食管鳞状上皮细胞癌中P53基因突变的PCR—SSCP检测

应用ＰＣＲ－ＳＳＣＰ银染技术检测了４０例食管鳞状细胞癌Ｐ５３基因第５、６、７和第８外显子的突变情况。结果显示：１３例（３２．５％）有Ｐ５３基因突变，突变主要发生在第５和第８外显子，与癌分化程度无明显关系，但伴淋巴结癌转移病例中的Ｐ５３基因的突变频率明显高无转移者（Ｐ〈０．０５）。本研究提示，Ｐ５３基因突变存在于中，晚期食管癌中，并可能与食管鳞癌的进展与转移有关。     

p53基因第72位密码子突变与食管鳞癌临床病理特征关系的研究

目的研究p53基因第72位密码子（p53 codon72）突变与人食管鳞癌临床病理特征的关系。方法应用聚合酶链反应-限制性片段长度（PCR-RFLP）方法检测118例人食管鳞癌组织及癌旁正常食管黏膜组织的p53 codon72的突变及其差异，并分析其与食管鳞癌临床病理特征的关系。结果p53 codon72的Arg／Arg和Pro／Pro或Arg基因型在癌组织和癌旁正常食管黏膜组织的频率分别为11．0％和38．1％、4．2％和7.60A。p53 codon72Arg／Pro基因型在癌组织和癌旁正常食管黏膜组织分布差异具有显著性（χ^2=55.75，P〈0.01），p53 codon72突变与p53蛋白表达有关（χ^2=15．21，P〈0．01）；且p53 codon72的Pro等位基因与食管癌的TNM分期、分化程度和淋巴结转移均呈显著相关（P〈0．01）。结论抑癌基因p53第72位密码子突变在食管癌发生、发展中可能起重要作用。     

30例配对新鲜胃癌标本中p53基因突变的研究

目的：探讨胃癌标本中p53基因外显子5～8的突变情况及其与胃癌各临床特征的关系。方法：采用银染PCR—SSCP检测30例配对新鲜胃癌手术标本中p53基因外显子5～8的突变。结果：p53基因突变检出率为46．7％（14／30）,p53基因外显子5—8的突变率分别为10％、16．7％、23．3％、16．7％;p53基因突变与病人年龄、性别、原发灶部位及肿瘤分化程度之间差异没有显著性意义（P〉O．05）;但是,p53基因突变与胃癌临床分期、淋巴结转移之间差异有显著性意义（P〈O．05）,进展期胃癌p53基因突变高于早期胃癌,有淋巴结转移的胃癌组织中p53基因突变高于无淋巴结转移胃癌组织。结论：p53基因突变可能参与了早期胃癌一进展期胃癌的发生发展过程,并可能是胃癌发生淋巴结转移的机制之一。     

肺腺癌p53基因突变与临床病理特征关系的研究

目的探讨p53基因在肺腺癌中突变的频率、位置和在肺腺癌发生发展中的作用及与其临床病理特征的关系。方法聚合酶链式反应一单链构象多态性（PCR－SSCP）检测31例肺腺癌的P53基因第5～8外显子突变。结果14例（45％）出现P53基因5～8外显子突变，其中7、8外显子突变占73％。P53基因突变男性显著高于女性（P—0．003），在肿瘤≤3cm的病例p53基因突变率显著低于肿瘤＞3cm的病例（P=0．005）。p53基因突变与吸烟史、年龄、组织学类型、分化程度、淋巴结状况、国际病理TNM分期及瘤栓无显著性差异（P＞0．05）。结论肺腺癌中P53突变率为45％，主要分布在第7、8外显子，P53基因突变参与肺腺癌癌变的始动和腺癌的进展。     

食管鳞状细胞癌p53基因杂合性缺失和基因突变的相关研究

 目的 检测食管鳞状细胞癌（ESCC）中p53基因杂合性缺失（LOH），p53基因突变及蛋白表达的情况，分析其与临床病理和预后的相关性。方法 采用聚合酶链反应-单链构象多态性分析（PCR-SSCP）和免疫组织化学方法（SP）检测56例ESCC中p53基因LOH和p53基因蛋白的表达情况。结果 56例ESCC组织中p53蛋白阳性表达率为60.7 %（34／56），它与患者的年龄、性别和家族史无关（P＞0.05），有或无淋巴结转移的阳性率分别为81.0 %（17／21），48.6 %（17／35）；生存率低于3年组和高于3年组的p53阳性表达率为73.9 %（17／23），46.4 %（13／28），差异有统计学意义。p53基因LOH率为80.5 %（33／41），与患者的年龄、性别、家族史和有无淋巴结转移无关，与3年生存率有相关性（P＜0.05）。p53基因总突变率为76.8 %（43／56），突变位于17号染色体第4外显子者5例，第5外显子者23例，第6外显子者1例，第7外显子者4例，第8外显子者7例，有3例在内含子。p53基因突变／过度表达率为46.4 %（26／56），两种方法检测的符合率为55.4 %（31／56）。结论 p53基因在ESCC的发生、发展中可能发挥重要作用，伴有p53基因LOH／p53蛋白过度表达的3年生存率明显降低。p53蛋白阳性表达的ESCC更易发生淋巴结转移，可作为判断食管癌预后的参考指标之一。     

