p53、Rb、p16抑癌基因在胃粘膜上皮异型增生病变中表达的意义

目的：研究p53、Rb和P16 3个抑癌基因在正常胃粘膜→异型增生粘膜→胃癌发展过程中的表达状态及相互关系。方法：收集胃手术及胃镜活检标本共60例,镜下观察胃粘膜并选取不增生病灶、35例胃腺癌和32例正常胃粘膜中的表达情况。应用聚合酶链反应单链构象多态性分析技术（PCR-SSCP）对35例胃腺癌进行p16基因点突变检测。结果：p53阳性率在轻、中、重度异型增生病灶的分别为7.5%、35.1%、59.1%,胃癌组为68.6%,正常对照组中无表达。Rb蛋白阳性率在轻、中、重度异型增生病灶中分别为80.0%、89.3%、81.8%,胃癌组为57.1%,正常对照组为90.6%。p16蛋白在正常胃粘膜、异型增生粘膜和胃癌中普遍表达,3组间阳性率比较无显著性差异。p16基因PCR-SSCP分析示只有1例低分化腺癌出现异常单链泳动带。不同类型（隐窝型、腺瘤型、再生型）异型增生病灶组间、位于癌旁和良性病变旁的异型增生病组间p53、Rb和p16蛋白的阳性率无显著性差异。高-中分化、低分化、未分化胃癌组p53、Rb和p16蛋白的阳性率无显著性差异。结论：突变型p53蛋白的积聚在胃粘膜异型增生阶段已经开始,随着异型增生程度的加重逐渐增加,重度病变中p53表达率与胃癌组相似,提示p53基因突变是胃癌发生过程中的早期事件。Rb蛋白在癌变组中缺失率较异型增生显著,可能是胃癌发生过程中的较晚事件。推测在p53基因突变的基础上加以Rb蛋白的缺失最终导致胃粘膜上皮癌变。p16蛋白的表达在胃癌发生过程的3个阶段病变中无显著的变化,可能不起要作用。不同类型（隐窝型、腺瘤型、再生型）异型增生间、癌旁或良性病变旁的异型增生间,不同分化程度的胃腺癌组间这3种抑癌基因蛋白表达状态基本相同。     

丙型肝炎病毒感染与ras,p53基因表达的相关性

为了探讨丙型肝炎病毒（ＨＣＶ）感染与肝细胞癌（ＨＣＣ）的关系以及ＨＣＶ可能的致癌机理，采用免疫组织化学方法及巢式ＰＣＲ法检测了１３６例肝细胞癌等肝病组织中的ＨＣＶＮＳ３抗原、ＨＣＶＲＮＡ及Ｐ２１、Ｐ５３蛋白。结果表明，肝细胞癌及癌周肝组织中有ＨＣＶＮＳ３抗原及ＨＣＶＲＮＡ检出，支持ＨＣＶ与ＨＣＣ的关联。Ｐ２１在ＨＣＣ、肝炎后肝硬化、慢性肝炎、体质性黄疸各组中的检出率随病变的加重而逐渐增高，在ＨＣＣ的癌及癌周组织中Ｐ２１呈致密的过量表达，提示ｒａｓ癌基因的激活在ＨＣＣ的发生过程中起一定作用。Ｐ５３的阳性率较Ｐ２１低，但ｐ５３的突变似乎也是肝癌发生的协同因素之一。组织中Ｐ２１的过量表达与ＨＣＶＮＳ３抗原阳性检出呈正相关，ＨＣＶＮＳ３抗原与Ｐ２１的这种关联提示，ＨＣＶ感染作为ＨＣＣ的密切相关因素之一，可能通过激活某些癌基因或使某些抑癌基因突变而致肝细胞癌变     

早期肺癌病人血液中P53的克隆及其pEGFP-N1-p53的构建

首先用RT-PCR方法从早期肺癌病人的外周血中克隆出人抗癌基因p53,经测序确认为野生型后,通过酶切、连接、转化构建出扩增质粒pMD18-T-p53,然后构建出含增强型绿色荧光蛋白(EGFP)基因的真核表达质粒pEGFP-N1-p53.pEGFP-N1-P53经限制性内切酶Hind Ⅲ和BamH Ⅰ酶切,酶切产物电泳结...     

p53蛋白对HL-60肿瘤细胞凋亡率的影响

实验以人白血病HL-60细胞为实验对象,观察p53蛋白对HL-60细胞凋亡的影响。采用吖啶橙（AO）荧光染色法分析p53蛋白对HL-60细胞生长的影响,所呈现的量效和时效关系,运用形态学、吖啶橙荧光染色法、透射电子显微镜观察法检测细胞凋亡。     

Interaction of hepatitis B virus with tumor suppressor gene p53: its significance and biological function

