抑癌基因RASSF1A在结肠癌中的表达研究

目的探讨抑癌基因Ras相关区域家族1A（RASSFlA）基因在结肠癌组织以及相应正常结肠组织中的表达并分析其表达与结肠癌临床病理因素之间的关系。方法采用免疫组织化学SP法和Western blot法检测34例结肠癌手术标本和相应的正常结肠组织中RASSFlA蛋白的表达水平,应用RT-PCR法检测RASSFlAmRNA在结肠癌和正常结肠组织中的表达情况。结果①免疫组织化学法结果：结肠癌组织中RASSFlA蛋白表达阳性率明显低于其在正常结肠组织中的表达阳性率[35．3％（12／34）比97．1％（33／34）,P〈0．05]。结肠癌组织中RASSFlA蛋白的表达与肿瘤分化程度和TNM分期均有关（P〈0．05）,即高、中分化及TNM分期较低（Ⅰ＋Ⅱ期）者的RASSFlA蛋白表达阳性率较高P〈0．05）。②Western blot法结果：在34例结肠癌患者中RASSFlA蛋白表达水平明显低于其在相应的正常结肠组织中的表达水平[0．3168＋0．0196比0．9144±0．1776,P〈0．05];该结果与RT-PCR法检测到的RASSFlAmRNA表达情况基本一致[0．1589±O．2237和0．5723±0．1939,P〈0．05]。结论RASSFlA基因的失表达可能在原发性结肠癌的发生、发展中起重要作用,检测RASSFlA的表达情况可为结肠癌的早期诊断提供帮助。     

Fibril protein fragmentation pattern in systemic AL‐amyloidosis

Immunoglobulin light chain (AL)‐amyloidosis was one of the first types of amyloidosis discovered and still little is known about its pathogenic mechanisms. One major obstacle is the very heterogeneous condition; in fact, every patient could be considered to have their own disease since symptoms and outcome vary enormously. The reason for this is not known but intrinsic factors of the immunoglobulin light chain (LC) and the fact that every LC is unique seem to be important. Post‐translational modifications such as glycosylation and proteolysis are most certainly involved. By using western blotting, we studied in detail the proteolytic pattern in six patients with AL‐amyloidosis of kappa type with the aid of three peptide antisera against two domains in the constant segment and one conserved domain in framework 3 of the variable region. Materials from one to five organs were analysed. The result clearly demonstrates that the fragmentation pattern was similar in amyloid of different organs in one patient but differed greatly between patients. Full‐length, N‐, and C‐terminal fragments were detected with the three antisera. The results strongly support the hypothesis that proteolytic cleavage occurs after fibril formation. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

An alternative strategy to western blot as a confirmatory diagnostic test for HIV infection

BackgroundIn China, western blot (WB) is the recommended procedure for the diagnosis of HIV infection. However, this technique is time consuming and labor intensive, and its complexity restricts wide application in resource-limited regions.ObjectiveThe aim of this study was to evaluate the efficacy of a dry blood spots (DBS)–urine paired enzyme-linked immunosorbent assay (ELISA) test, instead of WB, for HIV antibody detection.Study designPlasma, DBS, and urine samples were collected from 1213 subjects from different populations. Two diagnostic testing strategies were conducted in parallel. The equivalence of the paired ELISA and WB strategies was assessed.ResultsA diagnosis of HIV was determined in 250 subjects according to the paired ELISA test, and in 249 according to the WB strategy. The discordant case was judged HIV-positive during follow-up. In total, 18 subjects were diagnosed with possible HIV using the paired ELISA test, among whom, 11 subjects tested negative with WB, and one was confirmed to be HIV-positive during follow-up. For the remaining 945 subjects, both strategies indicated a negative result. The kappa test indicated good conformity (kappa = 0.954) between the two diagnostic strategies.ConclusionThe DBS–urine paired ELISA could be applied as an alternative to WB in HIV diagnosis, which would be valuable in resource-limited regions owing to the associated affordability and ease of use.     

子宫肌瘤组织Transgelin基因表达及其临床意义的研究

目的:检测Transgelin基因mRNA及蛋白在子宫肌瘤中的表达,探讨Transgelin基因与子宫肌瘤发生、发展的关系。方法:采用逆转录-聚合酶链反应(RT-PCR)及蛋白质印迹法,检测30例子宫肌瘤组织、30例子宫肌瘤包膜外2 cm子宫肌层组织和10例正常子宫肌层组织中Transgelin基因mRNA及蛋白的表达。结果:Transgelin基因mRNA在子宫肌瘤组织中阳性表达率及相对含量分别为23.3%和50.57±20.16,均低于子宫肌瘤包膜外2 cm子宫肌层组织的100.0%和84.62±6.12以及正常子宫肌层组织的100.0%和85.99±6.99,P<0.001。子宫肌瘤包膜外2 cm子宫肌层组织和正常子宫肌层组织中Transgelin基因mRNA的相对含量差异无统计学意义,P>0.05。Transgelin蛋白在子宫肌瘤组织中阳性表达率及相对含量分别为16.7%和23.46±16.54,均低于子宫肌瘤包膜外2 cm子宫肌层组织的100.0%和61.08±8.73以及正常子宫肌层组织的100.0%和67.19±7.62,P<0.001。子宫肌瘤包膜外2 cm子宫肌层组织和正常子宫肌层组织中Transgelin蛋白的相对含量差异无统计学意义,P>0.05。结论:Transgelin基因可能是子宫肌瘤发生过程中的抑制因素,与子宫肌瘤的发生、发展可能有关。     

