表面增强激光解吸电离飞行时间质谱在卵巢癌中的应用

表面增强激光解吸电离飞行时间质谱(SELDI-TOF-MS)是近年出现的蛋白质研究新技术,其综合了蛋白芯片和质谱技术的优点,可快速、高通量捕捉血清、组织及体液中的特异性蛋白质。SELDI-TOF-MS已应用于卵巢癌的早期诊断、指导治疗及预后判断等研究。     

雌、孕激素对早孕蜕膜基质细胞中Mig和IP-10表达的影响

母胎平衡机制一直是生殖研究领域的热点和难点之一.有研究证实,母胎界面中表达的趋化因子-趋化因子受体系统在保持母胎平衡中起重要作用[1-2].CXC类趋化因子Mig和IP-10在早孕蜕膜上皮及基质细胞中高表达,其表达变化与蜕膜中免疫细胞聚集和滋养细胞生物学行为有关[3-4].     

乏氧通过腺苷A2受体调节人成熟树突状细胞骨桥蛋白的表达

目的 探讨乏氧调节人成熟树突状细胞(mDCs)骨桥蛋白(OPN)表达的机制.方法 免疫磁珠法分离人外周血CD14+单核细胞,分别在常氧和乏氧条件下经GM-CSF及IL-4体外诱导生成mDCs;ELISA检测培养上清中OPN水平,RT-PCR检测OPN基因水平表达;使用腺苷替代物(NECA)、特异性A2R激动剂(CGS21680)和特异性A2R拮抗剂(SCH58261)探讨A2R在调节人mDCs表达OPN中发挥的作用;利用重组人TGF-β1(rhTGF-β1)及抗TGF-β1单克隆抗体(anti-TGF-β1Mcab)检测TGF-β1在这一过程中发挥的作用.结果 乏氧显著增加mDCs对OPN mRNA及OPN蛋白的表达,A2R拮抗剂抑制乏氧对OPN的诱导作用;常氧条件下,腺苷替代物(NECA)和A2R激动剂均能显著上调mDCs对OPN mRNA的表达及OPN蛋白的分泌,A2R拮抗剂可特异性抑制A2R激动剂对OPN的诱导;A2R参与调控免疫调节因子TGF-β1水平,通过rhTGF-β1和阻断抗体证实TGF-β1

Abstract：

Objective To investigate the mechanism of hypoxia regulate osteopontin (OPN) secreting by mature dendritic cells (mDCs). Methods CD14 + cells were enriched using anti-CD14 immunomagnetic beads, for inducing to mDCs, CD14 + cells were cultured with GM-CSF and IL-4 in hypoxia or normoxiain vitro. Concentration of OPN and TGF-β1 in supernatant were detected by sandwich ELISA, OPN mRNA detected by RT-PCR. Approach regulating function of A2 R in expressing of OPN by mDCs by using NECA (surrogate of adenosine), A2R agonist (CGS21680), A2R antagonist (SCH58261) and investigate role of TGF-β1 in this process by using rhTGF-β1 and anti-TGF-β1 Ab. Results Hypoxia inreased the level of OPN and OPN mRNA in mDCs, and this effect could be reversed by A2 R antagonist. Under normoxia,both NECA and A2R agonist (CGS21680) could upregulate the level of OPN and OPN mRNA in mDCs significantly, but this positive effect could be reversed by A2 R antagonist. A2 R played a role in regulating TGF-β1, and confirmed TGF-β1 involved in regulation of OPN by using rhTGF-β1 and anti-TGF-β1 Ab. Conclusion High adenosine induce the generation of TGF-β1 through the A2R on mDCs, and then TGF-β1 raise the OPN secreting by mDCs.     

缺氧条件下HIF-1α对舌鳞癌细胞VEGF-C及FAS表达的影响

目的探讨缺氧条件下人舌鳞癌Tca8113细胞株HIF-1α对VEGF-C及FAS基因表达的影响。方法采用RT-PCR技术半定量检测人Tca8113细胞常氧和缺氧培养0.5、1、3、6、12和24h HIF-1α、VEGF-C和FAS基因的表达。结果缺氧条件下HIF-1α与VEGF-C基因表达呈先升后降的趋势,二者表达呈显著正相关性（rs=0.979,P〈0.01）。FAS基因表达呈上升趋势,与HIF-1α表达无明显相关性（rs=0.556,P〉0.05）。结论缺氧条件下Tca8113细胞中HIF-1α可上调VEGF-C的表达,促进癌细胞的转移;HIF-1α参与促进肿瘤细胞FAS的表达,影响舌癌细胞的凋亡。     

hCG对HTR8 SVneo滋养层细胞系MMP 2表达的影响与调控机制

目的探讨绒毛膜促性腺激素（hCG）对滋养层细胞系HTR8 SVneo侵袭活性的影响及其可能的调控机制。方法将培养的HTR8 SVneo细胞根据处理因素分为对照组、低浓度hCG（10?IU/mL）刺激组、高浓度hCG（100?IU/mL）刺激组,再将HTR8 SVneo细胞分为对照组、hCG（100?IU/mL）刺激组、DPCPX(A1R特异性阻断剂）+hCG（100IU/mL）联合刺激组。采用RT PCR检测各组HTR8 SVneo细胞基质金属蛋白酶 2（MMP 2）的表达变化,明胶酶谱检测各组HTR8 SVneo细胞MMP 2蛋白水平的分泌变化。 结果高浓度hCG（100?IU/mL）较低浓度hCG（10?IU/mL）刺激后的HTR8 SVneo细胞MMP 2的表达与分泌均上升（P<均0.05）;腺苷受体A1R的表达同时升高（P<0.05）。阻断A1R的作用通路后,MMP 2的表达与分泌均下降（P均<0.05）。 结论随着hCG浓度的升高, HTR8 SVneo细胞的侵袭性逐步增强,其调控机制为通过促进A1R的表达而使MMP 2的分泌升高。     

