大蹼铃蟾皮肤膜联蛋白Ⅱ相关蛋白的分离纯化及活性

通过阴离子交换、凝胶过滤和阳离子交换层析, 从大蹼铃蟾皮肤中纯化到一个表观分子量为33 kDa 的单链蛋白。N - 末端序列比较分析显示, 该蛋白与来自非洲爪蟾、红色原鸡和人膜联蛋白Ⅱ的N - 末端序列 相同的氨基酸分别占70 %、64 %和56 %。该蛋白具有以钙依赖的方式抑制专一性血小板膜糖蛋白Ⅵ受体激动剂 ———Stejnulxin 诱导洗涤人血小板聚集的生物学功能, 最大抑制率达48 %。结合其N - 末端序列BLAST 搜索结 果及其活性的钙依赖性, 推测该蛋白是与膜联蛋白Ⅱ相关的一类蛋白质。

Effects of subretinal implant materials on the viability, apoptosis and barrier function of cultured RPE cells

Background: Subretinal microphotodiode array (MPDA) is a type of visual prosthesis used for the implantation in the subretinal space of patients with progressive photoreceptor cell loss. The present study aimed to evaluate the effect of materials for MPDA on the viability, apoptosis and barrier function of cultured pig retinal pigment epithelium (RPE) cells.Methods: Primary culture of pig RPE cells was performed and 24 pig eyes were used to start RPE culture. The third passage of the cultures was plated on different materials for MPDA and MPDAs. The tetrazolium dye-reduction assay (MTT) was used to determine RPE cell viability. Flow cytometry was measured to indicate the apoptosis rates of RPE cells on different materials. RPE cells were also cultured on microporous filters, and the transepithelial resistance and permeability of the experimental molecule were measured to determine the barrier function.Results: The data from all the methods indicated no significant difference between the materials groups and the control group, and the materials tested showed good biocompatibility.Conclusions: The materials for MPDA used in the present study had no direct toxicity to the RPE cells and did not release harmful soluble factors that affected the barrier function of RPE in vitro.

眼镜王蛇泛素融合蛋白和核糖体蛋白L30的分子克隆与分析及大蹼铃蟾膜联蛋白A2样蛋白的纯化与活性研究

本论文分为两个部分。第一部分利用分子生物学手段对广西产眼镜王蛇（OPhiophagushannah）泛素融合蛋白基因和核糖体蛋白L30基因进行了克隆及分析；第二部分利用生物化学手段对大蹼铃蟾（Bombinamaxima）膜联蛋白A2样蛋白进行了初步研究。首先，从广西产眼镜王蛇毒腺中抽提总RNA，经mRNA纯化后构建眼镜王蛇毒腺。DNA文库，得到大概1.8*105个独立的克隆。从所构建的cDNA文库中，我们随机筛选200个克隆测序，得到两个在进化上高度保守的基因：泛素融合蛋白基因（GenBahk登录号为AF297036）和核糖体蛋白L30基因（GenBank登录号是AF297033）。前者cDNA的开放阅读框为387bp，后者为348bp。前者编码128个氨基酸残基组成的泛素融合蛋白前体；后者编码1巧个氨基酸残基组成的核糖体蛋白L30前体。由cDNA序列推导出的氨基酸序列分析表明，泛素融合蛋白前体包括N-末端的泛素结构域（76个氨基酸残基）和C-末端的核糖体蛋白L40结构域（52个氨基酸残基）。该蛋白为一高碱性蛋白，C末端含有一个"锌指"结构域。与16个物种比较的结果表明，眼镜王蛇与脊椎动物的泛素融合蛋白氨基酸序列相似度较高，具有高度的保守性。我们克隆到的眼镜王蛇泛素融合蛋白基因和核糖体蛋白L30基因为泛素家族和核糖体家族提供新的序列信息，有助于深入研究泛素蛋白和核糖体蛋白的结构、功能以及与其他物种的类似分子之间的相互关系。其次，通过阴离子交换，凝胶过滤和阳离子交换层析我们从大蹼铃蟾皮肤匀浆物中纯化了一个表观分子量为33000Da的单链蛋白，N-末端序列比较分析显示它与脊椎动物膜联蛋白AZ亚群有较高的同源性，与来自非洲爪蟾、红色原鸡和人膜联蛋白AZ序列的annexin核心的第一个重复区结构域序列一致性分别为78.9％、89％和68％，因此命名为大蹼铃蟾膜联蛋白AZ样蛋白（BAllLP）。BAllLP具有钙依赖性的抑制专一性血小板膜受体GPVI激动剂一Stejnulxin诱导人血小板聚集的生物学功能。

