KR101971860B1 - Method for controlling expression of poly immunoglobulin receptor protein by ribosome inactivation - Google Patents
Method for controlling expression of poly immunoglobulin receptor protein by ribosome inactivation Download PDFInfo
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Abstract
본 발명은 리보솜 불활성화를 통한 중합체 면역글로불린 수용체(poly immunoglobulin receptor; pIgR) 단백질의 발현 조절 방법에 관한 것으로서, 구체적으로는 리보솜 불활성화를 통해 pIgR의 mRNA 안정성을 감소시켜, pIgR 단백질 발현을 억제시키는 방법에 대한 것이다. 리보솜 불활성화에 의해, pIgR의 단백질 발현 억제, 마우스 장내 박테리아 부착 증가 및 인터페론 유도 pIgR의 mRNA 안정성 감소 효과를 확인할 수 있었다. 한편, 상기와 같은 결과는 동물실험 모델에서 균총에 대한 감수성을 항진시키는데 기여하여 특정 균총에 의한 기능 활성 연구에 활용될 수도 있다. The present invention relates to a method for regulating expression of a polymer immunoglobulin receptor (pIgR) protein through ribosome inactivation, and more particularly, to a method for regulating expression of a pIgR protein by reducing mRNA stability of pIgR through ribosome inactivation It is about the method. Inhibition of ribosomal inhibition of protein expression of pIgR, increased bacterial adhesion in mouse intestine, and reduced mRNA stability of interferon-induced pIgR were confirmed. On the other hand, the above results can be utilized for the study of functional activity by a specific microflora in the animal experimental model by contributing to enhance the susceptibility to the microflora.
Description
본 발명은 리보솜 불활성화를 통한 중합체 면역글로불린 수용체(poly immunoglobulin receptor; pIgR) 단백질의 발현 조절 방법에 관한 것으로서, 구체적으로는 리보솜 불활성화를 통해 pIgR의 mRNA 안정성을 감소시켜, pIgR 단백질 발현을 억제시키는 방법에 대한 것이다.The present invention relates to a method for regulating expression of a polymer immunoglobulin receptor (pIgR) protein through ribosome inactivation, and more particularly, to a method for regulating expression of a pIgR protein by reducing mRNA stability of pIgR through ribosome inactivation It is about the method.
점막 상피 면역 시스템은 독성 화합물 및 병원균을 포함하는 다양한 환경적 영향에 대응하기 위한 1차 방어선이다. 중합체 면역글로불린 수용체(poly immunoglobulin receptor; pIgR)는 점막에서 면연글로불린 A(immunoglobulin A; IgA)의 수송을 통하여 외부의 병원균에 대하여 1차적인 방어를 하는데 중요한 역할을 하는 것으로 알려져 있다. pIgR은 장관(intestinal tract) 및 폐의 점막 상피 세포 기저측막(basolateral membrane)에서 선택적으로 발현되는 막관통 당단백질(transmembrane glycoprotein)이다. 상피 내 pIgR 발현 감소는 IgA의 분비를 억제하는데, 이는 감염 및 염증의 위험을 증가시킨다. 즉, IgA 겹핍은 염증성 장 질환(inflammatory bowel disease; IBD)을 포함하는 장내 염증 질환에 대한 예측인자가 될 수 있다. 병원성 박테리아는 IgA-결핍 개체에서 점막 상피 세포에 쉽게 정착하고, 점막하(sub-mucosa) 부위로 이동하는데, 이를 통해 염증성 반응이 시작된다. 즉, IgA는 점막에서 균의 침투를 억제하고 정착을 어렵게 하기 때문에 특정 균총의 활성 연구 측면에서는 감수성을 방해하는 요소로 작용할 수 있다.The mucosal epithelial immune system is the primary line of defense against a variety of environmental effects, including toxic compounds and pathogens. Polymer immunoglobulin receptor (pIgR) is known to play an important role in primary defense against external pathogens through the transport of immunoglobulin A (A) in the mucosa. pIgR is a transmembrane glycoprotein that is selectively expressed in the intestinal tract and the basolateral membrane of the lung. Decreased expression of pIgR in the epithelium inhibits the secretion of IgA, which increases the risk of infection and inflammation. That is, IgA deficiency may be a predictor of intestinal inflammatory disease including inflammatory bowel disease (IBD). Pathogenic bacteria are easily immobilized in mucosal epithelial cells in IgA-deficient individuals and migrate to the sub-mucosa site, where inflammatory responses are initiated. In other words, IgA inhibits bacterial infiltration in mucous membranes and makes it difficult to fix, which may interfere with susceptibility in terms of the activity of certain microorganisms.
