猪圆环病毒2型感染PK-15细胞中外泌体的鉴定及其对细胞增殖和凋亡的影响

旨在分离感染PCV2的PK-15细胞分泌的外泌体,探讨其在PCV2感染淋巴细胞中的作用。提取PCV2感染PK-15细胞上清液中的外泌体,利用透射电镜观察外泌体形态、纳米颗粒跟踪分析测定外泌体粒径、Western blot检测外泌体标记蛋白分子（CD81、TSG101）;PKH67标记外泌体后检测细胞对外泌体的摄取;提取PCV2-外泌体基因组,检测PCV2 Rep和Cap基因;利用间接免疫荧光试验检测PCV2-外泌体感染PK-15细胞后PCV2病毒定位;通过绝对定量PCR检测PCV2-外泌体对淋巴细胞的感染率;利用CCK-8法检测淋巴细胞增殖;利用Annexin V-FITC/PI检测淋巴细胞凋亡率。透射电镜和外泌体粒径分析结果显示,PK-15细胞分泌的外泌体为双层膜的囊泡,直径30~200 nm;Western blot结果显示PK-15细胞分泌的外泌体存在特异性标记蛋白CD81和TSG101;外泌体摄取试验结果显示PCV2-外泌体能够被细胞摄取;PCR结果表明PCV2-外泌体基因组中含有PCV2 Rep和Cap基因;间接免疫荧光结果显示,与PCV2直接感染相比,PCV2-外泌体感染的淋巴细胞中,PCV2病毒拷贝显著升高,差异明显（P<0.01）,外泌体裂解后感染淋巴细胞能力与PCV2病毒无显著差异;PCV2-外泌体可显著抑制淋巴细胞增殖（P<0.01或P<0.05）;PCV2-外泌体显著提高淋巴细胞凋亡率。PCV2感染PK-15细胞的外泌体中携带病毒基因,感染淋巴细胞后使细胞增殖降低和细胞凋亡增加。     

细胞凋亡及其在昆虫生理学研究中的意义

从细胞凋亡的概念、发生特点、形态学、生化特征和生物学意义方面进行了综述,并在近年昆虫细胞凋亡研究的基础上,阐述了细胞凋亡在其生理学研究上的重要意义。     

睾酮诱导的小鼠骨髓巨噬细胞凋亡途径研究

用睾酮诱导,研究小鼠骨髓巨噬细胞(BMMs)凋亡过程中Fas/FasL途径上caspase-8表达的改变。以BMMs经L929条件培养基(LCM)诱导5d后,用流式细胞仪分选出F4/80阳性细胞,并将细胞分为两大组。第1组是空白对照组,睾酮(100nmol·L-1,下同)处理组,去除LCM组,去除LCM同时用睾酮处理组。第2组用FADD反义寡核苷酸(ASODN)转染BMMs后,重复第1组的4个处理组,并以FADD错义寡核苷酸(MSODN)转染后的睾酮处理组作为对照。处理12h后,用流式细胞仪检测巨噬细胞的凋亡情况,并通过RT-PCR、Real-timeRT-PCR和WesternBlot方法检测各组中caspase-8基因及蛋白的表达。结果显示,在缺少LCM或用睾酮处理时,睾酮可在体外诱导小鼠骨髓巨噬细胞凋亡,并伴随caspase-8的活化。FADD反义寡核苷酸能抑制睾酮诱导的小鼠骨髓巨噬细胞的凋亡,其下游的caspase-8表达也被抑制。     

上腹部手术患者手术和麻醉对中性粒细胞凋亡及bc1-2基因表达的影响

目的：研究手术与麻醉对上腹部手术患者的中性粒细胞(PMN)凋亡及Bcl—2基因表达的影响。方法：分离围术期上腹部手术患者外周血中性粒细胞，采用流式细胞仪分析培养24h中性粒细胞凋亡及bcl-2基因表达的情况。结果：与健康人相比，手术患者术前中性粒细胞调亡被明显抑制(P＜0．01)，bcl—2基因表达明显上调(P＜0．01)。而与手术患者自身术前相比，硬外麻醉后中性检细胞凋亡抑制及bcl-2表达上调变化不明显，但手术切皮后3h、术后24h、72h变化有显著的差异(P＜0．01)，且以术后24h达峰值。结论：外科创伤可导致明显的PMN凋亡抑制，而bcl—2基因表达上调可能是创伤后PMN凋亡抑制的分子机制之一，硬膜外麻醉对PMN凋亡及bcl—2基因表达影响不大。     