辐照诱导的人正常肝细胞系HL-7702细胞延迟效应

利用X射线辐照人正常肝细胞系HL-7702细胞,运用胞质分离阻滞微核法实验检测细胞微核率,Annexin V-FITC细胞凋亡检测试剂盒检测细胞凋亡率,细胞微核率和凋亡率随着辐照剂量的增加而显著增加。X射线照射后细胞传代培养,第7代时不同剂量辐照后子代细胞微核率和凋亡率同未辐照细胞相比已无明显区别。对不同剂量辐照后传代7代的细胞再次照射2.5Gy的相同剂量,发现它们细胞微核率和凋亡率存在明显差异,即初次受辐照剂量高的细胞,再次以相同剂量辐照后的微核率和凋亡率也高。这些结果表明,X射线辐照导致了HL-7702细胞基因组不稳定性这一辐射延迟效应,再次辐照使得辐射的延迟效应得以明显的表现。

电离辐射对Jurkat T细胞凋亡与坏死的影响

目的:研究电离辐射诱导Jurkat T细胞凋亡与细胞坏死的变化规律,探讨电离辐射作用后细胞的死亡模式与机理。方法:采用Annexin V-EGFP和PI双染、流式细胞术(FCM)检测不同剂量(0.075、0.500、1.000、2.000、4.000和6.000Gy)X射线照射后Jurkat T细胞凋亡与细胞坏死的时间-效应关系和剂量-效应关系。结果:时间-效应关系,2.000Gy X射线照射后,与假照组比较,Jurkat细胞凋亡百分率在照射后18h显著增加,至24h始终维持在较高水平(P<0.001);细胞坏死百分率于照射后4h显著增加,12h达峰值,至48h始终维持在较高水平(P<0.001)。剂量-效应关系,0.075Gy X射线照射后18h,Jurkat T细胞凋亡百分率和细胞坏死百分率与假照组相比较未见明显变化(P>0.05)。2.000~6.000Gy较大剂量X射线照射后18h,细胞凋亡百分率和细胞坏死百分率与假照组比较均呈剂量依赖性增加(P<0.001)。结论:2.000Gy以上剂量X射线可以诱导Jurkat T细胞发生凋亡和坏死,细胞坏死比细胞凋亡出现时间更早,持续时间更长。

Endothelial cells release phenotypically and quantitatively distinct microparticles in activation and apoptosis

Background: Endothelial cells (EC) shed endothelial microparticles (EMP) in activation and apoptosis. Objectives: We compared the antigenic expression of EMP species released during activation as compared to apoptosis, in three cell lines. Methods: EC from renal and brain microvascular (MiVEC) and coronary macrovascular (MaVEC) origin were incubated with TNF-alpha to induce activation, or deprived of growth factors to induce apoptosis. Antigens expressed on EMP and EC were assayed flow cytometrically and included constitutive markers (CD31, CD51/61, CD105), inducible markers (CD54, CD62E and CD106), and annexin V binding. Results: It was found that in apoptosis, constitutive markers in EMP were markedly increased (CD31>CD105), with a concomitant decrease in expression in EC. Annexin V EC surface binding and annexin V+ EMP were more sharply increased in apoptosis than in activation. In contrast, in activation, inducible markers in EMP were markedly increased in both EMP and EC (CD62E>CD54>CD 106). Coronary MaVEC released significantly less EMP than MiVEC. Conclusion: EC release qualitatively and quantitatively distinct EMP during activation compared to apoptosis. Analysis of EMP phenotypic signatures may provide clinically useful information on the status of the endothelium. (C) 2003 Elsevier Science Ltd. All rights reserved.