Abstract Objective To study the end of the of Galβ1 4GlcNAc polysaccharide molecule expression in nasopharyngeal cancer, lung cancer, liver cancer and colorectal cancer patients with serum law. Methods RCA I (Ricinus communis agglutinin I) affinity chromatography from normal human serum the RCA Ⅰ end of Galβ1 4GlcNAc glycoprotein, horseradish peroxidase labeled lectins Subtraction, 133 patients with cancer , 64 patients with benign disease and 240 patients with normal serum to detect. Nasopharyngeal cancer, liver cancer, and in patients with colorectal RCA I identified polysaccharide molecules significantly higher than normal subjects and patients with benign disease, the positive rate was 66.7%, 73.9%, 64.9%; seropositive patients with lung cancer was 19.4%. The polysaccharide molecule level and effective treatment of nasopharyngeal carcinoma and colorectal cancer patients was significantly decreased, the positive rate of decline. Conclusions Serum RCA Ⅰ end of the Galβ1 4GlcNAc polysaccharide molecules, of serum glycosylation abnormalities and tumors pathogenesis research for cancer patients, and can provide a reference for clinical diagnosis and evaluation of treatment effect indicators.
【Keywords】 RCA polysaccharide tumor
Under normal circumstances, the presence in the cell membranes and serum lectin recognition polysaccharide molecules, and the relatively stable level. When the malignant cells, the number and nature of the polysaccharide molecules also will be changed, and the reaction of lectin binding ability . Lectin is a class of non-immunogenic, able to identify the protein structure of the sugar chain binding specificity similar to the antigen-antibody reaction . Lectin as a probe, can detect the surface of the tumor cells, the cells and the level of serum polysaccharide. Based on the above characteristics, the experimental application binding RCA with the polysaccharide molecules specificity of the terminal Galβ1 4GlcNAc I [3,4], the affinity purified normal human serum the terminal Galβ1 4GlcNAc glycoprotein using lectin differential subtractor detecting nasopharyngeal , lung cancer, liver cancer, end in the serum of patients with colon cancer Galβ1 4GlcNAc polysaccharide molecule expression levels discussed in tumorigenesis and clinical significance.
1 Materials and methods
RCA Ⅰ purchased from Vector, RCA Ⅰ Sepharose4B purchased from Sigma, The horseradish peroxidase Roche import packing.
1.2 serum specimens collected
Cancer patients collect Zhujiang Hospital pathology confirmed 133 cases of nasopharyngeal carcinoma 42, which collected 31 cases of nasopharyngeal carcinoma patients before and after treatment serum, lung cancer, 31 cases, 23 cases of liver cancer, colorectal cancer 37 cases, including 33 cases of colorectal cancer The patients were collected before and after treatment serum. Also collected 64 cases of benign disease serum were taken from hospital inpatients. 240 patients with normal serum taken from the hospital examination normal. Aged between 20 to 76 years old. Serum collected at -70 ℃ storage, to be seized.
Preparation and horseradish peroxidase labeled 1.3.1RCA Ⅰ binding glycoprotein
Collect healthy people sera A, B, O 3 type geometric mixing 30ml, 2.3% Rivanol solution was mixed with serum albumin complex forming lumps, to remove albumin , and the removed liquid albumin 0.01 M PBS, dialyzed to remove one of the Rivanol molecule. Take the above-mentioned liquid mixture after the reaction with RCA Ⅰ Sepharose4B purified using affinity chromatography and RCA Ⅰ binding glycoprotein . After the collection of the above-described glycoprotein, respectively, dialyzed, and concentrated. Horseradish peroxidase labeled glycoprotein, taken RCA Ⅰ affinity purified glycoprotein 5mg, 10mg of horseradish peroxidase labeled  by a conventional method.
1.3.2 serum RCA I recognition polysaccharide molecule detection
Taken RCA Ⅰ dissolved in a diluted solution of the lectin, and the concentration of 0.0125mg/ml coated 96-well ELISA plate, 100 μl per well, overnight at 4 ° C in PBS buffer wash 1, were added to the serum 100μl reaction 4h PBS then washed 3 times by adding horseradish peroxidase-labeled glycoprotein, each hole 100 μl 4 ° C the reaction 4H, washed 3 times with PBS, TMB the H2O2 chromogenic 10min, after terminating the reaction, the enzyme-linked immunosorbent detector wavelength of 450nm measured OD values .
The 1.3.3 experimental control, and the establishment of quality control curve
Take affinity purified glycoprotein positive control instead of serum; with PBS instead of serum as negative control; instead of serum dilution of different concentrations of glycoprotein quality control; unchanged other steps.
1.3.4 Statistical analysis
SPSS10.0 statistical software, using analysis of variance and χ2 test.