The mechanism of the interaction of hepatitis B virus (HBV) with tumor suppressor p53 and its role in the hepatocar-cinogenesis have been studied by PCR-directed sequencing, gel shift assays and in situ ultraviolet cross-linking assay. The biological function of the interaction of HBV with p53 gene was investigated by co-transfection of chloramphenicol acetyltransferase ( CAT) reporter gene. p53 and HBV DNA. and quantitative PCR. Among the 16 primary hepatocellular carcinoma (PHC) samples. 13 were HBV-DNA positive. 10 HBxAg positive and 9 p53 protein positive. The p53 gene point mutation was found in 5 samples, one of which had a G to T substitution located at codon 249. After analyzing the HBV genome by a computer program, a p53 response element binding sequence was found in HBV genome at upstream of enhancer I. from 1047 to 1059 nucleotides. This sequence could specifically bind to p53 protein, increase p53 protein accumulation in the PHC cells and stimulate the transactivating activity of p53 and HBV replication . The results also revealed that HBxAg could combine with p53 protein to form a complex in the cells and enhance CAT expression. Immunocytochemical staining showed that p53 protein complex was located in the cytoplasm and the process of p53 entry to nuclei was. in part, blocked. From our results, we conclude that the mutation of p53 gene at codon 249 is infrequent in HBV-associated PHC. the DNA-protein binding between HBV and p53. and the protein-protein binding between HBxAg and p53 might lead to the reduction or inactivation of p53 protein, which in turn resulting in HBV-associated hepatocarcinogenesis.     

DNA甲基化在肿瘤形成中的作用（综述）

DNA甲基化改变是肿瘤细胞中常见的现象,DNA甲基化与肿瘤的发生有密切关系。从以下几方面对此做一综述。（1）简介哺乳动物细胞的DNA甲基化;（2）DNA甲基化与肿瘤基因突变;（3）肿瘤DNA甲基化的基因外作用,其中包括：原癌基因的低甲基化和抑癌基因的高甲基化。     

胃癌中p53基因失活及其蛋白产物的免疫组化分析

应用ＰＣＲ－ＳＳＣＰ银染，Ｓｏｕｔｈｅｒｎ杂我及免疫组化等方法同步研究２５例胃癌标本的ｐ５３基因突变，杂合性丢失及蛋白持表达，在２２例胃癌中同时获得有关ｐ５３基因突变和杂合性丢失的检测结果，被检出ｐ５３基因突变的５例胃癌中，２例伴有杂合性丢失，３例仅有ｐ５３基因突变。     

Effects of enhancing p21 waf1 on proliferation and expression of G1cyclins andCDKs in human breast cancer cells

The cyclin-dependent kinase inhibitor p21( waf1/cip1/sdil) is an important negative regulator in control of cell cycle. Its functions of inhibiting cancer cell growth and its effects on expression of G1 phase cyclins and related CDKs are a worthy topic for study. The plasmid expressing p2l with high level was transformed to human breast cancer cells, and the expression of p2l in cells was enhanced, then the cell growth rate, anchorage-independent growth and tu-morigenecity were tested, at the same time the expression levels of cyclinD1, CDK4, cyclinE and CDK2 were analyzed by Northern blot. The results showed that since the expression of p21 was enhanced in the cell, the rate of cell growth and anchorage-independent growth was inhibited, tumorigenecity was suppressed, the level of expression of cyclinE and CDK2 decreased while that of cyclinDl and CDK4 was not affected. It is suggested that the enhanced expression of p21 markedly inhibits the proliferation and lessens the tumorigenecity of breast cancer cells, and that p2l expression is not related to that of cyclinDl and CDK4, but affects the expression of cyclinE and CDK2 .     

PTEN抑癌基因与胃癌关系的研究进展

PTEN基因是近年发现的新抑癌基因,目前已经证实其与多种肿瘤的发生发展有密切的关系。在许多肿瘤中都有PTEN基因表达异常,且其表达异常与肿瘤发生发展的生物学行为有一定相关性。胃癌是我国常见的多发性的恶性肿瘤之一,在胃癌组织中PTEN通过多种途径表达降低。PTEN的突变和异常表达与胃癌的发生、发展、浸润、转移显著相关,可能成为检测胃癌的有效指标之一。     

子宫颈癌抑癌基因P16第一外显子CpG岛异常甲基化

利用限制性核酸内切酶-PCR方法分析33例肿瘤组织和15例正常组织抑癌基因p16第一外显子SacⅡ和SmaⅠ酶切位点甲基化,应用SPSS软件进行统计分析。结果显示,18例子宫颈癌在抑癌基因p16第一外显子SacⅡ位点甲基化,8例子宫颈癌在SmaⅠ位点甲基化。正常组织仅有两例在抑癌基因p16第一外显子SacⅡ位点甲基化,正常组织在SmaⅠ位点没有甲基化。另外,抑癌基因p16第一外显子SacⅡ和SmaⅠ酶切位点甲基化易发生在肿瘤的早期。抑癌基因p16第一外显子SacⅡ和SmaⅠ酶切位点甲基化与子宫颈癌相关。     