리보솜 불활성화는 급성 및 만성 점막 염증을 유발하고, IBD를 포함하는 인간 장내 상피 염증 질환의 병리 인자인 것으로 알려졌다. 리보솜-불활성화 화합물에 대한 전신성 급성 반응은 전-염증성 사이토카인 폭풍에 의한 심각한 패혈증 유사 증상과 밀접하게 관련되어 있다. 이론적으로, 리보솜 불활성화는 장내 및 전신성 염증 모두를 촉발시키는데, 이는 상피 및 다른 면역 관련 세포에서 전-염증성 사이토카인의 생산을 유도하는데 따른 것이다. Libosome inactivation causes acute and chronic mucosal inflammation and is known to be a pathologic factor of human intestinal epithelial inflammatory disease including IBD. The systemic acute response to ribosome-inactivating compounds is closely related to severe sepsis-like symptoms caused by pre-inflammatory cytokine storms. In theory, ribosome inactivation triggers both intestinal and systemic inflammation, which results in inducing the production of pro-inflammatory cytokines in epithelial and other immune-related cells.
본 발명의 목적은 리보솜 불활성화를 통한 중합체 면역글로불린 수용체(poly immunoglobulin receptor; pIgR) 단백질의 발현 조절 방법에 관한 것이다. It is an object of the present invention to provide a method for regulating expression of a polymer immunoglobulin receptor (pIgR) protein through ribosome inactivation.
상기 목적을 달성하기 위하여, 본 발명은 리보솜 불활성화를 통해 pIgR mRNA의 안정성을 감소시켜 pIgR 단백질의 발현을 억제하는 것을 특징으로 하는 pIgR 단백질의 발현 조절 방법을 제공한다.In order to achieve the above object, the present invention provides a method for regulating the expression of pIgR protein, wherein the expression of pIgR protein is inhibited by decreasing the stability of pIgR mRNA through ribosome inactivation.
본 발명은 리보솜 불활성화를 통한 중합체 면역글로불린 수용체(poly immunoglobulin receptor; pIgR) 단백질의 발현 조절 방법에 관한 것으로서, 구체적으로는 리보솜 불활성화를 통해 pIgR의 mRNA 안정성을 감소시켜, pIgR 단백질 발현을 억제시키는 방법에 대한 것이다. 리보솜 불활성화에 의해, pIgR의 단백질 발현 억제, 마우스 장내 박테리아 부착 증가 및 인터페론 유도 pIgR의 mRNA 안정성 감소 효과를 확인할 수 있었다. 한편, 상기와 같은 결과는 동물실험 모델에서 균총에 대한 감수성을 항진시키는데 기여하여 특정 균총에 의한 기능 활성 연구에 활용될 수도 있다. The present invention relates to a method for regulating expression of a polymer immunoglobulin receptor (pIgR) protein through ribosome inactivation, and more particularly, to a method for regulating expression of a pIgR protein by reducing mRNA stability of pIgR through ribosome inactivation It is about the method. Inhibition of ribosomal inhibition of protein expression of pIgR, increased bacterial adhesion in mouse intestine, and reduced mRNA stability of interferon-induced pIgR were confirmed. On the other hand, the above results can be utilized for the study of functional activity by a specific microflora in the animal experimental model by contributing to enhance the susceptibility to the microflora.
도 1은 리보솜 불활성화에 의한 마우스 장내 pIgR의 발현 억제 결과를 나타낸다.
도 2는 리보솜 결합 에니소마이신(ANS)에 의한 마우스 장내 박테리아 부착 증가 결과를 나타낸다.
도 3은 장 상피세포에서 리보솜 결합 조성물의 농도에 따른 pIgR 단백질 발현 감소 결과를 나타낸다.
도 4는 장상피세포주에서 다양한 리보솜 결합 조성물에 의한 pIgR 단백질 발현 감소 결과를 나타낸다.
도 5는 리보솜 결합 데옥시니발레놀(DON)에 의한 pIgR mRNA 안정성 감소 결과를 나타낸다.
도 6은 리보솜 불활성화가 pIgR mRNA 안정성을 감소시키는데 있어, HuR 단백질과의 관련성을 확인한 결과를 나타낸다.
