The Empirical Study Of AhoA's Effect On The Migration Of HCC

Liver cancer is a kind of common and frequent tumor, which is a participating course in the generation and development of carcinoma, with multiple factors, multiple procedure and multiple genes. Transferrance and virulence are the two significant biological characteristic of the malignant cell. In order to provide some original methods and moduses of controlling the cell migration and scatter, we tries to fath the migratory mechanism of the malignant cell. The RhoA oncogene , have been revealed in recent years, are closely related to various malignant tumours. RhoA, belonging to Rho family, is one member of the ras superfamily, whose encoding production, namely RhoA protein, pertaining to micromolecular mass GTP conjugated protein family, in GTP conjunctive active form and GDP conjunctive unreactive form. It exist in the latter form in normal cells and the switch of the two forms enables them to educe functions, paralleling"molecular switch", the diminutive G protein in activated state being able to activate the wink dromo-iter to work functions, which is in the resemblance of process during which the Ras genic mutation induces its continous activations. If Rho genes constitution transform and handicap the GTP hydrolyzing and keep in the state of GTP's continous conjugated activation, which can enhance their multifunctions GTP's continous mutants of constitution such as RhoA(G14V), RhoA(Q63L), etc. Rho family proteins participate in the various vital process of regulating cells, including actin's recombinating the cystoskeleton, cell adhesion, cell movement, the advancement of cycle, cellular fission and genetic transcription, etc. The discovered RhoA downstream effect molecules comprehend ROCK, PKN, Citron, PI-3, etc. These molecules mediate the function of RhoA. Accordingly, diminutive G protein is considered the critical wink protein of the cell migration.cAMP is the important second messenger. Protein Kinase A(PKA) is the Protein Kinase cAMP depends, which mediates the signal transduction of cAMP. Such as PKA plays on important role in accommodating cystoskeleton and cell migration. PKA has been confirmed it can inhibit RhoA's functions recently. Lysophosphatidic acid(LPA) is excreted by blood disc, collagenoblast, cancer cell, and cellula adiposa, which is a multifunctional phosphatide messenger, acting on critical part in the generation and development of many disease such as cardia cerebrovassular disease, nephronia and tumour. In order to react all kinds of ecto-stimulus, LPA can delivery from vairies cells, including impaired endodermis agent, activated collagenoblast, intercapillary cells, phlegmasia cell. LPA produce by means of autocrine, paracrine, which determine LPA's much biological effect together with all kinds of disease closely. The investigation detects LPA can irritate RhoA protein to be active.In the experiment, we utilizes HepG2 cell as the pink of the hepatocelluar carcinoma cell(HCC), family, and L-O2 cell the normal liver cell's pink and studies the expressions of RhoA in the two kinds of cells and observes RhoA's function on the migration of cancer cell when the RhoA is activated or inactivated.Objective: To study the expression of RhoA protein in the HepG2 cells and L-O2 cells; To approach the role RhoA plays in the process of controlling hepatocelluar carcinoma cells'migrations by agitating and rivalrying RhoA protein's function and by surveying the migrations of the malignant cell; and to offer the feasibility and preliminary experimental foundation for the further study of transfusion mechanism of the HCC.Methods: The researching objects are HepG2 cell(hepatocelluar carcinoma cell line) and normal liver cell line L-O2 cell, which are culture in high glucose DMEM supplemented with 10% FBS. We observes the morphology of HepG2 cells and L-O2 cells in the inverted microscople, and completes the staining of HE in exponential phase of growth of HepG2 cells and L-O2 cells; The expression of RhoA protein in HepG2 cells and L-O2 cells is detected by immunocytochemistry of SABC, biomedicine image analysis system complete sereology analysis of immunocytochemistry photo and yield average optical(AO); We detects the invasion capabilities of HepG2 cells and L-O2 cells with Millicell cabins and detects the transport number after excitomotor and antagon separately and cultivating four hours. We lists the experimental data with the methods of mean±standard deviation and analysizes the data, deploying SPSS10.0 software and t-test. P﹤0.05 denotes to have statistics significance.Results: (1)Observation of morphology: HepG2 cells and L-O2 cells grow well in experimental phase of growth, the transparenty is good, the refraction is strong, the outline is unsharpness, and can observe the cells of dividing phase. (2)The immunocytochemistry staining of RhoA is strong in HepG2 cells, their positive product present buffy, it mostly distributes in endochylema, but the immunocytochenmistry staining of RhoA is very weak in L-O2 cells, its phanerosis is very diffcult; image analysis denotes average density of immunocy to chemistry stain: L-O2 cells, 0.092±0.010; HepG2 cells, 0.398±0.018. Their average optical shows distingwished disparity through being analysized in statistics analysis(P﹤0.05). (3)The Millicell detection shows that the number of HepG2 cells is much larger than that of the L-O2 cells after going through the hole added to the Lysophosphatidic acid (LPA ) is much larger than the number of cells going through the hole added to the cAMP. Twenty high power fields are chosen respectively at random to count the cells going through the cell cabin holes: L-O2: 74.30±7.59; HepG2: 131.40±7.26; They have significant difference in statistic analysis(P﹤0.05); HepG2 cells plus excitomotor: 192.00±7.59; HepG2 cells plus antagon: 65.10±4.61; They olso have significant difference in statistics analysis(P﹤0.05).Conclusion: Both the HepG2 cells and L-O2 cells have the expression of RhoA protein. However, the expression level of RhoA in HepG2 is much higher than that in L-O2. we also discover that the number of HepG2 cells mults more greatly with the detection of Millicell cabin than that of L-O2 cells through the hole, and that the number of HepG2 cells mults obviously and predominantly if added to RhoA excitomotor and decreases, obviously if added to RhoA antagon. So it is much possible that RhoA plays important role in the generation and extension of liver cancer.