冬虫夏草菌丝粗多糖对SP2/0细胞凋亡和p53表达的影响

观察冬虫夏草菌丝粗多糖对SP2／0细胞凋亡诱导和p53基因表达的影响。在体外用粗多糖处理SP2／0细胞,MTT法检测其对肿瘤细胞增殖抑制作用,Hochest33342荧光染色观察细胞凋亡的形态学变化,DNA琼脂糖凝胶电泳检测细胞凋亡,流式细胞仪检测细胞的周期,实时荧光定量PCR检测p53基因的表达情况。结果发现,与对照组相比,冬虫夏草粗多糖可抑制SP2／0细胞增殖,诱导出现典型凋亡形态变化,电泳出现特有梯形条带,引起细胞周期阻滞,使p53基因的表达明显改变。这提示冬虫夏草粗多糖对SP2／0细胞生长的抑制,可能与诱导凋亡和声53基因表达的改变有关。     

TNF-α对培养大鼠脂肪细胞凋亡的影响

以5、10、15ng／mL 3种剂量TNF—α作用体外培养的大鼠脂肪细胞0、12、24、36h和48h，采用荧光染色计数、流式细胞仪检测脂肪细胞凋亡率；透射电镜观察脂肪细胞超微结构变化；琼脂糖凝胶电泳检测DNA断裂情况。结果表明，TNF—α对脂肪细胞具有诱导凋亡作用，凋亡率在一定范围内具有剂量和时间依赖性，以10ng／mL TNF—α作用36h为最佳；电镜下观察可见脂肪细胞形态改变，出现染色质凝聚、边集和凋亡小体形成等特征性变化；DNA电泳检测可见处理24h以后出现“梯状”条带，作用0h和12h未见“梯状”条带。     

桦木酸对胸腺淋巴细胞凋亡的影响研究

研究桦木酸(betulinic acid,BA)对淋巴细胞凋亡的影响。采用体外培养的方法,以地塞米松(Dexamethasone,Dex)诱发胸腺淋巴细胞凋亡为模型,实验分为空白对照组、Dex组和BA(5μg/m L)组,用荧光显微镜进行细胞形态学观察,通过琼脂糖凝胶电泳观察细胞凋亡过程中DNA的裂解,用流式细胞仪分析并测定细胞凋亡率。结果表明,BA可降低Dex诱导的淋巴细胞凋亡。提示BA对Dex诱导的淋巴细胞凋亡具有干预作用,在一定程度上起到预防性的保护作用。     

两株H5N1亚型禽流感病毒NS1基因诱导细胞凋亡的研究

为了研究H5N1亚型禽流感病毒NS1基因诱导的细胞凋亡作用，我们将两株H5N1亚型禽流感病毒A／Duck／Guangdong／40／2000（H5NI）（简称DKGD／40／00）和A／Duck／Guangxi／35／2001（H5NI）（简称DKGX／35／01）的NSI基因克隆到真核表达载体plRES2-EGFP，转染Hela细胞后，应用荧光显微镜检测转染表达效率，应用TUNEL和流式细胞仪观察NSI基因产生的细胞凋亡作用。结果两株病毒NSI蛋白表达均能诱导Hela细胞凋亡，凋亡率无明显差异。表明单一的NS1并不能完全阐明凋亡的产生与病毒毒力强弱和病毒扩散之间的关系。     

甘草提取物(GC-X)体外诱导人宫颈癌HeLa细胞凋亡作用的研究

为了探讨甘草提取物(GC-X)体外对HeLa细胞的促凋亡作用,试验采用MTT比色法观察了GC-X对HeLa细胞增殖的抑制情况,AO/EB双荧光染色法和透射电镜观察了细胞凋亡的形态学变化,流式细胞仪测定了各组细胞的凋亡率。结果表明:GC-X体外对HeLa细胞的生长、增殖的抑制作用具有剂量和时间依赖性,其48 h的IC50仅为11.24μg/m L;AO/EB双荧光染色和透射电镜观察可见,GC-X作用后He La细胞均出现典型的凋亡细胞形态;流式细胞仪检测GC-X对HeLa细胞的诱导凋亡作用呈现明显的浓度依赖性。说明GC-X可抑制HeLa细胞的增殖并具有诱导细胞凋亡的作用。     

亚慢性铝中毒对雏鸡大脑神经细胞凋亡的影响

采用连续腹腔注射固定体积、不同浓度梯度三氯化铝(AICl3)水溶液法,建立不同程度的亚慢性铝中毒雏鸡模型,通过吖啶橙/溴化乙锭双荧光染色法、琼脂糖凝胶电泳法和流式细胞术对雏鸡大脑神经细胞凋亡进行观测.结果表明:①荧光显微镜下可见,染铝组雏鸡大脑神经细胞发生典型的凋亡和坏死变化,细胞凋亡指数增加,极显著高于对照组(P<0.01),且随AlCl3浓度增加而加重.②染铝组雏鸡大脑神经细胞G0/G1期细胞数量增加,G2/M和S期细胞数量减少,细胞增殖指数降低,细胞凋亡率升高,且随染铝剂量的增多而加重,与对照组比,组间差异均极显著(P<0.01).③各染铝组雏鸡大脑琼脂糖凝胶电泳检测出现典型的DNA梯形带.上述结果说明,铝可引发雏鸡大脑神经细胞DNA损伤,抑制其增殖,诱导其凋亡.     

