이 연구는 의학, 서울, 울산 대학교 의과 대학 학생 연구 그랜트 (13 ~ 14)과 아산 생명 과학 연구소, 서울, 한국에서 보조금 (2014-464)에 의해 지원되었다.

1. 세포주 <ol><li> 6 웰 플레이트에 문화 10 6 텔로 머라 제 - 불후의 인간의 각막 상피 세포 (인체 각막 상피 세포) : 기관지 상피 성장 매체 (BEGM)를 사용하여 5 % CO 2 분위기로 37 ° C 배양기에서 (밀도 1039.9 셀 / mm 2)까지 그들은 95 %의 합류에 도달합니다. </li></ol> 2. 웨스턴 블롯 분석 상피 스크래치 상처를 작성 후 <ol><li> 킬 멸균 200 μL 피펫 합류 배양 된 인체 각막 상피 세포의 잘의 표면을 가로 질러 팁 생물 안전 캐비닛 접시 당 네 번 (클래스 II, 유형 A2)와 5 % CO 2 분위기로 37 ° C 배양기에서 별도로 인체 각막 상피 세포를 배양 1, 5, 10, 30, 60 및 120 분. </li><li> 각막 상피 ​​상처 후 배양 시간에 따라 여섯 가지 샘플에 대해 하나의 6 웰 플레이트를 사용합니다. </li><li> 1X PBS로 부상 HCEC의 단일 층을 세 번 세척 한 후 각 웰에 2.0 ml의 BEGM를 추가합니다. </li><li> (2)를 사용하여 인체 각막 상피 ​​세포를 분리ml의 0.25 % 트립신 - 에틸렌 디아민 5 분 동안 잘 당 아세트산 (EDTA), 15 ML 튜브에서 5 분 900 XG에 원심 분리기 및 흡인 트립신 EDTA는 1 ml를 피펫을 사용하여. </li><li> PBS 1 배 1 ml의 인체 각막 상피 ​​세포를 일시 중단하고 1.5 ML 튜브로 전송. </li><li> 15 초 동안 10,000 XG에 원심 분리기 인체 각막 상피 ​​세포 및 흡인 1X PBS는 1 ml를 피펫을 사용하여. </li><li> 100 μl의 빙냉 용해 완충액에서 재현 탁 인체 각막 상피 세포 (10 mM 트리스, 10 mM의 NaCl을 2 mM의 EDTA, 25 mM의 NaF로부터, 2 mM의 나트륨 3 VO 4,1 밀리미터 페닐 메탄 설 포닐 플루오 라이드 [PMSF], 프로테아제 억제제 [A 펩 스타틴 1 μM, 1 μM의 류 펩틴, 0.1 μM 아프로 티닌] 0.5 % 트리톤 X-100의 pH 7) 및 그들을 잘 섞는다. </li><li> 세포 용해를 유도하는 얼음에 30 분 동안 세포를 인큐베이션. </li><li> 원심 분리기 15 분 동안 10,000 XG에 4 ° C에서 해물을하고 신선한 1.5 ml의 튜브 (90 μL 씩)에 상층 액을 전송하고 -80 ° C에 저장합니다. </li><li> 브래드 포드 (PR)을 사용하여 세포 용 해물의 전체 단백질 농도를 결정otein 분석. (9) </li><li> 나트륨 도데 실 설페이트 - 폴리 아크릴 아미드 겔 전기 영동 (SDS-PAGE)를위한 10 % 또는 12 % 아크릴 아마이드 겔에 부하 30 μg의 총 세포 단백질을 전기 영동 이동은 4 ℃에서 1 시간에 대해 200mA 전류 니트로 셀룰로오스 필터에 단백질 밴드 분리 C는 웨스턴 블롯 분석에 사용할 수 있습니다. </li><li> 5 % 차단 니트로 셀룰로오스 여과막 비 인산화 HSP27 대해 기본 토끼 폴리 클로 날 항체를 추가로 1 시간 동안 트윈 -20 (TBST)를 TBS 버퍼에서 탈지유 (1 : 1000 희석) 또는 인산화 HSP27 대해 기본 토끼 다 클론 항체 (1 : 1000 5 % 소 혈청 알부민에 희석) (BSA), 및 진탕 기에서 4 ℃에서 하룻밤 부화 막. </li><li> TBST로 10 분간 3 회 세척 한 후, 5 %의 BSA (10,000 희석 1) 양 고추 냉이 퍼 옥시 다제 - 결합 염소 항 - 토끼 항체를 사용하여 면역 반응성 밴드를 검출한다. </li><li> 서쪽 블로 팅 루미놀 시약에 멤브레인을 품어 (6-7m실온에서 1 분 동안 5cm 막 × 10cm) 당 리터. </li><li> 시약 용액으로부터 막을 제거, 플라스틱 시트 보호에 과잉 흡수 수건 액체과 장소를 제거합니다. </li><li> 안전한 빛 어두운 방에서 작업, 단백질면이 위를 향하도록 필름 카세트에 덮여 막 배치합니다. </li><li> 막 위에 X 선 필름을 넣고 1 분 동안 노출. </li></ol> 3. siRNA를 형질 분석 (10) <ol><li> 문화 5 × 10 5 세포에서 인체 각막 상피 세포 / 잘가 95 %의 합류에 도달 