DHPLC检测林州市高龄组和低龄组居民食管癌p53基因外显子突变谱的比较研究

目的：研究我国食管癌高发区林州市居民高龄食管癌患者与低龄食管癌患者中抑癌基因p53全部编码外显子基因突变谱的情况，比较两组患者中p53基因突变的差异。方法：留取林州市〉70岁和〈40岁、低年龄组食管癌患者食管癌新鲜标本51例，提取DNA，PCR扩增p53第2～11外显子。DHPLC进行突变的筛查。突变样本进行DNA纯化测序分析。结果：51例患者中，p53至少有1个外显子基因突变的36例，突变率为70．6％。高年龄组突变率为63．3％（19／30），低年龄组突变率为80．9％（17／21），两者比较无统计学意义（P〉0．05）。p53有2个外显子基因突变的10例，突变率为17．6％。高年龄组突变率为26．7％（8／30），低年龄组突变率为9．5％，两者比较无统计学意义，P〉0．05。结论：p53基因突变是林州市居民食管癌发生过程中的一个发生频率很高的事件。林州市居民的p53外显子突变谱中，共有15种突变类型，共计59个突变位点，其中最多的突变类型是插入碱基C。在p53基因第2～8外显子与内含子交界的非编码区有大量的基因突变。     

大肠癌p53基因突变和染色体异倍体表达与淋巴结转移的关系

目的：研究大肠癌转移与p53基因突变及染色体倍性之间的关系。方法：采用特异合成引物对p53基因7－8外显子进行PCR扩增,结合单链构象多态性分析（SSCP）和银染技术,检则22例大肠癌手术标本p53基因的突变;用流式细胞仪（FACS）分析其染色体的倍性。结果：在有淋巴结转移10例大肠腺癌中,p53基因突变者占6例（60％）;在无淋巴结转移的12例大肠腺癌中,p53基因突变者占1例（8．3％）。在有p53基因突变的7例大肠腺癌中异倍体者6例（85．7％）;无p53基因突变的15例大肠癌中异倍体者6例（40％）。在有淋巴结转移的10例大肠癌中,p53基因突变及DNA异倍体共同表达者5例（50％）;无淋巴结转移的12例大肠腺癌中p53基因突变及DNA异倍体共同表达者1例。结论：大肠癌p53基因突变及DNA异倍体共同表达与淋巴结转移密切相关。     

胃癌组织中p53基因突变及p53和mdm2蛋白表达的研究

目的：探讨mdm2和p53基因异常在胃癌发生发展中的作用以及两者相关性。方法：应用免疫组化技术检测58例胃癌组织以及相应癌旁组织中mdm2和p53蛋白的表达；PCR-SSCP银染技术检测p53基因exon5～8突变情况。结果：胃癌组p53和mdm2蛋白阳性率分别为86．21％（50／58）和29．31％（17／58），癌旁组织组p53和mdm2蛋白均为阴性，胃癌组p53和mdm2蛋白阳性率明显高于癌旁组织组，两两间差异有统计学意义，P=0．0000、P=0．0001。胃癌组织中p53和mdm2蛋白表达率与肿瘤大小、组织学类型、分化程度、淋巴结转移以及患者年龄等无显著相关性，P〉0．05。mdm2蛋白阳性表达与p53蛋白过表达呈显著正相关,X^2=11．1839，P=0．0008，r=0．4391。2例胃癌组织检测到p53基因突变，突变均位于exon5，58例相应癌旁组织均禾检测到p53基因突变。结论：mdm2和p53蛋白异常表达与胃癌发生有关，p53基因突变可能并非胃癌组织中p53蛋白异常累积的主要原因，mdm2蛋白在胃癌发生发展中的作用可能与p53蛋白密切相关。     

