人类恶性畸胎瘤PA-1细胞的p53基因结构、功能状态及其意义

目的:探讨人类恶性畸胎瘤PA-1细胞株p53基因的结构、功能状态及其意义。方法:采用DNA碱基序列分析、FASAY（酵母细胞中单个等位基因的功能分析）及Western blot（蛋白印迹分析）等方法对经20余年407-445继代培养的PA-1细胞株p53的基因结构、功能状态进行了研究。结果:RT-PCR产物DNA定向序列分析显示具有野生及突变2个带（p53密码子239突变）,FASAY结果也显示p53一个等位基因是有活性的（野生的）,而另一个是非活性的（突变的）。Western blot未检测出突变的p53基因蛋白,而p21蛋白的表达水平比正常成纤维细胞低。结论:人卵巢恶性畸胎瘤PA-1细胞株经20年的继代培养后,一个p53等位基因发生错义突变,另一个仍然是野生型的。仅一个p53等位基因发生突变,不足以引起细胞的染色体不稳定性。因此,研究PA-1细胞稳定核型的维持结构,在确定有关染色体的稳定性与不稳定性的基因的方面是非常重要的。     

p53蛋白三维空间结构改变与人骨肉瘤的关系

目的 研究骨肉瘤中p53基因突变及其蛋白三维结构的改变,旨在分析p53基因突变的分子机制及其与骨肉瘤发生、发展的关系.方法 应用PCR-SSCP、DNA序列分析以及相关分析软件重构p53蛋白三维结构等技术对43例骨肉瘤进行研究.结果 将16例p53基因突变病例分为A组(p53基因DNA结合位点发生突变或缺损组)与B组(非DNA结合位点突变组或同义突变),A组的远处转移率明显高于B组(P<0.01),组织学分化低于B组(P<0.05),且中位生存时间(10.3个月)低于B组(34.3个月).结论 p53基因碱基的突变可引起相应氨基酸的改变,导致p53蛋白三维空间构象改变.p53基因DNA结合位点的突变可影响其蛋白的生物学功能,并促进骨肉瘤的发生、发展.     

p53与肿瘤的形成,治疗和预后

p53基因作为抑制基因在约50％的人类肿瘤中被检测出来,其蛋白产物p53蛋白与特异性DNA序列结合并激活转录。转录激活导致p53增加,进而诱导蛋白p21、WAF1／CIP1产生,通过p21与。cyclin－cdk复合物结合来抑制cdk活性,从而达到控制细胞从GI期进入S期的目的．也可通过p21与PCNA结合抑制DNA复制。p53在肿瘤形成中的作用50－55％的人类肿瘤中发现有p53突变。这些突变完全淘汰不能与特异DNA序列结合的P53蛋白。P53的细胞周期抑制功能需要p53转录活性,至少有些p53的凋亡活动不需要有赖于p53的基因产物。这可能意味着,被选p53靶基因的…     

个体性反义RNA对乳腺癌细胞突变p53基因表达的特异性封闭作用

目的研究针对p53基因突变的个体性反义RNA对乳腺癌突变p53基因的特异性封闭作用。方法通过免疫细胞化学LSAB法染色、聚合酶链反应-单链构象多态性分析及测序确定人乳腺癌细胞系MDA-MB-231中p53突变位点,构建针对该突变点的反义表达载体并制备其反义RNA。原位杂交技术检测反义RNA与细胞内突变p53基因的特异性结合;在阳离子脂质体介导下将反义RNA转染细胞;以免疫细胞化学LSAB法染色及细胞生长抑制率观测反义RNA作用时效;Western blot检测p53蛋白的表达;四甲基偶氮唑盐法检测转染细胞的生长活性;流式细胞术检测转染细胞的细胞周期分布;原位缺口末端标记法检测转染细胞的凋亡情况。结果人乳腺癌细胞系MDA—MB-231的p53第8外显子（exon8）有突变,据此位点构建反义表达载体[pGEM3zf（＋／-）p53exon8]。原位杂交结果显示反义RNA（ASp53exon8＇RNA）在细胞质内有阳性杂交信号;于转染后48h反义RNA封闭效果最显著,突变p53蛋白（mt-p53）的表达受抑制,细胞增殖能力下降,G2／M期阻滞。结论针对p53基因突变位点制备的个体性反义RNA可特异性封闭乳腺癌细胞突变p53基因的表达。     

