Aβ_(25-35)诱发乙酰胆碱酯酶高表达成神经细胞瘤细胞凋亡(英文)

目的:研究Aβ_(25-35)引起乙酰胆碱酯酶高表达细胞株SC42凋亡与乙酰胆碱酯酶活性的关系。方法:用Aβ_(25-35)处理后,倒置相差显微镜观察细胞形态;噻唑蓝(MTT)法检测细胞存活率;核酸电泳观察DNA;分光光度测定乙酰胆碱酯酶活力。结果:Aβ_(25-35)1μol/L处理24-48小时后,细胞活力显著下降,细胞形态也发生明显的变化,DNA出现梯形条带,而乙酰胆碱酯酶的活力则显著上升,在48小时达到了正常水平的1.7倍,用不同浓度乙酰胆碱酯酶抑制剂石杉碱甲预处理后,明显抑制Aβ_(25-35)损伤48小时引起的乙酰胆碱酯酶活力上升,而对细胞活力下降没有明显的改善作用。结论:在SC42细胞中,Aβ_(25-35)能引起SC42细胞凋亡同时升高乙酰胆碱酯酶活性,并证明细胞凋亡与乙酰胆碱酯酶的水解活性无直接关系。     

SD大鼠骨髓基质细胞体外培养及分化研究

目的 研究SD大鼠骨髓基质细胞(marrow stromal cells,MSCs)的生物学特性及其成骨和成脂分化能力.方法 处死16只6月龄SD大鼠动物,非连续密度梯度离心分离大鼠MSCs,传代后分别在成骨及成脂培养基中诱导培养14天,观察细胞形态,绘制细胞生长曲线,通过组织特染对MSCs的成骨,成脂表型进行鉴定,计数阳性染色细胞百分比.结果 SD大鼠MSCs在体外具有成骨细胞分化潜力,油红0染色显示成脂诱导培养基可定向诱导其分化.结论 本实验成功分离SD大鼠MSCs且保持分化能力.     

大黄素对体外培养新生大鼠颅骨成骨细胞的影响

目的　观察大黄素(emodin)对体外培养新生大鼠颅骨成骨细胞增殖、分化作用特点及量效关系,并观察大黄素对骨保护蛋白(Osteoprotegerin,OPG)mRNA表达的影响。方法　在新生大鼠颅骨成骨细胞体系中加入不同浓度的大黄素,加药后 1、2、3d用MTT法检测细胞增殖, 3、5、7、9d用PNPP法检测细胞内碱性磷酸酶含量,加药后 7d检测细胞内OPGmRNA表达的量, 24d检测细胞上清中钙和细胞内羟脯氨酸含量。结果　大黄素能促进大鼠颅骨成骨细胞ALP活性,实验中最佳作用浓度为 1×10-6 mol·L-1,最佳作用时间是 7d。大黄素能增加细胞内羟脯氨酸含量,促进OPGmRNA的表达。结论　大黄素可促进成骨细胞分化,增加OPGmRNA的表达。     

虫草素体外抑制NB4细胞增殖与诱导细胞凋亡作用研究

目的观察虫草素体外对急性早幼粒细胞性白血病细胞（NB4细胞）的抑制增殖和诱导凋亡作用。方法将0.5,1.0,1.5,2.0 mg•mL 1虫草素在体外与NB4细胞共同培养,采用细胞计数、噻唑蓝（MTT）法检测不同浓度虫草素在24,48,72 h对NB4细胞增殖的抑制作用;用细胞瑞氏染色法、Hoechst33258荧光染色法、DNA亚二倍体检测法和线粒体膜蛋白检测法观察虫草素诱导NB4细胞凋亡的作用。结果与对照组比较,不同浓度虫草素均能显著抑制NB4细胞增殖,并呈剂量和时间依赖的量效关系;不同浓度虫草素作用24 h后均能显著促进NB4细胞凋亡。结论虫草素在体外能明显抑制NB4细胞增殖,并诱导细胞凋亡。     