도 7은 세포 내에서 pIgR 전사체와 결합하는 HuR 단백질을 측정하기 위한 RNA 면역침전분석 결과를 나타낸다.
도 8은 리보솜 불활성화에 의한 pIgR 억제 모식도를 나타낸다.
도 9는 인간 점막 상피세포에서 리보솜 불활성화-매개 점막 면역질환 발병 기작의 추정 모식도를 나타낸다. Fig. 1 shows the results of inhibiting the expression of pIgR in the mouse by ribosome inactivation.
Figure 2 shows the results of increased intestinal bacterial adhesion by ribosome-bound enisonamycin (ANS).
FIG. 3 shows the results of decreasing the expression of pIgR protein according to the concentration of the ribosome binding composition in the intestinal epithelial cells.
FIG. 4 shows the results of reduction of pIgR protein expression by various ribosome binding compositions in intestinal epithelial cell lines.
Figure 5 shows the results of reduced pIgR mRNA stability by ribosome-bound deoxynil valenol (DON).
Fig. 6 shows the result of confirming the relationship with the HuR protein in reducing the stability of pIgR mRNA by ribosome inactivation.
FIG. 7 shows the result of RNA immunoprecipitation analysis for measuring HuR protein binding to pIgR transcript in a cell.
Figure 8 shows a schematic diagram of pIgR inhibition by ribosome inactivation.
Fig. 9 shows a hypothetical model of the mechanism of onset of ribosome inactivation-mediated mucosal immune disease in human mucosal epithelial cells.
본 발명자들은 리보솜 불활성화가 점막에서 IgA 수송에 영향을 미칠 수도 있다고 가정하고, 장내 상피 및 장세포(enterocytes)에서 리보솜 불활성화에 따른 pIgR 수준을 측정하였다. 상피 pIgR 수준의 변화는 점막 방어 및 IgA 수준에 상당한 영향을 미쳤다. 즉, 리보솜 불활성화에 따른 상피 IgA 수송의 조절 기작이 인간 점막-관련 질환과 관련되어 있음을 확인하고 본 발명을 완성하였다. We hypothesized that ribosome inactivation might affect IgA transport in the mucosa and measured levels of pIgR following ribosome inactivation in intestinal epithelium and intestinal cells (enterocytes). Changes in epithelial pIgR levels significantly affected mucosal defense and IgA levels. That is, it has been confirmed that the regulatory mechanism of epithelial IgA transport by ribosome inactivation is related to human mucosa-related diseases, and the present invention has been completed.
본 발명은 리보솜 불활성화를 통한 중합체 면역글로불린 수용체(poly immunoglobulin receptor; pIgR) 단백질의 발현 조절 방법을 제공한다.The present invention provides a method of modulating the expression of a polymer immunoglobulin receptor (pIgR) protein through ribosome inactivation.
상세하게는, 상기 상기 pIgR 단백질의 발현 조절은 pIgR mRNA의 안정성을 감소시켜 pIgR 단백질의 발현을 억제할 수 있다. 보다 상세하게는, 상기 pIgR mRNA의 안정성 감소는 HuR 단백질의 세포질 내 유출 억제를 통한 것일 수 있다.In particular, the expression control of the pIgR protein may reduce the stability of the pIgR mRNA and inhibit the expression of the pIgR protein. More specifically, the decrease in stability of the pIgR mRNA may be through inhibition of intracellular flux of the HuR protein.
상세하게는, 상기 리보솜 불활성화는 데옥시니발레놀(deoxynivalenol; DON), 에니소마이신(anisomycin; ANS), 니발레놀(nivalenol; NIV) 또는 15-아세틸 DON(15-acetyl DON)을 처리하여 유도할 수 있으나, 이에 제한되는 것은 아니다.In detail, the ribosome inactivation is performed by treating deoxynivalenol (DON), anisomycin (ANS), nivalenol (NIV) or 15-acetyl DON (15-acetyl DON) But is not limited thereto.
상세하게는, 상기 pIgR 단백질의 발현은 RT-PCR, 경쟁적 RT-PCR(Competitive RT-PCR), 실시간 RT-PCR (Real-time RT-PCR), 웨스턴 블랏팅, ELISA(enzyme linked immunosorbent asay), 유세포분석(Flow Cytometry) 및 조직면역염색으로 이루어진 군에서 선택된 어느 하나를 이용하여 측정할 수 있으나, 이에 제한되는 것은 아니다.Specifically, expression of the pIgR protein can be detected by RT-PCR, competitive RT-PCR, real-time RT-PCR, Western blotting, enzyme linked immunosorbent assay (ELISA) Flow cytometry, tissue immuno staining, and the like, but the present invention is not limited thereto.
