血管内皮细胞生长抑制因子N端部分缺失对生物活性的影响

血管内皮细胞生长抑制因子 (vascularendothelialcellgrowthinhibitor,VEGI)是近年来发现的一类TNF家族新成员 ,具有抑制内皮细胞增殖与新血管生成的作用。为了探讨VEGI蛋白N端对其活性的影响 ,进行了VEGI与TNF家族成员基于结构知识的一级结构序列联配 ,在此基础上构建、表达了N端缺失 43与 5 1个氨基酸的VEGI突变体。N端缺失 43个氨基酸的VEGI(VEGI131)表达量占总菌体蛋白质的 2 5 .2 % ,N端缺失 5 1个氨基酸的VEGI(VE GI12 3)表达量为 2 7.8% ,纯化后纯度分别为 92 .5 % (VEGI131)和 91.6 % (VEGI12 3)。VEGI131可明显抑制人脐静脉内皮细胞 (HUVEC)增殖 ,IC50 约为 35mg/L ,而VEGI12 3在同样条件下几乎无抑制作用 ,野生型VEGI即N端缺失 2 3个氨基酸的VEGI(VEGI151,由作者实验室制备 )IC50 约为 2 7mg/L。鸡胚绒毛尿囊膜血管生成实验证明 ,VEGI151可使主血管数显著减少 ,VEGI131可使主血管变细 ,毛细血管数减少 ,而VEGI12 3 与对照组相比无明显变化。体内外研究显示VEGI活性从高到低依次为 :VEGI151>VEGI131>VEGI12 3。结果提示 :VEGI的N端前 43个氨基酸对活性无明显影响 ,而第 44～ 5 1位氨基酸对其生物活性的发挥具有重要作用。     

新型血管内皮细胞抑制因子VEGI_(151)的基因克隆表达及其活性

血管内皮细胞生长抑制因子 (vascularendothelialcellgrowthinhibitor,VEGI)是近年发现的一类肿瘤坏死因子超家族成员 ,具有抑制内皮细胞增殖的作用。从人脐静脉内皮细胞株 (ECV30 4)克隆到其基因 ,构建N端缺失 2 3个氨基酸的表达载体 ,并通过原核表达系统进行表达 (命名为VEGI151) ,表达量为 2 5 .5 % ,纯化后纯度达92 .5 %。通过生物学效应检测 ,发现VEGI151可明显抑制无血清培养基中内皮细胞的增殖 ,2 4h时VEGI151对内皮细胞的IC50 为 10mg/L ,0 .6 13mg/L时使内皮细胞在 36h内完全凋亡。通过检测体外培养肿瘤细胞 (A5 49、HepG2、Hela等 )的存活率 ,未发现明显的增殖或抑制效应。提示VEGI是一种主要作用于血管内皮细胞 ,在新生血管性疾病及肿瘤的治疗中有潜在的应用前景。     

Laminin‐421 produced by lymphatic endothelial cells induces chemotaxis for human melanoma cells

Melanoma has a high tendency to metastasize to lymph nodes, which is one of the clinicopathological factors to indicate poor prognosis. Recent investigations have shown the importance of lymphangiogenesis in lymph node metastasis in a variety of human tumors including melanoma. However, molecular mechanism of lymphatic metastasis is still poorly defined. We examined influence of interactions between normal lymphatic endothelial cells (LECs) and melanoma cells on cell migration. Medium conditioned with LEC (LEC‐CM) contained chemotactic and chemokinetic activities for human melanoma cell lines. The chemotactic activity was fractionated in more than 100 kDa, and inactivated by heat‐treatment. The chemotactic activity of LEC‐CM was abolished by immunodepletion with anti‐laminin‐1 antibody. And immunoprecipitation and Western blot analyses revealed that LEC‐CM contained laminin‐421. When melanoma C8161 cells were treated with function‐blocking antibodies to integrin α3 or α6, their chemotactic responses to LEC‐CM were markedly reduced. Furthermore, the knock‐down of tetraspanin CD151 weakened the chemotactic responses of C8161 and MeWo cells to LEC‐CM. These data suggest that laminin‐421 secreted by LEC possibly facilitates lymphatic metastasis through the induction of chemotaxis of melanoma cells.     

