猪骨髓基质干细胞低温生物学渗透特性的研究

以猪为材料利用细胞计数仪法研究了骨髓基质干细胞的渗透特性,包括细胞的等渗体积、低渗或高渗溶液中细胞的平衡体积及细胞的不可渗体积.结果表明,在等渗条件下,骨髓基质干细胞的平均体积为5248.4μm~3,相当直径d为21.6μm;细胞体积随溶液渗透压的变化规律符合Boyle van’t Hoff关系式,据此得到猪骨髓基质干细胞的不可渗体积V_b=0.36Vi,为1902.5μm~3.     

体外诱导成年大鼠骨髓间充质干细胞分化为 5-羟色胺敏感性神经元

体外诱导成年大鼠骨髓间充质干细胞分化为具有神经元表型和部分功能的细胞。在对Woodbury化学诱导法作改良的基础上,加用全反式视黄酸对骨髓间充质干细胞作预诱导。诱导3h后,细胞开始表现神经元的形态特征,细胞折光性增强,形成收缩的双极或多极胞体和细长突起。细胞可以维持神经元样存活72h以上。诱导5h后,对免疫染色的细胞用DAPI进行复染,(92.4±6.9)%的细胞表达神经元特异性烯醇化酶。诱导24h后,(93.9±5.2)%的细胞表达成熟神经元的标志物神经丝M H。在给予5-羟色胺刺激时可以产生与神经元相似的胞内钙离子峰,且免疫组化证实5-羟色胺1A受体在干细胞上表达微弱,但在分化后的神经元中表达较强。实验不仅从形态、细胞标志物而且从功能上证实诱导后的细胞为5-羟色胺敏感性神经元。     

Dissociation of Intracellular Ca Release and Ca Entry Response to 5-Hydroxytryptamine in Cultured Canine Tracheal Smooth Muscle Cells

The relationship between the agonist-sensitive Ca²⁺ pool and those discharged by the Ca²⁺-ATPase inhibitor thapsigargin (TG) were investigated in canine tracheal smooth muscle cells (TSMCs). In fura-2-loaded TSMCs, 5-hydroxytryptamine (5-HT) stimulated a rapid increase in intracellular Ca²⁺ ([Ca²⁺]_i), followed by a sustained plateau phase that was dependent on extracellular Ca²⁺. In such cells, TG produced a concentration-dependent increase in [Ca²⁺]_i, which remained elevated over basal level for several minutes and was substantially attenuated in the absence of extracellular Ca²⁺. Application of 5-HT after TG demonstrated that the TG-sensitive compartment partly overlapped the 5-HT-sensitive stores. Pre-treatment of TSMCs with TG significantly inhibited the increase in [Ca²⁺]_i induced by 5-HT in a time-dependent manner. Similar results were obtained with two other Ca²⁺-ATPase inhibitors, cyclopiazonic acid and 2,5-di-t-butylhydroquinone. Although these inhibitors had no effect on phosphoinositide hydrolysis, Ca²⁺-influx was stimulated by these agents. These results suggest that depletion of the agonist-sensitive Ca²⁺ stores is sufficient for activation of Ca²⁺ influx. Some characteristics of the Ca²⁺-influx activated by depletion of internal Ca²⁺ stores were compared with those of the agonist-activated pathway. 5-HT-stimulated Ca²⁺ influx was inhibited by La³⁺, membrane depolarisation, and the novel Ca²⁺-influx blocker 1-{β-[3-(4-methoxyphenyl) propoxy]-4-methoxyphenethyl}-1H-imidazole hydrochloride (SKF96365). Likewise, activation of Ca²⁺ influx by TG also was blocked by La³⁺, membrane depolarisation, and SKF96365. These results suggest that (1) in the absence of PI hydrolysis, depletion of the agonist-sensitive internal Ca²⁺ stores in TSMCs is sufficient for activation of Ca²⁺ influx, and (2) the agonist-activated Ca²⁺ influx pathway and the influx pathway activated by depletion of the inositol 1,4,5-trisphosphate-sensitive Ca²⁺ pool are indistinguishable.     

Mesenchymal stem cells inhibit lymphocyte proliferation by mitogens and alloantigens by different mechanisms

Human mesenchymal stem cells (MSCs) have immuno-modulatory properties. They inhibit T-cell proliferation to mitogens and alloantigens in vitro and prolong skin graft survival in vivo. We found that MSCs inhibited the proliferation of peripheral blood lymphocytes (PBLs) to phorbol myristate acetate (PMA), suggesting that MSCs exert an inhibitory effect downstream of the receptor level. We analyzed cytokine profiles of PBLs co-cultured with MSCs. MSCs increased interleukin (IL)-2 and soluble IL-2 receptor in mixed lymphocyte cultures (MLCs), while IL-2 and IL-2R decreased in phytohemagglutinin (PHA)-stimulated PBL cultures. MSCs inhibited IL-2 induced proliferation, without absorbing IL-2. IL-10 levels increased in MLCs co-cultured with 10% MSCs, while the levels were not affected in PHA cultures. In MLCs inhibited by MSCs, antibodies against IL-10 further suppressed proliferation but had no effect in PHA cultures. Addition of indomethacin, an inhibitor of prostaglandin-synthesis, restored part of the inhibition by MSCs in PHA cultures. However, indomethacin did not affect MSC-induced inhibition in MLCs. To conclude, our data indicate that MSC-induced suppression is a complex mechanism affecting IL-2 and IL-10 signaling and may function differently, depending on T-cell stimuli. Prostaglandins are important in the inhibition by MSCs when the T cells were activated by PHA, but not alloantigens.     

