Expression of human insulin gene wrapped with chitosan nanoparticles in NIH3T3 cells and diabetic rats
Abstract
Aim: To study the expression of human insulin gene wrapped with chitosan nanoparticles in NIH3T3 cells and diabetic rats.
Methods: pCMV.Ins, an expression plasmid of the human insulin gene, was constructed. In total, 100 μg pCMV.Ins wrapped with chitosan nanoparticles (chitosan–pCMV.Ins) was transfected to NIH3T3 cells and diabetes rats through lav age and coloclysis, respectively. The transfected cells were grown in Dulbecco's modified Eagle's medium, containing G418, for 72 h after transfection. The clones were selected and continued to grow in G418 medium for 24 d. The expression of human insulin was detected by immunohistochemistry. Human insulin in the culture medium of transfected cells was measured. Fasting blood glucose and plasma human insulin of diabetic rats were measured for 5 d after transfection. RT–PCR and Western blotting were performed to confirm the expression of the human insulin gene in diabetic rats.
Results: Approximately 10% of NIH3T3 cells transfected by chitosan–pCMV.Ins expressed human insulin. Human insulin in the culture medium of NIH3T3 cells transfected by chitosan–pCMV.Ins significantly increased compared with that of the control group (P<0.01). Fasting blood glucose levels of the lavage group and the coloclysis group decreased significantly in 5 d (P<0.01) in comparison, while plasma insulin levels were much higher (P<0.01). The human insulin gene mRNA and human insulin were only detected in the lavage and the coloclysis groups.
Conclusion: The human insulin gene can be transfected and expressed successfully by chitosan–pCMV.Ins in NIH3T3 cells and diabetes rats, which indicates that chitosan is a promising, non-viral vector for gene expression.
Keywords:
Methods: pCMV.Ins, an expression plasmid of the human insulin gene, was constructed. In total, 100 μg pCMV.Ins wrapped with chitosan nanoparticles (chitosan–pCMV.Ins) was transfected to NIH3T3 cells and diabetes rats through lav age and coloclysis, respectively. The transfected cells were grown in Dulbecco's modified Eagle's medium, containing G418, for 72 h after transfection. The clones were selected and continued to grow in G418 medium for 24 d. The expression of human insulin was detected by immunohistochemistry. Human insulin in the culture medium of transfected cells was measured. Fasting blood glucose and plasma human insulin of diabetic rats were measured for 5 d after transfection. RT–PCR and Western blotting were performed to confirm the expression of the human insulin gene in diabetic rats.
Results: Approximately 10% of NIH3T3 cells transfected by chitosan–pCMV.Ins expressed human insulin. Human insulin in the culture medium of NIH3T3 cells transfected by chitosan–pCMV.Ins significantly increased compared with that of the control group (P<0.01). Fasting blood glucose levels of the lavage group and the coloclysis group decreased significantly in 5 d (P<0.01) in comparison, while plasma insulin levels were much higher (P<0.01). The human insulin gene mRNA and human insulin were only detected in the lavage and the coloclysis groups.
Conclusion: The human insulin gene can be transfected and expressed successfully by chitosan–pCMV.Ins in NIH3T3 cells and diabetes rats, which indicates that chitosan is a promising, non-viral vector for gene expression.