할 때까지 BEGM를 사용하여 5 % CO 2 분위기로 37 ° C 배양기에서 6 웰 플레이트. </li><li> 10 또는 50 nM의를 만들 수 감소 혈청 배지를 형질 전환 (희석 요인이 41 또는 14.3이었다) 100 ㎕를 감소 혈청 매체와 형질 전환 시약 (2.5 또는 7.5 μl를) 희석과 HSP27 별을 용해하고 100 μL에 제어 siRNA를 스크램블 HSP27 별 및 제어 siRNA의 스크램블. </li><li> 100 μ 믹스100 ㎕의 희석 된 형질 전환 시약 L 개의 siRNA의 용액 (1 : 1 비) 실온에서 15 분 동안 혼합물을 배양한다. </li><li> 세포에의 siRNA - 지질 단지를 추가합니다. 그 후 4 시간 변화 매체 후 37 ℃에서 2 일 동안 세포를 BEGM를 완료하고 부화합니다. </li><li> 4.7 절 4.1에서 설명 된 바와 같이, 웨스턴 블로 팅에 의해 형질 전환 된 세포를 분석 할 수 있습니다. </li></ol> siRNA를 형질 세포 (11) 4. 웨스턴 블롯 분석 <ol><li> HSP27 특정 추출하고 100 μl의 빙냉 용해 완충액 (10 mM 트리스, 10 mM의 NaCl을 2 mM의 에틸렌 디아민 테트라 아세트산 (EDTA), 25 mM의 NaF로부터, 2 mM의 나트륨 3 VO를 이용한 생물학적 안전 캐비넷 제어 siRNA를 형질 인체 각막 상피 세포를 스크램블링 4, 1 ㎜ 페닐 메탄 설 포닐 플루오 라이드 (PMSF), 프로테아제 저해제, 및 0.5 % 트리톤 X-100, pH가 7). </li><li> 세포 용해를 유도하는 얼음에 30 분 동안 세포를 인큐베이션. </li><li> 10,000 XG에 펠렛 해물을 15 분 동안하고 신선한 1.5 ML의 욕조에 상층 액을 전송ES (90 μl의 분취 량) 및 -80 ° C에 저장합니다. </li><li> 브래드 포드 (Bradford) 단백질 분석을 이용하여 세포 용 해물의 단백질 농도를 결정한다. (9) </li><li> 10 % 또는 12 % 아크릴 아마이드 겔에 총 세포 단백질의 동일한 양의로드 샘플은 SDS-PAGE에 젤 대상 및 전기 영동 4 ° C에서 1 시간을 위해 200mA의 전류 니트로 셀룰로오스 필터로 분리 된 단백질 밴드를 전송 웨스턴 블롯 분석에 사용합니다. </li><li> , (1000 희석 1) (1000 희석 1) 인산화 된 Akt 5 % 블록 니트로 셀룰로오스 여과막 인산화 비 인산화 HSP27에 대한 일차 항체를 추가 1 시간 동안 트윈 -20 (TBST)를 TBS 버퍼에서 탈지유 Akt의 인산화되지 않은 (1 : 1000 희석, 세포 생존 마커로 사용됨), BCL-2 관련 X 단백질 (1 : 1000 희석, 프로 - 세포 사멸 단백질로 사용), 및 글리 세르 알데히드 -3- 포스페이트 탈수소 효소 (GAPDH 1 5 % 소 혈청 알부민으로 로딩 대조군으로 사용한 200 희석) (BSA), 및쉐이커에 4 ° C에서 하룻밤 멤브레인을 품어. </li><li> 세척 후 TBST로 3 회, 각 10 분간 세척을 5 % BSA에서 (10,000 희석 1) 양 고추 냉이 퍼 옥시 다제 - 결합 염소 항 - 토끼 항체를 사용하여 면역 반응성 밴드를 검출한다. </li><li> 실온에서 1 분 동안 웨스턴 블롯 루미놀 시약의 막 (6-7 mL의 10cm × 5cm 막) 부화. </li><li> 시약 용액으로부터 막을 제거, 플라스틱 시트 보호에 과잉 흡수 수건 액체과 장소를 제거합니다. </li><li> 안전한 빛 어두운 방에서 작업, 단백질면이 위를 향하게와 필름 카세트에 덮여 막 놓습니다. </li><li> 멤브레인 위에 X- 선 필름을 넣고 1 분 동안 노출. </li></ol> 5. 스크래치에 의한 세포 이동 (12)의 방향 상처 분석 평가 <ol><li> 생물 안전 캐비닛에서의 합류 문화의 잘의 표면을 멸균 피펫 팁을 드래그하여 상처를 만들HSP27 특정 siRNA를 형질 또는 스크램블 제어 siRNA를 형질 인체 각막 상피 ​​세포. </li><li> 바로 부상 후 부상 후 24 시간 동안 5 % CO 2 분위기로 37 ° C 배양기에서 BEGM 문화에서 그들을 1X 인산 완충 식염수 (PBS)로 두 번 세포를 씻어 유지한다. </li><li> 24 시간 부상 후 100 배의 배율로 직립 현미경을 사용하여 HCEC 이미지를 촬영하고 배경은 필터를 사용하여 병합 수행 이미지 분석 소프트웨어 명령어. </li><li> 선택 측정 명령을 사용하여 초기 상처의 끝과 끝에서 수직으로 커버 할 수있는 동일한 크기의 다각형과 관심 (AOI)의 영역을 정의하고, 각 샘플의 상처 지역에 세 가지 다른 AOI을 결정합니다. </li><li> 자동 카운트 / 크기 측정 메뉴 옵션을 사용하여 각 분야에서 세포 수를 계산. </li></ol> 세포 사멸 6. 