| Cell adhesion is an important life phenomenon which widely exist in almost all kinds of organism. It participates in modulating many physiological and pathological cell function. Many adhesion molecules located at the surface of cell member participate together to accomplish cell adhesion. Its form including adhesion cell to cell and cell to extracellular matrix.In past several years, because of involving contact inhibition of tumor cell, gap junction communication and signal conduction between cells, tumor invasion and metastasis, cell adhesion molecules become another new research hotspot in tumor prevention and cure domain.By now there were many reports about research of cell adhesion molecules in past years, but most of them only limited to examination of cell adhesion molecules in HCC, and relativity between expression change and tumor cell invasion. As for mechanism to bring these change is not clear.A new found gene, cell adhesion regulator gene, afford a new way to research the regulating mechanism on tumor cell adhesion molecules. CAR gene was located on the long arm of chromosome 16q. its cDNA was 464bp long with a tyrosine phosphorylation site at the extreme 3' end. It was considered to be a signal conduction molecule that function s in the regulation of integrins involved adhesion between cell and extracellular matrix.Foreign research indicated there were mutation or absence of CAR gene on different level in many malignant tumor, such as colorectal cancer and breast cancer. And these changes were positively correlation with tumor's invasion and metastasis. Thus it was considered to be a tumor-suppressor gene, which may reversely regulate tumor growth, differentiation, invasion and metastasis, through affecting cell adhesion, cell gap junction communication and signal conduction.Research about CAR gene and HCC has limited to few foreignreports. Because of high level of allelic loss on 16q, in the region of CAR, the relation between CAR gene and HCC may consanguineous. Some scholars determined the expression of CAR mRNA in 30 cases of HCC. All tumor samples showed amounts of expression that were lower in different level, compared with those found in their matching controls. The amounts of expression were obviously related to a-fetoprotein, histological differentiation, intrahepatic metastasis rate, 5-years relapse rate. There was no related report in our country by now.Through this study, we firstly determined the expression of CAR mRNA in several human hepatocellular cell-lines, which were often used in laboratory, offering academic base for further research on mechanism of this gene.Materials and MethodsA normal adult hepatocellular cell-line, L02 and three human hepatocellula cell-lines, SMMC-7721, HepG2 and Bel-7404, were cultivated in RPMI1640 routinely.Total RNA was extracted from cell lines using TRIzol one-step method, then the specimens were examined on quality. According foreign literatures, we design a pair of specific primers respectively for CAR and P-actin. The cDNA was synthesized from 2 ug of total RNA using MMLV transcriptase with random hexamers. The cDNA was amplified with the PCR. The intending PCR products was long as 318bp for CAR and 254bp for (3-actin. PCR was carried out under the following conditions: denaturation at 94@ for 2 min; annealing at 60@ for 30 sec; extension at 72@ for 30 sec; 35 cycles for CAR and 20 cycles for p-actin.10ug PCR products were electrophoresed in a 2% agarose gel, and the images were scanned and saved. Then the light density of bands were analyzed using Band leader Ver3.00, a electrophoretic image analysis software. The light desity ratios of CAR/P-actin mean the corresponding amounts of CAR mRNA expression.ResultsExamined by ultraviolet spectrophotometer, the A260/A280 ratios were all higher than 1.8, it mean extracted total RNA had good pureness, and had little protein pollution. Examined by 1.5% formaldehyde denaturation agarose gel electrophoresis, two bands could be clearly shown, which were18SrRNA...
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