이하에서는, 본 발명을 한정하지 않는 실시예에 따라 본 발명을 상세히 설명한다. 본 발명의 하기 실시예는 본 발명을 구체화하기 위한 것일 뿐 본 발명의 권리범위를 제한하거나 한정하는 것이 아님은 물론이다. 따라서, 본 발명의 상세한 설명 및 실시예로부터 본 발명이 속하는 기술분야의 전문가가 용이하게 유추할 수 있는 것은 본 발명의 권리범위에 속하는 것으로 해석된다. Hereinafter, the present invention will be described in detail with reference to embodiments which do not limit the present invention. It should be understood that the following embodiments of the present invention are only for embodying the present invention and do not limit or limit the scope of the present invention. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.
<< 실시예Example 1> 리보솜 불활성화에 의한 마우스 장내 1> Mouse intestine by ribosome inactivation pIgR의pIgR 발현 억제 Suppression of expression
1. 실험방법1. Experimental Method
리보솜 결합 조성물에 의한 리보솜 불활성화를 통해 마우스 장내 pIgR 발현 변화를 살펴보기 위하여 25mg/kg의 농도로 리보솜 결합 조성물인 데옥시니발레놀(deoxynivalenol; DON) 또는 에니소마이신(anisomycin; ANS)을 경구 투여하였다. 24 시간 후, 장을 적출하여 고정하고 횡단면을 절단 후 pIgR 특이적 항체(Santa Cruz, 미국)를 반응시켰다. 그 다음 DAB 및 헤마톡실린(Hematoxylin) 염색을 통하여 단백질 발현 양을 확인하고, 이를 Histo-Quest software를 통하여 정량화하였다. 마우스 장내의 pIgR mRNA 변화를 확인하기 위하여 적출한 장을 RNA 추출 키트 RiboEX(GeneAll, 한국)에 넣고 바로 질소에 얼린 다음 homogenization beads (Becton Dickinson, 미국)를 이용하여 균질화시켰다. 이후 RNA의 분리는 RiboEX 에 제공된 메뉴얼을 참고로 수행하였으며, UV-Vis 형광분광광도계(UV-Vis fluorescence spectrophotometer; Thermo, 미국)을 사용하여 정량화하였다. 분리된 RNA는 TOPscript RT DryMIX 키트(Enzynomics, 한국)를 사용하여 키트에 제공된 메뉴얼을 참고로 cDNA로 변환 시킨 다음 각각 특이적인 primer를 사용하여 PCR을 한 후 정량화 하였다. 대조군으로 GAPDH(glyceraldehyde 3-phosphate dehydrogenase)의 mRNA 발현량을 이용하였다. To examine the expression of pIgR expression in the mouse intestine through ribosome inactivation by the ribosome binding composition, deoxynivalenol (DON) or anisomycin (ANS), a ribosome binding composition, was orally administered at a concentration of 25 mg / . After 24 hours, the intestine was harvested and fixed, and the cross sections were cut and reacted with a pIgR specific antibody (Santa Cruz, USA). The amount of protein expression was then determined by DAB and hematoxylin staining and quantified using Histo-Quest software. In order to confirm the change of pIgR mRNA in the mouse intestine, the extracted sheet was placed in an RNA extraction kit RiboEX (GeneAll, Korea), immediately frozen in nitrogen, and then homogenized using homogenization beads (Becton Dickinson, USA). Separation of RNA was performed with reference to the manual provided in RiboEX and quantified using a UV-Vis fluorescence spectrophotometer (Thermo, USA). The isolated RNA was converted into cDNA by using the TOPscript RT DryMIX kit (Enzynomics, Korea) and the quantities were obtained by PCR using specific primers. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) mRNA expression level was used as a control.
PCR에 수행된 각 프라이머는 (주)코스모진텍(한국)에 의뢰하여 합성하였으며, 각 프라이머의 서열정보는 하기 표 1과 같다.Each primer that was performed in PCR was synthesized by asking Cosmosintech (Korea). Sequence information of each primer is shown in Table 1 below.