应用PCR-SSCP银染技术分析胃癌p53基因的点突变

目的 分析胃癌中p53基因第5－8外显子点突变的发生及其意义。方法 应用聚合酶链反应－单链构象多态性分析（PCR－SSCP）和银染技术，对40例胃良性疾病和30例胃癌标本中p53基因第5－8外显子的点突变情况进行检测。结果 40例胃良性疾病均无点突变发生，30例胃癌中有12例发生p53基因的点突变，突变率为40．00％（12／30）。结论 p53基因突变与胃癌的发生密切相关；PCR－SSCP银染技术不仅灵敏安全，而且简便、快速、重复性好，可用于临床的基因诊断。     

人食管鳞状细胞癌中p53基因突变在浙江与河南两地的差异

目的 更深入了解人食管鳞状细胞癌中p53基因突变的地域差异及临床特征之间的差异。方法 从中国食管鳞癌的高发区河南林县以及浙江省收集共92例人食管鳞状细胞癌（ESCC）标本，使用免疫组化、PCR—SSCP分析和DNA测序的方法分析p53肿瘤抑制基因。结果 p53基因的突变或过表达分别为30.4％和51.1％（P〈0．05）。p53的过表达与肿瘤的转移和患者存活期相关，两个不同地域收集的肿瘤标本中发现了不同的突变模式。结论 p53的突变或过表达在食管鳞癌的发展中可能起到了重要的作用致癌因子的各种不同组合，可能是两个地区人群中基因组的不稳定性所致，这种不稳定性在某种水平上决定了不同地区不同的发病率.     

41例人脑胶质瘤组织中p53基因突变的研究

背景与目的:p53抑癌基因在细胞周期的调控、维持细胞基因组的完整性、诱导细胞分化和凋亡中起着重要作用。胶质瘤尤其是星形细胞肿瘤中,经常发生p53基因突变。本研究探讨人脑胶质瘤中p53突变及与胶质瘤发生发展的相关性。方法:应用聚合酶链反应-单链构象多态性分析(polymerase chainreaction-single-strand conformation polymorphism,PCR-SSCP)及DNA测序技术对41例不同类型人脑胶质瘤p53基因突变进行检测。结果:通过PCR-SSCP检测发现在41例人脑胶质瘤组织中有17例(41.5%)呈现p53基因的单链构象多态性改变,均位于5～8外显子,突变例数依次为7(41.2%)、1(5.9%)、4(23.5%)、5(29.4%)。Ⅲ、Ⅳ级胶质瘤中p53基因突变率明显高于Ⅰ、Ⅱ级肿瘤(58.3%vs.17.6%,P<0.01);DNA序列分析显示,17例PCR-SSCP检测阳性的肿瘤p53基因相应外显子均存在基因点突变或缺失,其中错义突变13例,占76.5%;同义突变2例,占11.8%;移码突变2例,占11.8%;碱基突变以G→A或A→G最多,占55.6%。结论:胶质瘤中p53突变多发生于第5、8外显子,基因突变类型以错义突变为主,该基因的突变与胶质瘤的发生及恶性进展过程相关。     

Frequency and characterization of p53 mutations in primary and metastatic human prostate cancer

We have studied the frequency of mutations in the p53 gene in human prostate cancer. The investigated material consisted of 20 primary-tumor tissue specimens, obtained by transurethral resection and tissue specimens of 15 lymph-node metastases, obtained at total prostatectomy. The applied methods encompassed immunohistochemistry on frozen sections, using the monoclonal antibody PAb 1801, and single-strand conformation polymorphism (SSCP) analysis, after amplification of single exon sequences by PCR, on exons 5 to 8 of the p53 gene. The mutations, leading to aberrantly migrating bands in the PCRSSCP analysis, wereidentified by direct sequencing of the PCR product. Immunohistochemical and PCR-SSCP analysis were completely confirmative. In the primary tumors, mutations were found in 10% of the specimens (codons 232 and 273), and in lymph-node metastases in 15% of the specimens (codons 248 and 273). In one case (codon 273), the same mutation was found both in the primary tumor and in the lymph-node metastasis. Our results show that p53 mutations are infrequent in both primary and metastatic prostate tumors. In addition, they indicate that there is no strict correlation between p53 mutation and tumor metastasis.     

p53 tumor suppressor gene and ras oncogene mutations in hypopharyngeal squamous cell carcinomas

