Purification of pluripotent embryonic stem cells using dielectrophoresis and a flow control system

Pluripotent stem cells (PSCs) such as embryonic stem cells and induced PSCs can differentiate into all somatic cell types such as cardiomyocytes, nerve cells, and chondrocytes. However, PSCs can easily lose their pluripotency if the culture process is disturbed. Therefore, cell sorting methods for purifying PSCs with pluripotency are important for the establishment and expansion of PSCs. In this study, we focused on dielectrophoresis (DEP) to separate cells without fluorescent dyes or magnetic antibodies. The goal of this study was to establish a cell sorting method for the purification of PSCs based on their pluripotency using DEP and a flow control system. The dielectrophoretic properties of mouse embryonic stem cells (mESCs) with and without pluripotency were evaluated in detail, and mESCs exhibited varying frequency dependencies in the DEP response. Based on the variance in DEP properties, mixed cell suspensions of mESCs can be separated according to their pluripotency with an efficacy of approximately 90%.     

shASPP2 H22稳转肝癌细胞系的构建及ASPP2敲低对H22移植瘤小鼠血管生成的影响

摘要 目的：构建小鼠shASPP2 H22稳转肝癌细胞系,观察ASPP2敲低对血管生成的影响。方法：针对小鼠ASPP2基因设计了3个不同的shRNA干扰序列（Y18421,Y18422,Y18423）及1个对照序列（GL427NC2）,采用双酶切（Age Ⅰ和EcoR Ⅰ）及质粒连接构建重组质粒,使用菌落PCR和测序比对进行鉴定;使用293T细胞将各重组质粒包装慢病毒并测定滴度;将 shASPP2和对照慢病毒质粒转染H22细胞,采用流式细胞术测定转染效率;采用qRT-PCR、Western Blot法观察shASPP2慢病毒对H22细胞ASPP2的干扰效果;采用CCK8法观察ASPP2敲低对H22细胞增殖的影响;采用Western Blot法观察ASPP2敲低对H22细胞及上清VEGF表达和分泌的影响;采用细胞注射法建立小鼠ASPP2敲低H22细胞皮下移植瘤模型,游标卡尺法观察肿瘤体积大小,采用活体激光共聚焦观察肿瘤血管生成情况,采用Western Blot法观察肿瘤组织VEGF的表达。结果：双酶切、菌落PCR和测序鉴定结果表示各重组质粒构建成功;各重组质粒经慢病毒包装后,测定显示Y18421、Y18422、Y18423和GL427NC2慢病毒质粒的滴度分别为3.40×10⁸ TU/mL、4.08×10⁸ TU/mL、5.49×10⁸ TU/mL和1.7×10⁹ TU/mL;Y18421、Y18422、Y18423及GL427NC2慢病毒质粒转染效率分别为：86.2 %、69.6 %、60.8 %和76.9 %。与GL427NC2 H22细胞相比,Y18421 H22细胞的ASPP2 mRNA及蛋白的表达明显降低（P<0.01,P<0.05）;Y18421细胞在培养24,48,72 h后增殖速率显著增加（P<0.0001,P<0.001,P<0.01）;Y18421细胞及上清的VEGF表达显著升高（P<0.001,P<0.01,P<0.05）。与GL427NC2 细胞移植瘤相比,Y18421细胞移植瘤体积明显增大（P<0.05）,总血管长度显著增加（P<0.05）,VEGF蛋白的表达明显上调（P<0.05）。结论：小鼠shASPP2 H22稳转肝癌细胞系构建成功,ASPP2敲低可能通过上调VEGF的表达促进小鼠H22细胞移植瘤血管生成。     

