acts as a tumor suppressor. Scrib and Dlg colocalize and overlap with Lgl in epithelia; activity of all three genes is required for cortical localization of Lgl and junctional localization of Scrib and Dlg. scrib, dlg, and lgl show strong genetic interactions. Our data indicate that the three tumor suppressors act together in a common pathway to regulate cell polarity and growth control.     

三氯化铬对大鼠胰岛细胞活性及胰岛素分泌的影响

为了研究三氯化铬与大鼠胰岛细胞共培养对胰岛细胞活性与胰岛素分泌水平的影响,并对其影响机制进行探讨,以台盼蓝染色法和MTT法检测胰岛β细胞活性,以不同浓度葡萄糖刺激胰岛素释放评价三氯化铬对胰岛细胞功能的影响,同时以RT-PCR检测胰岛细胞的抗凋亡基因bcl-2的表达。结果发现,随培养液中三氯化铬浓度的增加,胰岛活性随之增加,凋亡率降低,但2.0μmol/L的活性有所降低,凋亡率与对照组没有显著差异(P>0.05)。低糖和高糖浓度环境中试验组与对照组的胰岛素分泌没有显著差异(P<0.05),RT-PCR发现试验组的bcl-2表达显著高于对照组(P<0.05)。2.0μmol/L CrCl3明显降低胰岛细胞的凋亡率,提高其存活率,改善胰岛功能。     

Research on the Mechanism of Epithelial Cell Exfoliation in Bladders of Prenatal and Postnatal Mice

[Objective] This research aimed at studying the mechanism of the occurrence of epithelial cell loss in bladders of prenatal and postnatal mice. [Method] Bladder epithelia of 1-2-day-old newborn Kunming mice and mice at later period of embryonic development were acquired, and paraffin sections of such epithelia were then made for Mallory's coloration and ordinary optical microscopic observation. The acquired bladder epithelia were made into frozen sections, which were then colored by DAPI and labeled in situ by Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling(TUNEL) for fluorescent microscopic observation. DNA electrophoresis and TUNEL in situ labeling were conducted on the acquired urine exfoliated cells. [Result] The results showed that the complete three layers of cells occurred in 20-day-old mouse embryos, whereas no superficial cells resided in the bladder epithelia of newborn mice and no cells exfoliating were observed. Cells exfoliating from the bladder epithelia of newborn mice were observed, and they exhibited the phenomenon of apoptosis. There were apoptosis existed in the urine cells of newborn mice. [Conclusion] Therefore, the results showed that a few days before and after their birth, mice witnessed the process of cell exfoliation in their bladder epithelia and the exfoliated cells showed typical characteristics of apoptosis.     

角蛋白启动子荧光素酶表达载体的构建及其表达活性分析

角蛋白14(K14)和角蛋白5(K5)主要在毛囊基质和复层鳞状上皮基底层的角质细胞里表达。这两个启动子已被广泛应用于毛囊特异表达外源基因的转基因小鼠模型的研究中,为了能将其应用于转基因绵羊生产,探讨基因对绵羊毛囊发育的影响,从人基因组中克隆得到K14和K5启动子,利用不同的报告基因在细胞水平分析各启动子的表达活性。结果表明,这两个启动子在不同细胞系中都能驱动外源基因的表达,而且K14启动子在小鼠皮肤细胞系JB6-C41中的表达活性高于其他细胞。     

Malignant activation of a K-ras oncogene in lung carcinoma but not in normal tissue of the same patient

A single genetic alteration, a guanine-to-cytosine transversion, is responsible for the acquisition of malignant properties by K-ras genes of two human tumor cell lines established from carcinomas of the bladder (A1698) and lung (A2182). As a consequence, arginine instead of the normal glycine is incorporated into the K-ras-coded p21 proteins at amino acid position 12. This mutation creates a restriction enzyme polymorphism that can be used to screen human cells for transforming K-ras genes. This approach was used to identify the mutational event responsible for the malignant activation of a K-ras oncogene in a squamous cell lung carcinoma of a 66-year-old man; this point mutation was not present in either the normal bronchial or parenchymal tissue or in the blood lymphocytes. Hence, malignant activation of a ras oncogene appears to be specifically associated with the development of a human neoplasm.     

An apical-membrane chloride channel in human tracheal epithelium

The mechanism of chloride transport by airway epithelia has been of substantial interest because airway and sweat gland-duct epithelia are chloride-impermeable in cystic fibrosis. The decreased chloride permeability prevents normal secretion by the airway epithelium, thereby interfering with mucociliary clearance and contributing to the morbidity and mortality of the disease. Because chloride secretion depends on and is regulated by chloride conductance in the apical cell membrane, the patch-clamp technique was used to directly examine single-channel currents in primary cultures of human tracheal epithelium. The cells contained an anion-selective channel that was not strongly voltage-gated or regulated by calcium in cell-free patches. The channel was also blocked by analogs of carboxylic acid that decrease apical chloride conductance in intact epithelia. When attached to the cell, the channel was activated by isoproterenol, although the channel was also observed to open spontaneously. However, in some cases, the channel was only observed after the patch was excised from the cell. These results suggest that this channel is responsible for the apical chloride conductance in airway epithelia.     

