| Objective: Breast cancer is one of the malignant tumors with the highest incidence for female and dose serious harm to women's health for it's incidence is becoming higher and higher. At present, there is no better method to diagnose breast cancer. Tumor marker is of importance in making an early diagnosis , choosing a better therapy project , inspecting recurrance and metastasis and evaluating prognosis and tract study of breast cancer. During past tens of years , the study on gene's change and function in breast cancer development, prognosis and metastasis has been the focus in this field. Tyrosine kinase is a kind of key protein during signal transduction of breast tissue cells, which has deep correlation with tumor development and growth. Recently, syk(spleen tyrosine kinase), a new tyrosine protein kinase, was found to be an important suppressor factor for the growth and metastasis of breast cancer cell. In this research, Syk protein was extracted and purified from Sf21 cells transfected by syk gene to preparation of rabbit anti-Syk antibody. Immunohistochemical staining was used to detect Syk protein's expression and RT-PCR was used to determine the contants and differences of SYK gene in the normal breasttissues and breast cancer tissues in order to demonstrate its effect on the growth, development and prognosis of breast cancer; The relationship between Syk protein and syk gene and pathological grade, clinical stage, and lymph node status was also studied . Such studies would provided theoretical evidence for the early diagnosis and prognosis assessment of breast cancer.Methods1 Extract and purify Syk protein : SO 1 cells were infected with recombinant syk gene. After 48h of incubation at 28°C, the transfected cells were collected and sonicated on Sonfier sonicator on ice. Filtered cell extract was loaded onto a Reactive Yellow-3 resin column and Toyopearl AF-Heparin-650M column respectively. The character of Syk protein in the fractions were identified by SDS-PAGE, Western blotting and IEF.2 Prepare rabbit anti-Syk antibody: Male rabbit were immunized with recombinant mouse Syk protein to prepare antibody against Syk. The purity and specificity of the antibody were analyzed by Western blot.3 Detect the expression of Syk protein in breast tissues: Malignant and nonneoplastic breast tissues samples from 44 women were obtained from Department of Surgery Fourth Hospital of HeBei Medical University from December 2003 to February 2004. All patients were female with median age 45. Immunohistochemical S-p method was used to detect thecontants of Syk in the malignant and normal tissues of breast.4 Detect the expression of syk gene in breast tissues: Samples from 31 women were obtained from December 2002 to Feburary 2003. RT-PCR was used to detect the contants of syk gene.5 Statistic Methods: Statistic analysis was performed with SPSS software 10.0.Results1 Obtained 225mg protein containing Syk from Sf21 cells (2.5><109) extract. There were two subpopulations in the elution of Reactive Yellow-3 resin column with the MW 72KDa. Then applied on Toyopearl AF-Heparin-650M column and obtained two pure subpopulations. The results of SDS-PAGE and Western blotting showed the two protein bands at approximately 72 kDa, corresponding to SYK, whereas different pi bands in IEF.2 The titer of serum anti-Syk antibody was 1:6400 (A450H.03) 6 weeks after the immunization.The SDS-PAGE and Western blot analysis showed that there was a obvious protein band with Mr72000, confirming the specificity of obtained anti-Syk antibody.3 It was displayed by immunohistochemical staining with anti-Syk antibody that in normal breast tissue Syk was highly expressed , whereas its expression was negative or low in carcinoma tissues. There is statistic relationship between the differences and the histological grade (PO.05).4 The expression of syk gene detected by RT-PCR: There are significant differences in the expression of syk between the malignant and nonneoplastic breast tissues. (p<0.01) . The expression of later is lower than the former, syk can be detected in all normal breast tissues, but only 50% in tumor tissues. There is statistic relationship between the differences and the following factors: histological grade , clinical stage, metastasis of lymph node (P<0.01).5 The absent of syk gene's expression may be occurred during transcription.Conclusions1 The yield of Syk is 8 mg from 2.5 billion cells and the purity is >95%. The subpopulations have the same MW and differentpi-2 The rabbit anti-Syk antibody with high titer and specificity hasbeen prepared.3 There are higher expression of Syk protein and syk gene in normal breast tissues than that in breast cancer tissues whose expression is lower or absent. There are significant differences between them, which indicated that syk may be one of tumor suppressor factors. It can keep normal breast cells from abnormally proliferating. Lower expression and absent of syk gene will cause cancer cells to grow rapidly.4 There are also negative relationships between the expression of Syk protein and syk gene and clinical stage, histological grade and lymph node status, which show the awful prognosis and...
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