电镜和荧光显微技术在细胞凋亡研究中的应用

目的：探讨荧光显微技术在检测细胞凋亡中的作用。方法：应用拓扑异构酶Ⅱ抑制剂VP-16及化学毒物叠氮钠分别诱导HL-60细胞凋亡和死亡，用透射电镜和经Hoechst 33258染色的荧光显微镜对凋亡及死亡动态观察和定量分析。结果：HL-60细胞在VP16处理后，荧光显微镜下可见核浓缩、染色质凝集、核碎裂等凋亡特征；而叠氮钠诱导的细胞死亡则出现核溶解、核染色质弥散，但无上述改变；两者电镜的观察与荧光显微镜的改变一致；凋亡细胞及坏死细胞荧光显微镜下呈不同的形态特征。对处理的不同时间点细胞的荧光显微镜观察发现：处理后4h核形态开始变化，有核浓缩的细胞比例在处理8h达高峰，然后下降，核碎裂细胞的比例在24h达高峰，约占80％，表明核浓缩发生在核碎裂之前。结论：荧光显微镜技术可明确观察判断细胞凋亡及坏死，并可进行动态及定量分析，是良好的凋亡检测方法。     

Myocardial ultrastructure in the isolated rabbit heart exposed to dopamine, dobutamine, isoprenaline, G-strofanthin, xamoterol and hypoxia.

The isolated spontaneously beating heart was used for comparing the effects of hypoxia and positive inotropic drugs on myocardial ultrastructure. Hypoxia gives a significant decrease in the volume fractions of mitochondrial cristae relative to the total mitochondrial volume (Vvmcristae) and a significant increase in the volume fraction of mitochondrial matrix relative to the total mitochondrial volume (Vvmmatrix), but changes in volume fractions of mitochondria (Vvmitochondria) and myofibrils (Vvmyofibrils) were absent. Significant changes in ultrastructure in hearts treated with dopamine (0.6 microM), dobutamine (90 nM), G-strofanthin (0.25 microM), xamoterol (32 nM) and isoprenaline (0.15 microM or 15 nM) were absent. Furthermore, myocardial effects in the isolated rabbit heart without exposure to any treatment showed a significantly decrease in oxygen consumption after 90 min. and a significant decrease in frequency of contractions after 120 min. perfusion time, but no change in contractility was seen. We conclude that this experimental model is useful in studies of positive inotropic drugs.     

Enhancement of spontaneous fibrinolytic activity in diabetic retinopathy.

The fibrinolytic system was studied in 96 patients with type I diabetes mellitus. Patients were grouped according to their degree of retinopathy; 38 patients with no evidence of retinopathy, 28 patients with background retinopathy and 30 patients with proliferative retinopathy. Thirty healthy individuals served as controls. The basal fibrinolytic activity as measured by clot lysis time and t-PA activity was increased in diabetic patients. This was associated with low levels of plasminogen activator inhibitor. Increased levels of D-dimer in diabetic patients further indicate enhanced in vivo fibrinolysis. The increase in fibrinolytic activity was highest in diabetics without retinopathy, and decreased with increasing retinopathy. Endothelial release of t-PA after venous occlusion was not different between controls and all diabetic groups. These findings suggest that in type I diabetics the fibrinolytic system is in an activated state. With worsening of retinopathy this increase in fibrinolytic activity diminishes.     

Morphofunctional changes in the small intestine of children with food allergy

Children with food allergy underwent allergologic and gastroenterologic examinations using esophagogastroduodenojejunoscopy. Impairment of the jejunum in the form of jejunitis associated with function abnormality as shown by the loading tests was revealed. It is concluded that the given patients: group should be examined for the morphofunctional status of the upper gastrointestinal tract.     

Characterization of contraction-mediating prostanoid receptors in human hand veins: effects of the thromboxane receptor antagonists BM13,505 and AH23848

