结直肠癌患者血清p53抗体与肿瘤p53蛋白表达关系的研究

目的 探讨结直肠癌患者血清p5 3抗体与肿瘤组织p5 3蛋白表达的关系 ,评价其能否间接反映p5 3基因的突变。方法 对 13 2例手术治疗的结直肠腺癌患者和 3 6例非肿瘤患者进行血清p5 3抗体定性检测。结直肠癌标本行p5 3蛋白免疫组化染色。结果 结直肠癌组中 5 3例 (4 0 .2 % )血清p5 3抗体阳性 ,而对照组仅 1例 (2 .9% )阳性 (P <0 .0 1)。在 75例p5 3蛋白染色阳性者中 5 1例 (68.0 % )血清p5 3抗体阳性 ;而 5 7例p5 3蛋白染色阴性的患者中 ,仅 2例 (3 .5 % )血清p5 3抗体阳性。结直肠癌血清p5 3抗体阳性率与肿瘤组织p5 3蛋白免疫组化染色阳性反应有明显关系 (P<0 .0 1)。结论 血清p5 3抗体能反映结直肠癌p5 3蛋白的阳性表达 ;检测血清p5 3抗体是间接判定P5 3基因突变的一种可靠而简便的方法。

Preparation of monoclonal antibody to P53 and its clinical application

Objective：The aim of this study was to prepare monoclonal antibody against P53, a kind of tumor suppressor protein,and use the antibody initial y in clinical immunoassay. Methods：Monoclonal antibody was prepared and identified via the classic protocol of monoclonal antibody preparation. Identified monoclonal antibodies were purified by af inity chro-matography. Antibody titer was determined by enzyme linked immunosorbent assay （ELISA）. The specific binding activity of antibody was detected by Western blotting and immunohistochemistry. Results：Three strains of monoclonal antibodies named 1P15, 2P37 and 3P40 were obtained and purified by af inity chromatography. The purity of antibodies was higher than 90%. The titers of antibodies were more than 1：6000. Western blot and immunohistochemistry assay showed that the specific antibody can combine with endogenous P53 protein in the tumor celllines and determine the expression of P53 in tumor tis-sue. Conclusion：Three strains of monoclonal antibodies with high af inity to P53 were successful y established, which can be used for detecting the expression of P53 in tumor cells or tissue.

p53 antibodies,metallothioneins,and oxidative stress markers in chronic ulcerative colitis with dysplasia

AIM:To investigate the role of p53 antibodies (p53Abs),metallothioneins (MTs) and oxidative stress markers in the early detection of dysplasia in chronic ulcerative colitis (UC).METHODS:The study included 30 UC patients,15 without dysplasia (group Ⅱ) and 15 with dysplasia (group Ⅲ),in addition to 15 healthy volunteers (group Ⅰ,control subjects).The enzyme-linked immunosorbent assay technique was used to measure serum p53Abs and MTs,while advanced oxidation protein products (AOPPs),and reduced glutathione (GSH) levels were measured by spectrophotometric method in all subjects.RESULTS:In group Ⅱ and group Ⅲ compared to group Ⅰ,there were significant increases in serum levels of AOPPs (145.94 ± 29.86 μmol/L and 192.21 ± 46.71 μmol/L vs 128.95 ± 3.06 μmol/L,P < 0.002 and P <0.001,respectively),MTs (8.18 ± 0.35 μg/mL and 9.20 ± 0.58 μg/mL vs 6.12 ± 0.25 μg/mL,P < 0.05 and P < 0.05,respectively),and p53Abs (20.19 ± 3.20 U/mL and 34.66 ± 1.34 U/mL vs 9.42 ± 1.64 U/mL,P < 0.001 and P < 0.001,respectively).There were significantly higher levels of AOPPs (P < 0.05) and p53Abs (P < 0.001) in UC patients with dysplasia compared to those without dysplasia,while MTs showed no significant difference between the 2 groups (P > 0.096).In contrast,GSH levels showed a significant decrease in both patients' groups (1.87 ± 0.02 μmol/mL and 1.37 ± 0.09 μmol/mL vs 2.49 ± 0.10 μmol/mL,P < 0.05 and P < 0.05 in groups Ⅱ and Ⅲ,respectively) compared with group Ⅰ,and the levels were significantly lower in group Ⅲ than group Ⅱ (P < 0.05).There was a positive correlation between AOPPs and both MTs (r=0.678,P < 0.001) and p53Abs (r=0.547,P < 0.001),and also between p53Abs and MTs (r=0.739,P < 0.001).There was a negative correlation between AOPPs and GSH (r =-0.385,P < 0.001),and also between GSH and both MTs (r=-0.662,P < 0.001) and p53Abs (r=-0.923,P < 0.001).CONCLUSION:Oxidative stress and oxidative cellular damage play an important role in the pathogenesis of chronic UC and the associated carcinogenetic process.p53Abs levels could help in early detection of dysplasia in these conditions.

