简单、实用、高效的细胞培养技术

通过近几年的实践,笔者对我国兽医生物制品生产中传统的细胞培养工艺进行了一些改进,包括用洗洁精代替其他洗液,用自行研制的营养液代替常规营养液用于细胞培养,改变培养时的转速,改变细胞消化液,改进细胞的保存方法.这些方法均成功应用于生产,取得了良好的效果.     

小麦干湿交替条件下的水分利用效率与生物学性状

在防雨棚盆栽试验条件下,以“A115”、“4185”为材料,用干湿交替方法研究了3种水分组合下小麦的水分利用效率与生物学性状的相关关系。结果表明产量水平的水分利用效率与株高、穗数、单株生物量、光合速率、蒸腾速率和产量成显著正相关;与除此而外的其他被测生物学性状不相关或相关不显著。叶片水平的水分利用效率与气孔导度和蒸腾速率成极显著负相关关系,与穗粒数和分蘖数成显著负相关关系;与其他被测性状不相关或相关不显著。     

High density lipoprotein subclasses and activities of protein kinase C

AIM: To investigate the relationship between high density lipoprotein (HDL) subclasses and the binding activities of hepatic cell and extra-hepatic cell HDL receptor and intracellular protein kinase C (PKC). METHODS: Using purified pre-β1 HDL and apoE-deficent HDL3 to react with human smooth muscle cells (SMC) and HepG2 cells, then the activities of pre-β1HDL and apoE-deficent HDL3 binding to SMC and HepG2 cells, and the effects of this two HDL subclasses on PKC activities in SMC and HepG2 cells were observed . RESULTS: The results showed that the value of Kd of pre-β1HDL binding to SMC HDL receptor was not only significantly lower than that of apoE-deficient HDL3 (P<0.05), but significantly lower than pre-β1 HDL binding to HepG2 HDL receptor (P<0.05). Cell PKC system was all activated by binding of the two HDL subclasses with SMC HDL receptor, and the effect of pre-β1HDL appeared to be stronger than apoE-deficent HDL3. No change in HepG2 cell PKC activity by binding the two HDL subclasses with HepG2 HDL receptor was observed. CONCLUSIONS: The results indicate that the ability of pre-β1HDL to promote efflux of cholesterol from extra-hepatic cells is stronger than that of apoE-deficent HDL3, and it seems that plasma pre-β1 HDL mainly interacts with extra-hepatic cell HDL receptor, subsequently, activates PKC signal transduction system and promotes the intracellular cholesterol efflux from cells.     

Role of PKC and MMPs in the pathogenesis of the rat experimental atherosclerosis and the action of Danshen injection

AIM: To study the effects of protein kinase C on the expression of MMPs that may play an important role in the formation of atherosclerosis and the possible mechanism of Danshen injection to treat atherosclerosis. METHODS: 50 Sprgue-Dawley (SD) rats were divided into three groups randomly: control group (group C), model group (group M) and Denshen treatment group (group D). The serum was collected to measure the level of cholesterol, triglyceride, low density lipoprotein cholesterol（LDL-ch）. The expression of PKC and MMPs were measured by immunohistochemistry. Light microscope and electron microscope were also used. RESULTS: ① The cholesterol, triglyceride, LDL-ch concentrations in group M and group D were significantly higher than those in group C (P<0.01), cholesterol, triglycerideand LDL-ch contents were lower in group D than group M (P<0.01). ② The expression of PKC, MMP-2, MMP-9 in group M were significantly higher than those in group C (P<0.01), and were significantly lower in group D than those in group M (P<0.01). The expression of PKC was correlated with MMP-2 and MMP-9. ③ Light microscope showed that there were plaques in intima and serious calcification, necrosis, obviously irregular thickness in media of group M, but slightly in group D. ④ Electron microscope showed that smooth muscle cells of group M were necrosed , collage grew abundantly and alined disorderly, most of the endothelial cells exfoliated, but slightly in group D. CONCLUSIONS: ① MMPs play an important role in the generation and development of atherosclerosis. ② The activation of PKC may take part in the formation of atherosclerosis and it may be the upstream mechanism of the expression of MMPs. ③ The reduced expression of MMPs and PKC is a part of mechanism for Danshen to prevent and treat atherosclerosis.     

