The Purification Methods Of ACRP30 And The Studies Of Tumor Inhibition On ES-2 In Vitro

Adiponectin is adipose cytokines which is secreted by adipocyte, it was isolated firstly from the rat adipocytes by Scherer in 1995, and then it was named as adiponectin by Arita in 1999. Research on Adiponectin largely focused on obesity and diabetes disease. Studies were showed that Adiponectin concentration in plasma was related to abdominal fat content, changes of Adiponectin would be linked to abdominal obesity, insulin resistance and atherosclerosis lipid profile. Recently many researchers found that Adiponectin leveles were associated with some tumors. Clinical epidemiological. studies indicted that the low Adiponectin levels increased the risk of endometrial cancer, breast cancer, stomach cancer and prostate cancer. As Adiponectin leveles were related to a variety of tumor-associated reproductive system, it is inferable that Adiponectin plays a role on ES-2 ovarian carcinoma cell line in vitro.The purpose of this study was to use genetically engineered bacteria including adiponectin gene and globular domain of adiponectin expressing a lot of aimed protein, to select a suitable purification protein method, to confirmed the inhibition of proliferation, migration and cell cycle on ES-2 ovarian cancer cell line by adiponectin.With selecting experimental conditions of protein expression, we found a suitable method to amplife a large number of gene-engineering bacteria and purify expressed protein. Purificating was based on specific affinity of His labels and Ni~+ using His-bind purification column. Different purification methods were used, for the full-length adiponectin protein tie mainly in the form of soluble protein and spherical domain of adiponectin protein lie mainly in the form of inclusion bodies. The full-length adiponectin was directly purified by purification column and spherical domain of adiponectin was indirectly purified by denatured affinity chromatography. Inhibiton of ES-2 ovarian carcinoma cell line was studied by human adiponectin recombinant in vitro, MTT test (tetrazolium salt colorimetric test) was used to inhibit cell proliferation, scratch experiment was used to affect cell migration by adiponectin, flowing cytometry technology was used to detect inhibit cell cycle.Through selecting suitable experimental conditions, we got a tot of genetically engineered bacteria and aimed proteins. The protein concentration of the full-length and globular domain of adiponectin is 0.51 mg/ml and 0.77 mg/ml.Proliferation of ES-2 ovarian cancer cell line was inhibited obviously by adiponectin and this effect was dose-dependent using MTT test (tetrazolium salt colorimetric test). Cell mitotic was arrested in G1 phase by adiDonectin, "limit point" was inhibited, and replication of cell DNA and chromosome was prevented. Inhibition of tumor cell proliferation by adiponectin was further confirmed. Through scratch experments, tumor cell migration was inhibited by adiponectin, which was positive correlated to adiponectin concentrations. It is showed that Adiponectin will reduce the movement of tumor cells and inhibit tumor cell migration, thereby suppress tumor metastasis.