家蚕蛹蛋白酶解产物对肿瘤细胞MGC-803的增殖抑制活性鉴定

以不同蛋白酶酶解制备家蚕蛹蛋白酶解产物(SPPHs),采用MTT比色法检测其对人胃腺癌细胞MGC-803的增殖抑制活性。不同蛋白酶酶解制备SPPHs对MGC-803的增殖抑制作用存在显著差异(P0.05),其中家蚕蛹蛋白以Alcalase 2.4L酶解的产物(AH)对MGC-803的增殖抑制活性最高,当AH的质量浓度为0.25 mg/m L时,水解度(DH)=25%的AH的抑制活性最高,对MGC-803细胞的增殖抑制率可达到92.74%。Alcalase2.4L酶解家蚕蛹蛋白产物的分子质量也显著影响其对MGC-803细胞的增殖抑制活性,其中分子质量5 k D组分的抑制率高达91.13%,将该组分经葡聚糖(G-25)凝胶层析后分离出4个组分(Ⅰ、Ⅱ、Ⅲ、Ⅳ),组分Ⅱ对MGC-803细胞的增殖抑制率最高(27.5%),但与未分离样品相比,抑制效率显著降低,而其他3个分离组分则无抑制作用。研究结果提示,蚕蛹蛋白酶解产物对肿瘤细胞的体外增殖有抑制作用,并且这种抑制作用可能是由多种组分协同完成。     

利巴韦林对猫细小病毒诱导的细胞凋亡抑制作用

为了研究利巴韦林对感染猫细小病毒(FPV)细胞的影响,试验将猫肾细胞(F81细胞)随机分为对照组、病毒组、药物组,采用MTT法检测细胞的生长曲线,Hochest 33258染色观察细胞的细胞核形态,流式细胞仪检测细胞周期。结果表明:Hochest 33258染色试验中药物组与病毒组差异不显著(P0.05);药物组细胞的增殖活性始终高于病毒组,但低于对照组;流式细胞仪检测利巴韦林治疗后48小时G0/G1期细胞增多,S期细胞减少,G2/M期细胞变化不大;72小时G2/M期细胞增多;药物组与病毒组细胞的流式检测在G1峰的左侧均出现典型的亚G1峰,但各时间点药物组细胞的凋亡指数均明显低于病毒组。说明利巴韦林能抑制FPV诱导的细胞凋亡,具有一定的治疗指导意义。     

柞蚕蛹虫草水提物对人乳腺癌细胞MCF-7增殖和凋亡的影响

研究柞蚕蛹虫草水提物（Aqueous extract from cordycep militaris of antheraea pemyi,AEoAPC）对人乳腺癌细胞MCF-7生长抑制与凋亡的作用.将不同浓度的AEoAPC分别作用于人乳腺癌细胞MCF-7 24、48、72 h后,应用倒置相差显微镜观察人乳腺癌细胞MCF-7的形态变化,噻唑蓝比色法测定细胞生长抑制效应,流式细胞术检测细胞周期变化及凋亡率.结果表明：AEoAPC能显著抑制人乳腺癌细胞MCF-7的增殖,且在一定的浓度范围内呈时间和浓度的依赖性;形态学观察发现,细胞形状不规则、变暗、皱缩;FCM检测结果显示,作用72 h后G0-G1期（DNA合成前期）的细胞分布有所增加,使细胞阻滞于G2期（DNA合成后期）和S期（DNA合成期）;0.8g/L AE0-APC在24、48、72 h凋亡率分别为8.65％、14.20％、26.30％,其凋亡程度与时间呈正相关.说明AEoAPC能明显抑制人乳腺癌细胞MCF-7的生长,诱导其细胞凋亡.     

