树突状细胞研究进展

树突状细胞 ( Dendritic cells,DC)是体内功能最强的一类抗原提呈细胞 ,参与机体对某一抗原的初次免疫应答过程 ,还可影响偏向性免疫应答。其功能和表型具有多样性。本文就 DC近年来的研究进展做一综述     

Effects of probucol on the proliferation of rat vascular smooth muscle cells stimulated by basic fibroblast growth factor and hydrogen peroxide

AIM: To investigate the effect of probucol on proliferation of rat vascular smooth muscle cells(VSMC) stimulated by basic fibroblast growth factor (bFGF) and/or hydrogen peroxide(H₂O₂). METHODS: Effects of probucol on VSMC proliferation and DNA synthesis stimulated by bFGF and/or H₂O₂ were observed by means of MTT test, cell number count and [3H]-TdR incorporation. RESULTS: ①Probucol significantly inhibited proliferation and DNA synthesis in VSMC stimulated by bFGF and/or H₂O₂, with dosage-dependent manner. Cell number, A value and [3H]-TdR incorporation in group probucol+bFGF and group probucol+H₂O₂ were reduced by 40.0%, 39.1%, 45.5% and 46.9%, 45.0%, 39.5%, respectively, compared with group bFGF and group H₂O₂ (P＜0.05, P＜0.01, respectively). ②Pretreatment of VSMC with probucol for 24 h prior to bFGF and/or H₂O₂ stimulation exhibited significant inhibiton of VSMC proliferation and DNA synthesis, but after prestimulation by bFGF and/or H₂O₂ for 24 h, probucol had no influence on VSMC proliferation and DNA synthesis (P＞0.05). CONCLUSION: Probucol dramatically inhibited proliferation and DNA synthesis in VSMC stimulated by bFGF and/or H₂O₂, but had no inhibitory effect on the cell proliferation prestimulated by bFGF and /or H₂O₂.     

Changes in human atrial myocyte dimension among different ages

AIM:To observe the changes in right atrial myocyte dimensions with myocardial development. METHODS: Cell length, width, length/width and cross-sectional area were measured in right atrial myocytes isolated from 12 weaning , 10 young , 13 adult human hearts. RESULTS: Cell length, width, length/width and cross-sectional area, at weanling group were (70.2±1.3) μm, (8.0±0.2) μm, (9.0±0.2) and (50.9±2.6) μm², respectively, at young group were (93.5±1.6) μm, (11.7±0.3)μm, 8.1±0.2, (109.7±5.8) μm², and (100.9±2.2) μm, (12.1±0.3) μm, 8.5±0.2, (119.0±5.5) μm² for adult group. Clearly, young myocyte lenght, width, cross-sectional area were greater than that of myocytes at weanling group(P＜0.01) but adult myocytes length/width and cross-sectional area increase slightly compared with young group. The length/width ratio has no significant change through myocyte maturity, which is between 8-9. CONCLUSIONS: Our data suggest that myocyte dimensions expect length/width ratio have increased progressively with maturity, the length/width ratio is preserved in atrial myocyte during the period of normal myocyte growth from weanling to adulthood, and these changes in myocyte dimensions are similar with ventricular myocytes changes from other mammalian species.     

Association of Sphingosine‐1‐phosphate (S1P)/S1P Receptor‐1 Pathway with Cell Proliferation and Survival in Canine Hemangiosarcoma

Background

Sphingosine‐1‐phosphate (S1P) is a key biolipid signaling molecule that regulates cell growth and survival, but it has not been studied in tumors from dogs.

Hypothesis/Objectives

S1P/S1P₁ signaling will contribute to the progression of hemangiosarcoma (HSA).

Animals

Thirteen spontaneous HSA tissues, 9 HSA cell lines, 8 nonmalignant tissues, including 6 splenic hematomas and 2 livers with vacuolar degeneration, and 1 endothelial cell line derived from a dog with splenic hematoma were used.

Methods

This was a retrospective case series and in vitro study. Samples were obtained as part of medically necessary diagnostic procedures. Microarray, qRT‐PCR, immunohistochemistry, and immunoblotting were performed to examine S1P₁ expression. S1P concentrations were measured by high‐performance liquid chromatography/mass spectrometry. S1P signaling was evaluated by intracellular Ca²⁺ mobilization; proliferation and survival were evaluated using the MTS assay and Annexin V staining.

Results

Canine HSA cells expressed higher levels of S1P₁ mRNA than nonmalignant endothelial cells. S1P₁ protein was present in HSA tissues and cell lines. HSA cells appeared to produce low levels of S1P, but they selectively consumed S1P from the culture media. Exogenous S1P induced an increase in intracellular calcium as well as increased proliferation and viability of HSA cells. Prolonged treatment with FTY720, an inhibitor of S1P₁, decreased S1P₁ protein expression and induced apoptosis of HSA cells.

Conclusions and clinical importance

S1P/S1P₁ signaling pathway functions to maintain HSA cell viability and proliferation. The data suggest that S1P₁ or the S1P pathway in general could be targets for therapeutic intervention for dogs with HSA.     

Establishment and Identification of One Marc-145 Cell Line Stably and Highly Expressing Porcine CD163

This study was attempted to generate one Marc-145 cell line stably and highly expressing porcine CD163 (pCD163) and set the foundation for PRRSV isolation and vaccine production.CD163 was shown to be a cellular receptor capable of mediating infection of PRRSV non-permissive cell lines.The pCD163 gene was amplified by RT-PCR from porcine alveolar macrophages and cloned into the eukaryotic expression vector pCI-neo, then the positive plasmid pCI-pCD163 was transfected into Marc-145 cells.After selecting with G418 and subcloning for 3 times, Marc-145 cell line expressing pCD163 was established.IFA results indicated that the fluorescence of pCD163-Marc cells was significantly brighter than Marc-145 cells;Western blotting results indicated that the pCD163-Marc cells could express higher levels of CD163 and the expression level was 8.7 times higher than Marc-145 cells.The pCD163-Marc cell line could be stably passaged for 20 passages and the expression level of CD163 was similar with different passages, which would be a valuable tool for facilitating virus propagation and vaccine production.     

