| Chapter1Identification of the Plasmids and the VirusesObjectives:To identify the construction of the recombinant plasmid and its expression of target protein; to detect the bio-activity of the virus.Methods:1. The recombinant plasmids (pAAV-FGF21and pAAV-FGF21-GLP1) were digested with restriction enzymes (Xbaâ… and BamHI), and then were tested by gel electrophoresis.2. HEK293cells were transfected in vitro with the recombinant plasmids, pAAV-FGF21and pAAV-FGF21-GLP1, respectively. The cell culture supernatant was collected and both FGF-21protein and GLP-1protein in it were detected by Western Blotting and ELISA.3. HepG2cells were infected in vitro with virus rAAV-mCherry. The bio-activity of it was detected by observing the red fluorescence protein expression under fluorescence microscope.Results:l.With restriction enzyme digestion, pAAV-FGF21was cutted into two fragments. One fragment had a size of5200bp, and the other had a size of630bp, which matched the band size of the vector plasmid and the FGF-21gene, respectively. With restriction enzyme digestion, pAAV-FGF21-GLP1was cutted into two fragments. One fragment had a size of5200bp, and the other had a size of780bp, which matched the band size of the vector plasmid and the FGF-21-GLP-1gene, respectively.2. Expression of FGF-21protein was confirmed in the supernatant of cells transfected with pAAV-FGF21. Expression of FGF-21and GLP-1proteins was confirmed in the supernatant of cells transfected with pAAV-FGF21-GLP1.3. Red fluorescence was observed under fluorescence microscope in the cells infected with virus rAAV-mCherry.Conclusions:Recombinant plasmids (pAAV-FGF21and pAAV-FGF21-GLP1) were constructed correctly. And they can mediate the expression of corresponding protein/proteins in vitro. The rAAVs were of bio-activity. Chapter2rAAV-mediated Treatment of T2DM in db/db mouse model in vivoObjectives:To evaluate the efficacy of rAAV for treatment of mice with T2DM.Methods:The fasting blood glucose and insulin levels of db/db mice were examined. The increased fasting blood glucose and HOMA-IR levels were taken as the criterion of the type2diabetes mellitus. Db/db mice were randomized to four groups and treated with:group A (N.S.), group B (empty vector rAAV-mCherry), group C (rAAV-FGF21), and group D (rAAV-FGF21-GLP1). Normal mice C57/BL6were categorized as group H (N.S.). Fasting blood glucose level, body weight, food intake and fasting blood insulin level of all5groups were monitored after the administration. All mice were sacrificed10weeks post the administration. Target gene expression in liver was detected by RT-PCR and the islet beta-cell mass in pancreas was determined by immunofluorescence.Results:1. High blood glucose and insulin resistance were identified in db/db mice.2. In group D, fasting blood glucose level, body weight, food intake and fasting insulin level of mice were significantly reduced than that in group A (P<0.01), however, those had no significant change in group B and group C.3. Expression of the target gene was confirmed in mouse liver of group C and group D.4. In group D, beta-cell mass of mice had more fluorescent cells with higher fluorescent intensity, compared with that in group A.Conclusions:1. T2DM was spontaneously established in all db/db mice in our experiment within8weeks of age.2. Both rAAV-FGF21and rAAV-FGF-GLP1can mediate expression of target gene in db/db mouse liver in vivo.3. rAAV-FGF21-GLP1can significantly reduce fasting blood glucose level, body weight, food intake and fasting insulin level of db/db mouse, and increase its islet beta-cell mass. |
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