胶原海绵复合新生大鼠原代心肌细胞构建工程化心肌组织

目的:探索以胶原海绵为支架、新生大鼠原代心肌细胞为种子细胞,于体外构建工程化心肌组织的方法。方法:实验于2005-12/2006-11在解放军第四军医大学西京医院心内科实验室完成。Ⅰ型胶原海绵剪切成方形片状(2.0cm×1.4cm×0.2cm),经60Co照射消毒,于DMEM培养液中水化1h左右。另取1d龄SD大鼠心脏,剪成小碎块,然后用2.5g/L胰蛋白酶于37℃中消化,吸取上清至含胎牛血清的DMEM中,重复消化四五次,用差速贴壁法除去大部分成纤维细胞,将细胞沉淀用DMEM培养液以2×109L-1的密度悬浮备用。将上述的心肌细胞悬液1mL缓慢滴注于玻璃模型中的胶原海绵上,然后置于细胞培养中培养。肉眼及显微镜主要观察工程化心肌组织在培养期间的自发收缩情况,包括收缩的部位、强度、频率、一致性以及收缩随时间变化的情况。苏木精-伊红染色观察工程化心肌组织内胶原纤维的变化,细胞形态,胞核的形状及细胞之间的连接。免疫组织化学染色和透射电镜观察工程化心肌组织片的形态和功能。结果:①细胞接种于胶原海绵上1d后,细胞/胶原复合物的凝胶化过程基本完毕,体积保持恒定,维持至培养结束,第3天细胞/胶原复合物局部出现点片状自发收缩,第5天整个细胞/胶原复合物出现同步化自发收缩,收缩频率61～199次/min。2周后37.5%的工程化心肌组织的自发收缩活动减弱,但75%的工程化心肌组织的自发收缩活动持续至培养结束。②苏木精-伊红染色、免疫组织化学染色和透射电子显微镜显示,工程化心肌组织内细胞间连接广泛存在,细胞多呈纵向分布,胞核呈长圆形,胞浆内α-肌节肌动蛋白阳性,胞内肌原纤维排列整齐,可见到心肌特异性的肌小节结构和Z线,多数细胞具有分化的心肌细胞表型。结论:用新生大鼠原代心肌细胞为种子细胞、以Ⅰ型胶原海绵为支架材料,构建出的工程化心肌组织,于体外可长时间持续自发收缩,该细胞/胶原复合物的形态结构与生理功能均类似于成熟大鼠心肌组织。     

Identity of Leu-19 (CD56) leukocyte differentiation antigen and neural cell adhesion molecule

Neural cell adhesion molecule (N-CAM) is a membrane glycoprotein expressed on neural and muscle tissues that is involved in homotypic adhesive interactions. We have demonstrated that N-CAM also is expressed on hematopoietic cells, and is recognized by the anti-Leu-19 mAb. Leu-19 is preferentially expressed on NK cells and T lymphocytes that mediate MHC-unrestricted cytotoxicity, but is also present on some myeloid leukemia cell lines. On NK cells, T cells, the KG1a.5 hematopoietic cell line, and a neuroblastoma cell line, Leu-19 is a approximately 140-kD polypeptide with N-linked carbohydrates and abundant sialic acid residues. Sequential immunoprecipitation and peptide mapping demonstrated that the Leu-19 and N-CAM molecules expressed on leukocyte and neuroblastoma cell lines are similar structures. These findings suggest that the Leu-19 antigen on leukocytes may be involved in cell adhesion, analogous to the function on N-CAM on neural cells.     

