Expression Of NDRF2Gene In Gallbladder Carcinoma And Its Effect On Growth Of Gallbladder Carcinoma Cells

Chapter I Expression and clinical significance of NDRG2protein in gallbladder carcinomaObjective:We design to detect the expression of NDRG2protein in allbladder carcinoma; With the clinicopathological data collectied, we also analyze the factors that may affect the expression of NDRG2protein. Accordingly, we probe to explore the role and significance of the NDRG2protein in the development of gallbladder carcinoma.Methods:We collected130cases of gallbladder carcinoma specimens and para-carcinoma tissues treated in Department of General Surgery, Baogang hospital, from September2009to2011August. All these cases should have complete medical records informations and should not have gong through radiotherapy, chemothrapy or other adjvent therapy before surgery. All specimens were fixed in10%formalin, embedded in paraffin, and cut into4μm serial sections. Immunohistochemistry was used to observe the expression of NDRG2protein in these tissues. The relationship between the expression of NDRG2protein and the related clinicopathlogical parameters were analysed statistically in case-control study.Result:The positive rate of NDRG2in gallbladder carcinoma tissues was significantly lower than those in para-carcinoma tissues (P<0.005). In the gallbladder carcinoma group, the positive expression cases were mostly the ones with well-differentiated pathological type(P=0.008),which demonstrated few clinical evidence of lymph nodes or distant metastasis(P=0.03).Conclusion:The low expression intensity of NDRG2protein in gallbladder carcinoma tissues, which is closely relatied with the stage, grade and clinical prognosis of gallbladder carcinoma; The NDRG2protein may play an important role during the development from precancerous lesions to gallbladder cancer, so it helps to early diagnosis even in the stage of precancerous lesions by detecting the expression intensity of NDRG2protein. Chapter II Construction of eukaryotic expressing vector of human NDRG2and establishment of stable transfectant GBC-SD cell lineObjective:To construct eukaryotic expressing vector of NDRG2and transfect GBC-SD cells so as to establish stable GBC-SD cell line.Methods:The full length NDRG2cDNA fragment was amplified by PCR from the human Gallbladder carcinoma tissue cDNA library and was inserted into pcDNA3.1. After the identification of digestion and sequencing on the recombinant pcDNA3.1/NDRG2, we transfected the recombinant into GBC-SD cell by lipofectamineTM2000. After screening culture by G418, stable transfected GBC-SD cell line was established, and the expression of NDRG2was identified by RT PCR and Western blotting.Results:The eukaryotic expression vector pcDNA3.1/NDRG2was constructed, stable transfected GBC-SD cell line was established, and NDRG2protein was expressed successfully. Conclusion:The construction of the eukaryotic expression vector pcDNA3.1/NDRG2and the establishment of stable transfected GBC-SD cell line have provided solid experiment foundation for further studies on the function of NDRG2. Chapter Ⅲ The transfection of NDRG2gene affects the biological behaviors of human Gallbladder carcinoma GBC-SD cellsObjective:To detect the effects of NDRG2transfection on the growth of GBC-SD in vivo and in vivo.Methods:In vitro the cell growth and apoptosis of three groups were evaluated by MTT assay and flow cytometry. For in vivo tumorigenicity studies,1×106cells/site(1site/animal)from each of the respective cell lines were injected subcutaneously into SCID mice. A total of ten mice were inoculated with each cell population on day zero and the mice were observed daily to determine the latency period before detectable tumor growth. Once tumors were palpable, the tumor size was measured weekly for a period of28days to assess tumor growth rate. After completion of the Study the mice were euthaulzed,the tumors wore removed,and a section of each tumor was placed in formalin for histopathologic examination. Sections of the tumors wore detected to assess NDRG2expression in these tumor cells.Results:The apoptotic cell number of GBC-SD-NDRG2(74.19±5.75) was higher than GBC-SD-Ve(28.75±3.46)and GBC-SD (17.42±2.71). There was significant difference between GBC-SD-NDRG2and GBC-SD-Ve or GBC-SD(P<0.05), but there was no significant difference between GBC-SD-Ve and GBC-SD(P>0.05). The cell inhibitory rate of GBC-SD-NDRG2was higher than the GBC-SD-Ve, there was significant difference between them(P<0.05). The latency period for tumor formation for both of the cell lines is5days. The tumors grew quite rapidly in the null transfectants compared with the NDRG2-transfected cells over the next4weeks. At the time of euthanasia,.8the tumors from GBC-SD-Ve measured384.17±72.84mm3on average, but those firom GBC-SD-NDRG2were significantly smaller(almost half the size), measuring only178.13±29.75mm3°n average. The tumor inhibitory rate of GBC-SD-NDRG2was50.00%. NDRG2protein expression was positive in tumor cells of GBC-SD-NDRG2group.Conclusion NDRG2transfection can inhibit the growth of GBC-SD cells in vitro and in vivo. NDRG2gene may play an important role in generation and development of Gallbladder carcinoma.