유동 세포 계측법 분석 <ol><li> 문화 HSP27 특정 siRNA를 형질 및 제어의세포 BEGM 5 % CO 2 분위기로 37 ° C의 배양기에서 95 % 합류점에 도달 할 때까지 / 웰 6 웰 플레이트에 5 × 105 세포의 농도로 각 10 nM의 siRNA를 함유 인체 각막 상피 세포를 IRNA는 형질. </li><li> 1 ML의 피펫을 사용하여 5 15 ML 튜브에 분하고, 흡인 트립신 EDTA 900 XG에서 5 분, 원심 분리기 잘 당 2 ml의 0.25 % 트립신 - EDTA를 사용하여 인체 각막 상피 ​​세포를 분리합니다. </li><li> 차가운 PBS로 두 번 세포를 씻어 준 후 10 6 세포 / ml의 농도로 완충액 (0.1 M HEPES / NaOH로 [pH를 7.4, 1.4 M의 NaCl, 25 mM의 CaCl2를)를 결합 1X에서 세포를 재현 탁. </li><li> 5 ㎖의 배양 관 내로 세포 현탁액 (1 × 105 세포) 100 μL를 전송. </li><li> 5 μL를 형광 염료 - 복합 넥신 V 및 5 μL의 프로피 디움 아이오다 이드를 추가합니다. </li><li> 부드럽게 와동 세포 및 어둠 속에서 실온에서 15 분 동안이를 부화. </li><li> 흐름 C가 세포를 각 튜브에 1X 결합 버퍼의 200 μl를 추가하고 분석1 시간 내에 ytometer. </li></ol>

<ol>
	<li>Dua, H. S., Gomes, J. A., Singh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+epithelial+wound+healing.">Corneal epithelial wound healing.</a> Br. J. Ophthalmol. 78 (5), 401-408 (1994).</li><li>Estil, S., Primo, E. J., Wilson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis+in+shed+human+corneal+cells.">Apoptosis in shed human corneal cells.</a> Invest. Ophthalmol. Vis. Sci. 41 (11), 3360-3364 (2000).</li><li>Guay, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+actin+filament+dynamics+by+p38+map+kinase-mediated+phosphorylation+of+heat+shock+protein+27.">Regulation of actin filament dynamics by p38 map kinase-mediated phosphorylation of heat shock protein 27.</a> J. cell. Sci. 110, 357-368 (1997).</li><li>Park, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+heat+shock+protein+mRNAs+under+in+vivo+glutathione+depletion+in+the+mouse+retina.">Differential expression of heat shock protein mRNAs under in vivo glutathione depletion in the mouse retina.</a> Neurosci. Lett. 413 (3), 260-264 (2007).</li><li>Rane, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat+shock+protein+27+controls+apoptosis+by+regulating+Akt+activation.">Heat shock protein 27 controls apoptosis by regulating Akt activation.</a> J. Biol. Chem. 278 (30), 27828-27835 (2003).