Interleukin 2 activation of natural killer cells rapidly induces the expression and phosphorylation of the Leu-23 activation antigen

IL-2 potentiates both growth and cytotoxic function of T lymphocytes and NK cells. Resting peripheral blood NK cells can respond directly to rIL-2, without requirement for accessory cells or cofactors, and enhanced cytotoxicity can be measured within a few hours after exposure to this lymphokine. In this study, we describe an activation antigen, Leu-23, that is rapidly induced and phosphorylated after IL-2 stimulation of NK cells and a subset of low buoyant density T lymphocytes. Previously, it has been uncertain whether all NK cells or only a subset are responsive to IL-2. Since within 18 h after exposure to IL-2, essentially all NK cells express Leu-23, these findings indicate that all peripheral blood NK cells are responsive to stimulation by IL-2. The Leu-23 antigen is a disulfide-bonded homodimer, composed of 24-kD protein subunits with two N-linked oligosaccharides. Appearance of this glycoprotein on NK cells is IL-2 dependent and closely parallels IL-2-induced cytotoxicity against NK-resistant solid tumor cell targets.     

Hypocalcemia-like electrocardiographic changes after administration of intravenous fosphenytoin

Fosphenytoin is a prodrug that is metabolized by phosphatases to yield the antiepileptic drug phenytoin plus inorganic phosphate. Thus, fosphenytoin can theoretically alter the electrocardiogram by 2 mechanisms: the direct effects of phenytoin on cardiac conduction and on phosphate binding of calcium, which could indirectly alter cardiac conduction as a result of hypocalcemia. We report the case of a 23-year-old man, weight 73 kg, with a known but untreated seizure disorder who was given prophylactic fosphenytoin, 1500-mg phenytoin equivalents over 85 minutes by intravenous infusion. The patient was normocalcemic before drug infusion. Fosphenytoin produced electrocardiographic changes (prolongation of the ST segment and the QT interval and merging of the T and P waves) consistent with hypocalcemia, and these changes were associated with new-onset reductions in both total and ionized serum calcium concentrations. Plasma phenytoin concentrations were within the therapeutic range during the electrocardiographic changes, and the patient's blood pressure was stable. We interpret these findings as fosphenytoin-related electrocardiographic changes likely attributable to inorganic phosphate-induced hypocalcemia.     

High expression levels of IKK alpha and IKK beta are necessary for the malignant properties of liver cancer

IKK-NF-kappa B signaling is regarded as an important factor in hepatocarcinogenesis and a potential target for liver cancer therapy.Therefore,in this study,we analyzed the expression of mRNAs encoding components and targets of NF-kappa B signaling including IKK alpha,IKK beta,RANK,RANKL,OPG,CyclinD3,mammary serine protease inhibitor(Maspin),CyclinD1,c-FLIP,Bcl-x1,Stat3,Cip1 and Cip2 by real-time PCR in 40 patients with liver cancer.After statistical analysis,7 indices including IKK alpha,IKK beta,RANK,Maspi...     

Evaluation in vitro of synthetic curcumins as agents promoting monocytic gene expression related to β-amyloid clearance

Accumulation of amyloid-beta (Aβ) is one of the hallmarks of Alzheimer's disease (AD), and efficient clearance of Aβ by cells of the innate immune system may be an important mechanism for controlling or preventing disease onset. It was reported that peripheral blood mononuclear cells (PBMCs) of most AD patients are defective in the phagocytosis of soluble Aβ. Natural curcumins were shown to restore Aβ phagocytosis by AD PBMCs and to up-regulate the expression of key genes including MGAT3 and those encoding Toll-like receptors (TLRs). Bisdemethoxycurcumin (BDC), a minor component of natural curcumin, was shown to have the greatest potency for stimulating AD PBMCs. Because natural curcumins have inherent limitations with regard to physicochemical properties, synthetic curcumin analogues were developed that showed improved solubility, stability, and bioavailability. An in vitro system using human monocytic cell lines (U-937, THP-1) was used to evaluate analogues for the potency of innate immune cell stimulation. These cell lines showed responses to curcuminoids and to 1α,25-dihydroxyvitamin D3 (VD3) resembling those seen in human PBMCs. From more than 45 curcuminoids analyzed, the most potent compounds possessing enhanced pharmaceutical properties were identified. The most promising candidates included prodrug versions containing water solubility-enhancing amino acids and stability-increasing modifications near the central diketone. In vivo studies showed compound (5) substantially increased bioavailability by combining several promising structural modifications. Studies examining ex vivo phagocytosis of Aβ and bead particles in mouse microglia showed that BDC and several water-soluble analogues were quite effective compared to curcumin or an unnatural analogue. In vitro studies using monocytic cell lines reported herein complement those using human PBMCs and represent a routinely accessible and uniform cellular resource allowing direct comparisons between compounds.     