二甲双胍对糖基化终末产物诱导的成骨细胞氧化应激和凋亡的影响

目的:观察二甲双胍对糖基化终末产物(AGEs)诱导的大鼠颅骨成骨细胞内氧化应激产物活性氧簇(ROS)、细胞早期凋亡的影响.方法:分离培养大鼠颅骨成骨细胞,2 '7'-二乙酰二氯荧光素(DCFH-DA)作为荧光探针,流式细胞检测技术检测细胞内ROS水平,膜联蛋白V-异硫氰酸荧光素/碘化丙啶(Annexin V-FITC/ PI)双染色分析细胞早期凋亡率.结果:与未经葡萄糖处理的牛血清白蛋白(BSA)组对比,500 μg/mLAGEs显著促进成骨细胞内ROS形成和细胞凋亡(均P＜ 0.01);给予二甲双胍(100～500μmol/L)呈浓度依赖性抑制BSA组及AGEs组成骨细胞内ROS形成和细胞凋亡,浓度分别为500、400 μmol/L时,对细胞内ROS形成和细胞凋亡的抑制作用达到最强.结论:AGEs显著诱导原代成骨细胞内ROS的形成和细胞凋亡,而二甲双胍能够呈浓度依赖性抑制AGEs诱导的成骨细胞内ROS的形成和细胞凋亡,减轻AGEs对成骨细胞的损害.     

爱普列特对良性前列腺增生体外细胞模型的凋亡诱导作用

目的:拟建良性前列腺增生体外细胞模型,并探讨爱普列特(epristeride)对前列腺增生模型中前列腺细胞作用机制。方法:建立大鼠前列腺细胞体外培养方法,给予过量睾酮和多肽生长因子模拟病理环境,探讨诱发前列腺细胞增生的最佳刺激条件,MTT实验观察爱普列特对增生前列腺细胞增殖抑制作用,流式细胞术观察药物对前列腺细胞的凋亡诱导作用。结果:初步建立了良性前列腺增生体外细胞模型,培养液(含睾酮9.08×10-8mol.L-1)培养9 d后,模型组细胞密度显著增高,同时培养液中前列腺特异抗原含量提高。爱普列特作用72 h细胞的IC50值5.0×10-6mol.L-1,流式细胞术分析显示凋亡的特征性亚二倍体峰。结论:雄激素能显著促进体外培养的大鼠前列腺细胞增殖,爱普列特可通过诱导前列腺细胞凋亡发挥对前列腺增生疾病的治疗作用。     

马氏珠母贝糖胺聚糖对体外成骨细胞增殖、分化及矿化功能的影响

目的观察珠母贝糖胺聚糖在体外对新生大鼠颅骨成骨细胞增殖、分化及矿化功能的影响。方法应用MTT法、PNPP法、茜素红染色 (ARS)矿化骨结节及用图像分析仪计算骨结节的面积等方法观察细胞的增殖、碱性磷酸酶 (ALP)活性及矿化结节形成的影响。结果珠母贝糖胺聚糖 0 .0 0 8～ 0 .5 g·L- 1在不同时间MTT测得的A值均低于空白对照组 ,不促进细胞的增殖 ;各浓度组在培养 5d时 ,碱性磷酸酶的活性显著高于空白对照组 ;培养 31d后茜素红染色显示 ,0 .0 6 3g·L- 1组所形成的骨结节面积显著大于空白对照组。结论珠母贝糖胺聚糖对体外培养的SD新生大鼠颅骨成骨细胞有显著促进分化和矿化的作用 ,但不促进细胞的增殖。     

