人骨髓间充质干细胞分离培养及血管内皮生长因子对其产生的诱导分化作用

目的：目前有关骨髓间充质干细胞向内皮细胞诱导分化的研究较少。本实验分离和培养人骨髓间充质干细胞，用带有VEGF165的质粒转染人骨髓间充质干细胞，探讨血管内皮生长因子对其体外诱导分化的作用。 方法：实验于2005—04／2006—04在吉林大学人兽共患病教育部重点实验室完成。取成人的已排除血液系统肿瘤疾病的新鲜骨髓（自愿提供），采用Percoll梯度分离培养骨髓间充质干细胞，于倒置显微镜下观察细胞形态变化和生长情况。原代细胞培养至增殖接近融合状态时，单克隆培养法分离传代培养，扩增骨髓间充质干细胞。采用流式细胞术检测细胞免疫学表型。在原核细胞大肠杆菌DH5α中复制扩增和提取，纯化、克隆pcDNA3．0-VEGF165质粒。用脂质体转染法转染骨髓间充质干细胞：应用流式细胞术检测诱导后骨髓间充质干细胞免疫学表型变化j并采用免疫荧光染色鉴定转染情况，并设质粒空载和未转染的骨髓间充质干细胞为对照。 结果：人骨髓间充质干细胞原代培养1周后，造血细胞消失，贴壁细胞体积增大，呈现梭形外观，有粗大的细胞突起伸出。2周后细胞融合成单层，梭形突起变长，排列有明显的方向性，细胞排列成旋涡状、网状、辐射状。流式细胞术显示，人骨髓间充质干细胞免疫学表型CD44、CD29阳性，CD34、CD31、CD45阴性。VEGF165诱导骨髓间充质千细胞后CD44表达明显降低，CD31明显升高。免疫荧光染色显示，用FITC标记后的VEGF抗体使细胞显现绿色荧光，用cy3标记的CD31抗体使细胞显现了红色荧光。 结论：转染后的骨髓间充质干细胞细胞表型发生明显转变，CD31表达率明显增高，呈现典型的内皮细胞的表型特征，这说明骨髓间充质干细胞具有向内皮细胞分化的潜能。     

Effect of danazol on clotting factor levels, bleeding incidence, factor infusion requirements, and immune parameters in hemophilia

Several recent studies have reported conflicting results on the effectiveness of danazol, an attenuated androgen, in raising plasma levels of clotting factors VIII and IX in patients with hemophilia. We undertook a randomized, double-blind cross-over trial using 8 weeks' administration of danazol (D), 600 mg/d, and 8 weeks' administration of placebo (P) separated by 2 weeks of rest in 12 patients with hemophilia A and four patients with hemophilia B. Plasma factor VIII and IX levels, frequency and type of bleeding episodes, amount of factor concentrate infused, fibrinogen, fibrinolysis assays, antithrombin III, liver function, and immune parameters were followed. During the danazol phase a minimal increase was noted in the average clotting factor levels, an increase that, although statistically significant, was of hemostatically marginal magnitude. Significant increases in protein C and plasminogen levels, however, were observed during the danazol period, suggestive of danazol-mediated enhanced fibrinolysis. Clinically, bleeding frequency was significantly increased, and more clotting factor was consumed during the danazol period. Furthermore, eight episodes of hematuria and oral mucosal bleeding was reported during the danazol phase in contrast to only one episode of hematuria during the placebo phase, consistent with an enhancement of fibrinolytic activity with danazol. We conclude that danazol does not have a hemostatically significant effect on plasma levels of factor VIII and IX but may be associated with enhancement of fibrinolytic activity, resulting in increased bleeding frequency and requiring more clotting factor infusions. Therefore, danazol is not a viable alternative in the treatment of hemophilia.     

急性心肌梗死后外周血单个核细胞表面CD147与基质金属蛋白酶-9 mRNA表达的相关性

目的：基质金属蛋白酶在急性心肌梗死后的心室重构中起着重要作用，但其调节机制目前尚未明确。实验拟通过动物模型的建立及体外细胞培养，观察急性心肌梗死后单个核细胞表面CD147与心肌成纤维细胞基质金属蛋白酶-9 mRNA表达的关系。 方法：实验于2006—08／2007-06在河北省人民医院临床实验中心完成。实验材料：SD大鼠及SD仔鼠（出生1～3d）购自河北医科大学试验动物中心。实验过程中对动物处置符合动物伦理学标准。实验方法：①将30只大鼠随机分为急性心肌梗死组（n=15）和假手术组（n=15），假手术组只过线不结扎。流式细胞分析法检测大鼠术后24h外周血单个核细胞表面CD147表达。②选择SD仔鼠制备心肌成纤维细胞。将单个核细胞与心肌成纤维细胞以细胞数0．5：1，1：1，2：1混合培养24h后，半定量反转录一聚合酶联反应法检测基质金属蛋白酶-9 mRNA表达。当单核细胞与心肌成纤维细胞2：1混合时，加入CD147单克隆抗体1，2，4μL/L，培养24h后检测基质金属蛋白酶-9 mRNA表达。 结果：①急性心肌梗死后外周血单个核细胞表面CD147表达明显增加。②单个核细胞与心肌成纤维细胞混合培养，随着单个核细胞比例的增加，心肌成纤维细胞基质金属蛋白酶-9 mRNA表达增加。③在单个核细胞与心肌成纤维细胞2：1混合培养体系中，随着加入CD147单克隆抗体浓度的增加，基质金属蛋白酶-9 mRNA生成减少。 结论：急性心肌梗死后单个核细胞表面CD147表达明显增加，对心肌成纤维细胞基质金属蛋白酶-9生成起上游调节作用。     

Film tomography compared with film and digital bitewing radiography for proximal caries detection