Structural elucidation of the m157 mouse cytomegalovirus ligand for Ly49 natural killer cell receptors

Natural killer (NK) cells express activating and inhibitory receptors that, in concert, survey cells for proper expression of cell surface major histocompatibility complex (MHC) class I molecules. The mouse cytomegalovirus encodes an MHC-like protein, m157, which is the only known viral antigen to date capable of engaging both activating (Ly49H) and inhibitory (Ly49I) NK cell receptors. We have determined the 3D structure of m157 and studied its biochemical and cellular interactions with the Ly49H and Ly49I receptors. m157 has a characteristic MHC-fold, yet possesses several unique structural features not found in other MHC class I-like molecules. m157 does not bind peptides or other small ligands, nor does it associate with beta(2)-microglobulin. Instead, m157 engages in extensive intra- and intermolecular interactions within and between its domains to generate a compact minimal MHC-like molecule. m157's binding affinity for Ly49I (K(d) approximately 0.2 microM) is significantly higher than that of classical inhibitory Ly49-MHC interactions. Analysis of viral escape mutations on m157 that render it resistant to NK killing reveals that it is likely to be recognized by Ly49H in a binding mode that differs from Ly49/MHC-I. In addition, Ly49H+ NK cells can efficiently lyse RMA cells expressing m157, despite the presence of native MHC class I. Collectively, our results show that m157 represents a structurally divergent form of MHC class I-like proteins that directly engage Ly49 receptors with appreciable affinity in a noncanonical fashion.     

DAP10- and DAP12-associated receptors in innate immunity

Summary:  The DAP10 and DAP12 signaling subunits are highly conserved in evolution and associate with a large family of receptors in hematopoietic cells, including dendritic cells, plasmacytoid dendritic cells, neutrophils, basophils, eosinophils, mast cells, monocytes, macrophages, natural killer cells, and some B and T cells. Some receptors are able to associate with either DAP10 or DAP12, which contribute unique intracellular signaling functions. Studies of humans and mice deficient in these signaling subunits have provided surprising insights into the physiological functions of DAP10 and DAP12, demonstrating that they can either activate or inhibit immune responses. DAP10- and DAP12-associated receptors have been shown to recognize both host-encoded ligands and ligands encoded by microbial pathogens, indicating that they play an important role in innate immune responses.     

Natural killer cells in NOD.NK1.1 mice acquire cytolytic function during viral infection and provide protection against cytomegalovirus

Resting natural killer (NK) cells in nonobese diabetic (NOD) mice have impaired immune functions compared with NK cells from other mouse strains. Here we investigated how NOD NK cells respond after mouse cytomegalovirus (MCMV) infection, using NOD mice congenic for the protective NK gene complex from C57BL/6 mice. Compared with C57BL/6 mice congenic for the H2 gene complex from NOD mice (B6.g7), NOD.NK1.1 mice fail to control early infection with MCMV. After MCMV infection, however, NOD.NK1.1 NK cells demonstrate increased cytolytic function, associated with higher expression of granzyme B, and undergo robust expansion. One week after infection, NOD.NK1.1 NK cells control MCMV replication as effectively as B6.g7 NK cells, even in the absence of T cells and B cells. Thus, the impaired cytotoxic function of NK cells in NOD mice is alleviated by viral infection, which enables NOD NK cells to efficiently control MCMV infection.