Nontoxic virus nanofibers improve the detection sensitivity for the anti-p53 antibody, a biomarker in cancer patients

The presence of anti-p53 antibody in serum is a biomarker for cancer. However, its high sensitivity detection is still an issue in cancer diagnosis. To tackle this challenge, we used fd phage, a human-safe bacteria-specific virus nanofiber that can be mass-produced by infecting host bacteria in an error-free manner, and genetically engineered it to display a peptide capable of recognizing and capturing anti-p53 antibody on its side wall. We employed the resultant phage nanofibers as a capture probe to develop a modified version of the enzyme- linked immunosorbent assay （ELISA） method, termed phage-ELISA. We compared it to the traditional ELISA method for the detection of anti-p53 antibody, p53-ELISA, which uses recombinant wild-type p53 protein to capture anti-p53 antibody. We applied phage-ELISA to detect anti-p53 antibody in an experimental group of 316 patients with various types of malignant tumors. We found that a detection rate of 17.7% （56 positive cases） was achieved by phage-ELISA, which was comparable to the detection rate of 20.6% for p53-ELISA （65 positive cases）. However, when both phage and p53 were combined to form antibody-capturing probes for phage/p53-ELISA, a detection rate of 30.4% （96 positive cases） was achieved. Our work showed that owing to the combined capture of the anti-p53 antibody by both phage nanofibers and p53, the phage/p53-ELISA achieved the highest diagnostic accuracy and detection efficiency for the anti-p53 antibody in patients with various types of cancers. Our work suggests that a combination of nanofibers and antigens, both of which capture antibody, could lead to increased detection sensitivity, which is useful for applications in the life sciences, clinical medicine, and environmental sciences.

The effect of Tagalsin on mice with transplanted H22 Hepatocarcinoma

Objective： The aim of our study was to explore the inhibitory effect of Tagalsin on murine transplanted tumour and its anti-tumour mechanisms. Methods： Animal models were established by transplanting H22 hepatoma cells to the left oxter of mice, and ten days later they were randomly divided into five groups： blank control group （edible oil）, positive control group （HCFU） and Tagalsin group, including low-dose, middle-dose and high-dose group. All mice were killed 24 h after medication, during which observation was conducted concerning survival conditions, body weight changes, spleen weight and tumor weight of tumor-bearing mice; the spleen index and the tumor inhibitor rate （IR） were calculated and pathological changes of tumor-bearing mice were observed by HE dye. Apoptosis factors p53 and Survivin mRNA were detected by reverse transcription polymerase chain reaction （RT-PCR）. Results： Tagalsin can inhibit hepatoma growth effectively without influencing spleen index and body weight, the tumor inhibitor rate （IR） of low, middle and high dose group of Tagalsin were 15.81%, 36.75% and 74.79% respectively, the tumor inhibitor rate （IR） of HCFU were 73.93%. Apoptosis cells could be found from the specimen of the positive control group and Tagalsin groups. Reverse transcription polymerase chain reaction （RT-PCR） results showed that positive control group’s and Tagalsin treatment groups’ p53 gene expression enhanced significantly and Survivin gene expression dropped comparing with blank group （P 0.05）. Conclusion： Tagalsin can inhibit growth of the H22 hepatoma cells significantly, the mechanism of anti-tumor effect may work by up-regulating p53 expression and down-regulating Survivin expression. Tagalsin may be considered as a potential candidate for chemoprevention.