The effects of prostaglandin F2 alpha, prostaglandin E1, prostaglandin E2 and the thromboxane A2 analogue U46619 were determined in ring segments of human hand veins. All prostanoids except prostaglandin E1 elicited contraction. The order of potency was U46619 greater than prostaglandin F2 alpha greater than prostaglandin E2. The thromboxane receptor antagonists BM13,505 and AH23848 both caused a parallel rightward displacement of the concentration-response curve for U46619 without depression of the maximum contraction, suggesting competitive antagonism. Schild plots for both antagonists yielded regression lines with slope indices not significantly different from unity. The pA2 values for BM13,505 and AH23848 were 7.9 and 8.4 respectively. Both antagonists also effectively inhibited prostaglandin F2 alpha-induced contractions. However, AH23848 significantly reduced the maximum response, and the results with BM13,505 gave no clear indication of the type of inhibition. In vein segments submaximally contracted by 5-hydroxytryptamine, prostaglandins E1 and E2 produced a biphasic response with a relaxation at low and a contraction at high concentrations. Prostaglandin F2 alpha and U46619 failed to elicit relaxation under these conditions. However, in the presence of either thromboxane receptor antagonist, prostaglandin F2 alpha but not U46619 produced a relaxation. The results are compatible with the presence of at least two prostanoid receptors in human hand veins, a contraction-mediating thromboxane receptor and an as yet unclassified receptor eliciting relaxation. U46619 was a potent agonist at the thromboxane receptor and prostaglandin E1 and E2 preferentially stimulated the relaxation-mediating receptor, whereas prostaglandin F2 alpha appeared to be active at both receptor sites.     

Hip fractures in urban population in Finland.

We studied prospectively the occurrence of hip fractures among the over 49-year-old urban populations (n = 88,206) in the cities Tampere, Jyv?skyl? and Kotka in 1989 and compared the results with a parallel Scandinavian multicentre study. During 1989, there were 266 inhabitants who were treated for a hip fracture, 213 of them were women. Women had in 132 cases a femoral neck fracture and in 81 cases a trochanteric fracture, and the corresponding numbers for men were 22 and 31. Among women, the total hip fracture incidence in Jyv?skyl? (n = 22) was the lowest when compared with the ones in Tampere (n = 47) and Kotka (n = 36). These incidences were on average lower than in the other Scandinavian cities studied in parallel. The all over ratio of femoral neck/trochanteric fractures was 1.1 in Tampere, 3.0 in Jyv?skyl? and 2.2 in Kotka and in Tampere, women had 9.3 times more femoral neck fractures than men. Our study showed that in Finland in three major cities the hip fracture incidence was lower than what has been similarly registered in corresponding urban populations in other Scandinavian countries. We also point out that the relative part of trochanteric fractures appears to be increasing.     

Lipoprotein lipase in guinea-pig adrenals: activity, mRNA, immunolocalization and regulation by ACTH.

Lipase activity in homogenates of guinea-pig adrenals was studied under conditions which exclude the hormone-sensitive lipase/cholesterol ester hydrolase. Antibody inhibition and chromatography on heparin-Sepharose showed that most of the activity was due to lipoprotein lipase (LPL), and that there was only a small amount of hepatic lipase activity. Northern blot analysis of total RNA demonstrated the same three adrenal LPL mRNA species (1.8, 3.1 and 3.5 kb) as were found in adipose tissue and heart. Hence, at least part of the LPL activity in adrenals is due to enzyme synthesized within the tissue. Immunolocalization showed that LPL was associated with the endothelium of blood vessels throughout the gland. In addition, there was cytoplasmic immunoreaction, suggesting that lipase was synthesized in a subpopulation of cells in the transitional zone between the fasciculata and reticularis layer of the cortex, particularly over lipid-filled cells. There was also intense immunofluorescence over scattered cells in the adrenal medulla. Treatment with an ACTH analogue depot (20 IU, i.m.) for 11 days induced a 12-fold increase in serum cortisol and increased adrenal weight 2.2-fold. The treatment induced increases in LPL mRNA (about twofold), LPL activity and in the number of cells in the adrenal cortex which gave an immunoreaction for LPL.     

Demonstration of endogenous lectins in synovial tissue.

We have recently shown that synoviocytes and extracellular matrices exhibit distinct patterns of carbohydrate expression. Their biological relevance is however not known. The purpose of the present study was to find out whether human synovial tissue would also show a specific receptor pattern for complex sugar molecules. Endogenous lectins were displayed by means of biotinylated neoglycoproteins and sulfated polysaccharides in paraffin-embedded material or cryosections. In addition to certain carbohydrate components that are known to be constituents of the carbohydrate part of cellular glycoconjugates, our panel included heparin and fucoidan, a sulfated fucose. Binding sites were shown using the avidin-peroxidase technique for light microscopy. The results were compared with immunohistochemical methods and enzyme histochemistry. Our study demonstrates that human synovial tissue contains a complex pattern of endogenous lectins depending on the different types of synovitis. The staining method we used in the investigation allows for precise localization of saccharide binding receptors and is therefore believed to be a reliable technique for further phenotypic characterization of synovial cells.