</li><li>Shin, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blocking+tumor+cell+migration+and+invasion+with+biphenyl+isoxazole+derivative+KRIBB3,+a+synthetic+molecule+that+inhibits+Hsp27+phosphorylation.">Blocking tumor cell migration and invasion with biphenyl isoxazole derivative KRIBB3, a synthetic molecule that inhibits Hsp27 phosphorylation.</a> J. Biol. Chem. 280 (50), 41439-41448 (2005).</li><li>Jain, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+phosphorylated+heat+shock+protein+27+during+corneal+epithelial+wound+healing.">Expression of phosphorylated heat shock protein 27 during corneal epithelial wound healing.</a> Cornea. 31 (7), 820-827 (2012).</li><li>Alekseev, O. M., Richardson, R. T., Alekseev, O., O'Rand, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+gene+expression+profiles+in+HeLa+cells+in+response+to+overexpression+or+siRNA-mediated+depletion+of+NASP.">Analysis of gene expression profiles in HeLa cells in response to overexpression or siRNA-mediated depletion of NASP.</a> Reprod. Biol. Endocrinol. 7, 45 (2009).</li><li>Park, H. Y., Kim, J. H., Lee, K. M., Park, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+prostaglandin+analogues+on+tear+proteomics+and+expression+of+cytokines+and+matrix+metalloproteinases+in+the+conjunctiva+and+corea.">Effect of prostaglandin analogues on tear proteomics and expression of cytokines and matrix metalloproteinases in the conjunctiva and corea.</a> Exp. Eye. Res. 94 (1), 13-21 (2012).</li><li>Voegeli, T. S., Currie, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=siRNA+knocks+down+Hsp27+and+increases+angiotensin+II-induced+phosphorylated+NF-kappaB+p65+levels+in+aortic+smooth+muscle+cells.">siRNA knocks down Hsp27 and increases angiotensin II-induced phosphorylated NF-kappaB p65 levels in aortic smooth muscle cells.</a> Inflamm. Res. 58 (6), 336-343 (2009).</li><li>Shi, B., Isseroff, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arsenite+pre-conditioning+reduces+UVB-induced+apoptosis+in+corneal+epithelial+cells+through+the+anti-apoptotic+activity+of+27+kDa+heat+shock+protein+(HSP27).">Arsenite pre-conditioning reduces UVB-induced apoptosis in corneal epithelial cells through the anti-apoptotic activity of 27 kDa heat shock protein (HSP27).