Expression of hepatocyte growth factor activator inhibitor type 1 in endothelial cells

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is an integral membrane Kunitz-type serine proteinase inhibitor initially identified as a potent inhibitor of hepatocyte growth factor activator (HGFA). HGFA is a serum proteinase that is critically involved in the activation of hepatocyte growth factor/scatter factor (HGF/SF) in injured tissue. Previous studies have shown that HAI-1 is expressed on the basolateral surface of various epithelial cells. In this study, we analyzed the expression of HAI-1 in human endothelial cells. Immunohistochemically, HAI-1 protein was observed in the endothelial cells of capillaries, venules and lymph vessels. On the other hand, arterial endothelial cells were poorly stained for HAI-1. Mesothelial cells on the serous surface were also positively immunostained. The endothelial expression of HAI-1 was also examined in cultured human endothelial cells of various origins, such as umbilical vein, microvessels and aorta. Notably, in accordance with the results of immunohistochemistry, HAI-1 mRNA and protein levels were high in the endothelial cells derived from umbilical vein and were hardly detectable in those derived from aorta. A low but distinct level of HAI-1 expression was also observed in endothelial cells from microvessels. As these HAI-1-positive endothelial cells also expressed MET tyrosine kinase, the specific receptor of HGF/SF, it is conceivable that HAI-1 might have an important regulatory role in the HGF/SF-MET signaling axis of endothelial cells, which could be involved in the process of angiogenesis.     

Comparisons of graph-structure clustering methods for gene expression data

Although many numerical clustering algorithms have been applied to gene expression dataanalysis,the essential step is still biological interpretation by manual inspection.The correlation betweengenetic co-regulation and affiliation to a common biological process is what biologists expect.Here,weintroduce some clustering algorithms that are based on graph structure constituted by biological knowledge.After applying a widely used dataset,we compared the result clusters of two of these algorithms in terms ofthe homogeneity of clusters and coherence of annotation and matching ratio.The results show that theclusters of knowledge-guided analysis are the kernel parts of the clusters of Gene Ontology (GO)-Clustersoftware,which contains the genes that are most expression correlative and most consistent with biologicalfunctions.Moreover,knowledge-guided analysis seems much more applicable than GO-Cluster in a largerdataset.     

内皮祖细胞及其在组织工程中的应用

目前,组织工程化血管的构建和工程化组织器官的血管化因内皮种子细胞的扩增能力不足和生物活性不强而受到限制。内皮祖细胞(EPC)是内皮细胞的前体细胞。出生后,EPC主要存在于骨髓,可向外周血液缓慢释放,参与机体缺血组织的血管重建和损伤血管的重新内皮化。现对EPC的来源、分布、表型特征、动员、分化、归巢、分离、培养与鉴定等生物学特性和EPC在组织工程中的应用进行了全面的综述,并指出目前存在的问题和研究方向。     

Alpha-smooth muscle actin expression identifies subpopulations of mouse lymph node non-hematopoietic cells

Significant attention has been given to the role played by non-hematopoietic cells in the immune organs, including the lymph nodes, in hopes of understanding the development, maintenance, and regulation of the immune system. However, the molecular and cellular characterization of non-hematopoietic cells is still in its infancy. Here we show that non-hematopoietic cells in mouse lymph nodes can be fractionated into previously unidentified subpopulations according to the transgenic reporter expression of alpha-smooth muscle actin (αSMA). αSMA⁺ non-hematopoietic cells were predominantly detected in gp38⁺CD31⁻ and gp38⁻CD31⁻ cells. Molecular expression profiles suggest similarities between αSMA⁺gp38⁺CD31⁻ and αSMA⁻gp38⁺CD31⁻ subpopulations and dissimilarities between αSMA⁺gp38⁻CD31⁻ and αSMA⁻gp38⁻CD31⁻ subpopulations. The results indicate that αSMA is a useful marker for further understanding the molecular and cellular characteristics of non-hematopoietic cells in the lymph nodes.     

植物β-半乳糖苷酶

β-半乳糖苷酶是一个与细胞壁降解相关的酶,广泛分布于植物组织中,参与一系列的生理生化过程,如植物的花粉发育、果实成熟及生长过程中多糖的裂解。目前,已从多种植物中分离到β-半乳糖苷酶基因。β-半乳糖苷酶基因属于多基因家族,随着研究的深入,其不同水平的转录本在不同植物的不同组织中被发现。但目前β-半乳糖苷酶在植物发育中确切的作用机制尚不明确。现介绍目前这一领域内细胞与分子生物学方面的研究进展,并结合所在课题组的研究结果进行相关探讨,为进一步研究β-半乳糖苷酶在植物中的作用机制提供新的线索。     

角质细胞生长因子研究进展

角质细胞生长因子(KGF)从属于成纤维细胞生长因子家族。KGF基因表达受多种细胞因子调控。KGF与受体KGFR特异性的结合发挥其多种生物学功能：参与组织、器官的发育;参与皮肤、胃、肠、肾、膀胱、肺等上皮的损伤修复;减少放、化疗所带来的副作用,具有损伤防护功能;KGF与肿瘤密不可分。     

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.