人乳铁蛋白对宫颈癌Hela细胞凋亡的影响

为了研究人乳铁蛋白对宫颈癌Hela细胞的生长抑制作用及其对细胞凋亡、p53蛋白表达和Caspase-3活性的影响,试验采用MTT方法检测细胞的生长抑制率、DNA片段化试验检测细胞凋亡、流式细胞术测定细胞周期、Western-blot检测p53蛋白表达、Caspase-3活性检测试剂盒检测Caspase-3的活性.结果表明:人乳铁蛋白对宫颈癌Hela细胞的抑制呈现时间和剂量依赖性,有73%的Hela细胞停滞在G0～G1期;Western-blot检测结果显示人乳铁蛋白可以上调p53蛋白的表达; 人乳铁蛋白作用48 h后Caspase-3的活性是0小时时的5.5倍,二者差异极显著(P<0.01).结果说明人乳铁蛋白能显著抑制宫颈癌Hela细胞生长,其抗肿瘤机制与诱导细胞凋亡、调控细胞周期、上调p53表达、活化Caspase-3有关.     

不同炮制方法对人参皂苷含量及肝癌细胞凋亡的影响

为了探讨不同炮制方法对人参皂苷含量及肝癌细胞凋亡的影响,采用HPLC法测定人参液中单体皂苷Rg1、Re、Rb1(三者合为总皂苷)和稀有皂苷Rg3的含量;MTT法检测不同人参提取液对人肝癌细胞HepG2的抑制率,用流式细胞仪检测细胞凋亡率。结果表明,在人参炮制过程中,人参总皂苷含量由0.56%下降到0.13%,稀有皂苷Rg3含量由0.17%增加到0.30%;抑制率检测结果表明,浓度为28.8 g/L时3种提取液对细胞的抑制作用最强,黑参组对肝癌细胞的抑制作用高于人参组和红参组;凋亡实验发现,黑参提取液对细胞的促进凋亡作用明显,人参提取液的促凋亡作用不明显。表明红参和黑参提取液可以抑制人肝癌细胞HepG2增殖并促进其凋亡,可能与参液中的稀有皂苷Rg3含量有关。     

乌龙茶多糖和多酚对肝癌细胞HepG2的协同抑制作用

为了探讨乌龙茶多糖、多酚对肝癌细胞HepG2细胞增殖及细胞周期的影响,以及两者联合使用对肝癌细胞HepG2协同杀伤抑制作用,通过四氮甲唑蓝(MTT)试验检测细胞的增殖抑制率,以观察乌龙茶多糖、多酚对肝癌细胞HepG2的增殖抑制作用;应用流式细胞仪检测细胞凋亡率。结果表明,乌龙茶多糖、多酚可以抑制肝癌细胞HepG2的生长、增殖;二者联合应用对肝癌细胞HepG2的杀伤作用显著强于2种药物单独使用,且经药物联合应用处理后的肝癌细胞HepG2的凋亡率要高于2种药物单独使用。说明乌龙茶多糖、多酚对肝癌细胞HepG2具有杀伤作用,乌龙茶多糖与多酚联合使用具有协同抗肿瘤的作用。     

连翘提取物FS-4体外诱导胃癌细胞SGC-7901凋亡作用的研究

为了探讨连翘提取物FS-4体外对胃癌细胞SGC-7901的促凋亡作用,试验采用MTT比色法观察了FS-4对胃癌细胞SGC-7901增殖的抑制情况,AO/EB双荧光染色法和透射电子显微镜观察了细胞凋亡的形态学变化,并通过流式细胞术测定细胞的凋亡率。结果表明：FS-4对胃癌细胞SGC-7901增殖的抑制作用具有剂量和时间依赖性,其36 h的半数抑制浓度(IC₅₀)仅为3.23μg/mL;FS-4作用后细胞均出现典型的凋亡细胞形态;FS-4对细胞的诱导凋亡作用呈现明显的浓度依赖性。说明FS-4可抑制胃癌细胞SGC-7901的增殖并具有诱导其凋亡的作用。     

