HIF-1α过表达慢病毒载体的构建及对心肌细胞Notch配体Jagged1表达的影响

目的构建低氧诱导因子1α(hypoxia-inducible factor 1α,HIF-1α)过表达重组慢病毒质粒并包装病毒,将病毒感染乳鼠心肌细胞,分析其对Notch配体Jagged1表达的影响。方法从cDNA文库中PCR扩增人HIF-1α编码序列并克隆入酶切线性化慢病毒载体GV218,转化感受态大肠杆菌细胞筛选出阳性克隆并测序鉴定。将HIF-1α过表达慢病毒质粒与病毒包装辅助质粒共转染工具细胞HEK293T,包装出慢病毒颗粒。原代分离SD乳大鼠心肌细胞,将HIF-1α过表达慢病毒感染心肌细胞,定量PCR分析Jagged1 mRNA水平的变化。结果成功扩增HIF-1α编码序列并正确连接入线性化载体GV218,获得阳性转化子并测序无误。将转化子质粒与病毒包装辅助质粒共转染HEK293T后,获得滴度为1×10~8TU/ml的慢病毒颗粒,Western blot法检测到HIF-1α-GFP融合蛋白。重组慢病毒感染乳鼠心肌细胞后,胞核中见GFP信号。与对照组相比,HIF-1α过表达5天后显著上调Jagged1 mRNA的水平。结论心肌细胞中HIF-1α可诱导Jagged1的表达。HIF-1α过表达重组慢病毒载体的成功构建,为研究缺血缺氧性心肌损伤反应的分子机制奠定了基础。     

利妥昔单抗对实验性自身免疫性葡萄膜炎小鼠的抗炎作用及其机制

目的　探讨利妥昔单抗（RTX）对实验性自身免疫性葡萄膜炎（EAU）小鼠的抗炎作用及其机制。 方法　取SPF级6～8周龄健康雌性C57BL/6J小鼠27只,使用光感受器间维生素A类结合蛋白1-20(IRBP_1-20)建立EAU小鼠模型,将EAU小鼠随机分为PBS对照组、50 mg·kg^-1 RTX实验组和100 mg·kg^-1 RTX实验组,每组各9只小鼠,PBS对照组小鼠腹腔注射PBS,50 mg·kg^-1 RTX实验组和100 mg·kg^-1 RTX实验组小鼠每千克体重分别腹腔注射50 mg RTX和100 mg RTX。造模后第21天,对各组小鼠进行眼底图像的采集与视网膜组织HE切片的制作,通过小动物视网膜成像仪采集各组小鼠眼底图像并参照Caspi临床评分标准进行炎症评分,将HE染色切片置于显微镜下观察并进行拍照,参照Caspi组织病理学评分标准进行评分。采用实时荧光定量PCR检测各组小鼠视网膜组织白细胞介素（IL）-1β、IL-6、IL-4和IL-10的mRNA相对表达量。采用SPSS 21.0软件进行统计学分析。结果　眼底图像显示:PBS对照组EAU小鼠眼底出现多发性血管炎,血管扩张,视网膜脉络膜出现多发病灶及少量线样病变;50 mg·kg^-1 RTX实验组EAU小鼠眼底发生轻度血管炎,视网膜出现个别局部病灶,无线样病变;100 mg·kg^-1RTX实验组EAU小鼠视网膜局部出现个别病灶,无线样病变。PBS对照组、50 mg·kg^-1 RTX实验组和100 mg·kg^-1RTX实验组EAU小鼠眼底图像Caspi临床评分分别为（1.944±0.808）分、（1.667±0.791）分、（1.667±0.829）分,三组间比较差异无统计学意义（P>0.05）。PBS对照组、50 mg·kg^-1 RTX实验组和100 mg·kg^-1RTX实验组EAU小鼠视网膜组织病理学评分分别为（1.563±1.116）分、（1.063±0.623）分、（1.250±0.655）分,三组间比较差异无统计学意义（P>0.05）。实时荧光定量PCR检测结果显示,50 mg·kg^-1 RTX实验组和100 mg·kg^-1 RTX实验组小鼠视网膜组织IL-6、IL-10 mRNA相对表达量以及IL-10/IL-6比值均高于PBS对照组（均为P<0.05）;100 mg·kg^-1 RTX实验组小鼠视网膜组织IL-6、IL-10 mRNA相对表达量以及IL-10/IL-6比值均高于50 mg·kg^-1 RTX实验组（均为P<0.05）。结论　腹腔注射RTX可一定程度缓解EAU小鼠视网膜组织的炎性病变,其机制可能与RTX提高小鼠视网膜组织IL-10/IL-6比值有关。     

消渴健脾胶囊质量标准研究

目的 建立消渴健脾胶囊的质量标准.方法 采用显微镜鉴别制剂中直接以粉末入药的白术、葛根、大黄、茯苓、车前子5味中药材的显微特征;采用薄层色谱(TLC)法定性鉴别栀子、土茯苓、大黄、茯苓、莱菔子、葛根6味药材的特征斑点;采用高效液相色谱(HPLC)法测定制剂中栀子苷的含量,色谱柱为Cosmosil C18柱(250 mm...     