To examine the potential role of p53 and ras gene mutations in hypopharyngeal tumorigenesis, twenty-eight primary hypopharyngeal carcinomas, obtained at biopsy or total pharyngolaryngectomy, were investigated. Exons 5 through 9 of the p53 gene and exons 1 and 2 of the H-, K-, N-ras gene were screened using a combination of immunohistochemistry and single-strand conformation polymorphism analysis of polymerase chain reaction products (PCR-SSCP). The targeted DNA sequences coding for p53 and ras were confirmed by direct DNA sequencing. Point mutations of p53 were found in 9 (32.1%) of the 28 cases, including one with a double mutation, 3 in exon 5, 1 in exon 6, 2 in exon 7 and 4 in exon 8. Positive nuclear immunostaining for p53 was evident in 14 (50.0%) lesions. Seven (25.0%) of the 28 demonstrated point mutations in the H-rns gene, and 11 (39.3%) showed positive cytoplasmic staining for I as. The 5-year survival rate was worse with than without p53 overexpression (p <0.05). The present results suggest that gene mutations, although they occur at a relatively low incidence, are involved in hypopharyngeal tumorigenesis with p53 expression being a prognostic factor.     

Serum anti-p53 in relation to mutations across the entire translated p53 gene in colorectal carcinomas

Previous reports have claimed that antibodies to mutated p53 protein indicate poor outcome in malignant disease. The mechanism behind this highly specific process is unclear, although it has been claimed that certain DNA alterations are prone to induction of immune response, since wild-type p53 is almost never immunogenic. The aim of the present analysis was to evaluate whether the presence of anti-p53 was statistically significantly related to any certain DNA alterations in the entirely expressed p53 gene in primary tumors of colorectal cancer. P53 serum antibodies were determined by an enzyme linked immunosorbant assay (ELISA). P53 antibodies were detected in serum of 24 of 88 patients (27%). Twenty-two of 24 (92%) sero-positive patients had mutations in their p53 gene while only 22 of 64 (34%) sero-negative patients had p53 mutations (p<0.01). Mutations were mainly missense with a trend to significantly higher frequency of deletions in sero-negative patients compared to sero-positive subjects (8/25, 32% and 2/22, 9% respectively, p<0.08). Mutations in sero-positive patients were mainly located in exon 5 and 7 and within conserved regions (17 of 22 mutations). In sero-negative patients missense mutations were usually located in exon 5, 7 and 8 being also most frequently located within conserved regions. Most of the p53 deletions in sero-negative patients were however located outside conserved regions (seven of eight deletions). There was no statistical difference between sero-positive and negative patients concerning the spectrum of mutations along the expressed gene. Our study demonstrates that p53 antibodies are usually related to p53 gene mutations but a mutational event is not sufficient to elicit self-immunization. Cellular protein binding to p53 or individual differences of major histocompatibility complex based presentation of p53 protein sequences by immune cells is therefore the most likely explanation between sero-negative and sero-positive patients.     

In primary human breast carcinomas mutations in exons 5 and 6 of the p53 gene are associated with a high s-phase index

A series of 121 human breast tumors was screened for point mutations in exons 5 through 8 of the p53 gene, by SSCP analysis. On the same tumor samples, the S-phase index (SPI) was determined by the incorporation of BUdR in fresh tissue. p53 mutations were observed in 29% of the cases. The frequency of point mutations for the individual exons was: exon 5, 10.0%; exon 6, 9.9%; exon 7, 7.1% and exon 8, 5.5%. Two mutations detected by SSCP were confirmed by sequencing the p53 cDNA. The presence of a p53 mutation, irrespective of its location, correlates (p = 0.003) with a high SPI. This association appears to primarily reflect mutations in exon 5 (p = 0.0002) and exon 6 (p = 0.05), since mutations in exons 7 and 8 failed to show any association. These results indicate that mutations in the p53 gene identify highly proliferating tumors, and that the position of the p53 mutation may have different effects upon the proliferative activity of tumor cells in vivo.     

PCR-TGGE法检测p53基因突变诊断肺癌淋巴结微转移

背景与目的：转移是影响肺癌患者预后的重要因素之一,但在淋巴结内存在少量癌细胞时常规的检测方法难以发现。本研究的目的是探讨检测肺癌相关淋巴结内微转移存在的敏感方法。方法：利用温度梯度凝胶电泳(temperature gradient gel electrophoresis,TFGGE)方法,对39例原发性肺癌患者的肿瘤组织及其相关的110枚淋巴结,进行p53基因(外显子5,6,7,8)的突变检测,并对部分淋巴结采用无间隔连续切片,证实微转移存在。结果：在39例肺癌患者中,23例原发肿瘤检测出p53基因突变,在相应的67枚淋巴结中,40枚淋巴结显示p53基因突变,且与原发灶变化一致;而在常规病理组织学检查时,仅26枚淋巴结存在肿瘤转移。原发肿瘤无p53基因突变者的43枚淋巴结和10枚非瘤患者淋巴结,均无p53基因突变。对常规病理组织学检查阴性,PCR-TGGE检测有p53基因突变的14枚淋巴结,进行无间隔连续切片,在4枚淋巴结中证实有微转移存在。结论：p53基因突变的检测发现了肺癌淋巴结中微转移的存在,可以作为常规病理组织学检查的补充。     