蛋氨酸脑啡肽对禽马立克细胞株（MSB-1）增殖的影响

实验旨在研究蛋氨酸脑啡肽(MENK)对禽马立克细胞MSB-1生长抑制作用和凋亡诱导效应。用MENK对禽马立克细胞MSB-1细胞株进行干预，采用光镜观察细胞密度及形态，MTT法测定细胞增殖抑制率，流式细胞术检测细胞凋亡。镜下观察可见在10-12 mol/l MENK的作用下，MSB-1细胞株数量降低，细胞形态渐圆钝，甚至出现皱缩，与对照组相比MENK对MSB-1细胞具有明显的生长抑制作用，抑制率为19%~46%(P<0.01)，诱导细胞凋亡率为（62.04±3.32）%(P<0.01)。MENK对MSB-1细胞株具有增殖抑制效应及凋亡诱导效应，是具有发展潜力的抗肿瘤药物。     

17-β-雌二醇对小鼠胸腺上皮细胞增殖的影响

为探讨17-β-雌二醇对小鼠胸腺上皮细胞增殖的影响及其分子机制,将小鼠胸腺上皮细胞系(MTEC1)经不同浓度和不同时间的17-β-雌二醇处理后,采用CCK-8法、Edu掺入法检测细胞增殖活性的变化;流式细胞术检测细胞周期的变化;DAPI染色法检测细胞核形态的变化;Western blot技术检测细胞周期相关基因CDK1及CyclinB1蛋白表达的变化。结果显示,雌激素显著抑制MTEC1细胞的生长,并呈时间和剂量依赖性;雌激素处理组G2/M期细胞显著增多,出现G2/M期阻滞;细胞形态变化明显,出现形状变大、多核、核染色质凝聚等现象;CDK1及CyclinB1的蛋白表达水平显著下调。结果表明,17-β雌二醇可以抑制MTEC1细胞的增殖,其机制可能是通过下调细胞周期正调节因子CDK1及CyclinB1的表达,使细胞阻滞于G2/M期。     

口蹄疫病毒诱导BHK-21细胞产生凋亡的研究

试验利用口蹄疫病毒感染BHK-21细胞,通过MTT法、Hoechst 33258染色、原位末端标记技术(TUNEL)、流式细胞术和链特异性荧光定量RT-PCR,分别就口蹄疫病毒对BHK-21细胞生长的抑制作用、凋亡细胞的形态学和分子生物学特征、凋亡峰的出现和细胞周期的变化以及口蹄疫病毒基因组在BHK-21细胞内的复制情况进行了检测。结果表明:口蹄疫病毒可抑制BHK-21细胞的生长并诱导其产生凋亡,呈现典型的凋亡细胞特征,出现细胞凋亡峰并且细胞周期明显被阻滞在G1/G0期,同时对凋亡率和口蹄疫病毒基因组复制的关系做了初步研究。     

华蟾毒精对小鼠免疫细胞活性的影响

为观察华蟾毒精（CBG）对小鼠免疫细胞活性的影响,本研究采用MTT法检测小鼠脾淋巴细胞的增殖情况,检测小鼠腹腔巨噬细胞的吞噬功能以及小鼠NK细胞对靶细胞的杀伤作用。结果显示,CBG在一定剂量范围内单独或者协同非特异性丝裂原（Con A或LPS）作用能够显著增强小鼠脾淋巴细胞的增殖,CBG单独作用可以显著提高小鼠腹腔巨噬细胞的吞噬功能,并能够显著提高小鼠NK细胞对靶细胞的杀伤作用,表明CBG能够提高小鼠免疫细胞的活性。     

华蟾毒精对B16及RMA-S细胞主要组织相容性复合体Ⅰ类分子及其相关基因表达的影响

试验旨在考察华蟾毒精（CBG）单体对B16和RMA-S细胞MHC-Ⅰ类分子及其相关基因表达的影响。通过MTT法检测CBG对B16和RMA-S细胞生长的影响;流式细胞术检测CBG对B16和RMA-S细胞表面MHC-Ⅰ分子表达的影响;荧光定量PCR检测CBG对B16和RMA-S细胞蛋白酶体相关基因（LMP2、LMP7）和ATP结合盒（ATP-binding cassette,ABC）转运蛋白（TAP1、TAP2）基因mRNA表达的影响。结果显示,CBG对B16和RMA-S细胞MHC-Ⅰ表达的影响均不显著(P＞0.05),但高浓度CBG可不同程度提高LMP2、LMP7、TAP1、TAP2基因mRNA表达水平。结果表明,CBG可能具有通过上调LMP2、LMP7、TAP1、TAP2基因表达水平来增强肿瘤自身免疫原性的潜能。     