2. 실험결과2. Experimental results
리보솜 기능장애를 가진 실험동물에서의 IgA 생산 변화가 장내 pIgR 발현과 관련될 수도 있다고 가정하고, 본 발명자들은 마우스 장내에서 리보솜 불활성화에 따른 상피 pIgR 발현을 측정하였다. 정상 내장 상피조직에서는 상대적으로 높은 수준의 pIgR이 발현되었으나, 리보솜 불활성화를 일으키는 리보솜 결합 조성물(ANS 또는 DON)을 위장관에 처리하면 pIgR 단백질의 발현이 억제되었다(도 1A). 또한, 리보솜을 불활성화시키는 ANS는 용량-의존적 방법으로 상피 pIgR mRNA 발현을 감소시켰다(도 1B).Assuming that changes in IgA production in experimental animals with ribosomal dysfunction may be associated with intestinal pIgR expression, we measured epithelial pIgR expression following ribosome inactivation in the mouse intestine. A relatively high level of pIgR was expressed in the normal intestinal epithelium, but the expression of the pIgR protein was inhibited when the ribosome binding composition (ANS or DON) causing the ribosome inactivation was treated in the gastrointestinal tract (Fig. 1A). In addition, ANS inactivating ribosomes reduced epithelial pIgR mRNA expression in a dose-dependent manner (Fig. 1B).
<< 실시예Example 2> 리보솜 불활성화에 의한 마우스 장내 박테리아 부착 증가 2> Increase in intestinal bacterial adhesion by ribosome inactivation
1. 실험방법1. Experimental Method
장내 존재하는 기존의 우점균으로부터 간섭을 최소화하기 위하여 5g/l 농도의 항생제(streptomycin)를 물에 섞어 24시간 동안 먹인 후 리보솜 결합 조성물인 에니소마이신(Anisomycin, ANS)을 25mg/kg의 농도로 경구 투여하였다. 24시간 후, 형광발색을 하는 EPEC-GFP (Enteropathogenic Escherichia coli)을 각 마우스에 109의 개수로 경구 투여하고, 4일 후에 장을 적출하여 고정하고 횡단면을 절단 후 형광현미경 (AXIO Imager. M2 ;ZEISS, 독일)으로 분석하였다.In order to minimize the interference from existing intestinal bacteria, 5 g / l of streptomycin was added to water for 24 hours, and anisomycin (ANS), a ribosome binding composition, was added at a concentration of 25 mg / kg Orally. After 24 hours, fluorescence-generating EPEC-GFP (Enteropathogenic Escherichia coli ) was orally administered to each mouse in a number of 10 9 , and after 4 days, the intestine was removed and fixed, and the cross section was cut and analyzed with a fluorescence microscope (AXIO Imager.M2; ZEISS, Germany).
2. 실험결과2. Experimental results
본 발명자들은 pIgR 결핍 또는 리보솜 기능장애를 가진 마우스 장내 상피에서 GFP-표지된 EPEC(Enteropathogenic Escherichia coli)의 분포를 관측하였다. pIgR에 대한 IgA 분비 효과를 측정하기 위해서, 이미 보고된 EPEC 감염 마우스 모델을 사용하였다(Infect Immun 73:1161-1170, 2005). 특히, C57BL/6J 마우스는 EPEC 감염에 민감하지만, 심각한 설사를 동반하지 않고도 EPEC 감염에 대한 상피 반응을 연구하기 위해 적합한 in vivo 모델이다. 우선, EPEC는 소장의 상피 내층에 콜로니를 형성함으로써 염증성 위장 질환을 유발한다. 실험이 진행되는 동안, 상피 표면상에 도입된 EPEC 확립 콜로니의 일부가 지속적으로 관측되고, 이의 장내 부착이 지속적으로 나타났다. 흥미롭게도, 대장 상피 표면으로의 박테리아 부착은 pIgR 결핍 또는 리보솜 불활성화에 의해 향상되었다(도 2). pIgR 결핍 또는 리보솜 불활성화된 마우스에서, EPEC의 지속적인 표면 부착은 대조군 마우스보다 빠르게 일어났다. 이는 내장 상피로의 박테리아 이동이 IgA 분비-매개 방어 기작의 붕괴로 인해 촉진된다는 것을 나타낸다. We found that GFP-labeled EPEC (Enteropathogenic Escherichia < RTI ID = 0.0 > The distribution of E. coli was observed. To measure the IgA secretion effect on pIgR, an already reported EPEC infected mouse model was used (Infect Immun 73: 1161-1170, 2005). In particular, C57BL / 6J mice are sensitive to EPEC infection, but are in vivo models suitable for studying the epithelial response to EPEC infection without accompanying severe diarrhea. First, EPEC causes inflammatory gastrointestinal diseases by forming colonies in the epithelium of the small intestine. During the course of the experiment, a portion of the EPEC-established colonies introduced on the epithelial surface was continuously observed and its intestinal attachment persisted. Interestingly, bacterial attachment to the surface of the colon epithelium was enhanced by pIgR deficiency or ribosome inactivation (Fig. 2). In mice lacking pIgR or ribosomes, persistent surface attachment of EPEC occurred faster than control mice. Indicating that bacterial migration into the visceral epithelium is facilitated by the collapse of IgA secretion-mediated defense mechanisms.