吲哚-3-原醇对猪血清诱导大鼠肝纤维化的治疗作用

研究吲哚-3-原醇(I3C)对猪血清诱导大鼠肝纤维化的治疗作用及其机制。腹腔注射猪血清制备大鼠肝纤维化模型,造模成功后用I3C治疗17天。采用HE和Masson三色染色法分别观察肝脏病理学和胶原含量改变;生化比色法测定肝组织羟脯氨酸(Hyp)含量;免疫组织化学法观察肝脏中α-平滑肌肌动蛋白(α-SMA)的表达。进一步培养大鼠肝星状细胞株HSC-T6,用13C处理24 h后,FITC-Annexin V/PI双重染色法检测细胞凋亡;实时荧光定量PCR法检测细胞凋亡相关蛋白Bax和Bcl-2的mRNA表达。结果显示,与模型对照组比较,各I3C治疗组的肝组织Hyp含量不同程度降低,肝细胞损伤减轻,胶原纤维沉积减少(P<0.01),α-SMA表达降低(P<0.01)。细胞实验显示,I3C可明显增加HSC-T6细胞凋亡率,升高Bax/Bcl-2的mRNA表达(P<0.05)。以上结果说明,I3C对猪血清诱导大鼠肝纤维化有一定治疗作用,可能与其诱导活化HSC凋亡继而促进基质胶原降解有关。     

诱生型一氧化氮合酶抑制剂对TNF—α诱导的人成骨细胞凋亡的保护作用

目的：探讨TNF－α对体外培养人成骨细胞的凋亡影响及iNOS抑制剂对其保护作用。方法：对流产胎儿颅骨分离培养人成骨细胞后分3组：对照组：TNF－α组（不同浓度），TNF－α加L－NMMA组。通过细胞计数绘制生长曲线，流氏细胞仪检测，电镜下观察超微结构的变化，三项指标检测成骨细胞凋亡情况。结果：TNF－α可诱导成骨细胞凋亡，并呈时间剂量依赖性，iNOS抑制剂L－NMMA可有效抑制TNF－α对成骨细胞的损伤作用。结论：iNOS抑制剂对TNF－α诱导的成骨细胞凋亡具有一定的保护作用。     

杜仲总黄酮对成骨细胞增殖及Ⅰ型胶原蛋白表达的影响

目的了解杜仲总黄酮对新生大鼠颅骨体外培养成骨细胞的增殖及合成胶原蛋白的影响。方法杜仲总黄酮以质量浓度10,100,200和400μg·mL-1分别加入从2 d的SD大鼠颅骨分离培养的成骨细胞中,用MTT法评价对成骨细胞增殖的影响;采用激光共聚焦扫描显微镜检测Ⅰ型骨胶原蛋白的表达情况。结果与对照组比较,质量浓度10~200μg·mL-1的杜仲总黄酮能明显促进成骨细胞的增殖(P<0.05),但并不能显著地促进成骨细胞合成Ⅰ型胶原蛋白。结论杜仲总黄酮能直接促进体外成骨细胞的增殖。     

离体和在体大鼠心肌细胞凋亡模型的建立

目的建立离体和在体心肌细胞凋亡模型。方法离体模型:原代培养大鼠心肌细胞进行饥饿处理24 h后,培养液换成Hank's平衡液,同时进行缺氧处理,30 min后恢复到正常氧状态再培养2 h,固定,TUNEL染色,计数凋亡细胞;细胞总DNA抽提,agrose凝胶电泳,观察DNA ladder的产生。在体模型:150 g左右的WKY大鼠麻醉,固定,气管插管,开胸,冠状动脉左前降支结扎30 min,再灌注2 h后,心脏取出,冰冻切片,进行TUNEL染色,观察危险区细胞凋亡。结果原代培养大鼠心肌细胞缺氧/复氧处理引起大量心肌细胞凋亡,TUNEL染色阳性,DNA电泳有明显梯状条带,在体大鼠心脏冠状动脉左前降支结扎/再灌注造成心肌梗死,梗死周边危险区有大量凋亡细胞产生。结论离体细胞缺氧/复氧处理和在体大鼠心脏冠状动脉左前降支结扎/再灌注都能导致心肌细胞发生凋亡,这两种凋亡模型具有操作简单、成功率高等特点,可以用于心肌梗死机制的研究以及保护心肌药物的筛选。     

