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Synthesis and characterization of polyethlyenimine conjugated with dexamethasone as an efficient gene carrier

Title
Synthesis and characterization of polyethlyenimine conjugated with dexamethasone as an efficient gene carrier
Other Titles
유전자 전달체로서의 Dexamethasone 이 접합된 Polyethylenimine 의 합성과 특성연구
Author
김현정
Alternative Author(s)
Kim, Hyunjung
Advisor(s)
이민형
Issue Date
2009-02
Publisher
한양대학교
Degree
Master
Abstract
Dexamethasone은 항염증효과를 지닌 글르코코르티코이드이고, 허혈성 심근세포를 세포사멸로부터 보호할 수 있다. Dexamethasone의 세포사멸 억제효과를 허혈성 질환 유전자 치료에 적용하기 위해, Dexamethasone을 접합한 Polyethylenimine (PEI-Dexa)을 합성하였고, 세포사멸억제효과를 지닌 유전자 전달체로서 평가하였다. PEI-Dexa는 낮은 분자량의 Polyethylenimine (PEI2K, 2kDa)으로 합성하였다. Luciferase assay로부터의 PEI-Dexa의 트렌스펙션 효율과 MTT assay로부터의 세포독성을 평가하였다. 세포사멸억제효과를 평가하기 위해, PEI-Dexa/DNA 복합체는 세포로 트렌스펙션 했고, 후에 과산화수소를 처리하였다. MTT assay로부터 세포 생존률과 Caspase-3 assay로부터 세포사멸 수준을 평가하였다. 쥐의 심근세포 (H9C2 cell) 로의 트렌스펙션 실험은 PEI-Dexa가 8/1 무게비율 (PEI-Dexa/DNA)에서 가장 높은 트렌스펙션 효율을 가진다는 것을 보여주었다. 이 비율에서, PEI-Dexa는 높은 분자량의 Polyethylenimine (PEI25K, 25kDa)과 PEI2K보다 더 높은 트렌스펙션 효율을 가졌다. 그리고, PEI-Dexa의 세포독성은 PEI25K보다 낮았다. 세포사멸억제효과를 평가하기 위해, PEI-Dexa/pSV-Luc복합체는 H9C2심근세포로 트렌스펙션 하였고, 그 후에 과산화수소를 처리하였다. 결과는, PEI-Dexa는 PEI2K와 비교했을 때 Caspase-3 활성을 감소시켰고, 세포 생존률을 증가시켰다. Heme Oxygenase-1 (HO-1) 유전자는 세포사멸로부터 허혈성 심근세포를 보호할 수 있다. 그러므로, pSV-HO-1은 PEI-Dexa를 사용하여 H9C2 cell에 트렌스펙션 했다. PEI-Dexa/pSV-HO-1 복합체가 트렌스펙션 된 세포는 PEI-Dexa/pSV-Luc복합체가 트렌스펙션 된 세포보다 과산화수로를 처리한 후에 낮은 Caspase-3활성을 가졌고, 높은 생존률을 보였다.\ Linear polyethylenimine (LPEI)는 Branched PEI (BPEI) 와 비교했을 때, 더 높은 유전자 발현과 낮은 세포독성을 가진 효과적인 비 바이러스성 유전자 전달체이다. 이번 연구에서는, LPEI는 증가 된 유전자 전달효율을 위해 Dexamethasone을 접합시켰다. Dexaemethasone은 글르코코르티코이드 수용체의 리간드이고, 수용체를 매개로 한 이동 으로부터 핵을 목표로 하는 유전자 전달에 유용하다. Dexamethasone이 접합된 LPEI (LPEI-Dexa)는 H9C2 심근세포와 A7R5 평활근 세포로 유전자 전달체로서의 기능을 평가했다. LPEI-Dexa는 2/1 무게비율 (LPEI-Dexa/DNA) 에서 트렌스펙션 효율을 가졌다. 이 비율에서, LPEI-Dexa는 LPEI, BPEI 25kDa과 Lipofectamine 2000보다 더 높은 트렌스펙션 효율을 가졌다. 그리고, LPEI-Dexa의 세포독성은 BBEI (25kDa)보다 낮았다. 결론으로서, PEI-Dexa는 세포사멸 억제효과를 가진 효과적인 유전자 전달체이고, 이것은 pSV-HO-1과 함께 세포사멸 억제 유전자 치료를 위해 유용할 것이다. LPEI-Dexa는 높은 트렌스펙션 효율을 가지는 유전자 전달을 위해 유용할 것이다.