<< 실시예Example 3> 리보솜 불활성화에 의한 3> by ribosome inactivation pIgR의pIgR 단백질 발현 억제효과 Inhibitory Effect on Protein Expression
1. 실험방법1. Experimental Method
리보솜 자극제에 의한 pIgR의 단백질 발현조절을 확인하기 위하여, HCT-8 인간 장내 상피세포를 5*10^5개의 수로 60mm 세포배양전용 플레이트에 48시간 동안 배양하였다. 그 다음, 20 ng/ml 농도의 인터페론 감마와 각 농도별 데옥시니발레놀(deoxynivalenol; DON)(Sigma, 미국) 및 에니소마이신(anisomycin; ANS)(Sigma, 미국)을 48시간 동안 처리 후 고정하여 pIgR 특이적 항체를 반응시켜 단백질 발현을 유세포분석기(FACS Canto II; Becton Dickinson, 미국)를 활용하여 분석하였다. HCT-8 human intestinal epithelial cells were cultured for 48 hours on a 60 mm cell culture dish with 5 * 10 < 5 > channels in order to confirm the regulation of protein expression of pIgR by a ribosome stimulant. Then, interferon gamma at a concentration of 20 ng / ml and deoxynivalenol (DON) (Sigma, USA) and anisomycin (ANS) at various concentrations (Sigma, USA) And the protein expression was analyzed using a flow cytometer (FACS Canto II, Becton Dickinson, USA) by reacting pIgR-specific antibody.
2. 실험결과2. Experimental results
pIgR 발현에 대한 리보솜 불활성화의 효과를 HCT-8 인간 장내 상피세포에서도 확인하였다. HCT-8 세포주는 염증성 질환 및 미생물 감염에 대한 장 세포 모델로서 널리 사용된다. 또한, HCT-8 세포의 기원인 소장의 회맹장(ileocecum)은 리보솜 불활성화와 관련된 발병에 가장 민감한 부위 중 하나이다. 하지만, 분리된 장내 상피 세포는 일반적으로 pIgR 발현이 미미한 수준인 것으로 나타났다. 따라서, 생리학적으로 염증성 내장 상피 환경을 촉진하기 위해서, 인터페론 감마(interferon gamma; IFNγ)와 같은 pIgR 촉진 내재성 인자를 추가하였다. 본 발명에서는 pIgR 발현을 증가시켜, 장내 상피 세포를 활성화하기 위해서 IFNγ를 첨가하였다. 리보솜 불활성화 스트레스 하에 놓인, IFNγ-유도 pIgR 단백질의 발현은 유세포분석을 통하여 측정하였다(도 3). in vivo 결과와 유사하게, 리보솜 불활성화 화합물(DON 또는 ANS)에 의한 pIgR 발현은 용량-의존적 방식으로 상당히 감소하였다(도 3). The effect of ribosome inactivation on pIgR expression was also confirmed in HCT-8 human intestinal epithelial cells. HCT-8 cell lines are widely used as intestinal cell models for inflammatory diseases and microbial infections. In addition, the ileocecum of the small intestine, the origin of the HCT-8 cells, is one of the most sensitive sites for the onset of ribosome inactivation. However, the isolated intestinal epithelial cells generally showed a minimal level of pIgR expression. Therefore, in order to promote physiologically inflammatory visceral epithelial environment, a pIgR-promoted intrinsic factor such as interferon gamma (IFN gamma) was added. In the present invention, IFN gamma was added to increase pIgR expression and activate intestinal epithelial cells. Expression of IFN gamma -induced pIgR protein under the ribosome inactivation stress was measured via flow cytometry (Figure 3). Similar to in vivo results, pIgR expression by ribosome-inactivating compounds (DON or ANS) was significantly reduced in a dose-dependent manner (Figure 3).