槲皮素对人食管癌Eca-109细胞的分化诱导作用

目的探讨槲皮素对人食管癌Eca-109细胞的分化诱导作用。方法将培养的Eca-109细胞分为两组:①加槲皮素组(Q组);②不加药物对照组(C组)。同时培养48h,收集各组细胞制备成细胞硝酸纤维素膜(NCM)标本及提取RNA制备RNA硝酸纤维素膜标本。分别对细胞硝酸纤维素膜(NCM)标本进行PCNA,VEGF的免疫斑点印迹阵列;对RNA硝酸纤维素膜标本EGFR,c-myc及wtp53的RNA斑点印迹阵列。印迹阵列皆用岛津薄层色谱扫描仪(TLC)进行波长523nm的扫描数值比较。结果①免疫斑点印迹显示:Q组的PCNA-IR,VEGF-IR的TLC扫描数值分别比C组低5.5倍,3.5倍;②RNA斑点印迹阵列显示,与C组相比:Q组的EGFR,c-myc的RNA斑点TLC扫描数值分别下调了7倍,2.2倍。而wtp53的RNA斑点TLC扫描数值则上调了2.2倍。结论槲皮素可下调作为肿瘤细胞恶性程度生物学指标的PCNA,EGFR,VEGF,c-myc等的表达,上调抑癌基因wtp53的表达,表明对人食管癌Eca-109细胞具有一定的诱导分化效应。     

FATP4在大鼠非酒精性脂肪肝中的表达及相关性

目的 探讨FATP4(fatty acid transport protein4)基因在大鼠非酒精性脂肪肝形成中的作用.方法 Wistar大鼠64只,随机分为正常对照组(C组)和高脂饮食组(F组),又各分2、4、8和12周组(其中每组各8只):HE染色光镜观察肝脏组织病理改变;硫代巴比妥酸法测定肝脏组织丙二醛(MDA)的含量变化:免疫组织化学和Western blot方法研究高脂饲料诱导的大鼠脂肪肝形成中FATP4表达变化.结果 高脂饲料组大鼠肝脏内MDA含量明显高于对照组,随时间延长,逐渐增加:随着脂肪肝程度的加重,MDA含量逐渐增强,肝细胞FATP4蛋白表达亦明显增强.结论 非酒精性脂肪肝大鼠肝细胞FATP4表达变化与脂肪肝引起的脂质过氧化损伤程度密切相关.     

TLR4受体在肝癌细胞株HepG-2的表达

目的：探讨TLR4受体在肝癌细胞株HepG-2和肝癌组织中的表达。方法：用RT-PCR和Western-blot方法检测肝癌细胞株HepG-2中TLR4基因的转录和蛋白的表达的情况,同时用免疫组化的方法检测肝癌组织中TLR4的表达的情况。结果：肝癌细胞株HepG-2中存在TLR4基因的转录和蛋白的表达。TLR4受体主要表达位于肝癌组织细胞的细胞膜上,其阳性率约为76.7%。结论：肝癌细胞株HepG-2和肝癌组织中均有TLR4的表达。     

ERK1/2通路在4-AP诱导大鼠肺动脉收缩中的作用

目的探讨细胞外信号调节激酶-1/2(ERK1/2)通路在4-氨基吡啶(4-aminopyridione,4-AP)阻断正常大鼠肺动脉平滑肌细胞(PASMCs)膜上电压依赖性钾通道(KV)所引起的肺动脉收缩中的作用。方法取正常鼠肺动脉制作肺动脉环,分别加入4-AP(KV通道阻断剂),PD98059/U0126+4-AP,比较肺动脉收缩的变化。同时培养肺动脉平滑肌细胞进行Western blot分析4-AP对ERK1/2的影响。结果①在血管环试验中,4-AP引起的肺动脉收缩有浓度依赖性;加入20mmol.L-1PD98059或2μmol.L-1U0126可以抑制4-AP引起的肺动脉收缩。②4-AP可刺激PASMCs ERK1/2蛋白磷酸化;③U0126可抑制4-AP引起的ERK1/2蛋白磷酸化。结论ERK1/2通路参与4-AP阻断正常大鼠肺动脉平滑肌细胞膜上电压依赖性钾通道(KV)引起肺动脉收缩。     

多寡核苷酸探针同步标记在Northern blot多基因分析中的应用

目的：建立一种多探针同时标记同步杂交检测多基因的简化的Northern blot方法.方法：通过T4多聚核苷酸激酶5＇末端放射性磷酸化标记法对Northern blot中多寡核苷酸探针同时标记,实现对同一张转移印迹膜上的多基因同步杂交显影检测.结果：与常规mRNA逐一检测的效果进行比较,同步简化法的各目的基因同时得到较好的检测.结论：同步简化法简化了实验过程,缩减了实验时间,节省了实验试剂,并使各基因表达丰度之间的比较具有更高的准确性,提高了Northern blot的检测效率和成功率.