结肠腺癌转移至牙龈1例报道及文献复习

0?引言 结肠癌是我国常见的消化道肿瘤之一,而口腔转移癌临床罕见,约占所有口腔恶性肿瘤的1％[1],可能发生在口腔软组织或颌骨.其中颌骨,尤其是下颌骨,比口腔软组织更易受累(2:1),在口腔软组织中,最常见的病变部位是牙龈(54％)[2].已有报道称牙龈癌转移来自肺、前列腺、直肠癌、肾上腺癌和乳腺等,结肠癌的牙龈转移报...     

过表达Notch1胞内域对c-Kit+骨髓间充质干细胞分化的影响

BACKGROUND: Activation of Notch signaling plays a critical role in stem cell differentiation, and this effect seems to be cell-type dependent. Little is reported on the role of activation of Notch1 signaling in the differentiation of c-Kit+ bone marrow mesenchymal stem cells. OBJECTIVE: To analyze the influence of activation of Notch1 signaling on the differentiation of c-Kit+ bone marrow mesenchymal stem cells. METHODS: The Notch1 intracellular domain (N1-ICD) was obtained from the cDNA library by PCR and cloned into BamHI/AgeI digested adenoviral GV314 plasmid to construct N1-ICD overexpressing shuttle plasmid, and the positive clones were verified by sequencing. N1-ICD shuttle plasmid and helper plasmids pBHGloxΔE1,3 Cre were used to co-transfect HEK293T cells to obtain N1-ICD overexpressing adenoviral particles (N1-ICD-Ad). The c-Kit+ subpopulation were isolated from bone marrow mesenchymal stem cells of the Sprague-Dawley rat femur via magnetic activated cell sorting. After transfection of the c-Kit+ BMSCs with N1-ICD-Ad adenovirus, we assessed the activation of Notch1 signaling and differentiation in c-Kit+ bone marrow mesenchymal stem cells by quantitative RT-PCR and immunofluorescent staining. RESULTS AND CONCLUSION: N1-ICD coding sequence was successfully generated from the cDNA library, and then was cloned into the linearized adenoviral vectors GV314. The resistant clones were verified by sequencing. With the assistance of packaging plasmids, recombinant N1-ICD-Ad adenovirus plasmids were successful packaged in HEK293T cells, and its title was 2×1012 PFU/L. c-Kit+ bone marrow mesenchymal stem cells with the purity of 91.6% were successfully isolated from the bone marrow mesenchymal stem cells of the Sprague-Dawley rat femur. Compared with the blank and negative controls, N1-ICD-Ad infection in the c-Kit+ bone marrow mesenchymal stem cells led to substantial accumulation of N1-ICD in the cytoplasm and nuclei, significantly unregulated expressions of Hes1 (a downstream gene of Notch) and cardiomyocyte differentiation genes Nkx2.5 and cTnT, significantly increased the expression of von Willebrand factor, an endothelial cell differentiation gene, and mildly increased the expression of smooth muscle 22α, a smooth muscle cell differentiation gene. These experimental results indicate that the activation of Notch1 signaling contributes to multi-lineages differentiation of c-Kit+ bone marrow mesenchymal stem cells, and the construction of N1-ICD overexpressing adenoviral vector makes the foundation for further research on the role of Notch1 signaling in stem cell biology.      

消渴健脾胶囊制备工艺、质量标准研究

目的 研究消渴健脾胶囊制备工艺、质量标准。方法 在单因素试验基础上，以提取时间、加水量、提取次数为影响因素，落新妇苷、栀子苷、水浸出物含量为评价指标，正交试验优化制备工艺。TLC法鉴别栀子、葛根、丹参、大黄，HPLC法同时测定绿原酸、葛根素、毛蕊花糖苷、大豆苷、落新妇苷、丹酚酸B、大豆苷元的含量。结果 最佳条件为提取时间1 h,加水量10倍，提取次数2次。TLC斑点清晰，阴性无干扰。7种成分在各自范围内线性关系良好(r≥0.999 4),平均加样回收率96.9%～100.5%,RSD 0.82%～2.62%。结论 该方法简便准确，可用于消渴健脾胶囊的质量控制。