钙稳态失衡在氯化镧诱粕CC-MSB1细胞凋亡中的作用

为研究钙稳态失衡在LaCl3诱导MDCC-MSB1细胞凋亡中的作用,将MDCC-MSB1细胞常规培养于RPMI-1640培养液中,加入终浓度为0．5、1．0、1．5、2．0、2．5、3．0、3．5和4．0mmol／L的LaCl3,继续培养24h后,应用MTT法检测细胞增殖抑制率,DNA Ladder法和TUNEL法检测细胞凋亡,以Fura-2为荧光探针检测细胞内[Ca^2＋]i的变化。在LaCl3浓度为0．5-4．0mmol/L范围内,细胞的增殖抑制率增加,细胞凋亡数量和细胞内[Ca^2＋]i呈升高趋势,并呈剂量一效应关系。结l果表明,LaCl3能抑制MDCC—MSB1细胞的增殖,并可能通过改变[Ca^2＋]i而诱导其发生凋亡。     

传染性法氏囊病病毒人工感染诱导靶细胞细胞凋亡的研究

本试验借助透射电镜技术、琼脂糖凝胶电泳、流式细胞术和荧光染色技术对传染性法氏囊病病毒变异 Ｅ 株人工感染诱导的雏鸡法氏囊淋巴细胞和体外培养法氏囊细胞的细胞凋亡进行了较为详细的研究。结果表明， Ｉ Ｂ Ｄ Ｖ 感染后早期阶段，雏鸡法氏囊淋巴细胞和体外培养法氏囊细胞呈现典型细胞凋亡的形态学特征和生化特 征。 Ｉ Ｂ Ｄ Ｖ 感染雏鸡后２４～４８ ｈ 和感染法氏囊培养细胞后４～２４ ｈ，法氏囊淋巴细胞凋亡细胞数量显著增加，而正常淋巴细胞数量相应减少，揭示 Ｉ Ｂ Ｄ Ｖ 人工感染可以诱导雏鸡法氏囊淋巴细胞和体外培养法氏囊淋巴细胞的细胞凋亡。     

动物细胞大规模培养中细胞凋亡检测方法的研究

为了探讨动物细胞大规模培养中细胞凋亡的检测方法,以不同浓度氯化铵诱导BHK-21细胞凋亡,每隔12 h收集细胞,采用台盼兰形态学排染法、吖啶橙和溴化乙锭双重荧光染色法、FITC-Annexin V/PI双染色荧光显微镜观察法、原位缺口末端标记法(TUNEL)、DNA凝胶电泳、流式细胞术(FCM)法进行细胞凋亡的检测。通过比较发现,不同方法检测结果都存在着显著性差异,但吖啶橙和溴化乙锭双重荧光染色法是一种简单、易行、准确、全面定性细胞凋亡的较为可靠的方法,而FITC-Annexin V/PI双染色流式细胞仪能特异地、准确地检出早期凋亡的细胞,是较为理想的凋亡定量检测方法。这2种方法的联合运用更适应于动物细胞大规模培养过程中细胞凋亡的检测。     

检测细胞凋亡的常用方法

细胞凋亡的主要形态学特征是凋亡小体的形成，过去常用普通光学显微镜和透射电子显微镜进行观察。实际上，细胞凋亡的发生远远早于典型的形态变化。怎样将病理形态和代谢结合在一起来观察细胞的凋亡，是多年来人们研究的重点。本文将近年来常用于细胞凋亡研究的十种新方法作以简介。这些方法包括碘化丙啶荧光染色法、吖啶橙荧光染色法。吖啶橙活细胞悬液染色法、Hoechst33258荧光染色法，简易末端标记法、5’末端^32P标记法、荧光原位末端标记技术、酶原位末端标记技术、PI染色的流式细胞分析术和Hoechst-P1染色的流式细胞分析术等。     

