효과적인 실험실연구 및 임상연구에 사용될 유전자 재조합 soluble RAGE 합성에 대한 연구

다국어 초록 (Multilingual Abstract)

Background: Recent studies have demonstrated that RAGE and its ligands may play an important role in mediating vascular inflammation and the subsequent development of atherosclerosis. In addition to membrane-bound RAGE, ager also produces a minor product that lacks the membrane anchor and the cytosolic signaling domain. This product is secreted into extracellular milieu as a soluble protein (sRAGE). Unlike RAGE that transmits signals to activate various cellular programs and causes inflammation, sRAGE functions as a natural decoy that binds RAGE ligands and prevents RAGE signaling. Because sRAGE is a minor product from alternatively spliced mRNA, direct purification from animal blood products produces only limited amounts. Recombinant sRAGE has been constructed and produced in E. coli and baculovirus systems for limited laboratory scale studies. However, a large scale of sRAGE production has not been achieved. This has prevented widespread basic and clinical studies of sRAGE and exploration of its therapeutic potentials. Results: We generated a version of a construct that expresses full-length sRAGE(tagged with T7 epitope tag) in laboratory cell lines. Western blotting and immunoprecipitation of cell culture medium collected from permanently transfected Chinese hamster ovary (CHO) cell showed that sRAGE is secreted into the medium. The fully released sRAGE from transfected CHO cells were treated with N-glycosidase (PNGase F) to confirm glycosylation of the generated sRAGE. Also, sRAGE constructs with mutations at the N25, N81 glycosylation site were subcloned and transiently transfected into CHO cells. The cell lysates were collected and the western blot analysis demonstrated difference in the molecular weight of the mutant RAGE compared to wild type. Also, the determination of in vitro half life of wild type sRAGE and double deglycosylation mutant sRAGE with cycloheximide chase assay showed that the wild type sRAGE had a half life of 8.6 hours, compared to 4.4 hours of the double deglycosylation sRAGE. For protein purification, the sRAGE was purified using an anti-T7 antibody agarose kit. The purified samples were resolved with silver stain to assess the purity of sRAGE. The results showed that sRAGE was purified with minimal contaminants using a single step purification. Coimmunoprecipitation of the purified sRAGE with RAGE ligand HMGB1 revealed binding of sRAGE with HMGB1. To determine whether sRAGE attenuates the RAGE ligand induced VSMC proliferation in vitro, young and old VSMC(passage 3-6) were treated with HMGB1, AGE and oxidized LDL and the proliferation assessed by BrdU cell proliferation assay. The results show that treatment with HMGB1 at 0.1 ug/ml was associated with significant increase in VSMC proliferation in old VSMC cells that was inhibited by pretreatment with sRAGE at dose range of 2,4 and 6 ug/ml. Also, treatment with AGE(4.0 ug/ml) and oxidized LDL(0.4mg/mL) were associated with significant increase in old VSMC proliferation that was attenuated by pretreatment with sRAGE. Coimmunoprecipitation of sRAGE with oxidized LDL revealed binding of sRAGE with oxidized LDL. .In vivo experiment using this particular construct of sRAGE was performed in carotid artery balloon injury model using Wistar rats. A total of 44 rats were divided into 4 groups according to drug dosage(group 1: saline, group 2 sRAGE 70 ng group 3: sRAGE 140ng group 4: sRAGE 210ng) and administered the drug intraperitoneally the day before the balloon injury and the 1st and 2nd day after the balloon injury. The results demonstrated that blockade of RAGE activation with delivery of sRAGE was associated with significant attenuation of balloon injury induced neointima hyperplasia with a dose dependent reponse. Conclusion: The results from this study show that we were able to develop a system of recombinant sRAGE production from mammalian cell system that was associated with good stability and functional activity in vitro and in vivo.