Apoptosis and anti-apoptotic heat shock proteins in canine cutaneous infundibular keratinizing acanthomas and squamous cell carcinomas

Cell stress and death are linked in the neoplastic process, and heat shock proteins appear to play an important role by inhibiting apoptotic pathways. The apoptotic rates in 9 canine infundibular keratinizing acanthomas (IKAs) and 17 canine squamous cell carcinomas (SCCs) were correlated with the immunohistochemical expression of caspase-3 and the antiapoptotic heat shock proteins Hsp27, 72 and 73. Apoptosis was evaluated using the terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL) method. The absence of a correlation between the TUNEL index and active-caspase-3 expression, a paucity of active-caspase-3-positive cells and Hsp72 over-expression were considered to be indicative of inhibition of apoptosis, and suggestive that inhibition of cell death plays a key role in oncogenesis and tumour growth of some canine skin neoplasms.     

Antiproliferative effects and apoptosis induction by probiotic cytoplasmic extracts in fish cell lines

Probiotic bacteria are known to exert a wide range of beneficial effects on their animal hosts. Control of intestinal homeostasis, inflammation suppression and a reduction in the incidence of cancer all rely on the antiproliferative potential of probiotics. In this paper, we assess the antiproliferative activity of probiotics in two teleost fish cell lines SAF-1, a fibroblast cell line and EPC, an epithelioma from carp. Cells were grown in the presence of cytoplasmic extracts obtained from two bacterial strains, Lactobacillus delbrüeckii subsp. lactis (LDL) and 51M6. Proliferation and apoptosis were measured after 4, 24, 48 or 72h in culture by the crystal violet or by double staining flow cytometry assays, respectively. Generally, LDL had stronger effects on cell growth than 51M6. Moreover, SAF-1 cells were more susceptible to growth inhibition than EPC cells. Apoptosis took place following growth inhibition, especially when LDL extracts were used. The results are discussed in terms of the biological significance of probiotic bacteria that naturally occur on the fish mucosal surfaces with an emphasis on how dose and species specificity may be determinant factors.     

Comparison of Tifton 85 and Coastal bermudagrasses for yield, nutrient traits, intake, and digestion by growing beef steers.

A study was undertaken to compare Tifton 85 (T85) and Coastal (CBG) bermudagrasses for effects of cultivar and age at harvest on yields of DM and digestible DM, in vitro digestion, nutrient content, cell wall composition, in situ digestion kinetics, and feed intake and digestion by growing beef steers. In Exp. 1, T85 and CBG forages staged for growth in May or July of 1993 were harvested at 3, 4, 5, 6, 7, and 8 wk from subplots. Tifton 85 bermudagrass had 7.1% greater DM yield, 18.2% higher (P < .05) digestible DM yield, and 7.1% greater IVDMD than CBG, and, after 5 wk of forage growth, IVDMD of both T85 and CBG decreased with increased age at harvest (P < .05). In Exp. 2, T85 and CBG forages staged for growth in July 1997 were harvested at 2, 3, 4, 5, 6, and 7 wk from subplots. Even though T85 had higher concentrations of NDF and ADF than CBG, T85 had 34.1% higher DM yield, 47.9% higher digestible DM, 55.0% higher digestible NDF, 91.7% higher digestible ADF, greater IVDMD, in vitro NDF and ADF disappearances, and higher in situ DM and NDF digestion (P < .05). Coastal bermudagrass had higher concentrations of lignin and lower concentrations of total neutral sugars, arabinose, glucose, and xylose than T85 (P < .05). In vitro digestibilities of DM, NDF, and ADF were lower and concentrations of ADF and lignin were greater for 7- vs 6-wk harvests of both T85 and CBG (P < .05). In Exp. 3, T85 and CBG forages staged for growth in July 1997 were harvested as hay at 3, 5, and 7 wk from .8-ha pastures and fed to 36 individually penned growing beef steers (initial BW = 244 kg) to quantify ad libitum intake without supplementation. Tifton 85 bermudagrass had lower concentrations of lignin and ether-linked ferulic acid and greater concentrations of NDF, ADF, hemicellulose, and cellulose than CBG (P < .05). Steers fed T85 had higher (P < .05) digestion of DM, OM, NDF, ADF, hemicellulose, and cellulose than steers fed CBG. Digestion of NDF, ADF, hemicellulose, and cellulose decreased (P < .05) with increased age at harvest for both cultivars. In conclusion, T85 produced more DM and had more digestible nutrients in vitro, in situ, and in vivo than CBG, and 3 and 5 wk of growth would be recommended ages to harvest either cultivar.     