柠檬醛对致敏大鼠腹腔肥大细胞脱颗粒和气道嗜酸粒细胞凋亡的作用

目的:研究柠檬醛对致敏大鼠肥大细胞脱颗粒的影响以及对大鼠气道嗜酸粒细胞凋亡的作用。方法:以卵蛋白致敏制备大鼠哮喘模型。通过大鼠被动肥大细胞脱颗粒,放免法测定肿瘤坏死因子α(TNF-α)和白细胞介素8(IL-8)的含量;密度梯度离心法分离支气管肺泡灌洗液中的嗜酸粒细胞,采用膜联蛋白和碘化丙啶联合标记法测定细胞凋亡,流式细胞仪检测细胞凋亡情况。结果:柠檬醛高、中剂量组肥大细胞脱颗粒数量以及TNF-α和IL-8的含量与模型组相比差异有非常显著意义(P<0.01);柠檬醛高、低剂量组都能明显提高嗜酸粒细胞的凋亡量,与空白对照组相比差异有非常显著意义(P<0.01)。结论:柠檬醛具有抑制肥大细胞脱颗粒作用并且促进嗜酸粒细胞凋亡。     

Effects of four types of hydroxyapatite nanoparticles with different nanocrystal morphologies and sizes on apoptosis in rat osteoblasts

Hydroxyapatite nanoparticles (nano‐HAP) have been reported to cause inflammatory reactions. Here, we aimed to compare the effects of four types of nano‐HAP with different nanocrystal morphologies (short rod‐like, long rod‐like, spherical or needle‐shaped crystals) and sizes (10–20, 10–30 or 20–40 nm) on growth inhibition and apoptosis in primary cultured rat osteoblasts. The osteoblasts was treated with the four types of nano‐HAP at various concentrations (20, 40, 60, 80 or 100 mg l^?1). The nano‐HAP specific surface area was detected using the Brunauer, Emmet and Teller method. The cell growth rate was detected using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay; apoptotic alterations and the level of reactive oxygen species in osteoblasts were measured using flow cytometry; and the amounts of apoptotic p53 and cytochrome c proteins were measured using western blotting. We observed that all four types of nano‐HAP inhibited the growth of osteoblasts in a dose‐dependent manner. These nano‐HAP significantly induced apoptosis in osteoblasts. Nano‐HAP with smaller specific surface areas induced lower apoptosis rates. The needle‐shaped and the short rod‐like particles induced greater cellular injury than the spherical and long rod‐like particles, respectively. The increased apoptosis rates were accompanied by increased p53 and cytochrome c expression. These findings indicate that nano‐HAP inhibit the activity of osteoblasts and also induce the apoptosis of osteoblasts in vitro. These findings also suggest that the nano‐HAP‐induced apoptotic pathway is mediated by a mitochondrial‐dependent pathway. Moreover, the sizes, morphologies and concentrations of nano‐HAP have significant effects on the apoptotic level. Copyright © 2011 John Wiley & Sons, Ltd.     

葛根素对缺氧性血管内皮细胞凋亡的保护作用

目的 研究葛根素(puerarin)对缺氧性血管内皮细胞凋亡的影响。方法 用NaCN合并无糖培养基造成培养的牛主动脉内皮细胞(BAECs)缺氧;用台盼蓝染色、末端脱氧核苷酸转移酶介导的dUTP缺口末端标记(TUNEL)、流式细胞仪计数和Hoechst 33342荧光染色法观察细胞受损和凋亡情况;用免疫细胞化学染色法观察细胞内Caspase-3的表达情况。结果NaCN合并无糖培养基可引起血管内皮细胞凋亡。葛根素可显著减少缺氧性内皮细胞凋亡,并对Caspase-3的表达有明显抑制作用。结论葛根素对缺氧条件下的血管内皮细胞有保护作用,此作用至少部分通过抑制Caspase-3的表达而实现。     

Lactoferrin inhibits apoptosis through insulin-like growth factor I in primary rat osteoblasts

Aim： Excessive apoptosis of osteoblasts is the major cause of low bone mass, and bovine lactoferrin （bLF）, an iron-binding glycoprotein, might protect osteoblastic cells from apoptosis induced by serum withdrawal. The aim of this study was to elucidate the mechanisms underlying the anti-apoptotic action of bLF in rat osteoblasts in vitro. Methods： Primary rat osteoblasts were incubated in the presence of varying concentrations of bLF for 24 h. The expression of insulin-like growth factor I （IGF-I） and IGF-I receptor （IGF-IR） was measured uisng RT-PCR and Western blotting. Cell apoptosis was examined with flow cytometry. siRNAs targeting IGF-I was used in this study.