<실시예 4> 장 상피세포에서 리보솜 결합 조성물에 의한 pIgR 단백질 발현 감소<Example 4> Reduction of pIgR protein expression by ribosome binding composition in intestinal epithelial cells
1. 실험방법1. Experimental Method
다양한 인간 장 상피세포주에서 데옥시니발레놀(deoxynivalenol; DON), 에니소마이신(anisomycin; ANS), 니발레놀(nivalenol; NIV) 또는 15-아세틸 DON(15-acetyl DON) 에 의한 리보솜 불활성화가 IFNγ에 의해 유도된 pIgR 단백질 발현조절을 확인하기 위하여 상피세포를 5*10^5 개의 수로 60mm 세포배양전용 플레이트에 48 시간동안 배양 하였다. 그 다음 20 ng/ml 농도의 인터페론감마와 1000 ng/ml 농도의 리보솜결합 조성물을 48시간 동안 처리 후 고정하여 pIgR 특이적 항체를 반응시켜 단백질 발현을 유세포분석기를 활용하여 분석하였다.Inhibition of ribosomes by deoxynivalenol (DON), anisomycin (ANS), nivalenol (NIV) or 15-acetyl DON in various human intestinal epithelial cell lines Was cultured for 48 hours in a 60 mm cell culture dish with 5 * 10 < 5 > channels in order to confirm IFN gamma-induced regulation of pIgR protein expression. Then, 20 ng / ml of interferon gamma and 1000 ng / ml of ribosome binding composition were treated and fixed for 48 hours and reacted with pIgR specific antibody to analyze protein expression using flow cytometry.
2. 실험결과2. Experimental results
인간 장내 상피세포주인 HCT-8, Intestine 407 및 HT-29에서 데옥시니발레놀(deoxynivalenol; DON), 에니소마이신(anisomycin; ANS), 니발레놀(nivalenol; NIV) 또는 15-아세틸 DON(15-acetyl DON) 에 의한 리보솜 불활성화가 IFNγ에 의해 유도된 pIgR 단백질 발현을 유의미하게 감소시키는 것을 확인하였다(도 4).Deoxynivalenol (DON), anisomycin (ANS), nivalenol (NIV), or 15-acetyl DON (NAD) in human intestinal epithelial cell line HCT-8,
<< 실시예Example 5> 리보솜 불활성화에 의한 인터페론 유도 5> Induction of interferon by ribosome inactivation pIgR의pIgR mRNAmRNA 안정성 감소 효과 Stability Reduction Effect
1. 실험방법1. Experimental Method
리보솜 자극제에 의한 pIgR mRNA의 안정성 기여도를 보기 위하여 HCT-8 인간 장내 상피세포를 2.5*10^5개의 수로 35mm 세포배양전용 플레이트에 48시간 동안 배양을 하였다. 그 다음 500 ng/ml 농도의 데옥시니발레놀(Deoxynivalenol)을 12시간 동안 전처리 후 20 ng/ml 농도의 인터페론 감마를 48시간 동안 처리하였다. 대조군 및 실험군에 전사 억제 인자인 액티노마이신 D(Actinomycin D) (Sigma, 미국)를 5 μM 농도로 처리를 하고 각각의 시간대에 회수하여 RNA 추출을 하였다. 이후 역전사 효소를 사용하여 추출된 남은 RNA를 cDNA로 변환시킨 다음 정량화하여 반감기를 계산하였다.HCT-8 human intestinal epithelial cells were cultured for 48 hours on a 35 mm cell culture plate with 2.5 * 10 ^ 5 channels for the stability contribution of pIgR mRNA by ribosomal stimulants. Then deoxynivalenol at a concentration of 500 ng / ml was pretreated for 12 hours and then treated with 20 ng / ml of interferon gamma for 48 hours. Control and experimental groups were treated with Actinomycin D (Sigma, USA), a transcription inhibitor, at a concentration of 5 μM and RNA was extracted at each time point. The remaining RNA extracted with reverse transcriptase was converted into cDNA and quantified to calculate half-life.
