Preparation Of Rabbit Anti-STAT5 Antibody And Study On The Role Of STAT5 In Carcinogenesis And Progression Of Human Breast Cancer

Objective: Signal transducers and activators of transcription 5(STAT5) is a kind of protein which plays a key role in signal transdution of cytokines and it has deep correlation with breast cancer growth and development. However, it is undefined that the action mechanism of STAT5 protein in carcinogenesis and progressin of human breast cancer. In this research, STAT5 protein was extracted and purified from Sf21 cells transfected by STAT5 gene to preparation of rabbit anti-STAT5 antibody. Immunohistochemical staining and Western blot were used to detect location and expression of STAT5 protein, respectively. RT-PCR was used to determine the differences of STAT5 gene in the normal breast and breast cancer tissues. JAK inhibitor I was used to study STAT5 protein expression in human breast cancer cell in order to demonstrate its effect on the growth, development and prognosis of breast cancer. Such studies would provided theoretical evidence for the early diagnosis and proper treatment project and prognosis assessment of breast cancer. Methods: 1. Extract and purify STAT5 protein : Sf21 cells were infected with recombinant STAT5 gene. After 48h of incubation at 28°C, the transfected cells were collected and sonicated by Branson sonicator on ice, its total protein were dissolved in ice cold extraction buffer. Filtered cell extract was loaded onto a Q Sepharose column, Heparin-Toyopearl column and Superdex G200 column respectively. The character of STAT5 protein in the fractions were identified by SDS-PAGE, Western blotting. 2. Prepare rabbit anti-STAT5 antibody: Rabbit were immunized with recombinant mouse STAT5 protein to prepare antibody against STAT5. The purity and specificity of the antibody were analyzed by SDS-PAGE and Western blotting respectively. 3. Detect the location of STAT5 protein in breast tissues: Malignant and nonneoplastic breast tissues samples from 75 and 20 women were obtained from Department of surgery Fourth Hospital of HeBei Medical University from September 2003 to April 2004. All patients were female with median age 45. Immunohistochemical S-P method was used to detect the contants of STAT5 in the malignant and normal tissues of breast. 4. Detect the expression of STAT5 protein and gene in human breast tissues: Samples from 44 women were obtained from December 2003 to May 2004. Western blotting and RT-PCR were respectively used to measure the expression of STAT5 protein and gene. 5. MTT method was used to evaluate that JAK inhibitor I effect on the proliferation of human breast cancer cell MCF-7 and MDA-MB-231. Cell-cycle and apoptosis were quantified byflow cytometry. Western blotting was used to detect protein levels of STAT5 and Bcl-xl in MCF-7 cell after treated with JAK inhibitor I. 6. Statistic Methods: Statistic analysis was performed with SPSS software 10.0. Results: 1. Extracted 493mg STAT5 protein from 2.5×109 Sf21 cells with being infected STAT5 gene. There was subpopulations in the elution of Q sepharose High performance column and Heparin-Toyopearl column with the MW 94KDa. Then it was applied by Superdex G200 and obtained a pure STAT5 protein. The results of SDS-PAGE and Western blotting showed the one protein bands at approximately 94 kDa, corresponding to STAT5. 2. The titer of serum anti-STAT5 antibody was 1: 204 800 (A450=1.01) after 6 weeks by immunization. The SDS-PAGE and Western blot analysis showed that there was a obvious protein band with Mr 94 000, confirming the specificity of obtained anti-Stat5 antibody. 3. It was displayed by immunohistochemical staining with anti-STAT5 antibody that in breast tissues STAT5 was expressed in nuclear and its numbers of positive cells were more than 5 percent, this was known as positive reaction; whereas STAT5 was expressed in cytoplasm, this was known as negative reaction. There is positive relationship between the numbers of STAT5 positive cells and the histological grade in breast cancer tissues (P<0.05).4. STAT5 protein levels in breast cancer tissues was markedly high compared with adjacent normal tissues (P<0.05). Overexpre...