| Invasion and metastasis are very important feature of tumor, tumor cells must degrade basement membrane and extracellular matrix (ECM). Heparan sulfate (HS) is the very capital compomant of ECM, and bond many growth factors and cytokines. Tumor cells can product the novel cloned Heparanase, degrade HS specifically and promote tumor cells to cross ECM. Heparanase also can release growth factors and cytokines, promote tumor growth, adhesion and angiogenesis. In addition, Heparanase is the only endoglucuronidase that can degrade HS of mammalian, so the correlation between Heparanase and tumor metastasis must be investigated and become the focal point of this area.We use molecular cloning technology, overcomed the difficulty that human Heparanase exist native complementary base in ORF region, amplified anterior segment and posterior segment of BamH I enzyme of Heparanase cDNA by reverse polymerase chain reaction (PCR) respectively, andconstructed sense and antisense eukaryotic expression vector of human Heparanase gene successful. The vectors were confirmed by restriction endonucleased digestion and identical with the Heparanase that in GenBank (AF144325) by auto sequencing. The reading frame was correct and suitable for expression. Then we transfected sense and antisense Heparanase gene vector into the human hepatocarcinoma cell line HepG2 and high metastasis hepatocarcinoma cell line HCC-P by Lipofect AMINE, empty vector was used as control. The transfected cells were selected in DMEM containing G418 for 60 days and the positive cell clones were attained. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis and immuno-histochemical assay were used for mRNA transcription and protein expression of Heparanase gene. Cell counted and MTT test were used to detect the condition of cells growth. The Heparanase mRNA expression level of cells transfected with sense Heparanase vector was significantly more than the cells transfected with antisense Heparanase vector or empty vector by RT-PCR, the mRNA level was inhibited in cells transfected with antisense Heparanase vector. Immunohistochemical assay also confirmed abundant Heparanase protein in cells transfected with sense Heparanase vector. The protein was expressed from cytoplasm to trans-membrane, and to outside of cell lines. But the condition of cell growth was not influenced by cell counted and MTT test. So we provided the good cells that can over express or inhibit Heparanase gene for advanced studying.To investigate the effect of sense and antisense human Heparanase gene, we constructed hepatocarcinoma metastasis model by administered transfected cells i.v. into nude mice. 30 days after injection, the lungs were removed,weighed, and surface metastastic nodes were counted by dissecting microscope. The lungs were fixed, decolorized, embedded in paraffin and stained with hematoxylin/eosin for histologic examination. Lung weight and metastastic nodes were significantly reduced in the mice injected with cells transfected antisense Heparanase gene (P0.01). Histologic examination revealed numerous large pulmonary metastases in lungs from the mice in sense group. In contrast, only a few small metastases could be seen in antisense group. The results confirmed that Heparanase can promote hepatocarcinoma cells metastasis, but antisense human Heparanase gene had significantly therapeutic effect on metastasis of hepatocarcinoma cells by inhibiting Heparanase gene expression.Then we subcloned sense human Heparanase gene into prokaryotic expression vector pRSET, transformed into E.coli BL21, Heparanase protein was expressed after IPTG induction. By immunizing rabbit with Heparanase protein obtained from above experiment, we produced Heparanase's polyclonal antibody. It is proved the Heparanase's polyclonal antibody had high titer, affinity and specificity and characterized the liter of antibody was 1:2000 by Western blotting. We tested the Heparanase protein expression of 25 human hepatocellular carcinoma (HCC) tissues by immunohisto...
|
PDF Full Text Request