中华蜜蜂工蜂碱性磷酸酶的纯化及部分性质研究

以中华蜜蜂的工蜂成体为提酶材料,经正丁醇提取、硫酸铵分级沉淀,0.1 5%NaCl溶液透析,得粗酶液.用Sephadex G-150葡聚糖层析柱纯化粗酶,获得提纯的碱性磷酸酶,提纯倍数为16.79,比活力达到135.85 U/mg.以对硝基苯磷酸二钠(PNPP)为底物,测定提纯后的碱性磷酸酶的理化性质.结果表明:该酶的最适温度为45℃,最适pH值为8.6,米氏常数(Km值)为0.97×10-3mol/L.甲醛、甲醇、乙醇和乙二醇对碱性磷酸酶均有抑制作用.     

Study on the activation of blood platelets by propylene-acidamide grafted polypropylene membrane in vitro

AIM: To evaluate the blood compatibility of a new bioartificial reactor membranous material (propylene-acidamide grafted polypropylene membrane, PP-g-AAm) in vitro. METHODS: Contacted PP-g-AAm membrane and PP (polypropylene) membrane with platelet-rich plasma in a swing bed, 37 ℃, to simulate the conditions in vivo, and another group of PRP without any membranes was set as control group. ELISA was used to study the expression of β-thromboglobulin, and flow cytometry was used to study CD62P and CD63 expression of the activated blood platelets after contacting the two kinds of membranes with PRP. Scanning electrical microscopy was used to study the configuration and numbers of platelet cells adhered on the membranes. RESULTS: After contacting with PRP 30 min, β-TG expression showed marked difference between the two kinds of material groups and the control group (P<0.01, P<0.05), and the difference between the two kinds of membrane groups was also significant (P<0.05). There were obviously differences on the expression of CD62P and CD63 between the two kinds of membranes after contacting with PRP for 30 min (P<0.05，P<0.01). When enlarged 10 000 times, the disfiguration of the platelet cells adhered on the two kinds of membranes after one hour were found by scanning electrical microscopy, and the numbers of platelets on the PP membrane were more than the PP-g-AAm membrane markedly. CONCLUSION: The PP-g-AAm membrane has better blood compatibility than the PP membrane.     

Influence of allogeneic cornea on human peripheral blood T lymphocyte subtype in vitro

AIM: To observe the immunoregulation of allogeneic cornea on the human peripheral blood T lymphocytes in vitro. METHODS: After Co-culture of human peripheral blood lymphocytes and allogeneic cornea in vitro, T lymphocytes were labeled by monoclonal antibody, and analyzed by fluorescent activated cell sorter (FACS). RESULTS: CD25 expression on T lymphocytes in control was 25.2%, after stimulated by the allogeneic cornea or PDB, CD25 expression on T lymphocytes was 56.8% and 80.9%, respectively. After stimulated by the allogeneic cornea, CD25 expression on CD4+ or CD8+ T lymphocytes were 67.3% and 52.3%, respectively. CONCLUSION: Allageneic cornea stimulates CD25 expression on human peripheral blood T lymphocytes, and the CD25 expression on CD4+ T lymphocytes is more prominent than CD8+ T lymphocytes.     

Effect of heme oxygenase on vascular remodeling in renal hypertension

AIM: To investigate the effect of heme oxygenase on vascular remodeling in renal hypertension. METHODS: Male Wistar rats were randomly divided into sham-operated, 2K1C （two-kidney one-clip） and hemin-induced groups. Four weeks after the treatments, the thickness of aortic media and HO enzymatic activity of the aorta were determined. Immunohistochemical staining was carried out to detect protein of HO-1 in the aorta. RESULTS: The blood pressure in 2K1C renal hypertension rats started to increase two weeks after the surgery and stabled at a high level at the 4th week. Hemin, an inducer of HO-1, markedly inhibited the increase in blood pressure. Aortic medium thickness of the 2K1C rats at 4th week was 27.5% thicker than that in the sham-operated rats. The thickness of aortic medium of the hemin-induced rats was 16.1% less than that in 2K1C group. At the 4th week after operation, protein level and enzymatic activity of HO-1 in aorta were higher than that in 2K1C group compared to those in the sham-operated group. CONCLUSION: Renal hypertension caused vascular remodeling and the activation of HO-1. HO-1 induction decreased the blood pressure of renal hypertension and reduced vascular remodeling.     

Effect of lactacystin and β-lactacystin on the activation and proliferation of T-lymphocytes

AIM: To evaluate the effects of lactacystin (LAC) and β-lactacystin (β-LAC), proteasome inhibitor, on the proliferation and activation of T lymphocytes. METHODS: Flow cytometry was used to analyse the proliferation and the expression of CD69, CD25 and CD3 in PHA activated T-lymphocytes. Furthermore, the expression of PA28 and IL-2 mRNA were assayed by competitive RT-PCR. RESULTS: (1) LAC and β-LAC significantly decreased the incorporation in PHA activated T-lymphocytes. (2) Although LAC and β-LAC did not affect the expression of CD69 at any time, they significantly inhibited the expression of CD25 (48 h, 72 h, P＜0.05). (3) In comparison with control, LAC and β-LAC significantly down-regulated the expression of PA28 and IL-2 mRNA (48 h, 72 h, P＜0.05).CONCLUSIONS: LAC and β-LAC significantly inhibit the proliferation and activation of T lymphocytes. Mechanisms involved are inhibition of CD25 and down-regulation of PA28 and IL-2 mRNA expression.     

