环维黄杨星D对NaCN致乳鼠心肌细胞损伤的影响

目的观察环维环杨星D(CVB-D)对心肌细胞缺氧性损伤的保护作用。方法利用NaCN诱导细胞内缺氧,建立心肌细胞缺氧损伤病理模型,从细胞存活率、LDH外漏释放、细胞凋亡率等方面,观察CVB-D对缺氧损伤心肌细胞的保护作用。结果 CVB-D能明显抑制NaCN造成的心肌细胞死亡、减少细胞内LDH的漏出(P0.05~0.01),并能减少缺氧诱导的心肌细胞凋亡。结论 CVB-D对缺氧损伤心肌细胞具有明显的保护作用,作用机制与清除氧自由基及降低细胞凋亡率有关。     

中药露蜂房蛋白对人红白血病细胞株K562细胞影响的实验研究

目的：研究中药露蜂房中蛋白成份抗白血病作用的机理，为中药露蜂房治疗白血病的临床应用和开发新的抗白血病药物提供实验和理论依据。方法：采用细胞培养和透射电子显微镜技术，观察中药露蜂房蛋白成份对人红白血病细胞（K562细胞）的生长抑制作用和形态结构的影响，采用免疫组织化学方法测定露蜂房蛋白对K562细胞凋亡相关信号转导蛋白NF-κBp65、β-catenin及iNOS表达的影响。结果：（1）与生理盐水对照组比较，露蜂房蛋白处理组白血病细胞生长饱和密度降低，细胞增殖活性减弱（P＜0．05）；（2）露蜂房蛋白处理组白血病细胞呈典型的凋亡形态学改变；（3）与生理盐水对照组比较，露蜂房蛋白处理组白血病细胞中NF-κBp65、β-catenin及iNOS表达减弱，NF—κBp65和β-catenin细胞核内转导减少（P＜0．05）。结论：中药露蜂房蛋白成份有明显抑制K562细胞的作用，其作用机制可能是通过调节凋亡相关信号传导因子NF-κBp65、β-catenin及iNOS的表达，从而诱导白血病细胞凋亡。     

Activation of Akt by mechanical stretching in human epidermal keratinocytes

Mechanical stretching represents an important part of the signaling in skin. We have previously demonstrated that mechanical stretching induced proliferative phenotypes in human keratinocytes, as shown in increased 5-bromo-2'-deoxyuridine (BrdU) incorporation, ERK1/2 activation, and keratin K6 induction. Here we have further investigated the antiapoptotic signals in human keratinocytes provoked by mechanical stretching in vitro. Keratinocytes were plated on flexible silicone supports to transmit mechanical stretching to keratinocytes, involving continuous stretching by +20%. Stretching of these cells for 15-30 min caused the phosphorylation and activation of Akt. Inhibition of mitogen and extracellular signal-regulated kinase (MEK1/2) with U0126 and phosphoinositide 3-OH kinase (PI 3-K) with Wortmannin attenuated Akt activation. The epidermal growth factor (EGF) receptor kinase inhibitor, AG1478, and calcium channel inhibitor, gadolinium (Gd3+), also inhibited Akt activation. In summary, our results demonstrate the activation of the Akt signaling pathway by mechanical stretching, generating not only proliferative but also antiapoptotic signals in human keratinocytes.     

Brain microvasculature and hypoxia-related proteins in Alzheimer's disease

Alzheimer's disease (AD) is a progressive, neurodegenerative disease of increasing incidence. The pathologic processes that underlie this disorder are incompletely understood, however, hypoperfusion/hypoxia is thought to contribute to disease pathogenesis. Hypoxia inducible factor 1-alpha (HIF-1α), a key regulator of cellular responses to hypoxia, is elevated in the microcirculation of AD patients. Cerebral hypoxia is a potent stimulus for vascular activation and angiogenesis. Microvessels isolated from the brains of AD patients express a large number of angiogenic proteins. Despite considerable data in human tissues regarding vascular expression of hypoxia-related angiogenic proteins, there is little information regarding these proteins in the brain vasculature of transgenic AD mice. The objectives of this study were to determine expression of HIF-1α, angiogenic proteins, angiopoietin-2 (Ang-2), and matrix metalloproteinase 2 (MMP2), and survival/apoptotic proteins (Bcl-xL, caspase 3) in the cerebromicrovasculature of AD transgenic mice and to determine the direct effect of hypoxia on cerebral endothelial expression of these proteins in vitro. Cultured brain endothelial cells were subjected to hypoxia for 4-6 h and analyzed by western blot and immunofluorescence. Our results demonstrated that HIF-1α is induced in cultured brain endothelial cells exposed to hypoxia and that expression of Ang-2, MMP2 and caspase 3 was elevated and the anti-apoptotic protein Bcl-xL decreased. Brain sections from AD and control mice showed that HIF-1α, Ang-2, MMP2 and caspase 3 are elevated and Bcl-xL decreased in the microvasculature of AD mice. These data suggest the cerebromicrovasculature is an important target for the effects of hypoxia in the AD brain.     