结肠癌细胞HCT116中p53的R213突变对靶基因p21表达的影响

目的确定在结肠癌细胞系中,p53的213位精氨酸突变后对其下游靶基因p21表达的影响。方法在p53缺失的结肠癌细胞系HCT116~(-/-)中转染p53的R213位突变的质粒;荧光素酶双报告基因系统检测p53靶基因启动子活性;EMSA检测p53与其靶基因p21的结合能力;real-time PCR和Western blot检测p53下游靶基因p21的表达。结果 p53蛋白213位精氨酸突变为赖氨酸后,明显降低了p53靶基因的启动子活性(P0.05);与靶基因p21启动子的结合能力明显降低(P0.05);并抑制了p21基因的表达(P0.05)。而213位突变为谷氨酰胺后,迁移率比突变前明显变快。结论在人的结肠癌细胞中,p53蛋白R213位的不同突变对下游靶基因p21有不同的影响,提示存在着不同的调控机制。     

p53AIP1基因转染对PC-3M人前列腺癌细胞生物学特性的影响

目的观测p53调节的凋亡诱导蛋白1(p53AIP1)对PC-3M人前列腺癌细胞的增殖、细胞周期、凋亡、侵袭及迁移的影响。方法构建真核载体pDC316-p53AIP1,并进行双酶切及PCR鉴定。在LipofectamineTM2000介导下转染至体外培养的PC-3M人前列腺癌细胞系中,Western blot法检测其表达,CCK-8法检测PC-3M细胞增殖情况,流式细胞术分析PC-3M细胞周期变化和annexinV-FITC/PI结合流式细胞术凋亡情况,TranswellTM实验检测p53AIP1对细胞迁移及侵袭能力的影响。结果成功构建了pDC316-p53AIP1真核表达载体,重组质粒转染PC-3M细胞后,Western blot法检测p53AIP1在PC-3M细胞中有表达,CCK-8结果显示表达的p53AIP1能抑制PC-3M细胞增殖(P0.05),流式细胞术检测细胞周期阻滞在S/G2-M期(P0.05)且p53AIP1能促进PC-3M细胞凋亡,TranswellTM侵袭和迁移实验显示p53AIP1使细胞侵袭迁移能力均降低(P0.05)。结论p53AIP1抑制PC-3M细胞的周期、增殖、侵袭及迁移,并能促进其凋亡。     

一个非综合征手足裂畸形家系的临床调查及遗传学分析

目的分析一先天性手足裂伴并指／趾畸形家系的临床表现，并从分子水平查找致病原因，为罹患家庭提供遗传咨询。方法通过X线检查资料及手足裂外观照片，对家系3代现存3例患者（共4例患者）进行临床分析，对3例患者用常规方法制备外周血淋巴细胞染色体标本，进行G显带核型分析。从7名家系成员（包含3例患者）外周血样品中提取基因组DNA。针对p63基因全部15个外显子及删rJ0b基因5个外显子进行引物设计合成、PCR扩增、回收纯化并测序。结果家系中现存3名患者均表现为双手中央分裂，其中1例患者双足呈楔形裂开，2例患者右足均为第3、4趾并指，皮肤黏连；G显带核型分析未发现染色体畸变；p63基因未检测到突变，删rJ0b基因的外显子5a中发现一个碱基突变c．1058C〉T。结论通过家系内息者临床表型分析，可将该疾病类型确定为非综合征手足裂畸形，且临床症状逐代加重。测序结果提示p63基因和删rJ0b基因关键区域内的点突变都不是引起该家系手足裂畸形的原因。     