Ultrastructure and function of human bone marrow mesenchymal stem cells

AIM: To study the cytoskeleton of mesenchymal stem cells (MSCs), the ultrastructure and function relationship by using atomic force microscope (AFM). METHODS: The ultrastructures and morphological feature of MSCs cultured for 1 d and 5 d were studied by AFM. RESULTS: The special structures that possess peculiar morphological characteristic of MSCs such as cytoskeleton, pseudopod, microfilament etc were identified by AFM, and these special structures are difficult to observe under electronic microscopy or other conventional optical microscopy. CONCLUSIONS: AFM is a powerful tool to study ultrastructures, morphological features, and cytoskeleton of stem cells in near physiological conditions. Its application prospect in cellular biology is extensive. The special cytoskeleton and other structures of MSCs observed above may represent the structural base of multi-differentiation potential of MSCs.     

Effect of oxymatrine on mouse allergic contact dermatitis induced by DNFB and lymphocyte proliferation stimulated by Con A

AIM: To explore the inhibitory effect of oxymatrine （OMT） on the allergic contact dermatitis （ACD） stimulated by dinitrofluorobenzene （DNFB） and its effects on the proliferation of the lymphocytes. METHODS: ① An ACD mouse model was established by stimulation with DNFB, and then the mice were injected intraperitoneally with different dosages of OMT， PBS and hydrocortisone （HCT） respectively, the swelling degree of their auricles was examined. ② Carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) dye and flow cytometer were used to examine the fluorescence intensity changes of lymphocytes stimulated by polyclonal stimulator ConA and OMT. RESULTS: ① compared with PBS group, OMT possessed the strong inhibitory effect on the ACD caused by DNFB in a dose-dependent manner, and its inhibitory effect was equivalent to the HCT of the same dosage with fewer side effects. ② In vitro experiments proved that OMT (500, 125 and 31 mg/L) had the ability to restrain the proliferation of lymphocytes of mouse. CONCLUSION: OMT possesses an inhibitory effect on the ACD induced by DNFB, and OMT is a kind of immunosuppressor.     

Study on animal intracorneal implantation model with xenogeneic swine corneal stroma

AIM: To study the immunogenicity and biocompatibility of xenogeneic swine corneal stroma as biological carrier for cornea reconstruction and to reconstruct corneal endothelial tissue with this carrier in vitro. METHODS: (1) The lymphocytes from the peripheral blood of F344 rats were immunologically labeled by anti-rat CD25-FITC and anti-rat CD4/CD8-PE, then determined by flow cytometry (FCM) at 12th， 90th day after intracorneal implantation with fresh and dehydrated swine corneal stroma. (2) The fresh and dehydrated grafts made of swine corneal stroma were implanted intralamellarly in corneas of New Zealand rabbits. Clinical examinations were performed monthly and histological examinations were made at 14th， 30th， 60th， 120th and 240th day. (3) The cat corneal endothelial cells were seeded on the Descemet's membrane of the dehydrated swine corneal stroma, then cultured in the medium with epidermal growth factor and laminin for 7 days. The morphology of reconstructed tissue was tested by microscope. RESULTS: (1) Compared to isograft group and negative control, the expression CD4＋CD25＋， CD8＋CD25＋ and CD4＋/CD8＋ of xenograft rat group implantation with swine corneal stroma did not appear significantly different in statistics (P>0.05). (2) In the total 12 rabbits, all the cornea grafts survived without rejection reaction， corneal haze or corneal neovascularization. The fresh grafts got transparent after 2 months, and the dehydrated grafts got transparent after 6 months. Histological study demonstrated both fresh and dehydrated stroma grafts had fused with rabbits'corneal stroma very well without lymphocytes infiltrating. (3) As shown in histological observations, the reconstructed cat corneal endothelial tissue was similar to the nature tissue. Cultured cat endothelial cells connected tightly to each other and attached to the Descemet's membrane closely. CONCLUSION: Swine corneal stroma has low immunogenicity and satisfying biocompatibility,it is an ideal biological carrier for cornea reconstruction in vitro.     

Effects of beta2-adrenergic blocker on cytosolic Ca2+ in isolated cardiomyocytes of rats with myocardial infarction

AIM: To investigate if beta2-adrenergic receptors result in more Ca2+ load after myocardial infarction (MI), the effects of beta2-adrenergic blocker on cytosolic Ca2+ (［Ca2+］i) were studied. METHODS: Male Wistar rats underwent a ligation of left coronary artery (n=9) or a sham operation (n=3). Cardiomyocytes were dissociated at 2, 4 and 8 weeks after MI and ［Ca2+］i was measured via fura-2 fluorescence. The response of cardiomyocytes to isoproterenol (1 μmol/L) in the presence or absence of atenolol (1 μmol/L), beta2-adrenergic blocker ICI118,551 (0.1 μmol/L) or propranolol (1 μmol/L) was examined. RESULTS: ICI118,551 suppressed the increase in ［Ca2+］i induced by isoproterenol at 4 and 8 weeks after MI (24.5%±5.7% vs 57.8%±13.2%, P<0.01; 12.2%±7.9% vs 44.6%±11.3%, P<0.01), but had no effects in control and 2 weeks post-MI groups. It decreased ［Ca2+］i in control and the three post-MI groups by 14.3%, 7.9%, 57.6% and 72.6%, respectively. Atenolol had suppressive effects only in control and 2 weeks post-MI groups (P<0.05). Propranolol had suppressive effects in control and all three post-MI groups (P<0.01). CONCLUSION: Beta 2-adrenergic blocker ICI118,551 exerts negative effects on ［Ca2+］i after MI, and the effects dramatically increase with the progression of MI.