Interaction of cytokines and growth factor in the regulation of verotoxin-induced apoptosis in cultured human endothelial cells

The role of CD77, inflammatory cytokines and endothelial cell growth factor (ECGF) in verotoxin (VT)-induced apoptosis in human umbilical vein endothelial cells (HUVECs) was studied. Apoptosis was detected using annexin V and propidium iodide staining. The expression of CD77 antigen was measured on a FACStar flow cytometer using specific monoclonal antibodies. Our experiments showed that HUVECs had very low initial levels of CD77 and were resistant to VT. Treatment with tumour necrosis factor alpha (TNF-alpha) resulted in a significant upregulation of CD77 expression and sensitized endothelial cells to VT-mediated apoptosis. HUVECs incubated with a combination of cytokines [TNF-alpha and interferon gamma (IFN-gamma), both 500 U/ml] showed more pronounced upregulation of CD77 expression (> sixfold at 48 h) and underwent apoptosis, suggesting that TNF-alpha and IFN-gamma have a synergistic effect on CD77 expression in HUVECs and can induce apoptosis without VT. Cells pretreated with TNF-alpha and IFN-gamma and incubated with VT showed the most pronounced (14-fold) increase in CD77 expression. ECGF had a partial protective effect against cytokine- and VT-induced apoptosis. Taken together, our data suggest that CD77 antigen is involved in the regulation of endothelial cell apoptosis.     

猴D型逆转录病毒（SRV）感染恒河猴诱导脑细胞凋亡

收集1990-03用我所首次在国内分离到的猴D型逆转录病毒(SRV)接种3只幼龄恒河猴(90101、90102、90103),经678d观察后处死的病理组织标本进行组织病理学观察.结果显示,3只实验猴网状内皮组织显微镜下呈现典型猴艾滋病(SAIDS)病理改变.其中1只实验猴(90103)的大脑组织除有片状变性坏死外,神经元周围星形胶质细胞和血管周围小胶质细胞还表现有明显的增生和凋亡,H.E形态以核浓缩的为主.     

TUNEL技术方法的探讨

探讨末端脱氧核苷酸转移酶介导的dUTP缺口标记技术（TUNEL）的染色方法,以期提高特异性与敏感性。采用以去势（经手术切除睾丸）后天数不同的大白鼠前列腺,用TUNEL法染色,观察不同时期的细胞凋亡水平,对经典的TUNEL法进行改良,将染色结果与TUNEL经典法和HE染色法进行对比。结果表明采用改良法后,阳性细胞明显增加,而且着色深,凋亡指数明显增高(P<0.01)。     

Mppa介导的光动力疗法诱导人卵巢癌细胞凋亡

目的：探讨Mppa（methyl pyropheophorbide-a）介导的光动力疗法(Mppa-PDT)抑制人卵巢癌SKOV3细胞活性、触发其凋亡的机制。方法：Mppa-PDT作用于人卵巢癌SKOV3细胞后,CCK-8法检测细胞活性;Annexin V-FITC/PI双染流式细胞术检测细胞凋亡率;DAPI染色观察细胞凋亡的核的形态学改变;DCFH-DA染色观察细胞内活性氧（ROS）的产生;单细胞凝胶电泳观察DNA损伤情况;Western blot检测P53、Caspase-3、Bax、Bcl-2蛋白的表达变化。结果：1.Mppa-PDT能显著抑制人卵巢癌SKOV3细胞的活性,其抑制作用具有一定的剂量依赖性（P<0.05）;2. Mppa-PDT诱导的人卵巢癌SKOV3细胞凋亡率显著高于对照组（空白对照、单药对照、单光对照）（P<0.05）,且空白、单药、单光三组对照组间凋亡率无明显差异（P>0.05）;3.LD50剂量的Mppa-PDT作用于人卵巢癌SKOV3细胞后,DAPI细胞核荧光染色可见到细胞核深染的凋亡细胞;DCFH-DA荧光染色发现Mppa-PDT组细胞内ROS水平高于三组对照组;单细胞凝胶电泳观察到Mppa-PDT组的DNA损伤情况高于三组对照组;Western blot检测发现P53、caspase-3、Bax蛋白升高,Bcl蛋白降低。结论：Mppa介导的光动力疗法能够显著抑制人卵巢癌SKOV3细胞活性并诱发其凋亡,且伴随有DNA损伤及线粒体凋亡途径的激活。     

δ阿片受体激活对血清饥饿致大鼠成骨细胞凋亡的影响及机制

 目的 利用体外培养大鼠成骨细胞,研究δ阿片受体激活对血清饥饿诱导成骨细胞凋亡的影响并探讨其机制。方法 培养大鼠成骨细胞,分组给药作用48h后,MTT法测定细胞存活率,流式细胞仪分析细胞周期,Annexin V-FITC/PI双标记法测定细胞凋亡率,Western blot测定PKC和Caspase-3表达。结果 给予δ阿片受体激动剂DADLE 1μmol?L-1 处理可显著抑制由血清饥饿导致的成骨细胞凋亡,表现为细胞存活率增加,凋亡率降低,G0/G1期百分比降低,G2/M百分比增加,Caspase-3表达下降,PKC表达增加;但这种现象可被Naltrindole所拮抗;并且给予10μmolL-1GF109203X亦可拮抗DADLE的这种抑制细胞凋亡的作用,表现为细胞存活率降低,凋亡率增加;G0/G1期百分比增加,G2/M期百分比降低;caspase-3表达增加;PKC表达下降。结论 DADLE激活δ阿片受体对血清饥饿诱导的成骨细胞凋亡起保护作用,且这种作用是通过PKC途径实现的。