Results： Treatment of bLF （0.1–1000 μg/mL） dose-dependently increased the expression of IGF-I and IGF-IR in the osteoblasts. Treatment with bLF （10, 100 μg/mL） markedly inhibited the osteoblast apoptosis （with the rate of total apoptosis of 70% at 10 μg/mL）, but the high concentration of bLF （1000 μg/mL） significantly promoted the osteoblast apoptosis. Knockdown of the IGF-I gene in osteoblasts with siRNA markedly increased the osteoblast apoptosis.

Conclusion： Lactoferrin （10 and 100 μg/mL） effectively inhibits apoptosis of primary rat osteoblasts by upregulating IGF-I expression.     

酸敏感离子通道阻断剂阿米洛利对佐剂性关节炎大鼠关节软骨细胞凋亡抑制作用的初步研究

目的：研究酸敏感离子通道阻断剂阿米洛利（Amiloride）对佐剂性关节炎（AIA）大鼠关节软骨组织的保护作用及机制。方法：将大鼠随机分为正常组、AIA模型组、阿米洛利（2.5、5.0、10.0mg·kg^-1.d^-1）组和阿司匹林（50mg/kg）对照组。弗氏完全佐剂（CFA）致AIA后第10天,大鼠出现继发性炎症,此时阿米洛利、阿司匹林灌胃给药,连续8d 正常组与模型组给予等容量的无菌注射用水。检测大鼠继发侧关节肿胀度,光学显微镜观察关节病理变化,透射电镜、Tunel法观察阿米洛利对AIA大鼠关节软骨细胞凋亡的影响,免疫组织化学技术测定阿米洛利对AIA大鼠关节软骨中的Ⅱ型胶原（COII）蛋白合成的影响,Alcian染色测定阿米洛利对AIA大鼠关节软骨中的PG合成的影响。结果：阿米洛利各剂量组对AIA大鼠的足肿胀未见明显影响 病理学观察关节软骨表面光滑,软骨细胞层次尚清晰,成熟软骨细胞数量增加 透射电镜观察软骨细胞核内异染色质轻度边集、细胞内粗面内质网轻度扩张、线粒体轻度肿胀,与模型组软骨细胞核膜皱缩、核染色质高度边集、核膜破裂、出现凋亡小体相比无明显细胞凋亡的特征;Tunel法原位检测显示凋亡阳性细胞数显著减少（P〈0.01） 免疫组化及Alcian染色检测显示关节软骨基质成分COII蛋白和PG的表达量增加。结论：ASICs阻断剂阿米洛利可通过抑制AIA大鼠关节软骨细胞凋亡发挥对关节软骨的保护作用。     

Capillary electrophoresis analysis of hydrogen peroxide induced apoptosis in PC12 cells

Apoptosis is a highly regulated and controlled process of cell death that plays a fundamental role in many biological processes. Abnormal apoptosis of cells is closely related to some diseases such as cancer. Development of a simple and effective method to detect apoptosis is of great importance. In the present paper, capillary electrophoresis (CE) method was applied to distinguish cell apoptosis and necrosis of rat pheochromocytoma (PC12) cells treated by hydrogen peroxide by characterizing the DNA fragmentation. Firstly, effects of separation conditions (voltage, polymer concentration, temperature and injection time) on DNA separation were studied using 100 bp DNA ladders as the analyte. Under optimal separation condition (polyacrylamide coated capillaries: 57.5 cm x 75 microm i.d., effective length: 50 cm; running buffer: 1x TBE containing 2% PVP and 1.2% HEC; separation voltage: 5 kV; temperature: 25 degrees C; electrokinetic injection: 10 kV x 10 s), CE was used to monitor the progress of hydrogen peroxide induced apoptosis of PC12 cells by analyze DNA fragmentation. It was found that normal, apoptotic and neurotic cells had distinct DNA fragmentation patterns analyzed by CE. The results by CE were tested by other current methods (DAPI nuclei staining, flow cytometry analysis and TUNEL) to detect apoptosis and correlated well with those methods. Results show that CE can distinguish cell apoptosis and necrosis, quantify the degree of apoptosis in cells and have advantages of high efficiency, fast sample analysis speed, minute sample consumption and reliable results, which provides an accessorial method in the research of multiple diseases with abnormal apoptosis such as cancer and neurodegenerative diseases.     