富硒乳酸菌对黄曲霉毒素B1诱导鸡小肠上皮细胞凋亡的影响

试验旨在探究富硒乳酸菌对黄曲霉毒素B₁(AFB₁)对鸡小肠上皮细胞(CSIEC)凋亡的影响。将体外培养的CSIEC细胞分为对照组(C)、AFB₁(300 μmol/L AFB₁)、AFB₁+0.01 Se(300 μmol/L AFB₁+0.01 μmol/L富硒乳酸菌,以Se计)、AFB₁+0.05 Se和AFB₁+0.1 Se组,待细胞长至60%~70%汇合时,AFB₁+0.01 Se、AFB₁+0.05 Se和AFB₁+0.1 Se组培养液更换为含对应Se浓度的培养液,对照组和AFB₁组更换为正常培养液,8 h后向含AFB₁组加入300 μmol/L AFB₁,继续培养24 h。用CCK-8法检测细胞存活率,ELISA法检测培养液中乳酸脱氢酶(LDH)的活性,TUNEL法检测细胞凋亡率,Western blotting检测线粒体通路和内质网通路相关蛋白Bcl-2、Bax、PERK、ATF4、CHOP、GRP78、p53和p21的表达。细胞存活率检测结果表明,与对照组相比,其余各组CSIEC存活率均极显著降低(P<0.01);与AFB₁组相比,加硒各组存活率均极显著升高(P<0.01)。LDH活性检测结果表明,与对照组相比,其余各组LDH活性均极显著升高(P<0.01);与AFB₁组相比,加硒各组LDH活性均极显著降低(P<0.01)。细胞凋亡率检测结果表明,与对照组相比,AFB₁组TUNEL阳性细胞数极显著升高(P<0.01),且与加硒各组差异均不显著(P>0.05)。Bcl-2/Bax表达量检测结果表明,与对照组相比,AFB₁组Bcl-2/Bax极显著降低(P<0.01),AFB₁+0.05 Se组显著升高(P<0.05)。内质网应激通路相关蛋白表达量检测结果表明,与对照组相比,AFB₁组PERK、CHOP、GRP78和p53的表达量均极显著升高(P<0.01);与AFB₁组相比,加硒各组PERK、GRP78表达量均极显著降低(P<0.01),AFB₁+0.05 Se和AFB₁+0.1 Se组CHOP和p53的表达量均极显著或显著降低(P<0.01;P<0.05)。因此,富硒乳酸菌能够缓解AFB₁诱导的CSIEC凋亡。     

茶多酚对鼠胎儿成纤维细胞凋亡的影响

为探究茶多酚(Tea polyphenols,TP)对体外培养鼠胎儿成纤维细胞(Mouse embryonic fibrolast,MEF)凋亡的影响。在MEF细胞培养液中添加质量浓度为0、10、40、70、100和150μg/mL的TP,分别培养24、48、72和96h,使用MTT法(3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐,噻唑蓝,MTT法)检测细胞活力;细胞培养72h,以RT-PCR检测B-细胞淋巴瘤/白血病-2原癌基因(B cell lymphoma leukemia-2,Bcl-2)、和Bcl相关的蛋白X(Bcl-as-sociated X protein,Bax)、半胱天冬酶-3(Caspase-3)的相对表达量。MTT结果表明:TP质量浓度为40μg/mL时细胞密度略高于其他组,但是没有显著差异(P0.05);质量浓度为100μg/mL时,对MEF的生长产生抑制作用(P0.05),细胞形态没有发生明显变化;当质量浓度为150μg/mL时对细胞生长抑制作用明显(P0.05),从形态观察细胞出现调亡;通过反转录结果表明,对Bax基因、Bcl-2基因和Caspase-3基因的分子转录水平没有影响(P0.05),表明质量浓度高于100μg/mL TP对MEF生长产生抑制作用,但对Bax基因、Bcl-2基因和Caspase-3转录分子机制无影响。     