Correlation of immunohistochemical staining and mutations of p53 in human hepatocellular carcinoma

Mutations of the p53 tumor suppressor gene are common in hepatocellular carcinomas (HCCs). Detection of mutations by sequencing provides more information than immunohistochemical staining, but the equipment needed and the time required make it less practical for use in large-scale studies or in studies in developing countries. The degree of correlation between results obtained with these two methods has been studied in various tumors but has not been well-established in human HCCs. Paraffin sections of HCCs of 28 patients from Qidong, China were immunohistochemically stained using monoclonal antibody to p53. In addition, exons 5-8 of the p53 gene were sequenced in these HCCs. Of the 28 HCCs, nine had 0-9% of nuclei stained for p53, and 19 had 50-95% stained. Mutations in p53 exons 5-8 were found in 17/28 (61%) HCCs, including 15 at codon 249 (exon 7), one at codon 198 (exon 6), and one at codon 175 (exon 5). Among these 17 cases with p53 mutations, 16 cases (94%) had 50-95% of nuclei stained. Among 11 HCCs with no mutations by sequencing, 8 were also negative by immunohistochemistry (0-9% of nuclei stained) (73%) (the five HCCs with no staining whatsoever all had wild-type p53). Immunohistochemical staining to detect p53 mutations in human HCCs detected most mutations that were detected by sequencing (94% sensitivity, 73% specificity), and this method is therefore suitable when sequencing cannot be performed.     

P53 tumour-suppressor gene mutations are mainly localised on exon 7 in human primary and metastatic prostate cancer.

Mutations in the p53 tumour-suppressor gene are among the most common genetic alterations in human cancers. In the present study we analysed the mutations in the p53 tumor-suppressor gene in 25 primary and 20 metastatic human prostate cancer specimens. DNA extracted from the paraffin-embedded sections was amplified by hot-start polymerase chain reaction, and p53 gene mutations in the conserved mid-region (exons 4-9) were examined using single-strand conformation polymorphism (SSCP) analysis and immunohistochemistry. In the present study, we used a novel hot-start PCR-SSCP technique using DNA Taq polymerase antibody, which eliminates primer-dimers and non-specific products. Because of this new technique, the results of PCR-SSCP showed very high resolution. Polymerase chain reaction products were sequenced directly for point mutations for the p53 gene. Mutations were found in 2 out of 25 primary prostate cancers (8%) and 4 out of 20 metastatic cancers (20%). Mutations were observed exclusively in exon 7 and not in exons 4, 5, 6, 8 or 9. Nuclear accumulation of p53 protein, determined by immunohistochemistry, correlated with the degree of metastasis in prostatic cancer.     

Effect of green tea on p53 mutation distribution in ultraviolet B radiation-induced mouse skin tumors

In the present study, administration of green tea to SKH-1 mice, via the drinking fluid, was found to significantly reduce the incidence and volume of ultraviolet B (UVB) radiation-induced skin tumors. Thirty-six skin tumors induced by UVB and 32 skin tumors induced by UVB, in mice treated with green tea in their drinking water, were collected and examined for the presence of mutations in the p53 gene. Polymerase chain reaction products from p53 exons 5-8 were screened by single- strand conformation polymorphism and direct sequence analyses. Eight of 36 UVB-induced tumors contained nine p53 mutations, with four in exon 5 and five in exon 8. In contrast, nine of 32 UVB-induced tumors in mice treated with green tea contained 11 p53 mutations, with two in exon 5, five in exon 6 and four in exon 8. All of the p53 mutations occurred at dipyrimidine sequences. These results were further corroborated by p53 immunohistochemistry. The most frequent mutations were C-->T or T-->C transitions, which are consistent with the genetic alterations caused by UVB exposure. Interestingly, mutations found in exon 6 of the p53 gene occurred only in tumors from the UVB/green tea group. Thus, the tumors observed in UVB/green-tea-treated mice have a different exon distribution of p53 mutations than tumors obtained from mice treated with UVB alone.