2. 실험결과2. Experimental results
IRF-1 또는 NF-κB를 통한 전사 조절은 리보솜 불활성화에 의한 pIgR 발현 억제를 설명할 수 없었으므로, 본 발명자들은 pIgR mRNA 안정성과 관련된 전사 후 조절에 대한 리보솜 불활성화의 효과를 확인하였다. IFNγ-촉진 조건하에서, 리보솜 불활성화는 pIgR mRNA 안정성을 상당히 감소시켰다(도 5). Since transcriptional regulation via IRF-1 or NF-κB could not account for inhibition of pIgR expression by ribosome inactivation, we confirmed the effect of ribosome inactivation on post-transcriptional regulation associated with pIgR mRNA stability. Under IFNy-promoting conditions, ribosome inactivation significantly reduced pIgR mRNA stability (Figure 5).
<< 실시예Example 6> 리보솜 불활성화에 의한 6> by ribosome inactivation HuRHuR 단백질 세포질 내 유출억제를 통한 pIgR PIgR through inhibition of protein cytoplasmic efflux mRNAmRNA 안정성 감소 Reduced stability
1. 실험방법1. Experimental Method
리보솜 불활성화가 pIgR mRNA 안정성을 감소시키는데, HuR 단백질이 관련되어 있는지 확인하기 위하여, 공벡터 또는 센스(sense) HuR 발현 플라스미드-형질감염된 HCT-8 세포에 20 ng/ml IFN-γ 존재 하에서, 500 ng/ml DON 또는 50 ng/ml ANS를 48시간 동안 처리하였다. 한편, 세포 내에서 pIgR 전사체와 결합하는 HuR 단백질을 측정하기 위해서 RNA 면역침전분석을 수행하였다. HCT-8 세포에 500 ng/ml DON (D) 존재 유무에 따라, 20 ng/ml IFN-γ (I)를 48시간 동안 처리하였다. 면역침전된 전사체는 실시간 RT-PCR로 측정하였다. *p < 0.05, 대조군과의 유의적 차이를 나타낸다. In order to confirm that the HuR protein is involved, ribosome inactivation reduces pIgR mRNA stability. In order to confirm that the HuR protein is involved, an open vector or sense HuR-expressing plasmid-transfected HCT-8 cells is seeded with 500 ng / ml DON or 50 ng / ml ANS for 48 hours. Meanwhile, RNA immunoprecipitation analysis was performed to measure the HuR protein binding to the pIgR transcript in the cells. 20 ng / ml IFN-y (I) was treated for 48 hours with or without 500 ng / ml DON (D) in HCT-8 cells. Immunoprecipitated transcripts were measured by real-time RT-PCR. * p <0.05, indicating a significant difference from the control group.
2. 실험결과2. Experimental results
mRNA 안정화에 주요한 기능을 하는 HuR 단백질이 비교적 긴 시간(12시간 이상)의 리보솜스트레스에 의해서 세포질로 유출이 제한되고 이로 인하여 pIgR의 mRNA 안정화가 저해되었다. 따라서 HuR 과발현 해주면 pIgR의 mRNA가 증가되는 것을 확인하였다(도 6). 또한, IFN-γ는 pIgR 전사체와 HuR 단백질의 결합을 상당히 증가시켰는데, 이는 리보솜 불활성화에 의해 pIgR이 완전히 억제된다는 것을 나타낸다(도 7).The HuR protein, which plays a major role in mRNA stabilization, was restricted to the cytoplasm by ribosomal stress for a relatively long time (over 12 hours), which inhibited the mRNA stabilization of pIgR. Therefore, it was confirmed that the mRNA of pIgR was increased by overexpressing HuR (FIG. 6). In addition, IFN-y significantly increased binding of HuR protein to pIgR transcript, indicating that pIgR is completely inhibited by ribosome inactivation (Fig. 7).
결론적으로, 도 8에서는 리보솜 불활성화에 의한 pIgR 억제 모식도를 나타냈고, 도 9에는 인간 점막 상피세포에서 리보솜 불활성화-매개 점막 면역질환 발병 기작의 추정 모식도를 나타냈다. In conclusion, FIG. 8 shows a schematic diagram of inhibition of pIgR by ribosome inactivation, and FIG. 9 shows a hypothetical model of the mechanism of the onset of ribosome inactivation-mediated mucosal immune disease in human mucosal epithelial cells.
상기와 같은 결과는 동물실험 모델에서 균총에 대한 감수성을 항진시키는데 기여하여 특정 균총에 의한 기능 활성 연구에 활용될 수도 있다. The above results can be used to study the functional activity of a particular microorganism by contributing to the sensitivity to the microorganism in the animal model.
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