酸性磷酸酶法检测体外培养细胞数

利用小鼠成纤维细胞系(NIH3T3)、小鼠骨髓瘤细胞系(SP2/0)、人大肠癌细胞系(LO-VO)和人白血病细胞系(K562),评价酸性磷酸酶(APA)法用于检测体外各类型细胞的增殖和杀伤作用。用直线回归分析光吸收度与每孔活细胞数的关系。结果表明,APA法能准确地反映检测的活细胞数(相关系数均>0.99)。本方法不仅能很好地检测表皮生长因子对细胞的增殖作用,也能够检测顺铂对体外细胞的杀伤作用。结果表明APA法简单、灵敏,可以用于上皮和间质等贴壁和悬浮生长的细胞计数。     

大鼠小脑缺血再灌注损伤的细胞凋亡及尼莫通的保护作用

采用四血管闭塞法制作全脑缺血再灌动物模型, 再灌后４８ 小时取小脑, 石蜡包埋切片。应用末端转移酶介导的缺口末端标记法原位检测到小脑皮质及小脑核有阳性反应的凋亡细胞, 表明细胞凋亡是迟发性神经元损伤的主要形式。缺血前３０ 分钟给以尼莫通能有效地减少细胞凋亡, 尼莫通对小脑缺血再灌注损伤有显著性保护作用     

大鼠脊髓不完全性损伤后前角运动神经元的酶细胞化学改变

以改良Ａｌｅｎ氏法造成Ｗｉｓｔａｒ大鼠不完全性脊髓损伤,采用神经学功能评分法评定大鼠运动功能,应用定量酶细胞化学方法观察脊髓前角运动神经元内乙酰胆碱酯酶（ＡＣｈＥ）和酸性磷酸酶（ＡｃＰ）活性变化。结果显示：１．脊髓损伤后大鼠运动功能障碍,随后逐渐恢复。２．前角运动神经元内ＡＣｈＥ活性减弱、ＡｃＰ活性增强;随后酶活性呈逐渐恢复,四周时ＡＣｈＥ活性基本恢复正常。结果说明：大鼠脊髓不完全性损伤后运动功能变化与前角运动神经元的功能状态具有较强的相关性;前角运动神经元在不完全性脊髓损伤运动功能恢复中起重要作用。     

局灶性脑缺血再灌损伤时神经细胞内Ca~(2+)时空动态变化的实验研究

本文用插线法制作局灶性脑缺血／再灌损伤模型,利用激光共聚焦扫描显微镜观察活体脑片细胞内Ｃａ２＋的分布及动态变化,结果表明：（１）缺血／再灌时间不同,梗塞面积不同,缺血４小时梗塞面积占同侧半球的１６．３％,缺血４小时再灌２０小时梗塞面积增加到２５．９％,缺血２４小时梗塞面积占同侧半球的６０．４％。（２）本文首次观察到在缺血４小时纹状体区域的Ｃａ２＋变化明显高于皮层,并且再灌后皮层及纹状体区域Ｃａ２＋的含量明显增加     

低强度激光血管照射治疗脑损伤的实验研究

本实验采用ＳＤ大鼠脑损伤模型,设对照组和照射组,采用低能量半导体激光器对大鼠股静脉进行血管外照射,水迷宫试验测定大鼠记忆功能,并测定脑组织和血ＳＯＤ、ＭＤＡ含量。结果发现低强度激光血管照射能促进照射组记忆功能的恢复,使ＳＯＤ活性提高,ＭＤＡ含量降低。这提示低强度激光照射改善了脑损伤后脑组织的缺血缺氧状态,从而减轻了氧自由基反应造成的继发性脑损害     

尼莫地平对急性脑缺血大鼠一氧化氮和自由基的影响

本文在大鼠双侧颈总动脉闭塞的不完全性脑缺血模型上,观察了尼莫地平在脑缺血中对一氧化氮（ Ｎ Ｏ） 和自由基的影响。发现尼莫地平显著降低脑缺血大鼠血清中乳酸脱氢酶（ Ｌ Ｄ Ｈ） 活性,丙二醛（ Ｍ Ｄ Ａ）含量,增加 Ｎ Ｏ 含量。结果提示：尼莫地平对脑缺血大鼠的保护作用可能与其抗脂质过氧化及增加 Ｎ Ｏ 有关。     

Hydrogen peroxide induces endothelial cell atypia and cytoskeleton depolymerization

Reactive oxygen intermediates induce cell injury in a variety of pathophysiological conditions. Human umbilical cord vein endothelial cell (HUVEC) cultures were exposed to 1 or 200 microM H2O2 for 15 min, and observed after 15 min, or 1, 4, 24, or 120 h. Factor VIII and the cytoskeletal proteins vimentin and tubulin were visualized immunocytochemically. Release of lactate dehydrogenase (indices of cell membrane injury) did not increase after H2O2 exposure; nor was cellular expression of factor VIII affected. 200 microM H2O2 induced cell contraction after 15 min which disappeared after 1 and 4 h, but was evident again after 24 h. Immediately after exposure, the filamentous structure of vimentin and tubulin disappeared, but normalized after 1 h. After 120 h, the cytoskeleton filaments were coarsened and disorganized, and an abundance of multinucleated giant cells were observed. Catalase (150 U/ml) abolished all effects of H2O2. One microM H2O2 did not induce any changes in HUVEC. Thus, the present concentrations of H2O2 did not induce cell necrosis or altered expression of factor VIII. Early, reversible cell contraction and depolymerization of cytoskeletal proteins were observed, followed by a delayed contraction and cell atypia after 200 microM H2O2.     