辛伐他丁对2型糖尿病大鼠血管内皮VCAM-1和MCP-1mRNA和蛋白表达的影响

目的了解辛伐他丁对2型糖尿病大鼠血管内皮细胞黏附分子-1(VCAM-1)和单核细胞趋化因子-1(MCP-1)的影响。方法30只SD大鼠随机分为正常组(NC)、糖尿病组(DMC)、辛伐他丁治疗组(DMT),每组各10只大鼠。喂养12周后抽血测定血脂全套,取主动脉标本以免疫组化和半定量RT-PCR法分别检测VCAM-1和MCP-1的mRNA和蛋白表达。结果DMT组大鼠血管内皮VCAM-1和MCP-1的mRNA和蛋白表达明显降低,与DMC组比较差异有统计学意义(P<0.05)。结论辛伐他丁可减少糖尿病大鼠血管内皮VCAM-1和MCP-1的表达,减轻血管内皮局部的炎症反应。     

小麦自噬相关基因ATG4和ATG8的原核表达及蛋白纯化

细胞自噬与植物生长、发育和逆境响应等过程密切相关。本研究构建了小麦重要自噬相关基因TaATG4a和TaATG8h的原核表达载体并导入大肠杆菌。IPTG诱导表达后的蛋白质SDS-PAGE电泳结果表明,两个基因在大肠杆菌中均能够高效表达,表达重组蛋白的分子量与预测分子量基本一致。利用Ni柱亲和层析方法进一步获得了纯度较高的重组蛋白,为今后的体外酶活分析、抗体制备和Western杂交等研究奠定了基础。     

Tumorigenicity in human melanoma cell lines controlled by introduction of human chromosome 6

Chromosome banding analysis of human malignant melanoma has documented the nonrandom alteration of chromosome 6. To determine the relevance of chromosome 6 abnormalities in melanoma, a normal chromosome 6 was directly introduced into melanoma cell lines. The resulting (+6) microcell hybrids were significantly altered in their phenotypic properties in culture and lost their ability to form tumors in nude mice. The loss of the chromosome 6 from melanoma microcell hybrids resulted in the reversion to tumorigenicity of these cells in mice. The introduction of the selectable marker (psv2neo) alone into melanoma cell lines had no effect on tumorigenicity. These results support the idea that one or more genes on chromosome 6 may control the malignant expression of human melanoma.     

Effects of palm fat powder and coated folic acid on growth performance,ruminal fermentation,nutrient digestibility and hepatic fat accumulation of Holstein dairy bulls

This study evaluated the effects of palm fat powder(PFP) and coated folic acid(CFA) on growth performance, ruminal fermentation, nutrient digestibility, microbial enzyme activity, microflora, hepatic lipid content and gene expression in dairy bulls. Forty-eight Chinese Holstein bulls((362±12.4) days of age and(483±27.1) kg of body weight(BW)) were assigned to four groups in a completely randomized design with a 2×2 factorial arrangements. Supplemental PFP(0 or 30 g PFP kg~(–1) dietary dry matter(DM)) and CFA(0 or 120 mg FA d~(–1) as CFA) were mixed into the top one-third of a total mixed ration. The study included a 20-day adaptation period and followed by a 90-day collection period. The lower(P0.01) feed conversion ratio with PFP or CFA addition resulted from the constant DM intake and the higher(P0.05) average daily gain. The higher(P0.05) ruminal p H, ether extract digestibility, microbial α-amylase activity, Butyrivibrio fibrisolvens copy, and expression of peroxisome-proliferator-activated receptor α(PPARα) and carnitine palmitoyl transferase~(-1)(CPT1), and lower ruminal total volatile fatty acids(VFA) concentration, acetate to propionate ratio, neutral detergent fibre(NDF) digestibility, copies of total protozoa and Ruminococcus flavefaciens, and expression of sterol regulatory element binding protein~(-1)(SREBP1) and acetyl-coenzyme A carboxylase α(ACACA) were observed for PFP addition. Supplementation with CFA increased(P0.05) ruminal total VFA concentration, acetate to propionate ratio, digestibility of DM, organic matter, crude protein and NDF, activity of cellobiase, pectinase and α-amylase, copies of selected microbial except for total protozoa, as well as expression of PPARα, but decreased(P0.05) ruminal p H, and expression of SREBP1 and ACACA. The PFP×CFA interaction(P0.05) was observed for ammonia N, hepatic TG content, and m RNA expression of CPT1 and FAS. There had no significant difference in hepatic TG content when CFA was supplemented in the diet without PFP addition, the lower(P=0.001) hepatic TG content was observed when CFA was supplemented in the diet with PFP addition. The higher(P0.05) m RNA expression of CPT1, and the lower(P0.05) m RNA expression of FAS and ammonia N concentration were observed when CFA was supplemented in diet either without or with PFP addition. The results indicated that supplementation of CFA in PFP diet was more effective on increasing hepatic CPT1 expression, and decreasing ammonia N, hepatic TG content and FAS expression than in diet without PFP. Supplementation with PFP or CFA improved growth performance of dairy bulls by promoting nutrient utilization, microbial enzyme activity, microflora, and hepatic gene expression.