8—Br—cAMP对人食管癌Eca—109细胞生长相关基因表达的影响

目的 探讨８－Ｂｒ－ｃＡＭＰ对人Ｅｃａ－１０９细胞与生长相关基因表达的影响。方法 体外培育的人食管癌Ｅｃａ－１０９细胞受８－Ｂｒ－ｃＡＭＰ处理后,用原位杂交,免疫组织化学及ＲＮＡ,蛋白质斑点印迹技术研究了与细胞生长相关的几种基因的表达变化。结果 ８－Ｂｒ－ｃＡＭＰ可减弱ＥＧＦＲ,Ｈ－ｒａｓ,ｃ－ｍｙｃ,突变型ｐ５３等原表达,增强野生型ｐ５３基因表达和ｐ２１＾ＷＡＦ１蛋白的表达。     

8-Br-cAMP对人食管癌Eca-109细胞生长相关基因表达的影响

目的 探讨８－Ｂｒ－ｃＡＭＰ对人Ｅｃａ－１０９细胞与生长相关基因表达的影响。方法 体外培育的人食管癌Ｅｃａ－１０９细胞受８－Ｂｒ－ｃＡＭＰ处理后,用原位杂交,免疫组织化学及ＲＮＡ,蛋白质斑点印迹技术研究了与细胞生长相关的几种基因的表达变化。结果 ８－Ｂｒ－ｃＡＭＰ可减弱ＥＧＦＲ,Ｈ－ｒａｓ,ｃ－ｍｙｃ,突变型ｐ５３等原表达,增强野生型ｐ５３基因表达和ｐ２１＾ＷＡＦ１蛋白的表达。     

ER阳性和ER阴性人乳腺癌细胞株p53,mdm—2及p21^WAF1蛋白表达…

目的 研究ＥＲ阳性和ＥＲ阴性人乳腺癌细胞株ｐ５３、ｍｄｍ－２和ｐ２１＾ＷＡＦ１蛋白的表达及其与细胞生物学特性的关系。方法 应用细胞培养、基因转染和免疫组化染色ＬＳＡＢ法等技术，检测ＥＲ阳性、表达野生型ｐ５３（ｗｔｐ５３）蛋白的ＭＣＦ－７细胞和ＥＲ阴性、表达突变型ｐ５３（ｍｔｐ５３）和ＭＤＡ－ＭＢ－２３１细胞以及ＥＲ转染阳性ＭＤＡ－ＭＢ－２３１细胞中ｐ５３、ｍｎｍ－２和ｐ２１＾ＷＡＦ１蛋白的表达水平     

ER阳性和ER阴性人乳腺癌细胞株p53、mdm-2及p21~(WAF1)蛋白表达的研究

目的研究ＥＲ阳性和ＥＲ阴性人乳腺癌细胞株ｐ５３、ｍｄｍ－２和ｐ２１ＷＡＦ１蛋白的表达及其与细胞生物学特性的关系。方法应用细胞培养、基因转染和免疫组化染色ＬＳＡＢ法等技术，检测ＥＲ阳性、表达野生型ｐ５３（ｗｔｐ５３）蛋白的ＭＣＦ－７细胞和ＥＲ阴性、表达突变型ｐ５３（ｍｔｐ５３）的ＭＤＡ－ＭＢ－２３１细胞以及ＥＲ转染阳性ＭＤＡ－ＭＢ－２３１细胞中ｐ５３、ｍｄｍ－２和ｐ２１ＷＡＦ１蛋白的表达水平，比较其与细胞生物学特性的关系。结果（１）ＭＣＦ－７细胞和ＭＤＡ－ＭＢ－２３１细胞ｐ５３蛋白的性质和分布明显不同，前者ｐ２１ＷＡＦ１和ｍｄｍ－２蛋白的表达水平明显高于后者（Ｐ＜０．０５），且前者的生物学特性较后者为好。（２）ＥＲ质粒转染ＭＤＡ－ＭＢ－２３１细胞后，其ｐ５３蛋白的表达水平降低（Ｐ＜０．０５），而ｍｄｍ－２蛋白的表达水平增加（Ｐ＜０．０５），生物学特性得以改善。结论乳腺癌细胞ＥＲ状态与ｐ５３和ｍｄｍ－２蛋白的表达水平以及生物学特性有关。     