Alterations in Ionized and Total Blood Magnesium After Experimental Traumatic Brain Injury

Experimental evidence suggests that magnesium plays a role in the pathophysiological sequelae of brain injury. The present study examined the variation of blood ionized and total magnesium, as well as potassium, sodium, and ionized calcium, after experimental fluid percussion brain injury in rats. Blood ionized magnesium concentration significantly declined from 0.45 +/- 0.02 to 0.32 +/- 0.02 mM by 30 min postinjury and stayed depressed for the 24-h study period in vehicle-treated rats. Blood total magnesium concentration was 0.59 +/- 0.01 mM and remained stable over time in brain-injured vehicle-treated animals. When magnesium chloride (125 micromol/rat) was administered 1 h postinjury, ionized magnesium levels were restored by 2 h postinjury and remained at normal values up to 24 h following brain trauma. Magnesium treatment also significantly reduced posttraumatic neuromotor impairments 1 and 2 weeks after the insult, but failed to attenuate spatial learning deficits. A significant positive and linear correlation could be established between ionized magnesium levels measured 24 h postinjury and neuromotor outcome at 1 and 2 weeks. We conclude that acute ionized magnesium measurement may be a predictor of long-term neurobehavioral outcome following head injury and that delayed administration of magnesium chloride can restore blood magnesium concentration and attenuate neurological motor deficits in brain-injured rats.     

Mitochondrial Respiratory Function and Cell Death in Focal Cerebral Ischemia

Pyruvate-supported oxygen uptake was determined as a measure of the functional capacity of mitochondria obtained from rat brain during unilateral middle cerebral artery occlusion and reperfusion. During ischemia, substantial reductions developed in both ADP-stimulated and uncoupled respiration in tissue from the focus of the affected area in the striatum and cortex. A similar pattern of change but with lesser reductions was seen in the adjacent perifocal tissue. Succinate-supported respiration was more affected than that with pyruvate in perifocal tissue at 2 h of ischemia, suggesting additional alterations to mitochondrial components in this tissue. Mitochondrial respiratory activity recovered fully in samples from the cortex, but not the striatum, within the first hour of reperfusion following 2 h of ischemia and remained similar to control values at 3 h of reperfusion. In contrast, impairment of the functional capacity of mitochondria from all three regions was seen in the first 3 h of reperfusion following 3 h of ischemia. Extensive infarction generally affecting the cortical focal tissue with more variable involvement of the perifocal tissue developed following 2 h of focal ischemia. Thus, mitochondrial impairment during the first 3 h of reperfusion was apparently not essential for tissue infarction to develop. Nonetheless, the observed mitochondrial changes could contribute to the damage produced by permanent focal ischemia as well as the larger infarcts produced when reperfusion was initiated following 3 h of ischemia.     

Dipeptidyl peptidase-4 inhibition prevents lung injury in mice under chronic stress via the modulation of oxidative stress and inflammation

Exposure to chronic psychosocial stress is a risk factor for various pulmonary diseases. In view of the essential role of dipeptidyl peptidase 4 (DPP4) in animal and human lung pathobiology, we investigated the role of DPP4 in stress-related lung injury in mice. Eight-week-old male mice were randomly divided into a non-stress group and a 2-week immobilization stress group. Non-stress control mice were left undisturbed. The mice subjected to immobilized stress were randomly assigned to the vehicle or the DPP4 inhibitor anagliptin for 2 weeks. Chronic stress reduced subcutaneous and inguinal adipose volumes and increased blood DPP4 levels. The stressed mice showed increased levels in the lungs of genes and/or proteins related to oxidative stress (p67^phox, p47^phox, p22^phox and gp91^phox), inflammation (monocyte chemoattractant protein-1, vascular cell adhesion molecule-1, and intracellular adhesion molecule-1), apoptosis (caspase-3, -8, -9), senescence (p16^INK4A, p21, and p53) and proteolysis (matrix metalloproteinase-2 to -9, cathepsin S/K, and tissue inhibitor of matrix metalloproteinase-1 and -2), and reduced levels of eNOS, Sirt1, and Bcl-2 proteins; and these effects were reversed by genetic and pharmacological inhibitions of DPP4. We then exposed human umbilical vein endothelial cells in vitro to hydrogen peroxide; anagliptin treatment was also observed to mitigate oxidative and inflammatory molecules in this setting. Anagliptin can improve lung injury in stressed mice, possibly by mitigating vascular inflammation, oxidative stress production, and proteolysis. DPP4 may become a new therapeutic target for chronic psychological stress-related lung disease in humans and animals.