Objective To establish hepatocellular carcinoma(HCC) cell lines which olig-expressed IGF1R gene stably.Methods An eukaryotic expressing vector pSUPER-IGF1R-siRNA that could block IGF1R expressing was transferred into hepatocellular carcinoma cell lines SMMC7721 and Hep3B with Lipofectamine 2000 reagents.After transferred,cells were selected with G418 to obtain positive clones.The expressions of IGF1R,cyclin D1 and cyclin B1 were detected by RT-PCR and Western-blot.Cell growth curve were painted.Results Two cell lines clones were screened olig-expressing IGF1R gene stably.The experimental cell lines grew more slowly than control cell lines and the expression of cyclin D1 decreased(P

Objective To establish and identify transgenic mice model that specifically express delta drosophila homolog-like 1 (DLK1) in liver for the functional study of DLK1 on liver differentiation,hepatopathy,and hepatocellular carcinoma.Methods The coding sequence of DLK1 cDNA was inserted downstream of mouse albumin gene enhancer/promoter to construct a liver-specific DLK1 expression recombinant vector.The DNA fragment of transgene digested from the recombinant vector by Pine I was transfected to Hepl-6 cells to verify the expression of DLK1 in vitro.Then the transgene fragment was microinjeeted into fertilized eggs of C57×CBA F1 mice and 20 transgenic founder mice were generated.The F1 mice of DLK1 transgenie founder mice were used to identify the expression of the transgene in liver and other tissues.Results RT-PCR and cellular immunofluorescence showed DLK1 expression when the transgene fragment was transfeeted into Hep1-6.For transgenic mice,RT-PCR and immunohistochemistry showed that DLK1 was specifically expressed in adult F1 mice liver.Conclusion Successfully established the liver-specific DLK1 expression transgenic mice model.

Objective To screen down regulated genes and find down regulated novel genes in gastric cancer. Methods Genes mRNA expression were detected between gastric cancer and normal gastric mucous membrane of five patients using cDNA microarray. Genes mRNA expression signals on hybridization membranes were analysized with computer software. Down regulated genes in gastric cancer were screened. cDNA suppression subtraction library was established by counterpart normal gastric mucous membrane mRNA(Tester) subtracting gastric cancer tissues mRNA(Driver) of five patients. After identification of the subtraction library, positive clones choosen randomly were sequenced , and down regulated novel genes in gastric cancer were screened. Some of the genes were identified by RT PCR.Results Down regulated genes in gastric cancer consist of 60 genes including tumor suppressing genes, apoptosis related genes, DNA replication and transcript or translate related genes, cell cycle related genes, cell migration related genes, etc. Two unknown gene fragments in gastric cancer were cloned. Conclusions Sixty down regulated genes in gastric cancer are confirmed. They are involved in gastric tumorigenicity and metastasis. cDNA subtraction library of gastric cancer was constructed successfully. Two down regulated novel gene fragments in gastric cancer was found.

OBJECTIVETo pursue insulin and islet-transplantation replacement therapy for type 1 diabetes based on engineered human non-beta cells which secrete mature insulin.

METHODSHuman proinsulin cDNA was cloned from its genomic gene and mutated by overlap extension PCR, introducing furin consensus cleavage sequences (Arg-Xaa-Lys/Arg-Arg). An expression vector encoding a genetically modified human proinsulin cDNA was generated and transduced to Hela, 293, and L02 cells by lipofectin-mediated DNA transfection. Following G418 screening, the surviving L02 cells were selected and enriched. Insulin levels in the supernatant and cells were evaluated using radioimmunoassay and immunofluorescence staining.

RESULTSThree sites in the insulin gene were mutated simultaneously. Insulin gene modified cells were able to express insulin at different levels: 8.45 - 188.00 microIU/24 h/2.0 x 10(6) Hela cells and 159.88 - 242.14 microIU/24 h/2.0 x 10(6) 293 cells for transient expression, and 2.56 - 61.95 microIU/24 h/2.0 x 10(6) from several L02 clones screened with G418. No insulin was released by control cells. Furthermore, immunofluorescence staining confirmed that proinsulin was stored as vacuoles in the cytoplasm of L02 cells.

CONCLUSIONA correctly mutated human proinsulin cDNA was obtained successfully, transfected and expressed efficiently in non-beta cells, lending support to the study of somatic gene therapy in diabetes mellitus.

Cell Line ; Chromatography, High Pressure Liquid ; DNA, Complementary ; genetics ; Fluorescent Antibody Technique ; Genetic Vectors ; genetics ; HeLa Cells ; Humans ; Insulin ; genetics ; metabolism ; Proinsulin ; genetics ; Radioimmunoassay ; Transfection