Design and Synthesis of Analogues of Marine Natural Product Galaxamide,an N-methylated Cyclic Pentapeptide,as Potential Anti-Tumor Agent in Vitro

Herein, we report design and synthesis of novel 26 galaxamide analogues with N-methylated cyclo-pentapeptide, and their in vitro anti-tumor activity towards the panel of human tumor cell line, such as, A549, A549/DPP, HepG2 and SMMC-7721 using MTT assay. We have also investigated the effect of galaxamide and its representative analogues on growth, cell-cycle phases, and induction of apoptosis in SMMC-7721 cells in vitro. Reckon with the significance of conformational space and N-Me aminoacid (aa) comprising this compound template, we designed the analogues with modification in N-Me-aa position, change in aa configuration from l to d aa and substitute one Leu-aa to d/l Phe-aa residue with respective to the parent structure. The efficient solid phase parallel synthesis approach is employed for the linear pentapeptide residue containing N-Me aa, followed by solution phase macrocyclisation to afford target cyclo pentapeptide compounds. In the present study, all galaxamide analogues exhibited growth inhibition in A549, A549/DPP, SMMC-7721 and HepG2 cell lines. Compounds 6, 18, and 22 exhibited interesting activities towards all cell line tested, while Compounds 1, 4, 15, and 22 showed strong activity towards SMMC-7221 cell line in the range of 1–2 μg/mL IC₅₀. Flow cytometry experiment revealed that galaxamide analogues namely Compounds 6, 18, and 22 induced concentration dependent SMMC-7721 cell apoptosis after 48 h. These compounds induced G0/G1 phase cell-cycle arrest and morphological changes indicating induction of apoptosis. Thus, findings of our study suggest that the galaxamide and its analogues 6, 18 and 22 exerted growth inhibitory effect on SMMC-7721 cells by arresting the cell cycle in the G0/G1 phase and inducing apoptosis. Compound 1 showed promising anti-tumor activity towards SMMC-7721 cancer cell line, which is 9 and 10 fold higher than galaxamide and reference DPP (cisplatin), respectively.     

铅和（或）镉对大鼠肾细胞的毒性损伤及硒的拮抗作用

为探讨铅和（或）镉的毒性机制及硒的拮抗作用,在大鼠肾细胞培养过程中添加不同浓度的铅、镉和硒,在显微镜下观察细胞形态,测定细胞凋亡率和细胞周期及上清液脂质过氧化指标。结果表明：（1）20μmol/L以上铅、镉单独染毒组细胞损伤严重,加硒后细胞的形态损伤有降低的趋势;（2）铅、镉单独和联合染毒组GSH-Px、SOD活性随染毒剂量的增加,呈逐渐下降趋势（P〈0.05）,而MDA含量和细胞凋亡率则呈升高趋势（P〈0.05）,具有明显的剂量-效应关系和剂量-时间关系;（3）在铅、镉单独和联合染毒组加硒,脂质过氧化指标呈下降趋势（P〈0.05）,说明硒对铅、镉及其联合暴露所致的脂质过氧化损伤有一定的保护作用（P〈0.05）。     

The effects of baicalein on canine osteosarcoma cell proliferation and death

Flavonoids are a group of modified triphenolic compounds from plants with medicinal properties. Baicalein, a specific flavone primarily isolated from plant roots (Scutellaria baicalensis), is commonly used in Eastern medicine for its anti‐inflammatory and antineoplastic properties. Previous research shows greater efficacy for baicalein than most flavonoids; however, there has been little work examining their effects on sarcoma cells, let alone canine cells. Three canine osteosarcoma cell lines (HMPOS, D17 and OS 2.4) were treated with baicalein to examine cell viability, cell cycle kinetics, anchorage‐independent growth and apoptosis. Results showed that osteosarcoma cells were sensitive to baicalein at concentrations from approximately 1 to 25 μM. Modest cell cycle changes were observed in one cell line. Baicalein was effective in inducing apoptosis and did not prevent doxorubicin cell proliferation inhibition in all the cell lines. The mechanism for induction of apoptosis has not been fully elucidated; however, changes in mitochondrial permeability supersede the apoptotic response.