表面增强激光解吸／离子化质谱技术在诊断下咽癌中的应用研究

目的探讨表面增强激光解吸／离子化质谱技术（SELDI-TOF-MS）在下咽癌早期诊断中的价值。方法采用弱阳离子（WCX2）蛋白质芯片结合表面增强激光解吸电离飞行时间质谱技术检测38例下咽癌患者和46例健康体检者的血清,建立预测模型。结果从下咽癌患者和健康人血清中筛选出4个明显表达差异的蛋白质峰,其分子量分别为4175、13265、3160和5804Da,均为下咽癌高表达。用其中的10例下咽癌患者和12例健康体检者样本作训练集和交叉验证后,再用筛选出的4个差异蛋白质峰建立诊断预测模型,然后对健康人和下咽癌患者样本进行盲法测试。结果显示,其准确率为89.3％（75／84）,敏感性为97．4％（37／38）,特异性为82．6％（38／46）。结论用SELDI-TOF-MS技术建立的诊断预测模型对区分下咽癌和正常人有较好的特异性与准确率,该技术为下咽癌的早期诊断及预后判断提供了新的手段。     

谷氨酰胺对人肺泡Ⅱ型上皮细胞热休克蛋白70表达的影响

目的评价谷氨酰胺对人肺泡Ⅱ型上皮细胞系A549细胞热休克蛋白（HSP）70表达的影响。方法取人肺泡Ⅱ型上皮细胞系A549细胞,在不含谷氨酰胺的DMEM培养液中孵育24h作为空白对照组（C组）,43℃孵育1h、37℃恢复4h作为阳性对照组（PC组）,不同浓度（2、4、8、12和16 mmol/L）谷氨酰胺的DMEM培养液中孵育24h作为不同浓度谷氨酰胺诱导组（Gln2组、Gln4组、Gln8组、Gln（12）组和Gln（16）组）,8mmol/L谷氨酰胺的DMEM培养液中孵育不同时间（1、2、6、12、24和48 h）作为不同时间谷氨酰胺诱导组（T1组、T2组、T3组、T4组、T5组和T6组）。分别采用RT-PCR和Western blot法检测HSP70 mRNA和蛋白的表达。结果与C组比较,PC组和不同浓度谷氨酰胺诱导组人肺泡Ⅱ型上皮细胞系A549细胞HSPTO mRNA和蛋白的表达均升高,Gln8组HSP70 mRNA和蛋白表达水平高于Gln2组、Gln4组、Gln（12）组和Gln（16）组（P＜0．01）,而与PC组相比差异无统计学意义（P＞0．05）。与C组比较,PC组和不同时间谷氨酰胺诱导组HSP70 mRNA和蛋白的表达均升高,T5组HSP70 mRNA和蛋白表达水平高于T1组、T2组、T3组、T4组和T5组（P＜0．01）,而与PC组相比差异无统计学意义（P＞0．05）。结论谷氨酰胺可明显上调体外培养人肺泡Ⅱ型上皮细胞系A549细胞HSP70 mRNA和蛋白的表达,并呈浓度和时间依赖性。     

人MUCI/Y基因真核表达载体的构建及其在COS/7细胞中的表达

MUCl/Y是1994年发现的新基因,其蛋白锚定于细胞膜。研究表明,MUCl/Y具有膜受体的特征,可能作为一种膜表面的重要分子通过ras信号传导通路参与细胞的恶性转化过程;进一步的动物实验表明,MUCl/Y的表达具有肿瘤特异性。由此推测,MUCl/Y可能是一种肿瘤主动特异性免疫治疗的理想靶分子,但迄今为止,MUCl/Y基因及其编码蛋白的功能尚未明确,因此,构建人MUCl/Y全长cDNA的真核表达载体,将对进一步研究该基因的功能以及以MUCl/Y为靶点的肿瘤核酸疫苗的制备,具有重要意义。     

Experiment studies of antitumor proliferation and metastasis of a new chinese herb AT-1

Objective: To study the effects of a new Chinese herb AT-1 on the tumor cell proliferation and metastasis in vitro.Methods: Tumor cell proliferation activity was tested by MTT. The ability of tumor cell invasion and migration was assayed by counting the number of tumor cells going throw matrigel. The expression changes of CD44 genes in PG cells treated with AT-1 were tested by FACS. Results:Compared with the control, the proliferation activity of the cells treated with the At-1 was restrained. The invasion and migration ability of PG cells and the expression of the cell adherence related gene CD44 was decreased treatment with AT-1. Conclusion: AT-1 is a new antitumor proliferation and metastasis agent. Its antitumor metastasis effect might be achieved by decreasing the expression of the cell adherence-associate gene CD44.     

HCA520, a novel tumor associated antigen,involved in cell proliferation and apoptosis

Objective: Tumor associated antigen encoding gene HCA520 (AF146019) was identified by screening a human hepatocellular carcinoma expressing cDNA library using SEREX technique. In this experiment we studied the effect of HCA520 on cell proliferation and apoptosis.Methods: Gene HCA520 was gained by PCR and transfected into 293 cells. The stable expression cells were obtained by G418 selection. The cell proliferation was measured by [^3H]-TdR uptake and apoptosis assay was measured by FACS. Results: Eukaryotic expression plasmid pcDNA3-HCA520 was constructed and its stable transfectants were obtained. Overexpression of HCA520 inhibited the cell proliferation and enhanced cell apoptosis after serum deprivation. Conclusion: HCA520 is a novel tumor associated antigen that can affect cell proliferation and apoptosis.