The role of endothelial insulin signaling in the regulation of vascular tone and insulin resistance

Insulin receptors (IRs) on vascular endothelial cells have been suggested to participate in insulin-regulated glucose homeostasis. To directly address the role of insulin action in endothelial function, we have generated a vascular endothelial cell IR knockout (VENIRKO) mouse using the Cre-loxP system. Cultured endothelium of VENIRKO mice exhibited complete rearrangement of the IR gene and a more than 95% decrease in IR mRNA. VENIRKO mice were born at the expected Mendelian ratio, grew normally, were fertile, and exhibited normal patterns of vasculature in the retina and other tissues. Glucose homeostasis under basal condition was comparable in VENIRKO mice. Both eNOS and endothelin-1 mRNA levels, however, were reduced by approximately 30-60% in endothelial cells, aorta, and heart, while vascular EGF expression was maintained at normal levels. Arterial pressure tended to be lower in VENIRKO mice on both low- and high-salt diets, and on a low-salt diet VENIRKO mice showed insulin resistance. Thus, inactivation of the IR on endothelial cell has no major consequences on vascular development or glucose homeostasis under basal conditions, but alters expression of vasoactive mediators and may play a role in maintaining vascular tone and regulation of insulin sensitivity to dietary salt intake.     

三种阳离子脂质体介导血管内皮细胞基因转染效率的比较

目的:评价不同阳离子脂质体介导基因转染血管内皮细胞的转染效率。方法:实验于2006-12/2007-02在中山大学生化实验室及广州市创伤外科研究所完成。采用增强型绿色荧光蛋白基因为报告基因,分别采用Lipofectin、Lipofectamine、Dosper3种不同的阳离子脂质体为载体,转染人脐静脉血管内皮细胞。在24孔板中,每孔加入人脐静脉血管内皮细胞悬液(1×106个细胞),各孔分别加入3种不同阳离子脂质体增强型绿色荧光蛋白质粒复合物,分别于培养24,48h后用荧光显微镜及流式细胞仪测定增强型绿色荧光蛋白在细胞内的表达及转染效率。结果:3种不同阳离子介导的增强型绿色荧光蛋白基因转染的人脐静脉血管内皮细胞内均有绿色荧光蛋白表达,24h后明显,48h后达高峰。Dosper介导组绿色荧光细胞百分比明显高于Lipofectin介导组及Lipofectamine介导组,差异有非常显著意义(P<0.01)。结论:Dosper介导的血管内皮细胞基因转染效率较高,较适合作为血管内皮细胞的基因转染载体。     

关节软骨脱细胞支架材料的制备

目的:探讨脱细胞关节软骨支架材料的制备方法,制备软骨理想的组织工程支架材料。方法:实验于2005-12/2006-08在兰州大学第二医院骨科研究所实验室完成。实验方法:利用冷冻干燥、化学去污剂等方法制备脱细胞的兔关节软骨。在无菌状态下取青紫蓝兔的新鲜兔关节软骨,剪成3.5mm×3.5mm,厚度0.2～2.0mm,在冷冻干燥器中冻干12h。在10g/L Triton X-100、Tris-HCl液内加入蛋白酶抑制剂-苯甲基黄酰氟,持续振荡48h后标本以双蒸馏水连续冲洗后置于DNase I酶和RNase A酶混合液中消化。置于10g/L Triton X-100、Tris-HCl液中洗脱。实验评估:①大体观察:肉眼观察脱细胞后关节软骨的外观形态。②组织学观察:将制备的脱细胞关节软骨行石蜡包埋切片,行苏木精-伊红染色、Massion三色染色并在光镜下观察。③扫描电镜观察:将制备的脱细胞关节软骨以戊二醛-锇酸双固定后,行扫描电镜观察。结果:①脱细胞后关节软骨外观形态:肉眼下可见正常关节软骨呈白色或淡黄色,脱细胞后关节软骨色呈灰白,半透明状,无光泽,外形与软骨相似并且仍维持了关节软骨的结构。②脱细胞后关节软骨的组织学变化:苏木精-伊红染色显示,软骨细胞消失,软骨巢分辨不清,在巢内没有蓝染的核物质,核细胞碎屑,只有红染为残留的细胞外基质。Massion三色染色显示,脱细胞后关节软骨内主要含有胶原纤维。③脱细胞后关节软骨的超微结构:扫描电镜下显示,脱细胞后关节软骨陷窝呈蜂窝状,未见到残余的细胞核、细胞器,残余的空穴高低不平。结论:经冷冻干燥、化学去污剂等方法可完整去除软骨中的细胞成分,保留胶原纤维等细胞外基质。     

壳聚糖纳米粒制备及表征与其抗肿瘤的生物学效应

目的:近期研究发现,壳聚糖纳米化后,不仅可改善其溶解性,还可提高其生物学功能。拟建立壳聚糖纳米粒的制备方法,并对壳聚糖纳米粒的表征及抗肿瘤生物学效应进行初步研究。方法:实验于2006-08/2007-05在浙江省医学科学院生物工程所完成。①建立壳聚糖纳米粒的制备方法:将壳聚糖粉末溶于乙酸溶液,用NaOH调节其pH为5,采用三聚磷酸钠为凝聚剂,进行离子交联来制备壳聚糖纳米颗粒。通过离心和冷冻干燥得到壳聚糖纳米粒粉末。②纳米粒的表征:经超声得到壳聚糖的悬浊液,用透射电镜来观察纳米颗粒的外观形态;用动态光散射仪来测定纳米颗粒的粒径大小与分布。③采用MTT法对壳聚糖纳米粒体外抗肿瘤生物学效应进行了初步研究。结果:①透射电镜下观测到了稳定、均一的颗粒;激光粒度分析仪测量发现纳米粒粒径大小在300nm左右,粒径分布较窄。②500mg/L的壳聚糖纳米粒对Hela细胞的抑制率为27%;对SMMC-7721细胞的抑制率为23%;对BGC-823细胞的抑制率为29%;对MCF-7细胞的抑制率最高,达55%。结论:建立的壳聚糖纳米粒的制备方法可靠,并证明其体外具有较好的抗肿瘤作用。     