一例火棉胶样儿TGM1基因的突变分析CSCD

目的对1例维吾尔族火棉胶样患儿的TGM1基因进行突变分析,寻找其病因。方法对患儿及其父母进行常规染色体G显带核型分析,应用芯片高通量测序方法对患儿进行鱼鳞病及鱼鳞病样皮肤病25个相关基因的检测。 结果染色体G显带核型分析结果显示患儿及父母核型均正常;芯片捕获高通量测序检测出患儿TGM1基因的c.919C〉T（p.Arg307Trp）和c.856C〉T（p.Arg286Trp）复合杂合突变;前者为已知致病突变,后者为临床意义未明突变。Sanger测序验证,患儿父亲携带TGM1基因c.856C〉T（p.Arg286Trp）杂合突变,未检出患儿母亲TGM1基因c.919C〉T、c.856C〉T的位点突变。结论TGM1基因c.919C〉T和c.856C〉T复合杂合突变可能是患儿的致病原因。     

表达BG4蛋白对人胃癌AGS细胞凋亡和端粒酶逆转录酶表达的影响

目的:构建G-四链体抗体BG4基因的真核表达载体p EGFP-N1-BG4,探讨转染该真核表达载体致BG4蛋白过表达对人胃癌细胞株AGS凋亡和端粒酶逆转录酶表达的影响。方法:以BG4基因原核表达载体p SANG10-BG4为模板,PCR扩增出含有BG4基因的目的片段,经TA克隆与p MD18-T载体连接,测序正确后,经Sac I和Pst I双酶切,与真核表达载体p EGFP-N1连接后转化感受态大肠杆菌DH5α,并进行酶切分析和序列测定,Western blot鉴定BG4蛋白表达;流式细胞术检测细胞周期;DAPI法及HE染色检测各组细胞凋亡;q PCR和Western blot检测空白对照组、p EGFP-N1组和p EGFP-N1-BG4组端粒酶逆转录酶mRNA和蛋白表达的变化。结果:酶切分析显示,目的基因条带与预期结果相符,DNA测序表明克隆的BG4基因序列正确,Western blot结果表明转染p EGFP-N1-BG4质粒可以成功表达BG4蛋白。转染p EGFP-N1-BG4质粒可抑制细胞进入S期。DAPI法及HE染色显示,p EGFP-N1-BG4组出现明显的新月形核和核固缩的细胞凋亡现象。q PCR和Western blot结果表明,p EGFPN1-BG4组端粒酶逆转录酶表达受到抑制。结论:用p EGFP-N1-BG4真核表达载体转染胃癌细胞株AGS可以抑制该细胞端粒酶逆转录酶表达,诱导细胞凋亡。     

Maintenance of near-diploid karyotype of PA-1 human ovarian teratocarcinoma cells due to death of polyploid cells by chromosome fragmentation/pulverization.

Chromosome instability (polyploidy or aneuploidy) is one of the characteristics of malignant tumors. Human teratocarcinoma cell line PA-1, which was established more than 10 years ago, consists of a majority of near-diploid cells and a minority of polyploid cells, indicating that it is karyologically very stable. In the present study we investigated this genomic stability from the view point of cytogenetics. Cleavages and breaks in the chromosome were found in the metaphase of PA-1 polyploid cells, accompanied by the formation of polynucleosomal DNA fragments. These findings were absent in the near-diploid cells. In addition, polyploid cells did not show colony-formation ability by in situ analysis of cytogenetics in each colony. Thus, the maintenance of the near-diploid karyotype in PA-1 cells may be due to a blockage in the M-phase of the polyploid cells by functional mitotic checkpoints, if any, leading to cell death due to inability to enter the next cell cycle.     

The human ovarian teratocarcinoma cell line PA-1 demonstrates a single translocation: analysis with fluorescence in situ hybridization, spectral karyotyping, and bacterial artificial chromosome microarray

Cell lines derived from tumors contain numerous chromosomal aberrations and are the focus of study in tumor evolution. The ovarian teratocarcinoma cell line PA-1 demonstrates a single chromosomal aberration: a reciprocal t(15;20)(p11.2;q11.2). A complete molecular genetic analysis was undertaken to characterize this cell line. The PA-1 cell line was studied with fluorescence in situ hybridization (FISH), spectral karyotyping (SKY), bacterial artificial chromosome (BAC) microarray, and Western blotting. Amplification of 20q is frequently implicated in both breast and ovarian cancer; this region contains a number of oncogenes including MDM2, ZNF217, and the ovarian tumor marker WFDC2 (alias HE4). FISH revealed gene amplification of AIB1 (now known as NCOA3) but not STK15 (now known as AURKA). Immunoblot analysis demonstrated 3.6-fold overexpression of the AIB1 protein product, but no elevation of the STK15. BAC cancer gene microarray analysis showed gene amplification of > or =1.20 for five oncogenes. The presence of a consistent single change in PA-1, the t(15;20)(p11.2;q11.2), suggests that the aberration is significant with respect to the transformation status of the cell line. This translocation appears to cause overexpression of AIB1 (and perhaps other proteins), which may provide an immortalizing effect on this cell line.     