국문 초록 (Abstract)

서론: 죽상동맥경화증의 주요 병태생리기전은 최근들어 혈관염증반응을 매개하는 cell receptor중에서 RAGE 및 그 ligand들의 중요성이 발표되고 있으며 RAGE를 매개로 한 혈관염증반응이 죽상동맥경화증의 병태생리에 중요한 역할을 한다는 연구결과들이 보고되고 있다. RAGE는 대부분 세포막에 부착된 cell membrane receptor로 존재하지만 일부는 membrane anchor와 cytosolic form이 없는 혈장용해성 RAGE(sRAGE)로 존재한다. Cell signaling을 매개하여 혈관염증을 유발하는 full length RAGE 와는 달리 sRAGE는 decoy receptor로 작용하여 혈중에 존재하는 RAGE ligand들과 결합하여 세포막 RAGE receptor와 결합하는 작용을 길항하는 역할을 한다. 본 연구자는 충분한 양의 recombinant sRAGE 생산할 수 있는 mammalian cell system을 개발하기 위해 laboratory mammalian cell line에서 sRAGE를 생산할 수 있는 sRAGE construct를 개발하고 in vitro 및 in vivo에서 그 효과를 입증하고자 하였다.
연구개발수행 내용 및 결과: 본 연구에서는 1020bp로 구성된 sRAGE Construct를 Chinese hamster ovary (CHO) cell에 permanent transfection 시킨 결과 지속적으로 cell medium으로부터 대량 sRAGE를 얻을 수 있는 시스템을 마련하였음. 여러 permanent transfection cell line medium으로부터 sRAGE의 secretion이 제일 많은 cell line을 선택하여 T7 agarose bead purification kit(Novagen, San Diego, CA, USA) 를 이용하여 purification을 하였고 glycosylation assay를 통해 cell medium과 lysate에 있는 sRAGE의 glycosylation여부를 확인하였음. 또한 N25, N81 glycosylation site의 deglycosylation mutant plasmid를 subcloning하여 transient transfection을 시행한후 wild type과 비교해본 결과 wild type과의 분자량 차이를 확인하였고 cycloheximide chase assay를 통해 RAGE wild type이 in vitro half life가 8.6시간인 반면에 double deglycosylation mutant는 in vitro half life가 4.4시간으로 glycosylation여부에 따라서 protein stability가 차이가 많이 남을 확인할 수 있었음. 또한 RAGE의 ligand인 HMGB1과 sRAGE가 상호작용하는지 coimmunoprecipitation을 시행하여 sRAGE와 그 ligand인 HMGB1의 binding여부를 확인하였음. BrdU cell proliferation assay 를 이용하여 RAGE ligand에 대한 VSMC proliferation 반응을 분석한 결과 old VSMC에서는 HMGB1 0.1ug/ml부터 baseline에 비해 유의하게 proliferation이 증가하였는고 sRAGE를 HMGB1과 함께 incubation한 경우 dose response는 관찰되지 않았지만 2ug/ml, 4ug/ml 및 6ug/ml의 용량에 대해서 BrdU incorporation이 억제됨을 확인할 수 있었음. AGE에 의한 stimulation에 대해서도 young VSMC은 proliferation이 baseline에 비해 증가하지 않은 대신에 old VSMC은 AGE 4.0 ug/ml의 농도에 대해서proliferation이 유의하게 증가됨을 확인할 수 있었음. 또한 oxidized LDL stimulation에 대해 VSMC의 proliferative response를 측정해본 결과 old VSMC에서는 0.4mg/ml이상의 oxidized LDL의 농도에서는 VSMC의 proliferation이 유의하게 증가하였고 그 proliferative response는 sRAGE 20ug/ml를 처치하였을때 유의하게 감소하였으며 oxidized LDL과 T7 tagged sRAGE를 coimmunoprecipitation결과 soluble RAGE가 oxidized LDL과 binding함을 확인할 수 있었다. 백서 경동맥풍선손상모델에서 sRAGE의 신생내막형성 억제효과를 입증하기 위해 총 44마리의 Wistar rat(350-380gm)를 대상으로 하였고 4개군으로 나눠 실험을 진행하였는데 실험약은 carotid balloon injury 하루전과 injury 다음날과 그 다음날까지 총 3차례 복강주(intraperitoneal injection)를 하였고 실험군은 sRAGE의 농도에 따라 1군(saline injection),2군(sRAGE 70ng), 3군(sRAGE 140ng), 4군(sRAGE 210ng)으로 나눠 연구를 진행하였다. 연구결