Dexamethasone is a potent glucocorticoid with anti-inflammatory effect. Dexamethasone can protect ischemic cardiomyocytes from apoptosis. To apply anti-apoptotic effect of dexamethasone to ischemic disease gene therapy, dexamethasone-conjugated polyethylenimine (PEI-Dexa) was synthesized and evaluated as an anti-apoptotic gene carrier. PEI-Dexa was synthesized with low molecular weight polyethylenimine (PEI2K, 2 kDa). Transfection efficiency and cytotoxicity of PEI-Dexa were evaluated by luciferase assay and MTT assay. To evaluate the anti-apoptotic effect, PEI-Dexa/DNA complex was transfected into the cells and the cells were treated with H₂O₂. Cell viability and apoptosis level were measured by MTT assay and caspase-3 assay, respectively. A transfection assay into H9C2 rat cardiomyocytes showed that PEI-Dexa had the highest transfection efficiency at an 8/1 weight ratio (PEI-Dexa/DNA). At this ratio, PEI-Dexa had higher transfection efficiency than high molecular polyethylenimine (PEI25K, 25 kDa) and PEI2K. In addition, the cytotoxicity of PEI-Dexa was lower than that of PEI25K. To evaluate anti-apoptotic effect, PEI-Dexa/pSV-Luc or PEI2K/pSV-Luc was transfected into the H9C2 cells and the cells were treated with H₂O₂. As a result, PEI-Dexa reduced caspase-3 activity and increased cell viability, compared with PEI2K. Heme oxygenase-1 (HO-1) can protect ischemic cardiomyocytes from apoptosis. Therefore, pSV-HO-1 was cloned and transfected into H9C2 cells using PEI-Dexa. The cells transfected with PEI-Dexa/pSV-HO-1 complex had lower caspase-3 activity and higher viability than the cells transfected with PEI-Dexa/pSV-Luc complex after the H₂O₂ treatment. Linear polyethylenimine (LPEI) has been shown to be an effective non-viral gene carrier with higher gene expression and lower toxicity than branched PEI (BPEI) with comparable molecular weight. In this study, LPEI was modified by conjugation of dexamethasone for improved gene delivery efficiency. Dexamethasone is a ligand to glucocorticoid receptor and useful for nuclear targeting in gene delivery by receptor-mediated translocation. Dexaemthasone conjugated LPEI (LPEI-Dexa) was evaluated as a gene carrier to H9C2 cardiomyocytes cell and A7R5 smooth muscle cell. LPEI-Dexa had keep transfection efficiency at an 2/1 weight ratio (LPEI-Dexa/DNA). At this ratio, LPEI-Dexa had higher transfection efficiency than LPEI, BPEI 25kDa and Lipofectamine 2000. In addition, the cytotoxicity of LPEI-Dexa was much lower than that of BPEI (25kDa). In conclusion, PEI-Dexa is an efficient gene carrier with anti-apoptotic effect and may be useful for anti-apoptotic gene therapy in combination with pSV-HO-1. And LPEI-Dexa may be useful for gene delivery with high transfection efficiency.
URI
http://dcollection.hanyang.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000053968https://repository.hanyang.ac.kr/handle/20.500.11754/145586
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GRADUATE SCHOOL[S](대학원) > BIOENGINEERING(생명공학과) > Theses (Master)
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