抗人CD3单链抗体/p53四聚功能域融合基因的构建及表达

目的：构建抗人CD3单链抗体(scFv)／p53四聚功能域融合基因，并进行真核表达及活性测定。方法：在已经构建抗人CD3 scFv和人IgG3上游铰链区／p53四聚功能域融合基因基础上，将抗人CD3 scFv克隆入载体pUC18／IgG3／p53中，构建抗人CD3 scFv／p53四聚功能域融合基因。经酶切鉴定及序列测定证实后，将融合基因克隆入真核表达载体pSecTag2-B中，并转染Hela细胞进行表达。表达产物纯化后，利用流式细胞仪进行活性测定。结果：获得了抗人CD3 scFv／p53四聚功能域融合基因，基因全长为882 bp，可编码294个氨基酸，与已发表的抗人CD3 scFv、人IgG3上游铰链区和人p53四聚功能域基因cDNA序列相一致。表达产物经SDS-PAGE和Western blot证实，为Mr约35000的特异蛋白条带。纯化后经流式细胞仪检测，可特异性地结合人外周血单个核细胞(PBMC)，亲和力高于scFv。结论：获得了可与PBMc特异性结合的抗人CD3 scFv四聚体，为进一步临床应用奠定了基础。     

p53 abnormality and chromosomal instability in the same breast tumor cells

To clarify the important role of the tumor-suppressor gene p53 in maintaining genetic integrity, we estimated chromosome instability and staining of overexpressed p53 protein in the same cells of five primary breast carcinomas. The method included both fluorescence immunohistochemistry and fluorescence in situ hybridization (FISH) on sections from formalin-fixed, paraffin-embedded breast cancer tissue. By using a centromeric FISH probe for chromosome 17 on interphase cells in these sections, we showed that cells with abnormal p53 protein expression had a statistically significant higher number of chromosome 17 than did cells with no p53 protein staining in the same samples as well as cells in four other tumor samples with no p53 protein staining. The samples identified positive for p53 abnormality by immunostaining were shown to have p53 mutation by constant denaturing gel electrophoresis analysis and DNA sequencing. These mutated samples were characterized by high DNA index, high S-phase, abnormal karyotype, and aneuploidy. The results strongly implicate p53 mutation as a cause for chromosomal instability and a crucial step in mammary carcinogenesis.     

人野生型p53 cDNA的克隆、表达和意义

目的：为探讨肿瘤细胞ｗｔ ｐ５３蛋白功能失活机制的研究,检测肿瘤患者血清抗ｐ５３抗体辅助临床诊断提供人ｗｔ ｐ５３蛋白。方法 将人ｗｔ ｐ５３基因ｃＤＮＡ片段插入到大肠杆菌表达载体ｐＱＥ－３０中,得到重组表达质粒ｐＱＥ３０－ｐ５３,用ｐＱＥ３０－ｐ５３重组质粒转化大肠杆菌Ｍ１５细胞,转化菌落经ＰＣＲ扩增和ＢａｍＨⅠ、Ｘｂａ Ｉ酶切证实ｐ５３基因插入。ＩＰＴＧ诱导大肠杆菌表达ｗｔ ｐ５３蛋白,通过Ｎ     