大麦(Hordeum vulgare L.)花药培养再生植株染色体的遗传分析和酯酶同工酶的研究

通过观察82株大麦花药培养再生植株根尖细胞有丝分裂和56株花粉母细胞（PMC）减数分裂过程中染色体的结构和分离情况。结果表明，单倍体和二倍体植株各为17．1％和35．4％，混倍体植株47．5％，在混倍体植株中，有二倍体、三倍体、四倍体甚至六倍体细胞和一些非整倍体细胞，但仍以单倍体、二倍体和四倍体细胞为主。根尖细胞有丝分裂过程中有染色体结构的变异，如染色体断片，环状染色体等。二倍体PMC减数分裂过程中染色体分离有异常现象，如染色体“桥”，但绝大多数能正常完成。而单倍体和四倍体不能正常完成，单倍体染色体分离以2：5或3：4为主，并在中后期Ⅰ看到染色单体分离现象，四倍体细胞染色体有多种联会形式。酯酶同工酶（EST）聚丙烯酰胺凝胶电泳（PAGE）结果表明，利用Estl座位不同等位基因的分离规律可检出再生植株中的纯合体与非纯合体。不同的二倍体花粉植株在性状上出现明显的分离现象，观察到二类性状有显著差异的植株，Ⅰ类和Ⅱ类，Ⅰ类占6．6％，Ⅱ类占93．4。     

激光扫描共聚焦显微镜检测Fluo-3 AM 负载大鼠心肌微血管内皮细胞最佳浓度和时间的探讨

为了探讨 Ca2+荧光探针 Fluo-3 AM 对大鼠心肌微血管内皮细胞(Rat Myocardial Microvascular Endothelial Cells RMMECs)最佳装载浓度和时间。将 Fluo-3 AM 分别稀释成1、2、3、4、5μM/L,每一浓度的探针分别装载细胞5、10、15、20 min,应用激光扫描共聚焦显微镜检测细胞内荧光信号的强度、位置、装载探针后细胞的形态来观察装载效果。结果表明：1～3μM/L的Fluo-3AM负载5~20 min均不出现荧光信号;4μM/L 的 Fluo-3AM 负载5~10 min 时即出现荧光信号;5μM/L 的 Fluo-3 AM 孵育细胞15 min后出现清晰且分布均匀的荧光信号。Fluo-3AM对贴壁生长的RMMECs负载效果主要受探针浓度和负载时间的影响。     

Marc-145细胞CD163基因克隆及其真核表达质粒构建

猪繁殖与呼吸综合征是危害养猪业重要传染病之一,CD163是猪繁殖与呼吸综合征感染易感细胞受体之一.从Marc-145细胞克隆出CD163基因并测序,再根据得到序列,应用Primer5.0软件设计引物,成功地扩增出带酶切位点CD163 ORF基因,通过Hind Ⅲ和Xho Ⅰ双酶切位点将CD163基因克隆入真核表达载体pcDNA3.1-myc-His中,获得重组质粒pcDNA3.1-CD163,并将重组质粒pcDNA3.1-CD163在BHK细胞上进行表达鉴定.结果表明,Marc-145细胞CD163蛋白在BHK细胞中得到了表达.这为进一步研究CD163受体在PRRSV感染Marc-145细胞过程中作用以及与其他受体之间相互作用提供试验数据.     

猪繁殖与呼吸综合征病毒GZ-KY1株的分离及ORF5基因的克隆分析

本试验首次采用PK-15细胞,通过同步接种的方法,成功分离到了1株猪繁殖与呼吸综合征病毒(PRRSV,GZ-KY1株),并扩增克隆了该毒株的ORF5基因。试验结果表明,该毒株ORF5基因全长603 bp,系美洲型,与国内强毒株的氨基酸一致性高于弱毒株,与经典美洲型毒株及国内弱毒株亲缘关系较远。     

高致病性猪繁殖与呼吸综合征病毒感染MARC-145细胞的差异蛋白

【目的】研究高致病性猪繁殖与呼吸综合征病毒（PRRSV）感染MARC-145细胞生长相关蛋白表达量的变化,明确差异蛋白的功能信息,为深入研究PRRSV病毒复制和致病机理奠定基础。【方法】采用差异蛋白质组学和Western blotting技术,通过建立双向凝胶电泳,对高致病性PRRSV感染MARC-145细胞后表达量差异蛋白质进行了胶内酶解、质谱分析鉴定及数据库检索,明确差异蛋白质的相关信息。【结果】得到7个表达稳定、重复性好的蛋白点,其中有5个蛋白点找到匹配蛋白,分别为热休克蛋白70、硫氧还蛋白、S100钙结合蛋白A6、3-磷酸甘油脱氢酶和内参肌动蛋白,其余2个蛋白点为未知蛋白。在MARC-145细胞感染PRRSV后,热休克蛋白70和S100钙结合蛋白A6表现为下调,而硫氧还蛋白、3-磷酸甘油脱氢酶和内参肌动蛋白表现为上调。功能预测分析发现,5个蛋白与MARC-145细胞抗应激、抗氧化、生长、凋亡和信号转导等作用息息相关。【结论】高致病性PRRSV感染MARC-145细胞后,能诱导细胞部分功能蛋白表达量的增加或减少。