| Primary liver carcinoma (PLC) is a seriously threatening malignant tumor which accounts for 5.4% of all cancers newly found and seizes above 500,000 lives annually in the world. With a high incidence in China, PLC ranks the second places on the list of mortality of malignant tumors and occupies about 53% of global population died from PLC. It has a constantly increasing trend on incidence and mortality in recent years in China. The therapeutic method against PLC is a complex mainly of surgical resection of local tumors combined with radiotherapy and chemotherapy. Accompanied with the progresses of recognitions of physical functions and anatomical structures of the live and the improvements of both imaging techniques and experimental tools, the excision rate has increased dramatically while the post-operation mortal rate has decreased markedly. However, for the reason that the onset of the PLC is insidious with no obvious or typical early symptoms and the advancement is quite rapid, lots of patients have been diagnosed at advanced stage missing the windows of resection. Meanwhile, there is no breakthrough on the radiotherapy and the chemotherapy. The metastasis and recurrence of the tumor also still remain to be solved.Based on the theory of modification of biological response introduced in 1980s, the fourth therapeutic model against tumor, bioimmunotherapy, has been built after the surgical resection, the radiotherapy and the chemotherapy. With recent progress of molecular biology and the rush of new biotherapeutics, the targeted therapy has become a new choice for the patients at advanced stage. Molecular targeted therapy as well as radioimmunity targeted therapy have achieved an exciting result and have gradually come to be the first choice on the advanced stage patients. The possible breakthrough on targeted therapy against PLC may include: detection of new target, development of multi-target drugs,emergence of complex immune targeted drugs of double-marker, targeted therapy combined with the other therapy.Adenosine triphosphatase (ATPase) used be thought as multiple subunits complex molecule of enzyme located in the inner membrane of chondriosome, mainly dedicated to the synthesis of ATP and energy metabolism. New researches have brought it forward that ATPase is also a kind of receptor protein existing in the surface of cells. The Membrane-expressed ATPase has not only the function of the synthesis and decomposition of ATP, but also plays an important role in series of physical activities: adjustment of serum cholesterol, angiogenesis of endotheliocytes and the modulation of pH value of interior of tumor cells. It has been widely and energetically focused on the ATPaseF1 abroad. But, there is no concern on the correlativity between ATPaseF1 and PLC. This paper set a primary research on the expression and biological function of ATPaseF1 in human PLC cells using prepared monoclonal antibody of ATPaseF1 domain. By the hybridoma technique, the monoclonal antibody of ATPaseF1 domain is prepared using ATPaseF1 of beef-heart chondrosome as antigen. After the specificity has been identified, the monoclonal antibody has been used to detect the expression in human PLC cells. Finally, the expression and biological function of ATPaseF1 in hepatoma HepG2 strain aimed at a new direction for the targeted treatment against PLC.ObjectiveThe expression of ATPaseF1 in human PLC tissue.The preparation of the monoclonal antibody of ATPaseF1 domain and the identification of he specificity. Confirmation of differences of the expression of ATPaseF1 in membrane between the strain of HepG2 and the strain of L-02.The effects of antibody of ATPaseF1 domain on the reproductive and immigration activity.Methods1 The expression of ATPaseF1 has been examined by immunohistochemical Envision method in 38 samples of PLC.2 The mitochondria F1-ATPase have been extracted from bovine heart and be used to immunize BALB/c mice. Spleen lymphocytes have been fused with SP2/0 myeloma cells. Hybridoma cells secreting monoclonal antibodies (McAb) have been produced by three-time limited dilution method. The monoclonal antibody in the ascites has been purified by affinity chromatography with protein A sepharose CL-4B and its purity has been detected by SDS-PAGE.3 The specificity of monoclonal antibody has been identified by western blot, cell inmunofluorescence and ELISA. The subtype of monoclonal antibody has been identified by ELISA- kit.4 The soucrose density gradient centrifugalization has been utilized to dissociate plasma membrane fraction and PIERCE-KIT reforming method has been used to extract pericellular membrane protein. The extracted protein has been validated by COX-1 MoAb so as to deplete the interference of mitochondrial membrane proteinum. Consequently, the distinction of the pericellular membrane expression of ATPaseF1 between HepG2 cell line and L-02 cell line has been detected by Western blot assay.5 The distinction of the pericellular membrane expression of ATPaseF1 between HepG2 cell line and L-02 cell line has been detected by cell inmunofluorescence and Flow cytometry detectiong.6 The effect of anti-ATPaseF1 antibody on reproductive activity of HepG2 cell line is evaluated by CCK-8 assay. The similar effect of anti-ATPaseF1 antibody on metastasis and migration ability of HepG2 cell line is surveyed by Wound-healing assay and Transwell migration assay.Results1 It is showed in immunohistochemistry analysis that 30 of the 38 cases of PLC tissue and 11 of the 38 cases of adjacent normal liver tissue have a middle to high expression of ATPase F1 (P<0.01).2 Not only the purified bovine heart mitochondria ATPaseF1 but also the hybridoma cell 5G10 stably secreting anti-hATPaseF1 monoclonal antibody are obtained. The antibody titer is 1:104 in the supernatant of hybridoma cell 5G10 and is more than 1:106 in the ascites. The monoclonal antibody in the ascites has been purified by using affinity chromatography with protein A sepharose CL-4B and its purity is above 95%.3 Mab5G10 belong to IgG1 hypotype which has been detected by Sigma Immune TypeTM Kit. Western blot has been performed both total protein of HepG2 cells and purified bovine heart mitochondria ATPaseF1 and all result in a single belt at 50KD without obvious cross reaction with other components. The cell immunofluorescence of A549 cells indicate that the monoclonal antibody can recognize ATPaseF1 in cytoplasm and has a special binding with cellular membrane in the dyeing of impermeable membrane. Thus the prepared monoclonal antibody has the activity to bind with natural antigen. That the monoclonal antibody can bind with ATPaseF1 and ATPaseF1βhas been proved by ELISA tests.4 The protein extracted from the HepG2 cell and the L-02 cell has been validated by COX-1 MAb so that the authenticity of the above extraction of pericellular membrane proteinum has been confirmed. There is positive expression of ATPaseF1 in the pericellular membrane protein of the HepG2 cell line while negative expression in that of the L-02 cell line in western blot assay.5 ATPaseF1MAb can bind specificly with the pericellular membrane protein of the HepG2 cell which has been confirmed by cell inmunofluorescenceand Flow cytometry detectiong. Meanwhile, there is no specifical binding of ATPaseF1MAb and the pericellular membrane protein of the L-02 cell.6 The results of CCK-8 assay indicate that ATPaseF1MAb compared with control group has no predominance depressant effect on the proliferation of HepG2 cell (P<0.01). The results of Wound-healing assay reveal that ATPaseF1MAb obviously restrain the migration of HepG2 cell (P<0.01). The results of Transwell migration assay note that ATPaseF1MAb obviously suppress the migration of HepG2 cells.ConclusionsThe expression of ATPaseF1 in PLC is relatively higher.A hybridoma cell line which can secret a novel functional mouse anti-human ATP synthase F1 has been developed successfully. There is a positive expression of ATPaseF1 in the pericellular membrane protein of HepG2 cell while a negative expression in that of the L-02 cell. ATPaseF1MAb can not obviously repress the proliferation of HepG2 cells, but may repress the migration of them. It maybe turns out to be a novel way for targeted therapy of PLC. |
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