重组腺病毒介导的野生型p53对人胰腺癌细胞凋亡的诱导作用

目的研究恢复外源性野生型（ｗｔｐ）ｐ５３的表达对人胰腺癌细胞生长和凋亡的作用。方法构建了一个复制缺陷型５型腺病毒ｗｔｐ５３表达载体Ａｄ５ＣＭＶｗｔｐ５３，含有人ＣＭＶ启动子，人野生型ｐ５３和ＳＶ４０ｐｏｌｙＡ信号，经腺病毒包装细胞２９３细胞包装、扩增后，转染胰腺癌ＰＣ２细胞。ＰＣＲ和免疫沉淀技术证实转染的细胞有外源ｗｔｐ５３基因的存在和表达。结果转染的ＰＣ２细胞的生长率和３Ｈ掺入率降低。原位凋亡检测、流式细胞术和ＤＮＡ凝胶电泳显示转染细胞凋亡明显增多。而ＰＣ２细胞和用Ａｄ５ｐＸＪ转染的细胞没有这些改变。结论复制缺陷型腺病毒是一种安全高效的基因转移载体，恢复ｗｔｐ５３的表达可以有效地诱导凋亡和抑制胰腺癌细胞生长     

Ⅲ期鼻咽癌p53蛋白表达与生物学行为及预后的关系及其与p53基因突变、潜伏膜蛋白-1表达的相关研究

目的：探讨Ⅲ期鼻咽分化型非角化性鳞癌中P53蛋白的表达与生物学行为及预后的关系,以及P53蛋白表达与P53基因突变和潜伏膜蛋白（LMP）－1蛋白表达的关系。方法：应用免疫组织化学EnVison法和聚合酶链反应－单链构象多态性分析（PCR－SSCP）检测58Ⅲ例鼻咽分化型非角化性鳞癌标本。结果：58份Ⅲ期鼻咽分化型非角化性鳞癌中,P53蛋白总阳性率为65．5％（3858）,生存期在5年以下的患者阳性率为82．1％（32／39）,5年以上患者31．6％（6／19）,两组间P53蛋白表达差异有显著性意义（P＜0．05）,P53蛋白表达在有预底侵袭组为74．4％（29／39）,无颅底侵袭组为47．4％（919）,两组间差异有显著性意义（P＜0．05）,PCR－SSCP检测15例P53蛋白阳性的病例,P53基因5－8外显子突变率为0（0／15）,LMP－1蛋白免疫组织化学总阳性率为72．4％（42／58）,与P53蛋白表达呈正相关关系（r=0.504)。结论：本组Ⅲ期鼻咽分化型非角化性鳞癌患者中P53蛋白表达与P53基因突变无相关关系。与LMP－1蛋白的表达相关,P53蛋白的表达阳性率随着患者生存期的延长而降低,有颅底侵袭组P53蛋白表达阳性率高于无颅底侵袭组。     

Apoptosis in rheumatoid arthritis: p53 overexpression in rheumatoid arthritis synovium.

DNA damage induces p53 tumor suppressor gene expression and protein production, which in turn facilitates DNA repair or apoptosis. Wild-type p53 protein has a short half-life, so it is rarely detected in non-neoplastic tissue. Because DNA fragmentation is abundant in the intimal lining in rheumatoid arthritis (RA) synovial tissue (ST) using in situ end-labeling (Firestein GS, Yeo M, Zvaifler NJ: Apoptosis in rheumatoid arthritis synovium. J Clin Invest 1995, 96:1631-1638), we assessed ST p53 expression. Immunohistochemical analysis of fixed RA synovium using antibody PAb 1801 showed prominent p53 staining in the cytoplasm and nuclei of intimal lining cells. Noninflammatory and osteoarthritis (OA) ST had significantly less p53 in the lining. These data were confirmed by Western blot analysis of ST extracts, with abundant p53 found in RA compared with OA. p53 expression in cultured fibroblast-like synoviocytes (FLS) was then examined. Flow cytometry on permeabilized cells showed that RA FLS constitutively express p53 protein. Western blots showed that RA FLS expressed significantly more p53 than either OA FLS or dermal fibroblasts. Immunohistochemistry of FLS cultured in chamber slides localized the p53 to the cytoplasm of most resting FLS, with nuclear staining in only 10.7 +/- 2.4%. Exposure to hydrogen peroxide for increased nuclear staining to 70.7 +/- 12.8% after 8 hours (P = 0.003). These data indicate that p53 is overexpressed in RA ST in the intimal lining, which is the primary site of DNA damage, and is constitutively expressed by FLS.