斑点杂交及RNA酶保护分析法检测反义HSP90基因转染细胞 …

目的：检测ＨＳＰ９０反义核酸转染细胞后反义ＲＮＡ的表达,方法：采用打点杂交及ＲＮＡ酶保护分析法,结果：ＨＳＰ９０反义核酸转染细胞ＡＨ－ＳＧＣ７９０１,ＡＨ－ＳＧＣ７９０１／ＶＣＲ,ＡＨ－ＨＣＣ７４０２及ＡＨ－Ｅｃ１０９有ＨＳＰ９０反义ＲＮＡ的表达。结论：ＥＳＰ９０反义ＲＮＡ在ＡＨ－ＳＧＣ７９０１,ＡＨ－ＳＧＣ７９０１／ＶＣＲ,ＡＨ－ＨＣＣ７４０２及ＡＨ－Ｅｃ１０９细胞的表达,为进一步研究ＨＳＯ９０     

硫代反义寡核苷酸体外对HDV的抑制作用

观察硫代反义寡核苷酸（Ｓ－ＡＳＯＤＮ）体外对ＨＤＶ的抑制作用。方法在ＨＤＶ／ＨＢＶ感染人胎肝细胞中加入不同浓度的针对ＨＤＶ ＳｔｅｍⅠ区６８４－６９８位核苷酸的１５聚Ｓ－ＡＳＯＤＮ,分别采用ＥＬＩＳＡ和斑点杂交法检测上清液中ＨＤＡｇ和细胞中ＨＤＶ ＲＮＡ。结果ＨＢｓＡｇ、ＨＤＡｇ在感染后第２天至第１６天均可测出,以第４天至第１２天达高峰,加入Ｓ－ＡＳＯＮＤ（２、４、６μＭＯＬ／ｌ）ＲＧ ２ＧＤ ,     

急性丙型肝炎患者免疫状况的研究

用间接免疫荧光法、酶联免疫吸附试验（ＥＬＩＳＡ）及ＬＤＨ释法，对１６例急性输血后丙型肝炎（抗－ＨＣＶ、ＨＣＶ－ＲＮＡ均阳性）患者，分别进行外周血单个核细胞（ＰＢＭＣ）的Ｔ细胞亚群计数、Ｔ４／Ｔ８比值、Ｔａｃ受体的检测及血清可溶性白细胞介素２受体（ｓＩＬ－２Ｒ）ＮＫ细胞活性的测定。并与正常人组比较，经ｔ检验发现，急性丙型肝炎患者的Ｔ４亚群所占百分比、Ｔ４／Ｔ８比值及ＰＢＭＣＴａｃ受体表达均明显低于正常人组（Ｐ＜０．０５），而ＮＫ细胞活性、血清ｓＩＬ－２Ｒ明显高于正常人组（Ｐ＜０．０５）。患者的这些免疫状态改变，可能对其发病机理的研究有一定意义。     

CHANGES OF 6-K-PGF_(1a) RELEASE FROM THE LUMINAL SURFACE OF DACRON SEEDED WITH AUTOLOOUS VENOUS TISSUE FRAGMENTS

ＣＨＡＮＧＥＳＯＦ６－Ｋ－ＰＧＦ１ａＲＥＬＥＡＳＥＦＲＯＭＴＨＥＬＵＭＩＮＡＬＳＵＲＦＡＣＥＯＦＤＡＣＲＯＮＳＥＥＤＥＤＷＩＴＨＡＵＴＯＬＯＯＵＳＶＥＮＯＵＳＴＩＳＳＵＥＦＲＡＧＭＥＮＴＳＣＨＡＮＧＥＳＯＦ６－Ｋ－ＰＧＦ１ａＲＥＬＥＡＳＥＦＲＯＭＴＨ...     

