氧化型低密度脂蛋白诱导U937细胞凋亡及其对p53、p21和Bcl-2表达的影响

为研究单核细胞源性泡沫细胞凋亡机制 ,以氧化型低密度脂蛋白诱导人髓系白血病细胞U937凋亡 ,观察p5 3、p2 1和Bcl- 2的表达。用流式细胞术和DNA断裂分析检测细胞凋亡 ;用细胞免疫荧光标记结合流式细胞术检测p5 3、p2 1和Bcl- 2蛋白表达 ,反转录聚合酶链反应显示p5 3、p2 1和Bcl- 2mRNA表达水平。结果表明氧化型低密度脂蛋白可致U937细胞凋亡 ,其作用具有浓度效应 ;氧化型低密度脂蛋白诱导U937细胞凋亡过程中 ,p5 3、p2 1mRNA和蛋白表达增加 ,Bcl- 2mRNA和蛋白表达减少。提示氧化型低密度脂蛋白可能通过上调p5 3、p2 1基因表达 ,下调Bcl- 2基因 ,导致U937细胞凋亡。     

氧化型低密度脂蛋白诱导U937细胞凋亡时泛素缀合酶和p53的表达

为研究氧化型低密度脂蛋白致U937细胞凋亡时泛素-蛋白酶体通路中泛素缀合酶、p53与泡沫细胞凋亡之间的关系，用氧化型低密度脂蛋白处理人单核细胞U937细胞，诱导其凋亡，采用DNA琼脂糖凝胶电泳和流式细胞术检测细胞凋亡，逆转录聚合酶链反应检测泛素缀合酶和p53的基因表达；流式细胞术分析泛素缀合酶、载脂蛋白B和P153的蛋白表达。结果发现，氧化型低密度脂蛋白可以诱导U937细胞凋亡，在U937细胞凋亡过程中泛素缀合酶表达降低，p53表达增高，而且细胞内载脂蛋白B也增加。结果提示，泛素缀合酶的表达降低可能使P53和栽脂蛋白B经泛素-蛋白酶体通路的降解减慢．从而引起P53和载脂蛋白B在细胞内积聚．导致U937细胞凋亡。     

氧化型低密度脂蛋白诱导U937细胞凋亡时细胞间粘附分子-1的表达时序

研究氧化型低密度脂蛋白诱导人单核细胞U937细胞吞噬脂质发生凋亡过程中对细胞间粘附分子－1表达的影响,用胸腺嘧啶核苷将U937细胞阻滞在G1期,流式细胞仪监测同步化处理效果;流式细胞术检测U937细胞泡沫化过程中细胞凋亡和细胞间粘附分子－1的表达;逆转录－聚合酶链反应分析细胞间粘附分子－1 mRNA的表达,结果显示,80mg/L氧化型低密度脂蛋白温育U937细胞48h可形成典型的泡沫细胞,72h可见凋亡细胞增多;氧化型低密度脂蛋白温育U937细胞12h即可检测到细胞间粘附分子－1表达,24h时达到最高值,48h略有降低,72h时则明显降低。结果表明,氧化型低密度脂蛋白可以促进U937细胞细胞间粘附分子－1表达,细胞间粘附分子－1的表达增强有利于巨噬细胞的趋化运动和对脂质的吞噬。     

沥水调脂胶囊对氧化型低密度脂蛋白诱导内皮细胞凋亡及Bcl-2、BaX、P53和Caspase-3蛋白表达的影响

目的：研究沥水调脂胶囊抑制内皮细胞凋亡的机制。以氧化型低密度脂蛋白诱导人脐静脉内皮细胞凋亡，观察Bcl-2,Bax,P53和Caspase-3蛋白表达。方法：用流式细胞术检测细胞凋亡并进行周期分析；用细胞免疫荧光标记结合流式细胞术检测Bcl-2,Bax,P53和Caspase-3蛋白表达水平。结果：沥水调脂胶囊对氧化型低密度脂蛋白诱导的内皮细胞凋亡有抑制作用。在此过程中沥水调脂胶囊上调了Bcl-2蛋白表达水平，对Bax蛋白表达未见明显影响，同时下调了P53和Caspase-3蛋白表达水平。结论：推测沥水调脂胶囊可能是通过上调Bcl-2蛋白表达，下调P53和Caspase-3蛋白表达抑制内皮细胞凋亡的。     