鼠伤寒沙门氏菌粘附素样糖蛋白的分离与鉴定

鼠伤寒沙门氏菌用去污剂NP-40处理，获得细菌外膜增溶液，经ConA-Sepharose4B亲和层析，甘露糖洗脱，得到了ConA结合糖蛋白，SDS-PAGE电泳显示出了5条蛋白带，分子量在15～43kD之间。用不同处理并借助扫描电镜观察细菌在雏鸡肠上皮的粘附情况，表明分离的ConA结合糖蛋白具有粘附素样作用，肠粘膜表面含乙酰氨基葡萄糖的糖复合物则可能是粘附素的受体。     

Evaluation of the effects of histone deacetylase inhibitors on cells from canine cancer cell lines

OBJECTIVE: To determine whether exposure of canine cancer cells to histone deacetylase (HDAC) inhibitors S(+)-N-hydroxy-4-(3-methyl-2-phenyl-butyrylamino)benzamide (OSU-HDAC42) or suberoylanilide hydroxamic acid (SAHA) results in increased histone acetylation and decreased cell viability and whether any changes in viability involve induction of apoptosis or alterations in progression of the cell cycle. Sample POPULATION: 9 canine cancer cell lines. PROCEDURES: Cells from 9 canine cancer cell lines were treated with dimethyl sulfoxide vehicle, OSU-HDAC42, or SAHA, then assays of cell viability were performed. Histone acetylation was assessed by use of western blot analysis. Apoptosis was assessed via ELISA to detect fragmentation of cytoplasmic nucleosomal DNA and western blot analysis to detect cleavage of caspase 3. Cell cycle analysis was performed by use of propidium iodide staining and flow cytometry. RESULTS-Concentrations of OSU-HDAC42 and SAHA required to achieve 50% inhibition of cell viability (IC(50)) were reached in cells of 6 and 4 canine cancer cell lines, respectively, and ranged from approximately 0.4 to 1.3 microM for OSU-HDAC42 and 0.6 to 4.8 microM for SAHA. Cells from T-cell lymphoma, mast cell tumor, osteosarcoma, and histiocytic sarcoma lines were most sensitive to HDAC inhibition, with IC(50)s of < 1 microM for OSU-HDAC42 and < 5 microM for SAHA. Induction of apoptosis was indicated via cleavage of caspase 3 and increases in cytoplasmic nucleosomes and the subG(1) cell population. CONCLUSIONS AND CLINICAL RELEVANCE: Micromolar concentrations of HDAC inhibitors OSU-HDAC42 and SAHA induced histone acetylation, cytotoxicity, and apoptosis in canine cancer cells. In general, OSU-HDAC42 was more potent than SAHA.     

