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1.
Screening of H-ras Gene Point Mutations in 50 Cases of Bladder Carcinoma   总被引:2,自引:0,他引:2  
Background Mutation converts the H-ras gene into an activated oncogene in about 10% of human bladder cancers. Codons 12 and 61 are the major "hot spots" for activation. A simple and accurate method to detect point mutations in these codons may be clinically useful for early diagnosis of bladder cancer.
Methods Bladder cancer samples from 50 patients, plus 10 samples of normal bladder mucosa, were analyzed for possible point mutation of the H-ras gene at either codon 12 or codon 61. The H-ras gene DNA segments that include these 2 codons were amplified by PCR methods, then the possible presence of a point mutation was evaluated at each codon by susceptibility of the respective DNA segments to digestion with the restriction enzyme and by dot blot hybridization assay. A bladder cancer patient who had an H-ras gene mutation was examined to see whether the mutation was also detectable in the cells released in the urine.
Results Definite or possible point mutations were found in 6 (1 2%) out of 50 bladder cancer patients, while no mutation was detected in normal mucosa. A point mutation could also be detected in cells isolated from the patient's urine sample.
Conclusion The prevalence of point mutations at codon 1 2 or codon 61 of the H-ras gene found in this study was similar to that previously estimated for human bladder cancer by DNA transfection assay. The method we have used for detecting point mutations of the H-ras gene provides a simple and highly accurate way to detect mutated cancer cells even in the urine. It may be clinically usable for early diagnosis of bladder cancer.  相似文献   
2.
目的运用构建的小发夹状RNA重组质粒pshRNA/H-ras,研究其对卵巢癌SK-OV3细胞株内源H-ras基因的抑制作用。方法应用基因克隆技术,设计含21bp H-ras基因编码序列片段及中间以4~5个bp间隔的反向重复序列,克隆至转录载体pTZU6 1上并行序列分析。转染重组质粒pshRNA/H-ras至SKOV3细胞中,分别采用RT-PCR、Western blot检测细胞内H-rasmRNA和蛋白表达的变化。结果成功构建发夹状RNA载体,经DNA序列鉴定为正确的目的序列。RT-PCR和Western blot结果表明,重组质粒pshRNA/H-ras1和pshRNA/H-ras2均能在基因转录水平和蛋白表达水平上明显抑制SKOV3细胞内源H-ras蛋白的表达,抑制率达67%以上。结论重组质粒pshRNA/H-ras可显著抑制SKOV3细胞内源H-ras基因mRNA的转录和相应蛋白质的表达,为进一步研究H-ras基因在卵巢癌细胞异常增殖中的作用打下基础。  相似文献   
3.
The correlation between the mutation at codon 61 of the H-ras gene and the expression of the Bcl-2 protein was investigated in naturally occurring hepatocellular proliferative lesions in B6C3F1 mice. Specimens of histologically diagnosed neoplastic or preneoplastic lesions of the liver, obtained from the control mice used for 2-year carcinogenicity studies, were examined by immunohistochemical techniques. All of 25 lesions confirmed to be hepatocellular carcinomas stained positive for the Bcl-2 protein. Three of 12 foci of cellular alterations, as well as 24 of 42 hepatocellular adenomas, stained weakly positive. Bcl-2 protein was expressed to a greater degree in hepatocellular carcinomas as opposed to adenomas and confirmed by Western blot analysis. Seven of 18 hepatocellular adenomas that stained positive for Bcl-2 and three of 16 hepatocellular adenomas that stained negative had a mutation at codon 61 of the H-ras gene. Overexpression of Bcl-2 protein is likely to enhance the malignant turnover of the neoplastic cells, following a mutation at codon 61 of the H-ras gene particularly. These findings suggest that Bcl-2 overexpression and the mutation at codon 61 of the H-ras gene may be critical factors in the development of naturally occurring hepatocellular tumors in B6C3F1 mice.  相似文献   
4.
7,12-Dimethylbenz[a]anthracene (DMBA) elicits leukemia in Long–Evans rats (LE). This leukemia is mostly erythroblastic and 30% of leukemias have total and partial trisomy of #2 chromosome and the rest have diploid karyotype. The common duplication site is in 2q26–q34 and N-ras gene is located in 2q34. 7,8,12-Trimethylbenz[a]anthracene (TMBA) also induces similar leukemias. These leukemias reveal a highly specific mutation of N-ras gene as in human leukemias. N-ras mutation is induced 48 h after DMBA treatment. Wild type N-ras allele is frequently lost in diploid leukemias but not in trisomy type. Therefore, a gene dosage problem related to the mutant N-ras gene is involved in development of leukemia. Some secondary genetic rearrangements involving abl and H-ras are also observed in cultured leukemia cells. DMBA-induced chromosome aberrations as well as leukemia are enhanced by erythropoietin and blocked by Sudan III given prior to DMBA treatment. This leukemia will provide an important tool for chemical carcinogenesis and leukemia studies.  相似文献   
5.