人野生型P^53基因与逆转录病毒载体的构建及转染

应用逆转录病毒为载体构建了含人野生型Ｐ＾５３基因的重组质粒，并成功地转染了ψｃｒｉｐ包装细胞。实验应用的为ＰＬＸＳＨ，１．８ｋｂ野生型Ｐ＾５３作为插入片断，通过Ｔ４ＤＮＡ连接酶进行连接。用「α－＾３２Ｐ」ｄＣＴＰ标记野生型Ｐ＾５３基因片断为探针进行原位杂交。     

p^53,p^21^WAF1蛋白在非小细胞肺癌中的表达及其临床意义

目的 探讨原发性非小细胞肺癌中的ｐ＾５３、ｐ＾２１＾ＷＡＦ１蛋白表达与临床病理及预后的关系。方法 应用免疫组织化学（ＳＰ法）方法。共检测非小细胞肺癌１４７例,其中腺癌６６例,鳞癌６３例,腺鳞癌１４例,大细胞癌４例。结果 ｐ＾５３蛋白总阳性率为６１．２％（９０／１４７）,腺癌为５７．６％（３８／６６）,鳞癌阳性率为６３．５％（４０／６３）,腺鳞癌为７１．４％（１０／１４）,大细胞癌２例阳性,ｐ＾２１     

膀胱移行细胞癌细胞周期调节相关基因表达与肿瘤生物学行为的关系

目的：探讨细胞周期调节相关基因p16INK4,p21WAF／CIP1,p53mlt在膀胱移行细胞癌中的表达与肿瘤增殖能力,病理分级及临床分期的关系。方法：应用免疫组织化学技术分析77例膀胱移行细胞癌组织p16INK4,p21WAF/CIP1,p53mlt基因的表达和增殖细胞核抗原（PCNA）表达情况,并与病理分级及临床分期之间进行综合分析。结果：p16INK4,p21WAF/CIP1,p53mlt在膀胱移行细胞癌中的表达及PCNA增殖指数与肿瘤的病理分级有关,与临床分期无关。p16,p21,p53阳性组与阴性组分别比较,其PCNA增殖数之间有差异性。多因素分析发现,p16INK4和p21WAF／CIP1阴性及p53mlt阳性组的PCNA值明显高于p16INK4和p21WAF／CIP1阳性及p53mlt阳性组,两者比较不同病理分级的阳性表达构成比亦有显著性差异。结论:联合检测p16INK4,p21WAF/CIP1,p53mlt基因的表达情况能充分反映膀胱移行细胞癌的增殖能力及生物学行为,对膀胱移行细胞癌患者的预后判断及治疗有指导意义。     

Frequent p16ink4a inactivation is an early and frequent event of intraductal papillary neoplasm of the liver arising in hepatolithiasis

Intraductal papillary neoplasm of the liver (IPNL) is a precursor lesion of intrahepatic cholangiocarcinoma (ICC) arising in hepatolithiasis. In this study, 98 foci of IPNL identified in 39 surgically resected hepatolithiatic livers were investigated for expression of p16INK4a, cyclin D1, p21WAF1/CIP1, p53, mouse double-minute 2 (MDM2), and pRb. In addition, methylation-specific polymerase chain reaction (MSP) for p16 INK4a promoter region was performed in these foci. Nonneoplastic bile ducts from 11 hepatolithiatic livers, 5 histologically normal livers, and 9 cases of nonpapillary conventional ICC were used as controls. Decreased expression of p16INK4A was seen in IPNL group 1 with mild dysplasia and continued along the progression of IPNL to ICC. The expression of cyclin D1, p21WAF1/CIP1,and pRb gradually increased along the progression of IPNL to ICC and became significantly high in IPNL of group 3 (carcinoma in situ). The expression of p53 and MDM2 was increased in IPNL group 3 and group 4 with evident invasive carcinoma. MSP revealed that 54.6% of 44 IPNL foci harbored p16INK4a promoter hypermethylation, and such foci were significantly correlated with decreased expression of p16INK4a protein. Ki-67 labeling index exhibited a stepwise increase from IPNL group 1 to group 4. We conclude that p16INK4a inactivation, due mainly to its promoter hypermethylation, is a frequent and early event of IPNL and may be responsible for genetic and epigenetic alterations of other cell cycle regulators in IPNL.     

