The Study Of Malignant Phenotype Of A549 Lung Carcinomacell Line Reversed By Human CC10 Gene

Part1 Expression of CC10 and its relation to apoptosis in thenon-small cell lung cancerObjectiveTo investigate the relationship between CC10 protein expression and apoptosis in non-small cell lung cancer(NSCLC) MethodsCC10 protein expression of 43 NSCLC paraffin-embeded samples and 20 benign lesion tissues were examined by using immunohistochemistry method. The results were analysed by HPIAS-1000. The 43 NSCLC apoptosis were detected by TUNELtechnique. analysing correlation between the 43 NSCLC apoptosis index (AI) and CC10 protein expression. ResultsThe positive rate of CC10 protein expression in lung cancer tissues was 46.51%(20/43), which was significantly lower than that in benign lesion tissues,85. 0 % (17/20) (P<0.01). The expression level of CC10 was related to tumor TNM stage, histological differenation and lymph node involvement, but was not related to sex, age, tumor diameterand and histological classification. The apoptosis index(AI) of CC10 protein expression positive in lung cancerwas 5. 585 dr 1. 7822%, the apoptosis index(AI) of CC10 protein expression negative in lung cancer was 2. 8565 ± 1. 3990%, obvious correlation was observed between CC10 protein expression andapoptosis cell index(Rs=-0.211,P<0.05). ConclusionCC10 protein expression was descressed in NSCLC. down-expression of CC10 may be high correlation with apoptosis in NSCLC. Part II To construct and identifyCC10 eukaryotic expression plasmidObjectiveTo construct and identify a eukaryotic expression plasmid pcDNA3.1-hCC10. provide a basis of sequent experiments MethodsA 273 bp cDNA fragment was amplified from the total RNA of normal lung tissue by reverse transcriptase-PCR(RT-PC R ) , To cut amplification product and pcDNA3.1 by incision enzyme BamHI and Hind I coupled reaction between purified amplification product and pcDNA3.1 segment was carried out through T4 DNA joinase. The recombinant plasmid was transferred in DH5a. Ecoli .We selected the positive clone ., eukaryotic expression plasmid was identitied .by the methods of double enzyme digesetion with HindIII/ Bam I and directly DNA sequceing. ResultsAfter the identification of two restriction enzymes analysis Hind IIIand Bam I and directedly DNA sequencing technique , We found the sequence of the clone gene was consistent with the CC10 exon 1 in Genebank, The hCC10 was inserted in pcDNA3.1-eukaryotic expression vector. ConclusionWe successfully constructed The pcDNA3.1-hCC10 expression plasmid, The recombinant plasmid cDNA3.1-hCC10 may serve as an effective tool for the study of tumorogenesis and tumor treatment. Part III Establishment of lung adenocarcinoma carcinoma A549 cell lines which stably expresses CC10 geneObjectiveEstablishment of lung adenocarcinoma carcinoma A549 cell lines which stably expresses exogenous CC10 gene MethodsA large volume of pcDNA31-hCC10 was acquired from DH5a. Ecol with Plasmid Maxiprep extract kit. A549 cell line in group A and group B were transfected with pcDNA3.1-hCC10 and pcDNA by liposome.respectively, group C was control group which transfected with nothing. A549 cell lines were selected by G418 Western blot was carried out to detect CC10 protein expression in A549 cell line in all groups. CC10mRNA expression on A549 cell line was detected by RT-PCR. ResultsCC10 mRNA and protein expression on A549 transfected with pcDNA3.1-hCC10(+) in group A were higher than those in group B and group C. ConclusionWe successfully established A549 cell line which high stably expressesed CC10 gene. Part IV The study of malignant phenotype of A549 lung carcinoma cell line reversed by human CC10 geneObjectiveTo study the effect of growth-inhibiting and apoptosis on A549 by exogenous CC10 in vitro and in vivo MethodsThe test were divided into 3 groups . group A and group B were transfected with pcDNA3.i-CC10 and pcDNA3.1 respectively, group C was negative group which transfected with nothing. cell growth- test, soft agar clonogenic assay were used to evaluate the effects of exogenous human CC10 gene on A549 cell growth.in Vivo. The growth of xenograft tumor experiment was observed to evaluate the effects by exogenous CC10in vitro Apoptosis. morphological on A 549 cell line were detected by eletricscope and Hoechst33258 staining; Apoptosis . degree were evaluated by the methods of AnnexinV-PI staining Flow cyometry analysis(FCS) and Western Blot, RT-PCR were used to determine . changes of cell cycle progression and the protein and mRNA expressionof cell cycle protein CyclinD₁ ResultsIn group A the A549 cell growth curves, clone formation rate and A549 lung xenograft cancer growth rate were obviously inhibited . Compared with group B and group C ,we also found more typical apoptosis morphological cell and high apoptosis In group A. By Flow cyometry and Western Blot, RT-PCR,we ubserved that more percentage of cell in G₁ phase and more gene expression of cell cycle protein cyclinD₁ than group B and group C. We also found that cyclinD₁ protein and mRNA expression were increasing with the time of CC10 gene transfected prolong, ConclusionHuman CC10 gene can reverse malignant phenotype of A549 lung carcinoma cell line in vitro and in vivo...