Mice with cardiomyocyte-specific disruption of the endothelin-1 gene are resistant to hyperthyroid cardiac hypertrophy

Endothelin 1 (ET-1), a potent vasoconstrictor peptide expressed by endothelium, is also produced in the heart in response to a variety of stresses. It induces hypertrophy in cultured cardiac myocytes but only at concentrations far greater than those found in plasma. We tested whether ET-1 generated by cardiac myocytes in vivo is a local signal for cardiac hypertrophy. To avoid the perinatal lethality seen in systemic ET-1-null mice, we used the Cre/loxP system to generate mice with cardiac myocyte-specific disruption of the ET-1 gene. We used the alpha-myosin heavy chain promoter to drive expression of Cre and were able to obtain 75% reduction in ET-1 mRNA in cardiac myocytes isolated from these mice at baseline and after stimulation, in vivo, for 24 h with tri-iodothyronine (T3). Necropsy measurements of cardiac mass indexed for body weight showed a 57% reduction in cardiac hypertrophy in response to 16 days of exogenous T3 in mice homozygous for the disrupted ET-1 allele compared to siblings with an intact ET-1 gene. Moreover, in vivo MRI showed only a 3% increase in left ventricular mass indexed for body weight in mice with the disrupted allele after 3 weeks of T3 treatment versus a 27% increase in mice with an intact ET-1 gene. A reduced hypertrophic response was confirmed by planimetry of cardiac myocytes. We conclude that ET-1, produced locally by cardiac myocytes, and acting in a paracrine/autocrine manner, is an important signal for myocardial hypertrophy that facilitates the response to thyroid hormone.     

Morphometry of human leukocytes

In order to establish quantitative models of leukocyte functions, several morphometric parameters on individual white cells are needed. These include the diameter, volume, and membrane area of the cells and their nuclei in the undeformed state. A stereologic method was used to obtain these quantities from transmission electron microscopy of random sections through human white blood cells (neutrophils, lymphocytes, monocytes, and eosinophils). In order to estimate possible artifacts due to preparation of the cells for transmission electron microscopy, a detailed comparison with light microscopy was made. The results show that undeformed white cells in isotonic solution are spherical with many membrane foldings and have a significantly smaller diameter than that measured on blood smears. A method of chemical fixation was employed so that the shrinkage due to fixation of the cells is below the resolution of light microscopic measurements. Further, it was shown that all leukocytes, including lymphocytes, have much more membrane area than is needed to cover their volumes, and this membrane area remains constant when the cell is hypotonically swollen.     

Treatment of persistent non-union of the humerus using a vascularized bone graft from the supracondylar region of the femur

Six patients with recalcitrant post-traumatic humeral shaft non-unions were treated using vascularized bone grafts from the supracondylar region of the femur. The initial state of injury showed that four fractures were closed, while two were open fractures. At the acute stage, five fractures were fixed using intramedullary nailing, while one was fixed with a plate. In all patients, a bony flap was placed on the cortex after decortication. The size of the harvested bone flap ranged between 4 x 2.5 cm and 5 x 3 cm. After fixation of the bony flap, the inferior genicular artery and vein were anastomosed to the deep brachial artery and vein. The mean time required to obtain radiographic bone union was 3.3 months. The only graft-site complication involved transient mild paresthesia in the saphenous nerve area in one patient. This procedure was useful for atrophic nonunions without substantial bone defects of the humeral shaft.     

CONCURRENT MYELITIS AND GUILLAIN-BARRÉ SYNDROME AFTER VARICELLA INFECTION

Myelitis and Guillain-Barré syndrome occurring concurrently after varicella infection is very rare. A 34-year-old man presented with progressive flaccid tetraparesis, facial palsy, respiratory failure, sensory loss and urinary incontinence one week after varicella infection. Clinical, imaging and electrodiagnostic studies supported the diagnosis of myelitis and Guillain-Barré syndrome. He improved with intravenous acyclovir and gammaglobulin.     

Asynchronism of the recovery of baroreflex sensitivity, blood pressure, and consciousness from anesthesia in rats

Anesthesia inhibits arterial baroreflex functions such as baroreflex sensitivity （BRS）. The main objective of the present study was to determine the time course of BRS recovery from anesthesia and to determine whether BRS recovery is synchronous with the recovery of consciousness and blood pressure （BP）. Experiments were performed in male Sprague-Dawley rats using different commonly used anesthetics at routine doses through intraperitoneal administration： （1） diazepam/ketamine,     

Synergism of atenoloi and nitrendipine on hemodynamic amelioration and organ protection in hypertensive rats

OBJECTIVE： This study was designed to investigate the possible synergism of atenolol and nitrendipine on blood pressure （BP） and blood pressure variability （BPV） reductions, baroreflex sensitivity （BRS） amelioration, and organ protection in hypertensive rats. METHOD： The dose was 20 mg/kg for atenolol, 10 mg/kg for nitrendipine and 20 ＋ 10 mg/kg for the combination of these two drugs. In an acute study, a single dose was given via a catheter previously inserted into the stomach in spontaneously hypertensive rats （SHR）.