Cell cycle regulators in bladder cancer: a multivariate survival study with emphasis on p27Kip1

Cyclin-dependent kinase inhibitors (CKIs) prevent cyclin-dependent kinases from phosphorylating critical substrates such as retinoblastoma gene protein (pRb), hence blocking the cascade of events leading to cell proliferation. Currently, the list of CKIs includes p21WAF1/Cip1, p27Kip1, p57Kip2 (the Cip/Kip family), p15/ INK4b, p16/INK4a, p18/INK4c, and p19/INK4d (the INK4 family). Among them, p27 plays a crucial role linking extracellular growth-regulatory signals to progression to or exit from the cell cycle. Unlike p53, p16, and Rb, mutations in Kip1 and WAF1 genes are distinctly rare in bladder cancer. We analyzed immunohistochemically the expression of p27 and other interacting G1 proteins (ie, p21, p16, pRb, p53) in 120 consecutive cases of transitional cell carcinomas (TCCs) and related it to proliferation rate, clinicopathologic parameters, and survival. p27 levels were significantly higher in low-grade (P = .001), superficial (Ta-T1) (P = .001), papillary (P < .001), and slowly proliferating TCCs (rs = -0.235, P = .05). p27 also positively correlated with p16 expression (rs = 0.212, P = .05). In univariate analysis, decreased p27 expression was associated with poor overall (P = .0109) and postrelapse (P = .0344) survival, especially if combined to increased Ki-67 expression (P = .0004 and P = .036, respectively). Furthermore, in multivariate analysis, Ki-67/p27 status had the strongest bearing on the overall survival of muscle-invasive TCCs (P = .0019). Our results indicate that low p27 expression is more common in poorly differentiated muscle-invasive TCCs and is a major player in cell cycle control in these neoplasms. More importantly, the combined Ki-67/p27 expression provides prognostic information beyond that provided by conventional parameters or other cell cycle-related proteins, concerning overall survival in muscle-invasive TCCs.     

Progressive deregulation of the cell cycle with higher tumor grade in the stroma of breast phyllodes tumors

We studied cell cycle-regulating proteins in phyllodes tumor pathogenesis by immunohistochemical analysis for Ki-67, cyclin A, cyclin D1, retinoblastoma protein (pRb), p53, p16INK4A, bcl-2, and p21waf1 in the epithelium and stroma of 40 primary (benign, 21; borderline, 8; malignant, 11) and 7 recurrent tumors of different grades. In most cases, the epithelium showed no altered expression of cell cycle regulators. Stromal overexpression of p16INK4A, p53, cyclin A, pRb, and p21waf1 correlated significantly with tumor grade. The number of altered proteins in stroma increased with higher grade and was accompanied by increased proliferation. Stromal cyclin A expression was the best separating marker between tumor grades. Correlations existed between stromal overexpression of p16NK4A and p21waf1, p16INK4A and p53, and p53 and pRb. No immunostaining differences were detected between primary tumors and recurrences. Four or more altered proteins and p53 expression in the stromal component were independent negative prognosticators for disease-free survival. The stromal component of mammary phyllodes tumors displays an increasing level of cell cycle deregulation with higher tumor grade; the epithelial compartment mostly remains inconspicuous. Several combinations of aberrantly expressed cell cycle proteins seem important in the stromal progression of phyllodes tumors. The number of stromal cell cycle aberrations and stromal p53 expression might predict clinical behavior.     

Differential cell cycle response of nontumorigenic and tumorigenic human papillomavirus-positive keratinocytes towards transforming growth factor-β1