miR-455-5p靶向SOCS3抑制RSV感染致气道上皮炎症反应的机制研究

摘要 目的：揭示miR-455-5p对呼吸道合胞病毒（RSV）感染致气道上皮细胞炎症反应的作用机制。方法：qRT-PCR检测30例健康体检儿童（健康组）、RSV感染轻症组（n=41）和重症组（n=31）患儿血清miR-455-5p水平。将16HBE细胞分为Control组、NC-agomir组、miR-455-5p-agomir组、NC-antagomir组、miR-455-5p-antagomir组。使用Lipofectamine 3000转染16HBE细胞后培养48 h后,分为Blank组、NC组（转染了NC-agomir的细胞）、RSV+NC-agomir组、RSV+miR-455-5p-agomir组,用RSV病毒液感染RSV+NC-agomir组和RSV+miR-455-5p-agomir组16HBE细胞,Blank组和NC组16HBE细胞正常培养。用CCK-8法和EdU法检测细胞增殖、TUNEL法检测细胞凋亡、ELISA法检测上清液中TNF-α、IL-6、IL-8水平,qRT-PCR检测miR-455-5p和SOCS3的mRNA水平,Western blot检测SOCS3、IFN-α、STAT1、STAT2、p-STAT1和p-STAT2的蛋白水平。结果：与健康组相比,轻症组和重症组患儿的血清miR-455-5p水平降低（P<0.05）。与轻症组相比,重症组的血清miR-455-5p水平降低（P<0.05）。与Control组和NC-agomir组相比,miR-455-5p-agomir组16HBE细胞的miR-455-5p水平、相对细胞活力和EdU阳性率升高（P<0.05）,TUNEL阳性率降低（P<0.05）,上清液中的TNF-α、IL-6和IL-8水平降低（P<0.05）,SOCS3 mRNA和蛋白水平降低（P<0.05）,IFN-α蛋白、STAT1和STAT2磷酸化水平升高（P<0.05）。与Control组和NC-antagomir组相比,miR-455-5p-antagomir组16HBE细胞的miR-455-5p水平、相对细胞活力和EdU阳性率降低（P<0.05）,TUNEL阳性率升高（P<0.05）,上清液中的TNF-α、IL-6和IL-8水平升高（P<0.05）,SOCS3 mRNA和蛋白水平升高（P<0.05）,IFN-α蛋白、STAT1和STAT2磷酸化水平降低（P<0.05）。与Blank组和NC组相比,RSV+NC-agomir组16HBE细胞的miR-455-5p水平、相对细胞活力和EdU阳性率降低（P<0.05）,TUNEL阳性率升高（P<0.05）,上清液中的TNF-α、IL-6和IL-8水平升高（P<0.05）,SOCS3 mRNA和蛋白水平升高（P<0.05）,IFN-α蛋白、STAT1和STAT2磷酸化水平降低（P<0.05）。与RSV+NC-agomir组相比,RSV+miR-455-5p-agomir组的miR-455-5p水平、相对细胞活力和EdU阳性率升高（P<0.05）,TUNEL阳性率降低（P<0.05）,上清液中的TNF-α、IL-6和IL-8水平降低（P<0.05）,SOCS3 mRNA和蛋白水平降低（P<0.05）,IFN-α蛋白、STAT1和STAT2磷酸化水平升高（P<0.05）。结论：miR-455-5p在RSV感染患儿血清中下调,上调miR-455-5p通过抑制SOCS3的转录和表达从而激活RSV感染的16HBE细胞中IFN-α介导的抗病毒反应。     

