A Study On The Expression Of Canstatin In The Eukaryotic Cell And Its Biological Effects On Lung Cancer

Angiogenesis is crucial for the growth and metastasis of solid tumor. Increased angiogenesis in lung cancer is associated with tumor progress and poor prognosis. Hence, inhibiting angiogenesis in tumor tissue may prevent tumor from growth and metastasis. Many inhibitors of angiogenesis have been recently discovered in succession. Such as canstatin, a 24-kD peptide derived from the C-terminal globular non-collagenous (NC1) domain of theα2 chain of type IV collagen, has previously displayed its character to inhibit proliferation of endothelial cells in vitro and suppress tumor growth in vivo. This study was therefore undertaken to determine if the canstatin gene could be expressed in the human lymphocyte and lung cancer A549 cells, and to evaluate the effect of canstatin on the growth and metastasis of lung cancer.Methods1. A recombinant vector pCMV-Script-CEAS-Cans being reconstructed with canstatin gene was transfected into A549 cells and human lymphocytes by electroporation respectively. The positive clone of transfected cells was screened by G418. Naked vector (pCMV-Script vector) was also transfected into the cells with the same way for comparison.2. PCR technique was used to examine the canstatin DNA in the transfected cells, and the expression of canstatin protein in cultural supernatant of the transfected cells was detected by SDS-PAGE assay.3. The cultural supernatant of the four kinds of transfected cells was concentrated with the way of ultrafiltration for tumor treatment test.4. The lung cancer cells of Lewis were subcutaneously inoculated to C57BL mice to make animal model of tumor. When the transplanted tumor on the mice had developed to 1cm3, 25 mice were randomly assigned into 5 groups. Then, these mice in 4 groups were injected with 0.2ml cultural supernatant of the lymphocytes and A549 cells that were transfected with pCMV-Script-CEAS-Cans vector or naked vector respectively, and 0.2ml NS was also used for the left group.(the former two as treatment group;the latter three as...