Human papillomaviruses (HPVs) are causative agents of a number of malignancies in humans, including cervical cancer. Their tumorigenic potential is linked to expression of the viral E6/E7 genes which can interfere with normal cell cycle control by targeting p53, p21WAF1, p27KIP1, and pRb. We show here that nontumorigenic and tumorigenic HPV-positive keratinocytes (HPK) exhibit striking differences in the response of cell cycle regulatory genes towards transforming growth factor beta-beta1. Treatment with this agent led to an efficient induction of p53 and the growth-inhibitory p15INK4 and p21WAF1 genes only in nontumorigenic HPKs and was linked to an efficient reduction in viral E6/E7 oncogene expression. This was associated with increased pRb levels, exhibiting sustained hypophosphorylation, and a permanent growth arrest in the G1 phase of the cell cycle. In contrast, tumorigenic HPKs exhibited only a modest rise in p53 protein levels and a substantially reduced induction of the p15INK4 and p21WAF1 genes, which was linked to a lesser degree of viral oncogene repression. In addition, tumorigenic HPKs rapidly resumed cell growth after a transient G1 arrest, concomitantly with the reappearance of hyperphosphorylated pRb. These results support the notion that the progression of HPV-positive cells to a malignant phenotype is associated with increased resistance to growth inhibition by transforming growth factor-beta1. This is linked in the tumorigenic cells to a lack of persistent G1 arrest, inefficient induction of several cell cycle control genes involved in growth inhibition, and inefficient repression of the growth-promoting viral E6/E7 oncogenes.     

The site of infection and ethnicity of the patient influence the biological pathways to HPV-induced mucosal cancer.

High-risk human papillomaviruses are the causative agents of cervical cancer and are also believed to be aetiologically involved in a subset of squamous cell carcinomas of the head and neck region, especially the tonsil. Cervical cancers arise through disruption of the pathways of p53 and the product of the retinoblastoma gene by the human papillomavirus oncoproteins E6 and E7. It is generally assumed that the same pathways are involved in human papillomavirus-induced carcinogenesis at other mucosal surfaces. However, the patterns of expression of cell cycle proteins targeted by human papillomavirus E6 and E7 in cancers from different anatomic sites have been inconsistent, due to either biologic or technological factors. In this study, 73 human papillomavirus, 16-positive cervical squamous cell carcinomas (35 from Australian and 38 from Chinese women) were analysed for the expression of p53, pRb, p16(INK4A), p21(CIP1/WAF1), p27(KIP1) and cyclin D1 by semiquantitative immunohistochemistry. Cervical cancers from Chinese women were found to be significantly more likely to overexpress p53, pRb, p21 and p27 than their Australian counterparts. These findings were compared with those from 31 human papillomavirus 16-positive tonsillar squamous cell carcinomas, all of Australian origin, tested using the same methodology. Comparisons of the tonsillar and combined cervical data showed that tonsillar cancers were significantly more likely to be p53-positive, whereas cervical cancers were significantly more likely to overexpress pRb, p16 and p27. When the tonsillar data were compared with cervical data from Australian women, the associations for p53 and pRb remained. These findings represent new evidence that the molecular pathways to human papillomavirus-induced mucosal cancer may be influenced by anatomic location and ethnicity.     

HIV-1感染引起组蛋白乙酰化和增加p21WAF1表达

目的：DNA甲基化和组蛋白乙酰化是基因表达调控的主要形式。人类免疫缺陷病毒（HIV-1）可引起T淋巴细胞DNA甲基化酶上调。本文旨在明确HIV-1对细胞周期依赖刺激酶抑制剂p21^WAF1表达的影响。方法：建立HIV-1感染的Hut78细胞系；以RT-PCR和Westem blotting 分析p21^WAF1表达情况；以亚硫酸氢钠修饰DNA和基因测序，研究p21^WAF1基因启动子甲基化，以Western blot-ting 和染色体免疫测定探究总组蛋白和与p21^WAF1基因启动子相关的组蛋白乙酰化水平。并以GST pull-down和免疫沉淀分析HIV-1导致乙酰化及乙酰化引起p21^WAF1过表达的可能机理。结果：HIV-1感染后，其反式激活蛋白Tat与辅助转录因子P/CAF、hGCN5结合，共同刺激组蛋白H3乙酰化。尽管p21^WAF1启动子部分区域有甲基化发生，但p21^WAF1表达仍上调。这可能与E2A对p21^WAF1的作用有关。结论：HIV-1感染可引起T淋巴细胞p21^WAF1基因的甲基化和乙酰化紊乱，导致p21^WAF1表达增强。