云芝多糖影响人宫颈癌HeLa细胞的增殖和凋亡的研究

研究云芝多糖(CVP)对宫颈癌HeLa细胞系的增殖、凋亡及产生途径。采用MTT法测定了CVP对HeLa细胞增殖的体外抑制作用,流式细胞仪检测凋亡细胞的比例,qRT-PCR检测P53、Bcl-2和Fas基因在细胞处理前后表达量的变化。结果显示：CVP以时间和剂量依赖的方式抑制HeLa细胞的生长。作用时间在24 h,48 h和72 h时对细胞抑制的抑制率分别为92%、95%和98%;当CVP浓度达到1.25 mg/L时,作用24 h、48 h和72 h时的IC50值分别为0.2507±0.01 mg/L、0.2720±0.04 mg/L和0.2736±0.03 mg/L;当CVP浓度为0.5 mg/L时,作用24 h和48 h后细胞凋亡率分别为26.36% 和63.81%;CVP处理HeLa细胞24 h后,Bcl-2基因的表达被显著下调。研究表明：CVP促进HeLa细胞凋亡,其作用机制与Bcl-2基因的表达下调有关,推测是通过Bcl-2介导的途径促进细胞凋亡。图4表1参30     

用计算机对人类TSPYl基因P53结合位点的鉴定

根据p53下游基因在其调节区域（启动子或内含子）含有与P53蛋白特异性结合的一致性序列5’-RRRCWWGYYYN（0-13）RRRCWWGYYY-3’，R—G或A，W—T或A，Y—C或T，N—A，C，T，G。用计算机对人类基因组中P53结合位点进行了研究，发现Y染色体上的TSPY1基因内含子中含有这样的一致性序列5’-GGGCTAGTTTtgGAGCTAGCCT-3’，意味着TSPY1基因有可能是一个p53下游基因。     

用计算机对人类TSPY1基因P53结合位点的鉴定

根据p53下游基因在其调节区域（启动子或内含子）含有与P53蛋白特异性结合的一致性序列5’ –RRRCWWGYYYN（013) RRRCWWGYYY3’,R=G或A,W=T或A,Y=C或T,N=A,C,T,G。用计算机对人类基因组中P53结合位点进行了研究,发现Y染色体上的TSPY1基因内含子中含有这样的一致性序列5’GGGCTAGTTTtgGAGCTAGCCT3’,意味着TSPY1基因有可能是一个p53下游基因。     

Cloning of theAspergillus awamori glucoamylase gene and expression inSaccharomyces cerevisiae

The A. awamori glucoamylase I gene was obtained by RT_PCR and inserted into the plasmid pAC1. We constructed an expression vector pAC1_GA, which was used to transform the yeast S. cerevisiae AS 2 1364 without auxotrophic marker. The transformant secreted glucoamylase into the medium efficiently and degraded starch. The glucoamylase activity of the culture filtrate is up to 8 4 U/mL.     

Influence of DNA methylation on transgene expression

DNA methylation plays an important role in gene expression in eukaryote. But DNA methylation of transgene usually leads to target gene silencing in plant genetic engineering. In this research, reporter gene b-glu- curonidase (GUS) gene ( uidA ) was introduced into tobaccos via Agrobacterium-mediated transformation method, and the foreign uidA gene became inactive in some transgenic tobaccos. No mRNA of uidA was detected in these plants by Northern blotting analysis, and DNA methylation of promoter region was found. The results indicated that gene silencing might be caused by DNA methylation of promoter.     

Construction and expression of retroviruses encoding dual drug resistance genes in human umbilical cord blood CD34+ cells

A series of retroviral vectors encoding human mdr1 gene alone as well as in combination with either human mgmt gene or human mutant Ser31-dhfr gene are engineered. The resultant retroviruses are used to transduce human umbilical cord blood CD34+ cells. It has been shown that expression of dual drug resistance genes in transduced cells confers a broad range of resistance to both kinds of corresponding drugs. These data suggest a rationale for the use of such double chemoresistance gene constructs in an in vivo model in which transduced hematopoietic cells will acquire multiple protection against the cytotoxic side effects of combination chemotherapy and may have future application in chemoprotection of normal tissues, thus killing tumor cells more effectively.     

Secretory expression of the fusion protein composed of human IL-2 and PreS antigen of hepatitis B virus in mammalian cells

In order to make entire HBV pmSAg secrete from mammalian cells, we conatmcted an eukaryotic expression vector by using leader sequence of human interleukin-2 (IL-2) as secretory signal peptide, and using high hydmphilic amino acids as the linker between IL-2 C end and preSAg N end. As a result, the IL-2preS fusing protein could be secreted from mamalian cells transfected with the reconstructed vector and the expression efficiency was identical to that of natural IL-2. It was considered that the retentive effect of preSlAg could be successfully bypassed. The results not only laid a theoretical and practical foundation for constructing specific gene vaccine against HBV persistent infection, but also supplied experimental evidence for studying modulation of protein secretory expression.