C-kit mutations and PKC crosstalks: PKC translocates to nucleous only in cells HMC⁵⁶⁰,⁸¹⁶

The human mast cell lines HMC‐1⁵⁶⁰ and HMC‐1^560,816 were used to study histamine release, Ca²⁺ signaling and protein kinase C (PKC) localization and expression, with phorbol 12‐myristate 13‐acetate (PMA). Both sublines carry activating mutations in the proto‐oncogene of c‐kit that cause autophosphorylation and permanent c‐kit tyrosine kinase activation. Both have the Gly‐560 → Val mutation but only the second carries the Asp‐816 → Val mutation. In this study, it was observed that the stimulation of PKC has different effects in HMC‐1⁵⁶⁰ and HMC‐1^560,816 and this would be related to the difference in activating mutations in both mast cell lines. PKC activation increases ionomycin‐induced histamine release in HMC‐1⁵⁶⁰. This article demonstrates an opposite histamine response in HMC‐1^560,816 cells, even though classical PKCs are the family of isozymes responsible for this effect in both cellular lines. Furthermore, it can be observed that upon cell stimulation with PMA, primarily cytosolic PKC translocates to the nucleous in HMC‐1^560,816 cells, but not in HMC‐1⁵⁶⁰ cell line. J. Cell. Biochem. 112: 2637–2651, 2011. © 2011 Wiley‐Liss, Inc.     

体外证据不支持5-氮胞苷诱导骨髓基质细胞向心肌细胞分化

目的探讨胞嘧啶类化学物质5-氮胞苷是否可诱导体外培养的骨髓基质细胞 (marrow stromal cells, MSCs)向心肌细胞分化.方法用不同浓度(3, 5, 10 μmol/L)的5-氮胞苷以各种方法(一次处理与反复处理)诱导原代及传代MSCs.采用免疫荧光及Western Blot技术检测心肌特异性肌球蛋白重链 (myosin heavy chain α/β, MHCα/β)及肌钙蛋白I(troponin I, Tn I)的表达;并用心室肌特异性肌球蛋白轻链(ventricular myosin light chain 2, MLC2v)启动子(250 bp)控制的增强型蓝绿色荧光蛋白(enhanced cyan fluorescent protein, ECFP)报告基因表达技术检测被诱导MSCs是否发生MLC2v的转录启动.上述检测技术的特异性和可靠性用培养的心肌细胞进行验证.结果在本试验最长观察时间内(30 d),各种浓度及各种方法5-氮胞苷诱导的MSCs培养物中未见细胞自发搏动、肌管形成、心肌特异性MHC和Tn I表达;亦无MLC2v转录启动阳性细胞出现.结论从转录启动到翻译后水平的证据均不支持体外5-氮胞苷处理可诱导MSCs表达心肌特异性蛋白.     

Strontium supports capacitation and the acrosome reaction in mouse sperm and rapidly activates mouse eggs

Extracellular Ca²⁺ is required for capacitation and fertilization in the mouse, but very little is known about the ability of other divalent cations to substitute for Ca²⁺. In this study, Sr²⁺, Ba²⁺, and Mg²⁺ were evaluated for their ability to support capacitation, the acrosome reaction, hyperactivated motility, and fertilization. Ba²⁺ proved to be ineffective, but Mg²⁺-containing medium was able to support capacitation to a greater extent than unsupplemented Ca²⁺-deficient media; despite this, Ca²⁺ was required for fertilization. In contrast, Sr²⁺ proved capable of substituting for Ca²⁺ in all events. Furthermore, Sr²⁺-induced responses were indistinguishable from the corresponding Ca²⁺-induced ones: Sperm capacitated at the same rate and underwent the acrosome reaction to the same extent. However, demonstration of sperm:egg fusion in Sr²⁺ required the use of zona-free eggs. This was due not to the inability of the sperm to penetrate the zona but to the very rapid activation and cortical granule release by eggs in response to Sr²⁺. When zona-intact eggs were used, the block to polyspermy had been mounted by the time sperm had penetrated the zona. A 15 min exposure to Sr²⁺ was sufficient to block sperm fusion, but a longer exposure was required to ensure the resumption of meiosis in eggs; such a response was surprising in that the eggs were freshly ovulated and not susceptible to activation by many different treatments. Thus Sr²⁺ can profoundly affect both gametes in the mouse: It substitutes completely for Ca²⁺ in sperm responses and rapidly activates eggs, possibly by displacing Ca²⁺ from intracellular stores into the cytoplasm, where the Ca²⁺ can then trigger the various events of activation.     

钙与植物乙烯反应的关系研究

研究了Ca2+ 对番茄(Lycopersicon esculentum Mill cv. Lichun)黄化幼苗乙烯反应的影响.通过测定不同Ca2+ 浓度条件下番茄黄化幼苗的\"三重反应\"、内源乙烯释放量、乙烯受体基因NEVER-RIPE(NR)表达量及胞内CaM含量的变化,结果发现,随着培养基中Ca2+ 浓度从0 mmol/L增加到3.8 mmol/L,番茄黄化幼苗的\"三重反应\"表型明显增强,内源乙烯释放量、NR基因的表达量及胞内CaM的含量都有不同程度的增加;当Ca2+ 浓度由3.8 mmol/L进一步增加到10 mmol/L时,番茄黄化幼苗\"三重反应\"表型受到抑制,内源乙烯释放量、 NR基因的表达量及胞内CaM的含量都有所下降.因此,Ca2+ 对番茄黄化幼苗\"三重反应\"的影响与Ca2+ 调节内源乙烯合成和乙烯受体基因的表达有关,而且Ca2+ 可能是通过CaM含量的变化来调节乙烯作用的.