miR-24-3p靶向HAP1基因对STZ诱导胰岛β细胞凋亡的影响

目的探讨微小RNA-24-3p(miR-24-3p)靶向亨廷顿蛋白相关蛋白(HAP)1基因表达对链脲佐菌(STZ)诱导胰岛β细胞凋亡的影响。方法培养小鼠胰岛β细胞NIT,分别将anti-miR-NC、anti-miR-24-3p、pcDNA、pcDNA-HAP1转染入NIT细胞,加入STZ诱导细胞;不做任何处理为Con组。采用实时荧光定量聚合酶链反应(qRT-PCR)检测miR-24-3p及HAP1 mRNA表达水平;采用蛋白免疫印迹(Western印迹)实验检测HAP1及B细胞淋巴瘤(Bcl)-2、Bcl-2相关X蛋白(Bax)、活化的半胱氨酸蛋白酶(cleaved-caspase)-3蛋白表达水平。双荧光素酶报告实验验证miR-24-3p的靶基因。流式细胞术检测各组细胞凋亡情况。结果 STZ处理后胰岛β细胞中miR-24-3p相对表达量明显高于Con组(P0.05),而HAP1 mRNA及蛋白表达均明显降低(P0.05);STZ可增加胰岛β细胞凋亡率,抑制miR-24-3p表达可降低胰岛β细胞凋亡率,还可上调抗凋亡蛋白Bcl-2表达,下调促凋亡蛋白Bax、cleaved-caspase-3表达;HAP1过表达与抑制miR-24-3p表达均可抑制STZ诱导的胰岛β细胞凋亡;HAP1是miR-24-3p的靶基因,miR-24-3p可负向调节HAP1表达;抑制HAP1表达可促进胰岛β细胞凋亡。结论 miR-24-3p表达可通过抑制靶基因HAP1表达进而促进STZ诱导的胰岛β细胞凋亡。     

氧化低密度脂蛋白对U937细胞凋亡的影响及其机制的研究

目的探讨氧化低密度脂蛋白(ox-LDL)对培养的单核巨噬系细胞株U937细胞凋亡的影响,并进一步探讨凋亡产生的机制。方法应用流式细胞术(FCM)检测细胞凋亡;Caspase-3、8、9的测定采用免疫组织化学技术。结果ox-LDL组细胞调亡率增加,并且随着作用时间的延长和浓度的增加,凋亡呈递增后递减变化,U937细胞在用ox-LDL培养后LOX-1和Caspase3、8、9表达都增加。结论ox-LDL可诱导U937细胞凋亡,ox-LDL诱导U937细胞凋亡既有线粒体通路,又包含了死亡受体。     

外源性Rb基因对U937细胞泡沫化过程中CD36表达的影响

为研究抑癌基因Rb基因在U937细胞泡沫化过程中的表达，应用重组腺病毒载体导入外源性Rb基因。采用逆转录聚合酶链反应和流式细胞术检测氧化型低密度脂蛋白致U937细胞泡沫化过程中清道夫受体CD36和Rb基因的表达和外源性Rb基因导入U937细胞后CD36和Rb基因的表达。结果发现，氧化型低密度脂蛋白致U937细胞泡沫化过程中，Rb mRNA和蛋白的表达随作用时间延长呈下调趋势，CD36 mRNA和蛋白的表达则呈上调趋势；随着外源性Rb导入U937细胞并表达，CD36表达呈下调趋势。结果提示，外源性Rb基因的导入可以下调清道夫受体CD36的表达。     