OBJECTIVES: The purpose of this study was to evaluate the efficiency of conventional tomography for proximal caries detection compared with conventional bitewing and digital intraoral radiography. METHODS: In this study, 48 extracted human posterior permanent teeth were used. Conventional bitewing radiographs, digital intraoral radiographs and conventional tomograms were obtained of the teeth. The presence or absence of caries was scored according to a five-point scale by two oral and maxillofacial radiologists. True caries depth was determined by histological examination. The diagnostic accuracy of each radiographic system was assessed by means of receiver operating characteristic (ROC) curve analysis. The mean of areas under the ROC curve (A(z)) was analysed by pairwise comparison of ROC curve. The interobserver agreement was evaluated using a t-test. RESULTS: The statistical analysis of A(z) scores has shown no significant difference for three imaging modalities (P>0.05). There was no statistically significant difference between interobserver agreement (P>0.05). CONCLUSIONS: This study has demonstrated that the diagnostic accuracy of conventional tomograms is comparable to that of conventional bitewing and digital intraoral radiographs for proximal caries detection.     

Effect of a High Fat Diet on Quantitative Features of Adipocytes in the Omentum: An Experimental, Stereological and Ultrastructural Study

Background: Omental adipose tissue specimens of female rats that were fed a high fat (HF) diet were evaluated stereologically and histopathologically. To our knowledge, there is no stereological study on numerical density, nuclear height and volume of adipocytes in omental adipose tissue in the female rat fed a HF diet in the literature. Method: 20 female Spraque Dawley rats were used in the study. 10 of the animals were fed HF diet consisting of 30% of calories from fat for 3 months. The remaining 10 rats, the control group, were fed a normal diet. After the experimental procedure, all animals were anesthetized and omental adipose tissues in the same area were dissected and fixed for the histochemical process using a mixture of 3% glutaraldehyde and 1% osmium tetraoxide in 0.1 M phosphate buffer. After embedding of tissues in araldite CY 212, semi-thin and thin sections were cut. The semi-thin sections were stained with toluidine blue. The physical dissector counting method was used for estimation of numerical density and nuclear height of adipocytes. Cavalieri principle was used for the estimation of adipocyte volume; volume fraction approach was applied to find the volume fraction of adipose tissue components. Results: The mean numerical density of adipocytes in the HF diet group was significantly higher than the control. The mean nuclear height of adipocytes was also very high in the HF diet group. The volume fraction of adipose mass was increased whereas the extracellular matrix volume fraction was reduced in the HF diet group compared to the controls. The mean volume of adipocytes in the HF diet group was also significantly higher than in the control group. At the light microscopy level, it was found that adipocytes were enlarged and gaining irregular shape in the HF diet group. Thicker basal lamina and electron dense lipid content were also found in this group at the electron microscopy level. Conclusion: Lipid content and number of adipocytes in the adipose tissue of HF diet rats were higher than in the controls. Thus, HF diet induces increase in body weight via both hypertrophy and hyperplasia of adipocytes.     

维生素K3粉末降解动力学

在温度为50～90℃,相对湿度为50～93%范围内,维生素K₃的变色速率方程为—dR/dt=k_RR,降解速率方程为d(C_o—c)/dt=k_C(c_o—c)^1/2,光解速率方程为—dR/dt=k_P(R_o—R)^-1。k_R和_C与热力学温度T和相对湿度RH的关系通式为k=k′exp(—E_A/RT)exp(m′RH)。在实验温度和湿度范围内,变色和降解的表观活化能平均值分别为105.51和130.72 kJ/mol。     

指数程序升温药物稳定性试验

介绍了一种新的程序升温(指数程序升温)药物稳定性预测加速试验方法及计算方法。在这一新的程序升温方法中,温度每升高10℃,升温速率将增大2～4倍:dT/dt=a^(T-T₀)/10·(dT/dt)₀,使药物在高温和低温范围内的降解程度尽可能一致,提高了试验准确度。采用单因素优选法和数值积分法处理试验数据,避免了任何近似处理,使计算结果准确可靠。与线性升温、倒数升温和对数升温加速试验进行了对比,结果表明,指数程序升温法的准确性优于其它3种升温法。     

Effect of High Fat Diet on the Volume of Liver and Quantitative Feature of Kupffer Cells in the Female Rat: A Stereological and Ultrastructural Study

Background The role of Kupffer cells (KCs) in nonalcoholic steatohepatitis (NASH) which is regarded as a major cause of cryptogenic cirrhosis of the liver was investigated using stereological methods and electron microscopy in the rat model. To our knowledge, there is no stereological study on the volume of liver, total number, numerical density, and nuclear height of KCs of liver in the female rat fed with a high fat diet (HFD) in the literature. Method 16 female Sprague Dawley rats were randomized into HFD and control group, with HFD and standard diet for 12 weeks, respectively. In this study, two basic research methods were used to analyze the samples. One was histopathological observation at both light and electron microscopic level. The other was stereological methods that consist of Cavalieri principle for liver volume estimation and physical disector method for estimation of numerical density and total number of KCs in the liver. Results Liver volume, both mean numerical density and total number of KCs, were statistically increased in HFD rats. Ultrastructurally, a significant decrease in the mean nuclear height of KCs in HFD rats was also found. In the control group, no abnormal change was observed, but in the HFD group, some changes such as diffuse steatosis, mononuclear cell infiltration, necrosis, fibrosis, accumulation of fat droplets and intra-cytoplasmic vacuoles, and swollen mitochondria with irregular membranes were observed in the hepatocytes. Conclusion The number and activity of KCs are increased significantly in NASH induced by HFD, and KCs might be involved in the pathogenesis of steatohepatitis as previously attributed as a major cause of cryptogenic cirrhosis of the liver.