恩诺沙星联合用药对MRC-5细胞生长抑制的研究

本研究旨在评价恩诺沙星分别与3种抗菌药（环丙沙星、氟苯尼考和磺胺二甲嘧啶）的联合毒性。选用MRC-5细胞作为细胞模型模拟抗菌药的混合污染对肺细胞的损害，设置多个浓度梯度和混合比例，采用CCK-8方法检测4种抗菌药单药对细胞生长的抑制率及恩诺沙星分别与3种抗菌药混合后对细胞生长的抑制率，而后使用Chou-Talalay方法拟合中值效应曲线，计算出联合指数（combination index，CI）。结果显示，在检测浓度范围内，4种单药对MCR-5细胞的生长抑制率随药物浓度增加均呈阶梯状上升，其中氟苯尼考对MCR-5细胞的生长抑制率较低（<4.5%）。3种二元药物组合的混合毒性均表现出了浓度依赖性和混合比例的依赖性，联合使用恩诺沙星与环丙沙星在高、中剂量组下对MRC-5细胞表现协同毒性（CI<1），极低剂量组下表现为抑制毒性（CI>1）；联合使用恩诺沙星与氟苯尼考对MRC-5细胞的毒性主要是相互抑制的（CI>1）；联合使用恩诺沙星与磺胺二甲嘧啶对MRC-5细胞的毒性可能表现相互促进（CI<1），也可能表现相互抑制（CI>1），具体表现与给药剂量和浓度比值有关。本研究表明，在抗菌药的毒性评价中，评估抗菌药的联合毒性是有必要的，使用Chou-Talalay方法可快速高效地完成细胞水平上对抗菌药联合毒性的评估。     

Evaluation of Joint Toxicity of Enrofloxacin and Three Antimicrobials on MRC-5 Cells

The aim of this study was to evaluate the combined toxicity of enrofloxacin with each of three antimicrobials (ciprofloxacin,florfenicol and sulfamethazine).MRC-5 cells were used as a cell model to simulate the damage of lung cells caused by mixed contamination of antimicrobials.Multiple concentration gradients and mixing ratios were set.CCK-8 method was used to determine the inhibition rate of cell growth caused by four antimicrobials and the inhibition rate of cell growth caused by enofloxacin mixed with three antimicrobials,respectively.Then Chou-Talalay method was used to fit the median effect plot and to calculate the combination index (CI) value.The results showed that the growth inhibition rates of MCR-5 cells caused by four single drugs went up in a step-like manner with the increase of drug concentration in the tested concentration range,among them,the growth inhibitory rate of MCR-5 cells by florfenicol was low (<4.5%).The combined toxicity of the three binary combinations showed a concentration-dependent and mixing-ratio dependence.Mixing enrofloxacin and ciprofloxacin showed synergistic toxicity (CI<1) on MRC-5 cells at high and middle concentration groups,and antagonistic toxicity (CI>1) at the very-low-concentration groups.Mixing ciprofloxacin and florfenicol mainly showed an inhibited toxicity (CI>1).Binary combination enrofloxacin and sulfamethazine might present either a synergistic joint toxicity (CI<1) or an antagonistic joint toxicity (CI>1) as the concentration and mixing ratio changing.This study showed that it was necessary to assess the combined toxicity of antimicrobials in the toxicity evaluation of antimicrobials.Using Chou-Talalay method,the joint toxicity of multiple antimicrobials could be quickly and efficiently determined at cellular level.     

In vitro retinoid-induced growth inhibition and morphologic differentiation of canine osteosarcoma cells

OBJECTIVE: To determine differentiation and growth inhibition effects of retinoids on canine osteosarcoma cells. SAMPLE POPULATION: 3 osteosarcoma cell lines established from osteosarcomas in dogs. PROCEDURE: Osteosarcoma cells were incubated with various concentrations of all-trans-retinoic acid and 9-cis-retinoic acid or control medium, counted daily for 10 days, and evaluated for morphologic changes. Synthesis of DNA was measured by use of a cell proliferation ELISA. To analyze effect of retinoids on colony formation on plastic dishes, cells were cultured for 14 days, fixed, and stained; number of colonies was counted. RESULTS: In a dose-dependent manner, both retinoids induced morphologic differentiation and growth inhibition in the 3 osteosarcoma cell lines and inhibited each cell's ability to form anchorage-dependent colonies. CONCLUSION AND CLINICAL RELEVANCE: Retinoids induced differentiation of osteosarcoma cells of dogs, resulting in altered expression of their malignant phenotype. Induction of differentiation by retinoids may have potential as an adjunctive treatment for osteosarcoma in dogs.