2.1RCA Ⅰ combined with the preparation of the glycoprotein
30ml serum albumin dialysate was removed from about 60ml. Coomassie blue staining measured received after RCA Ⅰ Sepharose4B purified by affinity chromatography purified glycoprotein content of 6.9 mg.
The binding glycoproteins mark in 2.2 RCA Ⅰ
Taken 5mg affinity purified glycoprotein with 10mg mixed horseradish peroxidase labeled, labeled ELISA titer 1:512 .
2.3 Serum RCA I identified the detection result of the polysaccharide molecules
Coated RCA Ⅰ constant and excess and serum certain circumstances, the serum content of the polysaccharide molecules higher, and RCA I After the reaction, the horseradish peroxidase labeled glycoprotein RCA Ⅰ identified fewer ELISA measurement value Etsu low, both the inverse relationship. SPSS10.0 statistical software and quality control curve control minus 1.64 standard deviation after the value of the average of the detection of the normal group, the As judgments positive threshold, see Table 1. Table 1 serum RCA Ⅰ in recognition the polysaccharide molecule detection results * with benign disease, the disease of benign disease ** compared with normal subjects 2.4 composition and test results
In 64 patients with benign disease, benign respiratory disease in 41 cases, positive in eight cases, including six cases of chronic pulmonary heart disease. 23 cases of patients with benign digestive diseases, positive in 4 cases, including three cases of patients with gallstones.
2.5 HCC patients test results
17 cases were positive in 23 cases of liver cancer patients, including 10 patients with distant metastases, three cases of intrahepatic diffuse lesions, four cases of stable disease.
2.6 in the treatment of 31 cases of patients with nasopharyngeal carcinoma (the put ± chemotherapy) before and after the test results of the polysaccharide molecules in the serum
OD value before and after treatment, and the positive rate increased OD values after treatment than before treatment, after treatment, the positive rate of lowering serum after the treatment of patients with nasopharyngeal carcinoma significantly lower than before treatment of the polysaccharide molecule, as shown in Table 2 . Table 2 NPC patients serum
2.7 the polysaccharide molecules detected in the serum of 33 patients with colorectal cancer before and after treatment results
OD value before and after treatment, and the positive rate of significant differences in OD values after treatment than before treatment, after treatment, the positive rate of reducing the polysaccharide molecules than before treatment significantly reduced after treatment in the serum of patients with colorectal cancer. In seven cases out of nine cases positive patients after treatment for patients with advanced, two cases of patients with distant metastases. Note the polysaccharide molecule in the treatment of uncontrolled remained positive, as shown in Table 3. Table 3 colorectal cancer patients before and after treatment serum RCA Ⅰ identified polysaccharide molecule detection results
It has now been found that the glycosylation process within the tumor cells to mutate, resulting in cells secreted or membrane glycoproteins or glycolipids in sugar chain sequences change , these sugar chains and cell adhesion, motility, molecular recognition and cell identification, the sugar chain structure changes may result in the identification and growth and metastasis of tumor cells evade the host immune system. Lectin sugar binding specificity can detect such changes . RCA Ⅰ capable of specifically recognizing the sugar chain having a terminal Galβ1 4GlcNAc sequence polysaccharide molecules. This study the characteristics of RCA Ⅰ study result, it was confirmed that the polysaccharide molecules present in the serum RCA Ⅰ recognition Galß1 4GlcNAc sugar chain having a terminal sequence, and the first use of the affinity chromatography method of separating the substance from the serum. The study found that the polysaccharide molecule level in nasopharyngeal carcinoma, liver and serum of patients with colorectal cancer was significantly higher positive rates were 66.7%, 73.9% and 64.9%. Positive rate and liver cancer AFP, CEA colorectal quite, but this experiment does not require preparation of antibodies, the method is simple. Nasopharyngeal carcinoma and colorectal cancer effectively treated the polysaccharide molecule level significantly decreased, the positive rate of decline in the treatment of uncontrolled, the polysaccharide molecule remained positive. The results of this study suggest that the polysaccharide molecule levels with cancer have a certain relationship, have a certain sense, the search for nasopharyngeal carcinoma and colorectal cancer markers, to determine the efficacy of treatment. In lung cancer patients, the positive rate of patients with benign disease of the polysaccharide molecule was no significant difference, indicating that the polysaccharide molecule has little significance for the clinical diagnosis of lung cancer.
In this study, the use of the principle of specific binding lectin and polysaccharide molecules, serum RCA Ⅰ end Galβ1 4GlcNAc polysaccharide molecules, can be used for tumor in patients with abnormal glycosylation and tumor pathogenesis, and may for the clinical diagnosis and Evaluation of treatment to provide reference indicators.