 我们对40例宫颈癌活检组织同时进行HPV、H-ras、p53基因的检测,应用GP-PCR技术检测了多型HPV的感染,发现阳性率为85%(34/40)。HPV16转化基因E7占61.8%(21/34)。此外,利用PCR-SSCP(单纯DNA构象多态性)分析方法,对宫颈癌中H-ras、p53基因点突变进行了检测,H-ras突变占22.5%(9/40),p53占2.5%(1/40),而H-ras突变在HPV感染阳性组织中占14.7%(5/34),其中3例的病程分期为Ⅲ期以上。结果表明:HPV16E7转化基因在宫颈癌的致癌过程中为关键基因,H-ras突变很可能与有关报道相一致,属于“迟发”事件,与肿瘤的恶性程度及浸润有关。而p535—6外显子基因突变在HPV感染的宫颈癌中发生率很低。  相似文献   
6.
妊娠早期小鼠子宫内膜原癌基因H-ras的表达   总被引:4,自引:0,他引:4  
目的 :本实验检测原癌基因 H-ras的转录和翻译水平在妊娠早期小鼠子宫内膜的表达规律。方法 :采用反转录聚合酶链反应 ( RT-PCR)、免疫转移印迹 ( Western-blot)和免疫斑点印迹 ( Dot-blot)法。结果 :原癌基因 H-ras在妊娠早期小鼠子宫内膜的表达呈现两个峰值 ,分别为妊娠的第 1和第 6、7天。结论 :提示原癌基因 H-ras参与了胚泡着床过程及胚胎的早期增殖与分化过程  相似文献   
7.
We examined the level of receptor binding in H- ras elements, using nuclear extracts derived from human endometrial and ovarian lesions and from adjacent normal tissue in gel retardation assays. We found increased binding of the glucocorticoid receptor (GR) to the H- ras GR element in more than 90% of endometrial tumors and in all ovarian tumors tested, as compared to the corresponding adjacent normal tissue. Additionally, we found elevated binding of the estrogen receptor (ER) in H- ras ER element in all pairs of ovarian tumor/normal tissue tested, whereas in ER-negative control breast tumor/normal tissue pairs, no differences in ER DNA-binding levels were observed. These results suggest that steroid hormone receptor binding could directly activate the H- ras oncogenic potency in human endometrial and ovarian lesions, providing additional evidence for the role of H- ras expression in hormonally responsive human cancers.  相似文献   
8.
Hyperplastic, preneoplastic and neoplastic urinary bladder lesions induced by bladder carcinogens and toxins in the rat were evaluated for immunoreactivity with polyclonal or monoclonal antibodies to H- ras p21 or binding to peanut lectin with avidin-biotin immunocytochemistry. A low proportion (<20%) of hyperplastic and neoplastic bladder lesions induced by N-butyl-N-(4-hydroxybutyl)-nitrosamine and fixed in Bouin's fixative only were immunoreactive on the cell membrane with the antibodies to H- ras p21. Lectin binding was found for these lesions, as well, even in formalin-fixed tissue and for lesions induced by other carcinogens, but not in regenerative bladder hyperplasias after cyclophosphamide exposure or in bladder exposed to bladder tumor promoters. The latter lesions were also not immunoreactive with antibodies to p21. Our results suggest that this relatively simple technique might be used for identification and screening of tumors for involvement of ras oncogenes and carcinogen initiation.  相似文献   
9.
人工免疫组化判定结果与灰度值相关性   总被引:5,自引:0,他引:5  
免疫组织化学是用特异性抗体显示组织化学成分的重要方法,是实验结果以显微图像的形式显示出来,目前,对免疫组化图像的分析方法主要仍主要为人工分析,该方法简便易行,对实验的标准化要求不高,但无法做精确的量化分析;随着计算机的广泛应用,一些学者开始探讨应用计算机图像处理分析免疫组化图像,这种方法在精确性、客观性、分析速度等方面具有巨大优势,已经成为免疫组化显微图像分析技术发展的一个趋势,但目前仍不完善.本研究将采用计算机图像分析系统测量免疫组化切片灰度,并与人工技术方法相比较,从而验证灰度测量与人工方法在免疫组化染色结果判定方面的相关性.  相似文献   
10.
This study used tissue samples from male B6C3F1 mice treated with ethanol in drinking water (0%, 2.5%, or 5%) for 4 or 104 weeks. We tested whether chronic alcohol drinking promotes oxidative stress in the liver and characterized the mutation profile of spontaneous and ethanol-induced tumors. We show that ethanol does not cause detectable oxidative stress in the liver at any time point and acts by promoting H-ras mutated cells.  相似文献   
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