Canine distemper virus causes apoptosis of Vero cells

Apoptosis of Vero cells infected with two canine distemper virus (CDV) vaccine strains was detected using TdT (terminal deoxynucleotidyl transferase)-mediated dUTP nick end-labelling (TUNEL), flow cytometric analysis, agarose gel electrophoresis and electron microscopy (EM). By TUNEL, apoptotic cells were found in CDV-Onderstepoort (CDV-Ond)-infected cells. DNA fragments isolated from infected cells were separated by agarose gel electrophoresis and a 'ladder' pattern appeared. EM observations demonstrated that the cells undergoing cytopathic effect (CPE) possessed morphological characteristics of apoptotic cells. Flow cytometric analysis indicated that CDV could induce apoptosis of Vero cells, but the percentages of the apoptotic cells were correlated with the CPE types. The strain showing the cell-rounding type of CPE produced a much higher percentage of apoptotic cells than CDV-Ond with the syncytium type of CPE (P < 0.01). It was concluded that CDV vaccine strains could induce apoptosis of Vero cells and the apoptosis was virus strain-dependent and cell-dependent. The mechanism remains to be studied.     

Tumor necrosis factor-alpha induces apoptosis in cultured porcine luteal cells

Some studies in mammalian species recently demonstrated that tumor necrosis factor (TNF)-alpha plays an important role for corpus luteum (CL) function by way of apoptosis during the estrous cycle. The objectives of this study were to clarify the induction of apoptosis in cultured porcine luteal cells by TNF-alpha treatment. Luteal cells prepared from porcine ovaries collected from crossbred mature gilts on Days 10-14 of the estrous cycle were isolated and examined as follows: 1) Flow cytometric analysis was carried out to determine apoptosis in cultured luteal cells. 2) Single-cell gel electrophoresis (comet assay) was performed to investigate apoptotic DNA fragmentation in luteal cells. The results of the flow cytometric analysis and comet assay demonstrated coincidentally that TNF-alpha induces DNA fragmentation in luteal cells causing apoptosis. These results revealed that TNF-alpha is an inducing factor of apoptosis in luteal cells.     

水泡性口炎病毒诱导BHK-21细胞的凋亡

采用HE、Giemsa染色、透射电镜以及DNA琼脂糖凝胶电泳等研究了水泡性口炎病毒（VSV）诱导BHK-21细胞凋亡的过程。结果显示：VSV感染BHK-21细胞后,光镜下可见细胞圆缩,细胞器固缩、核仁消失、染色质凝聚和核碎裂、凋亡小体出现;电镜下观察到染色质聚集形成典型的新月形,胞浆中充满大量空泡,细胞核因染色质凝聚也发生了空泡化;1％的琼脂糖凝胶电泳出现180-200bp整倍数的DNA梯形条带。结果表明,VSV诱导BHK-21细胞凋亡是其致细胞病变的主要表现形式之一。     

传染性法氏囊病病毒变异E株感染鸡细胞凋亡的研究

研究了传染性法氏囊病病毒（IBDV）变异E株人工感染28日龄SPF雏鸡后鸡法氏囊淋巴细胞的凋亡情况。电镜观察和DNA电泳分析结果表明,IBDV感染后12～48h,雏鸡法氏囊淋巴细胞出现典型细胞凋亡的形态学特征和生化特征;经流式细胞计检测和荧光染色观察,统计学分析表明,IBDV感染后24～48h,雏鸡法氏囊淋巴细胞凋亡数量显著增加（P<0.05或P<0.01）。试验结果揭示IBDV变异E株人工感染可以诱导雏鸡法氏囊淋巴细胞凋亡。     

蚯蚓寡肽对伪狂犬病毒感染细胞和 HeLa细胞凋亡的影响

利用细胞病变抑制法测定了蚯蚓寡肽的体外抗伪狂犬病毒（PRV）作用，分别用AO／EB染色法和DNA琼脂糖凝胶电泳法测定了蚯蚓寡肽对HeLa细胞凋亡的影响。结果显示，蚯蚓寡肽可以抑制PRV感染细胞，同时存在剂量效应和时间效应。蚯蚓寡肽浓度越大细胞病变程度越轻，各处理组在72h的细胞病变程度最轻。蚯蚓寡肽可以诱导HeLa细胞凋亡，且具有浓度效应，1．5mg／mL组、0．75mg／mL组的细胞凋亡率分别为60．20％和48．12％。     

JA1体外诱导HCC细胞凋亡的实验研究

采用噻唑蓝(MTT)还原法,测定了梯度浓度的某植物种子粗提物JA1对人肝癌细胞株HCC增殖作用的影响;同时采用流式细胞术、DNA凝胶电泳和透射电子显微镜技术,在体外观察了JA1对HCC细胞凋亡的诱导作用。结果显示,JA1可显著抑制肝癌细胞HCC的增殖,而且这种抑制有浓度依赖性和时间依赖性;流式细胞仪分析表明,经JA1作用后的肝癌细胞HCC检测标本中有明显的DNA低含量颗粒(“亚G1期”峰);凝胶电泳呈现出典型的DNA梯形条带;电镜下出现细胞凋亡典型的形态学改变。     