人参皂甙Rh2对人脑胶质瘤细胞株U251凋亡的影响及机制探讨

目的观察人参皂甙Rh2（GS-Rh2）对人脑胶质瘤细胞株U251凋亡的影响,并探讨其机制。方法人脑胶质瘤细胞株U251经GS-Rh2处理后,采用MTT法测算细胞抑制率,用流式细胞术测算细胞凋亡率,用RT-PCR检测U251中Bcl-2、Caspase-3 mRNA,用Western blot法检测U251中Bcl-2、Caspase-3蛋白。结果 GS-Rh2对U251具有抑制作用,并呈剂量依赖性;根据细胞增殖抑制曲线计算出GS-Rh2作用于细胞的抑制浓度（IC）,求得IC30、IC50、IC70值分别为10.6、22.3、70.2μg/ml;GS-Rh2可诱导U251凋亡,并呈剂量依赖性。在GS-Rh2作用下,U251中Bcl-2 mRNA、蛋白呈明显低表达,随着GS-Rh2作用时间延长,Caspase-3 mRNA、蛋白表达逐渐增高。结论 GS-Rh2可明显抑制U251增殖,诱导其凋亡,其机制与GS-Rh2下调U251中Bcl-2 mRNA及蛋白表达、上调Caspase-3 mRNA及蛋白表达有关。     

抗磷脂抗体对巨噬细胞摄取氧化型低密度脂蛋白及CD36表达的影响

为了探讨抗磷脂抗体对巨噬细胞摄入氧化型低密度脂蛋白的影响及其主要清道夫受体CD36抗原表达的调节 ,将培养的U937细胞与氧化型低密度脂蛋白 (80mg L)和抗磷脂抗体 (10 0mg L)孵育 ,用酶荧光法观察人类单核细胞株U937内脂质的变化、逆转录—聚合酶链反应和流式细胞术观察CD36抗原mRNA和蛋白水平表达的改变。结果发现氧化型低密度脂蛋白存在时细胞内胆固醇和胆固醇酯含量分别为 2 4 3± 9mg g细胞和 2 89± 12mg g细胞 ,流式细胞仪测CD36抗原为 2 5 % ,凝胶电泳分析CD36mRNA β actionmRNA为 1.0 5 ;在加入抗磷脂抗体后 ,胞浆内胆固醇和胆固醇酯明显增加 ,分别为 2 76± 10mg g细胞和 4 78± 13mg g细胞 ,流式细胞仪测CD36抗原为 37% ,凝胶电泳分析CD36mRNA β actionmRNA为 1.90 ;两组相比差异有显著性 (P <0 .0 5 )。以上提示抗磷脂抗体有促进U937细胞摄取氧化型低密度脂蛋白的作用 ,这一过程是通过在转录及蛋白水平上调CD36抗原表达而实现的     

雌激素对氧化低密度脂蛋白诱导单核细胞趋化因子受体CXCR2的影响

目的：研究17β-雌二醇对氧化低密度脂蛋白（oxLDL）诱导单核细胞合成趋化因子受体CXCR2的影响。方法：单核细胞的研究模型为人前单核细胞系U937细胞。流式细胞仪测定单核细胞趋化因子受体CXCR2的蛋白表达，逆转录PCR测定其mRNA水平。结果：oxLDL(50μg/ml)可明显促进U937细胞的CXCR2mRNA及蛋白表达；17β-雌二醇（50nmol/L）预处理抑制了oxLDL(50μg/ml)诱导的U937细胞CXCR2的mRNA及蛋白表达，且17β-雌二醇（5-500nmol/L）抑制oxLDL(50μg/ml)诱导U937细胞合成的CXCR2蛋白呈正向量效关系。结论：雌激素抑制oxLDL诱导U937细胞产生的趋化因子受体CXCR2表达。     