Apoptosis induced by β-amyloid25-35 in acetylcholinesteraseoverexpressing neuroblastoma cells

AIM: To examine the relationship between apoptosis induced by β-amyloid fragment 25-35 (Aβ25-35) and the activ-ity of acetylcholinesterase (ACHE) in AChE over-expresser-SC42 cells. METHODS: Cell survival was measuredby microscopy and MTT reduction; DNA laddering was observed by electrophoresis; AChE activity was determined by spectrophotometry. RESULTS: Aβ25-35 1 μmol/L exposure for 24-48 h caused a significant decrease in cell viability, along with changes in morphology and DNA fragmentation. AChE activity was affected in an inversemanner, increasing gradually to a level that was 1.7-fold higher than control at the 48-h time point. No change in thecytotoxicity of Aβ25-35 was observed when the increased AChE activities were effectively inhibited by huperzine Athroughout the 48-h exposure period. CONCLUSION: Although Aβ25-35 can induce apoptosis in SC42 cells andsimultaneously increase AChE activity, the capacity of AChE to hydrolyze acetylcholine is not involved in thisapoptosis model.     

Overexpression of acetylcholinesterase inhibited cell proliferation and promoted apoptosis in NRK cells

AIM: To study the potential function of acetylcholinesterase (AChE) in apoptosis through overexpression of AChE in Normal Rat Kidney (NRK) cells. METHODS: AChE activity was detected by the method of Karnovsky and Roots. Activated caspase-3 was analyzed by Western blotting and immunoflurescence with antibody special to activated caspase-3 fragment. The expression plasmids were constructed in pcDNA3.1 containing AChE gene or a fragment of AChE antisense that were got from RT-PCR. Stable expression cell lines were selected by G418 in cells transfected by lipofection. AChE expression was analyzed by RT-PCR and Western blotting. The proliferation rates of transfected cells were examined by the growth curve and cloning efficiency. MTT assay was used to analyze the cell viability. RESULTS: The proliferation rate of the cells transfected with AChE was retarded and the cloning efficiency was lower (28.2 %±3.1 % and 48.7 %±2.1 %) than cells transfected with vector (56.1 %±0.3 %) or AChE-antisense (77.7 %±2.2 %     

没食子儿茶素没食子酸酯对MPP~+诱导大鼠PC12细胞凋亡的拮抗作用

目的探讨没食子儿茶素没食子酸酯(EGCG)对MPP+诱导大鼠PC12细胞凋亡的拮抗作用。方法培养PC12细胞给予MPP+(900μmol.L-1)诱导细胞凋亡,实验分为6组:空白对照组、MPP+组、维生素E(10μmol.L-1)组和EGCG高(100μmol.L-1)、中(50μmol.L-1)、低(10μmol.L-1)剂量组。药物处理0.5h后,加入MPP+损伤细胞,4h后取细胞测定乳酸脱氢酶(LDH)漏出量,MTT法检测细胞活力,Hoechst33342荧光染色法和流式细胞术检测细胞凋亡,透射电镜观察凋亡细胞线粒体形态结构改变。结果MPP+处理后,细胞活力下降,LDH漏出量增加,细胞凋亡率增加,线粒体肿胀,出现空泡和嵴断裂等细胞凋亡征象。维生素E和不同剂量EGCG处理后,明显提高细胞活力,降低LDH漏出和细胞凋亡率,并减少MPP+引起的线粒体结构损伤。结论EGCG具有抑制MPP+诱导的大鼠PC12细胞凋亡作用,其作用与保护细胞线粒体结构的完整性有关。