Antineoplastic effect of the cyclooxygenase inhibitor meloxicam on canine osteosarcoma cells

A decisive role in cancer development has been attributed to cyclooxygenase-2 (COX-2) activity, but the significance of COX-2 inhibitors in cancer treatment still needs to be thoroughly investigated. We studied the influence of meloxicam, a non-steroidal antiinflammatory drug with preferential inhibitory effects on COX-2 compared to COX-1, on canine osteosarcoma (D-17) cells. We demonstrated that D-17 cells expressed mRNA and COX-2 protein. Treatment with meloxicam induced a time- and dose-dependent inhibition of cellular growth. To determine if apoptosis plays a role in meloxicam-induced cell death, we performed agarose gel electrophoresis and found a DNA-ladder pattern, typically seen in apoptosis, as well as early apoptotic changes by Annexin V tests. Furthermore, electron microscopy revealed ultrastructural alterations typical of apoptosis. Quantification of apoptotic cells by immunohistochemical staining of caspase 3 confirmed the results. However, further studies with meloxicam are necessary to assess its potential use for treatment of osteosarcomas in dogs.     

提取东北虎毛发DNA两种方法的比较研究

研究东北虎毛发DNA的水煮抽提法，并将传统的酚仿抽提法和水煮法进行比较。水煮法即通过对细胞的煮沸和冷却，使细胞破裂、蛋白质变性，从而获得用于PCR扩增的模板DNA。通过测定OD值并计算浓度和纯度，同时进行琼脂糖电泳检测、PCR扩增和PAGE电泳检测，分析2种方法提取东北虎毛发DNA的质量差异。结果表明：水煮法提取东北虎毛发中DNA是一种快捷、简便、经济、高效的方法。     

Caprine herpesvirus-1 (CapHV-1) induces apoptosis in goat peripheral blood mononuclear cells

Programmed cell death (PCD), or apoptosis, is initiated in response to various stimuli, including virus infection. A number of studies have shown that deregulation of apoptosis is an important feature of virus-induced immunosuppression for various viral diseases. In the present study, CapHV-1 was found to cause apoptosis in mitogen-stimulated as well as nonstimulated caprine peripheral blood mononuclear cells (PBMC). Apoptotic index, as quantified by fluorescent dyes, revealed a significant increase in the percentage of apoptotic cells at 24 and 48 h postinfection as compared to their respective noninfected controls. Apoptosis specific internucleosomal laddering in DNA from CapHV-1 infected PBMC was seen in agarose gel electrophoresis. No DNA fragmentation was observed in control noninfected PBMC. Virus-induced apoptosis was reduced by Z-VAD-FMK, an aspecific caspase inhibitor, by AC-DEVD-CHO (caspase-3-specific) and AC-VEID-CHO (caspase-6-specific) treatment. PCD in CapHV-1 infected peripheral blood mononuclear cells occurs at the G0/G1 phase of the cell cycle. However, penetration of virus particles and infection was not required for PCD, as UV-inactivated CapHV-1 induced apoptosis of mitogen-stimulated bovine peripheral blood mononuclear cells in vitro.     

Pasteurella haemolytica leukotoxin induced apoptosis of bovine lymphocytes involves DNA fragmentation

It has been reported that Pasteurella haemolytica leukotoxin (LKT) induces morphologic changes in bovine leukocytes consistent with apoptosis in vitro, but DNA fragmentation was not observed. We investigated whether bovine lymphocytes undergo DNA fragmentation during LKT-induced apoptosis. Bovine peripheral blood lymphocytes were isolated by density gradient centrifugation and exposed to LKT or inactive pro-LKT protein from a lktC- mutant strain. After exposure, DNA fragmentation in lymphocytes was quantified colorimetrically by diphenylamine assay and visualized by agarose gel electrophoresis. At high LKT concentrations, bovine lymphocytes were lysed, but at low concentrations, LKT caused DNA fragmentation characteristic of apoptosis. Maximal DNA fragmentation in bovine lymphocytes was induced by 0.1 TU ml(-1) LKT following 3 h exposure, but only background level of DNA fragmentation was observed with the inactive pro-LKT. Equine lymphocytes that are resistant to LKT intoxication did not show DNA fragmentation following exposure to LKT. Preincubation of LKT with a neutralizing anti-LKT monoclonal antibody inhibited LKT-induced DNA fragmentation. Electrophoresis of DNA from bovine lymphocytes treated with 0.1 TU ml(-1) LKT demonstrated the typical 'ladder' pattern of internucleosomal DNA cleavage, the hallmark of apoptosis associated with activation of endonucleases. LKT-induced DNA fragmentation was inhibited by 0.5 mM ZnCl2, an endonuclease inhibitor. The results indicated that LKT at low concentrations induced apoptotic cell death of bovine lymphocytes, which may play a role in initiation and persistence of P. haemolytica infection.