Structure of the Sec23p/24p and Sec13p/31p complexes of COPII

COPII-coated vesicles carry proteins from the endoplasmic reticulum to the Golgi complex. This vesicular transport can be reconstituted by using three cytosolic components containing five proteins: the small GTPase Sar1p, the Sec23p/24p complex, and the Sec13p/Sec31p complex. We have used a combination of biochemistry and electron microscopy to investigate the molecular organization and structure of Sec23p/24p and Sec13p/31p complexes. The three-dimensional reconstruction of Sec23p/24p reveals that it has a bone-shaped structure, (17 nm in length), composed of two similar globular domains, one corresponding to Sec23p and the other to Sec24p. Sec13p/31p is a heterotetramer composed of two copies of Sec13p and two copies of Sec31p. It has an elongated shape, is 28-30 nm in length, and contains five consecutive globular domains linked by relatively flexible joints. Putting together the architecture of these Sec complexes with the interactions between their subunits and the appearance of the coat in COPII-coated vesicles, we present a model for COPII coat organization.     

Immunohistochemical Expression of p16, p53, and p63 in Colorectal Adenomas and Adenocarcinomas

Purpose The aim of this study was to investigate the immunohistochemical expression of p16, p53, and p63 proteins according to some pathologic parameters related to colorectal adenomas and adenocarcinomas such as grade of dysplasia and histologic type. Methods Immunohistochemistry with the antibodies p16, p53, and p63 was performed in tubular, tubular-villous, and villous adenomas (n = 30) and in well, moderately, and poorly differentiated adenocarcinomas (n = 30). The p63-positive cases were submitted to double immunolabeling with the cytokeratin 5 (CK5). Results The p16 and p53 labelings were observed in some adenomas and adenocarcinomas but without any association with p63 expression, histologic type, or grade of differentiation of the neoplasm. P63 expression was found mainly in the villous adenomas and in the poorly differentiated adenocarcinomas. The poorly differentiated adenocarcinomas also exhibited coexpression of CK5 and p63. Conclusions Despite both p16 and p53 having been detected in colorectal neoplasms, they were not related to the different histologic variables nor to the expression of p63. However, p63 expression was closely associated with villous adenomas and poorly differentiated adenocarcinomas. Thus, p63 may represent a marker of poor differentiation in colorectal neoplasms. The coexpression of p63 and CK5 observed in this study could be related to divergent differentiation during the development of colorectal cancer, although further studies are warranted to refine the understanding of this process.     

Contribution of p53, p63, and p73 to the developmental diseases and cancer

Tumor suppressor TP53 gene is one of the most mutated genes in human genome. Inactivating somatic mutations and disruption of p53 protein have been described in almost all human malignancies. Its inactivation by germline mutation leads to the rare but severe familial precancerosis termed Li-Fraumeni syndrome. This syndrome is characterized by the early onset of different types of cancers including soft-tissue sarcomas, breast and brain cancers, leukemias, lung, laryngeal cancers, and adrenocortical carcinomas. The key role of p53 in tumor suppression has been confirmed in animal models as well. The p53 -knock-out and knock-in animals were born alive but were tumor prone. In the late nineties, two genes with high homology with TP53 were discovered, TP73 and TP63, respectively. Animal models showed that p73 is an important player in neurogenesis, sensory pathways and homeostatic control. The p63 is critical for the development of stratified epithelial tissues such as epidermis, breast, and prostate. Despite the structural similarities with p53, the function of these proteins in tumorigenesis is controversial. On one hand, there are evidences that both, p63 and p73-deficient animals are not tumor prone; on the other hand, there is evidence that such animals develop tumors later during their life. Unlike in TP53 gene, mutations in TP63 and TP73 genes are rare, however, germline mutations in TP63 are linked to the human developmental diseases. In this minireview, we describe the contribution of the p53, p63, and p73 to human pathology with emphasis on their different roles in development and tumorigenesis.     

