Objective To express a mutated insulin gene in HepG-2 cell line to further research of insulin gene therapy. Methods Native human insulin cDNA was obtained from fetus pancreas with RT-PCR. Furin consensus cleavage sequence was introduced into proinsulin cDNA with site-directed mutagenesis (overlap extension PCR), and the new sequence was named as INS/furin. Subsequently, INS/furin was subcloned into the multiple clone sites of plasmid p(G1RE)3BP-1Luc. The new plasmid p(G1RE)3BP-11?furin was identified with the method of enzyme digestion by Hind Ⅲ and EcoR V. HepG-2 cells were transfected with the plasmid p(G1RE)3BP-11?furin by liposome-mediated method. The transfected HepG-2 cells were incubated for 48h in a glucose-containing medium (25mmol/L), and then the conditioned media were collected and HepG-2 cells were harvested respectively. The expression of INS/furin mRNA in transfected HepG-2 cells was examined by RT-PCR, the regained DNA was sequenced and insulin in conditioned media was investigated by radioimmunoassay. Results Two enzymes, Hind Ⅲ and EcoR V, digested p(G1RE)3BP-11?furin, and 2 fragments with length of 260 bp and 4 700bp, were obtained. The 260bp fragment was identified as insulin/furin, indicating that the target gene had been successfully inserted in specific sites. RT-PCR showed that insulin/furin mRNA was expressed in transfected HepG-2 cell, and the regained DNA was confirmed as insulin/furin by sequencing; while insulin was detected by radioimmunoassay in conditioned media. Conclusion The recombinant mammalian expression plasmid p(G1RE)3BP-11?furin has been successfully constructed, and transfected into HepG-2 cells, which therefore may efficiently secrete bioactive insulin.

Objective To examine the association between the insertion/deletion polymorphism in intron 4 of ecNOS gene (ecNOS4b/a) and diabetic nephropathy (DN) in the Han people of Tianjin, China. Methods The genotypes of ecNOS4b/a polymorphism were detected by PCR-minisatellite DNA detection technique. 70 healthy volunteers, 48 patients with type 2 diabetes without DN, 35 patients with type 2 diabetes and DN without chronic renal failure (CRF), 45 patients with DN in type 2 diabetes with CRF, and 58 patients with non-DN CRF were investigated. Results (1) One case (a female DN patient without CRF) with a rare genotype, the heterozygote of 447 bp and 420 bp, was found. (2) The frequency of allele a in type 2 diabetes without DN group was higher than that in control nonsignificantly (x2 = 1. 672, P = 0. 196) . (3) The frequency of allele a in DN group was lower than that in type 2 diabetes without DN group nonsignificantly (x2 = 1. 082, P = 0. 298). (4) The allele frequencies were similar between the DN without CRF group and the DN with CRF group (Continuity correction x2 = 0. 002, P = 0. 967). (5) The frequency of allele a in DN with CRF group was lower than that in non-DN CRF group significantly (x2=4. 360, P = 0. 037). Conclusions (1) Allele a is not a risk factor of DN in the Han people of Tianjin; (2) The frequency of allele a is lower in the Han people of Tianjin than that in Japanese(4. 3% vs 10. 1% , x2 = 4. 898, P = 0. 027); (3)There are different roles of ecNOS in DN-induced CRF and non-DN CRF.