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.     

Relationship between serum concentrations of IL-18, IL-10, IL-6 and acute coronary syndrome

AIM: To examine the relation between serum concentrations of interleukin-18, interleukin-10, interleukin-6 and acute coronary syndrome (ACS). METHODS: Serum concentrations of IL-18, IL-10, IL-6 were measured in 17 patients with acute myocardial infarction (AMI), 30 patients with unstable angina pectoris (UAP), 15 patients with stable angina pectoris (SAP) and 20 controls by enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA).The relation between IL-18, IL-6 and IL-10 was compared. RESULTS: Serum concentrations of IL-18, IL-6 were significantly increased in the AMI and UAP groups in comparison with the SAP and control groups. Conversely, serum concentrations of IL-10 were significantly decreased in the AMI and UAP groups in comparison with the SAP and control groups. The correlation of concentrations of IL-18 and IL-6 had no significance; but the levels of IL-18 and IL-6 were negatively correlated with IL-10. CONCLUSION: Serum IL-18, IL-6 concentrations increase while serum IL-10 concentration decreases in patients with acute coronary syndromes. The inflammatory imbalance between IL-18, IL-6 and IL-10 may play an important role in the instability of atherosclerotic plaque.     

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.     

Effect of polyamines on intracellular calcium of the rat cardiomyocytes with anoxia and reoxygenation

AIM: To study the effect of extrogenous low concentration polyamine on cardiomyocyte calcium overload caused by anoxia and reoxygenation. METHODS: Enzymatically isolated rat ventricular myocytes were perfused with normal Tyrode solution for 8 min, then change to anoxia solution for 32 min, at last back to normal Tyrode solution perfusion for 8 min to establish the cardiomyocyte model of anoxia and reoxygenation. Spermine was added extracellularly to the bath before anoxia and spermine, spermidine or putrescine was added extracellularly after reoxygenation. Intracellular calcium fluorescence intensity (FI) was measured continuously by laser scan confocal microscope (LSCM). RESULTS: In the unstimulated state, exogenous spermine (1 mol/L) did not change resting ［Ca2+］i in the rat cardiomyocytes. Adding spermine before anoxia antagonized the ［Ca2+］i elevating caused by anoxia/reoxygenation. Adding spermine after reoxygenation also lowed the enhanced ［Ca2+］i caused by reoxygenation. Considering the potency of two conditions, the former was more efficacious than the later. Spermidine and putrescine also lowed the enhanced ［Ca2+］i caused by reoxygenation, but they were less efficacious than spermine. CONCLUSIONS: The results indicate that spermine given before anoxia or after reoxygenation, antagonized or lowed the cardiomyocytes calcium overload caused by anoxia/reoxygenation, but the later was weaker than the former. The order of potency of the polyamine lighten cardiomyocytes calcium overload caused by anoxia/reoxygenation was spermine>spermindine>putrescine.     

Effect of fluvastatin on migration of vascular smooth muscle cells from rats

AIM: To investigate the effect and mechanism of fluvastatin on the migration induced by platelet derived growth factor-BB (PDGF-BB) and endothelin-1 (ET-1) in cultured vascular smooth muscle cells (VSMCs). METHODS: Cultured VSMCs derived from spontaneously hypertensive rats (SHR) were used. Cell migration was determined by modified Boyden chamber assays. Intracellular free calcium (［Ca2+］i) was measured with fluorescent Ca2+ indicator Fura-2/AM. RESULTS: PDGF-BB and ET-1 significantly induced VSMCs migration, which was inhibited by pretreatment of VSMCs with fluvastatin (10-9-10-5 mol/L) in a dose-dependent manner, and the peak inhibition rate of migration induced by PDGF-BB and ET-1 was over 86.67%. Fluvastatin also attenuated the increase in ［Ca2+］i induced by PDGF-BB and ET-1, with a peak inhibition rate of 86.76% and 65.32%, respectively. CONCLUSION: PDGF-BB and ET-1 promote migration of VSMCs from SHR．Fluvastatin may have direct inhibitory effects on cell migration induced by PDGF-BB and ET-1. The increase in ［Ca2+］i may acts as intracellular signaling in the migration in response to PDGF-BB and ET-1 in VSMCs.