Wnt通路中蓬松蛋白DNA甲基化对骨质疏松患者骨髓间充质干细胞成骨分化影响

摘要 目的：探讨蓬松蛋白（DVL）DNA甲基化对骨质疏松（OP）患者骨髓间充质干细胞（BMSCs）成骨分化及Wnt通路的影响。方法：分离、培养OP患者的BMSCs,成骨诱导培养BMSCs 0、7、14、21天,观察BMSCs细胞形态变化,检测碱性磷酸酶（ALP）染色及活性,检测茜素红染色及钙结节形成,荧光定量聚合酶链式反应（RT-PCR）及免疫印迹检测远端缺失同源盒5（Dlx5）、核心结合蛋白因子2（Runx2）、成骨细胞特异性转录因子（OSX）、Ⅰ型胶原蛋白（Colla Ⅰ）表达。检测DVL1、Wnt、糖原合成酶激酶3（GSK3）、β连环蛋白（β-catenin）表达及DVL DNA甲基化水平。在成骨诱导培养基中加入甲基转移酶抑制剂5-Aza,将BMSCs分为对照组（Control组）、甲基转移酶抑制剂组（5-Aza组）、甲基转移酶抑制剂+si-NC组（5-Aza+si-NC组）、甲基转移酶抑制剂+si-Wnt组（5-Aza+si-Wnt组）,依次进行ALP活性测定,茜素红染色及钙结节形成测定。RT-PCR检测Dlx5、Runx2、OSX、Colla 1水平,免疫印迹检测Dlx5、Runx2、OSX、Colla Ⅰ、DVL1、Wnt、GSK3、β-catenin的蛋白表达量,并检测DVL DNA甲基化水平。结果：成骨诱导后BMSCs具有强的ALP活性和矿化结节生成能力,且随着培养时间的增长,BMSCs细胞ALP活性和矿化结节生成能力增强,Dlx5、Runx2、OSX、Colla Ⅰ mRNA水平和Wnt、GSK3、β-catenin、DVL1表达升高,DVL DNA甲基化水平降低（P＜0.05）。5-Aza组较Control组ALP染色加深,活性增强,钙结节形成增多（P＜0.05）,Dlx5、Runx2、OSX、Colla Ⅰ mRNA及蛋白表达、Wnt、GSK3、β-catenin、DVL1表达升高,DVL DNA甲基化水平降低（P＜0.05）。5-Aza+si-Wnt组较5-Aza+si-NC组ALP染色变浅,活性降低,钙结节形成减少（P＜0.05）,Dlx5、Runx2、OSX、Colla Ⅰ mRNA及蛋白表达、Wnt、GSK3、β-catenin、DVL1表达降低,DVL DNA甲基化水平升高（P＜0.05）。结论：DVL DNA甲基化可以通过抑制Wnt/β-catenin信号通路抑制OP患者BMSCs成骨分化。     

ULTRASTRUCTURE,DEVELOPMENT AND CYTOPLASMIC ROTATION OF SETA-BEARING CELLS OF COLEOCHAETE SCUTATA (CHLOROPHYCEAE)1

Part of the cytoplasm, which always contains the plastid, of seta-bearing cells of Coleochaete scutata Bréb. rotates clockwise about the base of the seta. Many golgi bodies, vesicles and much endoplasmic reticulum occupy the bridges between the rotating central core of cytoplasm and the stationary peripheral layer of these cells. The setae, which grow from their base, are devoid of organelles other than vesicles and elongate mitochondria. At irregular intervals along the thin seta wall are annular thickenings containing callose. Microtubules which encircle the base of the seta disappear on treatment with colchicine. This drug had no effect on the speed of rotational streaming or the growth rate of existing hairs but did inhibit the development of new setae. Cytochalasin B slowed, but did not stop, streaming after 3 h exposure. However caffeine, but not EDTA, EGTA or the Ca ionophore A23187, reversibly inhibited cyclosis. The mechanism of cytoplasmic rotation is discussed in the light of these drug treatments and the presence of actin in the alga.     

Maladaptive innate immune training of myelopoiesis links inflammatory comorbidities

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Bacillus thuringiensis crystal toxin: Ultrastructural studies of its effect on silkworm midgut cells

Bacillus thuringiensis crystal toxin induced a cytoplasmic response in columnar cells within 1 min after ingestion although external symptoms were not exhibited by larvae until 15 min after ingestion. Microvilli became less consistently uniform in diameter; their organized internal microfilaments were disrupted and disappeared. The cisternae of rough endoplasmic reticulum were enlarged and denuded of ribosomes. By 5 min after ingestion, microvilli of some columnar cells disappeared entirely and gross ultrastructural changes were observed in other regions of the cells. Up to 5 min after ingestion there were few, if any, ultrastructural changes observed within goblet cells. Mitochondria in columnar cells were swollen but did not exhibit the condensed configuration reported by other workers. Both the buffer system used in the fixation medium and its osmolarity influenced the changes in the ultrastructure of midgut cells exposed to B. thuringiensis crystal toxin.