Enhanced protection of Ins-1 beta cells from apoptosis under hypoxia by delivery of DNA encoding secretion signal peptide-linked exendin-4.
In this study, we developed an expression system of exendin-4, a glucagon-like peptide (GLP-1) analog, using a secretion signal peptide (SP) to facilitate exendin-4 secretion. For delivery of the exendin-4 expression system, high-molecular-weight polyethylenimine (25 kDa, PEI25k), low-molecular-weight polyethylenimine (2 kDa, PEI2k), and polyamidoamine (PAMAM) dendrimers were evaluated as gene carriers to Ins-1 beta cells. As a result, PEI25k showed the highest transfection efficiency. For the construction of the exendin-4 expression vector, DNA coding the SP sequence was inserted upstream of the exendin-4 cDNA, resulting in the construction of pbeta-SP-Ex-4. Transfection assay showed that the secretion level of exendin-4 increased in the pbeta-SP-Ex-4 transfected cells, compared with the pbeta-Ex-4 transfected cells. To identify the beta-cell protection effect of pbeta-SP-Ex-4 delivery, the Ins-1 beta cells were transfected with pbeta-SP-Ex-4 or pbeta-Ex-4 and incubated under normoxia or hypoxia. An MTT assay showed that the pbeta-SP-Ex-4 transfected cells had higher beta-cell viability than the pbeta-Ex-4 transfected cells under hypoxia. In addition, the pbeta-SP-Ex-4 transfected cells exhibited lower caspase-3 activity than the pbeta-Ex-4 transfected cells. Therefore, PEI25k/pbeta-SP-Ex-4 complex may be useful to protect isolated beta cells from apoptosis during transplantation.
Ah Kim
Hyun
H
Department of Bioengineering, College of Engineering, Hanyang University, Seoul, Korea.
Lee
Suyeon
S
Park
Jeong-Hyun
JH
Lee
Sanghyun
S
Lee
Byung-Wan
BW
Ihm
Sung Hee
SH
Kim
Tae-il
TI
Kim
Sung Wan
SW
Ko
Kyung Soo
KS
Lee
Minhyung
M
D017209:Apoptosis / Q000187:drug effects*; D001672:Biocompatible Materials / Q000008:administration & dosage; D053148:Caspase 3 / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004247:DNA / Q000627:therapeutic use; D050091:Dendrimers; D000077270:Exenatide; D005822:Genetic Vectors / Q000138:chemical synthesis; D006801:Humans; D000860:Hypoxia / Q000139:chemically induced / Q000188:drug therapy*; D050417:Insulin-Secreting Cells / Q000187:drug effects* / Q000378:metabolism; D010455:Peptides / Q000235:genetics* / Q000627:therapeutic use; D011073:Polyamines / Q000008:administration & dosage; D011094:Polyethyleneimine / Q000008:administration & dosage; D021382:Protein Sorting Signals / Q000235:genetics*; D014162:Transfection / Q000379:methods; D014688:Venoms / Q000235:genetics* / Q000627:therapeutic use
D001672:Biocompatible Materials; D050091:Dendrimers; C104700:PAMAM Starburst; D010455:Peptides; D011073:Polyamines; D021382:Protein Sorting Signals; D014688:Venoms; D011094:Polyethyleneimine; D004247:DNA; D000077270:Exenatide; D053148:Caspase 3