Chromosome 17p and p53 changes in lymphoma

The clinical course of lymphoma patients in whom rearrangements or deletions of the short arm of chromosome 17 (17p) were evident by cytogenetics was rapidly progressive with a short survival. The gene for the protein designated p53 resides in 17p. We studied four lymphoma cell lines derived from human tumours, and 25 tumour samples of patients with lymphomas, for any evidence of p53 genomic changes by Southern blot technique. The four cell lines and four of the 25 tumour samples showed numerical changes of chromosome 17 or structural abnormalities of 17p (translocations or deletions). Allelic loss of the p53 gene was found in two of the four cell lines, and one of these in addition showed a rearrangement of the 3' end of the gene. Of the four tumours known to have chromosome 17 abnormality, one specimen showed allelic loss of the p53 gene. None of the remaining tumour samples showed any significant change. These studies suggest that acquisition of changes in the short arm of chromosome 17, which may be interrelated with the p53 gene, may carry a poor prognosis in patients with non-Hodgkin's lymphoma.     

Immunodominant epitopes of HIV-1 p17 and p24.

Immunodominant antibody-binding sites were mapped using overlapping synthetic peptides of the structural proteins p17 and p24 of human immunodeficiency virus type 1 (HIV-1). Using sera from HIV-1-infected individuals at a variety of disease states, three major epitopes were identified within p17 and one within p24. Antibodies which recognized these epitopes were present in all risk groups throughout all stages of HIV infection, regardless of the presence of high levels of serum p24 antigen.     

p73和p51基因在结直肠癌中的表达和意义

背景：p53基因是一种肿瘤抑制基因，其家族成员p73和p51在结构上与p53具有高度同源性，影响细胞转录和凋亡的功能与p53相似。目的：研究p73和p51基因在结直肠癌中的表达及其与细胞凋亡和肿瘤临床病理特征的关系，探讨两者在结直肠癌发生、发展中的可能作用。方法：以逆转录聚合酶链反应（RT—PCR）检测60例结直肠癌组织和相应癌旁组织中p73、p51mRNA表达，以原位末端标记（TUNEL）法检测细胞凋亡。结果：结直肠癌组织p73、p51AmRNA表达阳性率显著高于相应癌旁组织（p73：71．7％对5．0％，P〈0．01：p51A：46．7％对11．7％，P〈0．01）：p51B mRNA在结直肠癌组织与相应癌旁组织中的相对表达量无明显差异（0．7318±0．3628对0．6836±0．3516，P〉0．05）。p73、p51A mRNA表达阳性者肿瘤细胞凋亡指数分别显著低于p73、p51A mRNA表达阴性者（p73：3．2％±2．5％对5．5％±2．8％．P=0．003；p51A：2．6％±2．3％对4．9％±2．7％，P=0．001）。p73mRNA表达与结直肠癌的分化程度、TNM分期和淋巴结转移相关（P〈0．05），p51A mRNA表达仅与淋巴结转移相关（P〈0．05）。结论：结直肠癌中p73、p51A基因表达上调，两者可能通过抑制肿瘤细胞凋亡而参与了结直肠癌的发生、发展。p73过表达可能与结直肠癌预后不良有关。     

p21和p27基因多态性与妇科肿瘤的相关性研究

目的探讨p21和p27基因多态性与妇科肿瘤遗传易感性的关系。方法采用聚合酶链反应—限制性片段长度多态性方法,分析100例妇科肿瘤患者和95例健康对照者的p21基因3′非翻译区（3′UTR）和p27基因第109密码子多态性位点的基因型。结果妇科肿瘤患者p21基因突变型TT频率为26.00%,对照者为16.84%,两者相比,P〈0.01;年龄〈46岁组肿瘤患者的p21基因突变型（CT＋TT）频率为86.84%,≥46岁组为59.52%,两组相比,P〈0.01;中低分化组妇科肿瘤患者的突变型CT和TT基因型频率为76.47%,高分化组为46.67%,两组相比,P〈0.05;妇科肿瘤患者与对照者的p27基因型总体分布及V和G等位基因频率相比,P均〈0.01。结论p21基因3′UTR多态性和p27基因V109G多态性与妇科肿瘤的遗传易感性有关。