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hPDX1 유전자의 삽입에 의한 직접 췌도세포 분화 KCI 등재
Transdifferentiation of α-1,3-Galactosyltransferase Knock Out (GalT KO) Pig Derived Bone Marrow Mesenchymal Stromal Cells (BM-MSCs) into Pancreatic Cells by Transfection of hPDX1
pp.249-255
Diabetes mellitus, the most common metabolic disorder, is divided into two types: type 1 and type 2. The essential treatment of type 1 diabetes, caused by immune-mediated destruction of β-cells, is transplantation of the pancreas; however, this treatment is limited by issues such as the lack of donors for islet transplantation and immune rejection. As an alternative approach, stem cell therapy has been used as a new tool. The present study revealed that bone marrowderived mesenchymal stromal cells (BM-MSCs) could be transdifferentiated into pancreatic cells by the insertion of a key gene for embryonic development of the pancreas, the pancreatic and duodenal homeobox factor 1 (PDX1). To avoid immune rejection associated with xenotransplantation and to develop a new cell-based treatment, BM-MSCs from α-1,3-galactosyltransferase knockout (GalT KO) pigs were used as the source of the cells. Transfection of the EGFP-hPDX1 gene into GalT KO pig-derived BM-MSCs was performed by electroporation. Cells were evaluated for hPDX1 expression by immunofluorescence and RT-PCR. Transdifferentiation into pancreatic cells was confirmed by morphological transformation, immunofluorescence, and endogenous pPDX1 gene expression. At 3∼4 weeks after transduction, cell morphology changed from spindle-like shape to round shape, similar to that observed in cuboidal epithelium expressing EGFP. Results of RT-PCR confirmed the expression of both exogenous hPDX1 and endogenous pPDX1. Therefore, GalT KO pig-derived BM-MSCs transdifferentiated into pancreatic cells by transfection of hPDX1. The present results are indicative of the therapeutic potential of PDX1-expressing GalT KO pig-derived BM-MSCs in β-cell replacement. This potential needs to be explored further by using in vivo studies to confirm these findings.
재료 및 방법
1. 시약 및 배양액
2. 중간엽 줄기세포(Mesenchymal Stromal Cells, MSCs)의 추출및 배양
3. pEGFP-hPDX1 Vector 백터 구축
4. hPDX1 Vector가 삽입된 세포 생산 및 검정
5. 면역 형광염색에 의한 PDX1 발현 확인
6. Reverse Transcription Polymerase Chain Reaction(RT-PCR)에 의한 Exogenous와 Endogenous PDX1 mRNA 발현 확인
결 과
1. hPDX1 Vector가 삽입된 세포 구축
2. hPDX1 Vector가 삽입된 세포의 분화
3. 면역 형광염색에 의한 hPDX1 발현 확인
4. RT-PCR에 의한 Exogenous와 Endogenous PDX1 mRNA 발현확인
고 찰
결 론
REFERENCES