</a> J. Cell. Physiol. 206 (2), 301-308 (2006).</li><li>Shen, E. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+corneal+epitheliotrophic+capacity+among+different+human+blood-derived+preparations.">Comparison of corneal epitheliotrophic capacity among different human blood-derived preparations.</a> Cornea. 30 (2), 208-214 (2011).</li><li>Song, I. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat+shock+protein+27+phosphorylation+is+involved+in+epithelial+cell+apoptosis+as+well+as+epithelial+migration+during+corneal+epithelial+wound+healing.">Heat shock protein 27 phosphorylation is involved in epithelial cell apoptosis as well as epithelial migration during corneal epithelial wound healing.</a> Exp Eye Res. 118 (1), 36-41 (2014).</li></ol>

저자는이 연구에서 언급 된 모든 자료 나 방법에는 금융 또는 소유권 관심이 없습니다.

In this present study, we evaluated the potential role of HSP27 in corneal epithelial wounding using in vitro approaches. The critical steps involved siRNA transfection for HSP27 knock-down to observe the function of HSP27 in cells subjected to stress. Notably, a role for HSP27 was revealed by these experiments in epithelial cell migration and apoptosis during corneal epithelial wound healing. Unlike previous studies10 that used rat HSP27-specific siRNA to transfect vascular smooth muscle cells, we us...

그들은 동시에 윤부 각막 상피 기저 층에서 세포로 대체하는 동안 각막 상피 세포 (CECs)이 지속적으로, 눈물 막으로 흘려된다. (1) 다양한 외부 스트레스가 CECs의 세포 사멸 및 박리를 유도 할 수 있습니다. 2 열 충격 단백질들 (HSPs) 고도로 보존되어 있으며, 분자의 크기에 따라 두 개 패밀리로 분할 될 수있다. (3) 최대 HSP 계열은 HSP90, HSP70, 및 HSP60 및 작은 가족 HSP27 포함을 포함한다. 4 HSP27의 인산화는 세포 생존에 중요한 역할을하는 것으로 알려져 때문에 굴지의 리모델링이 단백질의 역할의 세포 이동이 필요합니다. 5-7 따라서, 우리는 상피 상처 치유의 체외 모델에서 CEC 마이그레이션 및 세포 자멸사에서 HSP27 인산화의 잠재적 인 역할을 테스트하려고했습니다. RNA 간섭 (RNAi의) 작거나 짧은 간섭 RNA를 하나를 사용하여 (siRNA의)은 GE있다잠재적으로 허용하는 두 기초 및 응용 생물학 nerated 관심이 관심의 유전자의 발현이 노크 다운 될 수 있습니다. (8) 여기서, 우리는 CEC 상처 치유와 세포 자멸사에 대한 HSP27의 기여를 평가하기 위해 HSP27 특정 siRNA를 사용했다. 세포에서 유전자 노크 다운에 대한 기존의 RNAi 방법은 siRNA를을 만드는 조립 될 수있는 두 개의 수정되지 않은 21 메르 올리고 뉴클레오티드를 포함하는 합성 RNA 이중 가닥을 사용합니다. 우리가 본 연구에 사용 된 siRNA의 RNAi의 세포를 형질하는 간단하고 효율적인 방법이며,이 시약은 다양한 불멸화 세포주와 함께 작동한다. 이 연구에서는, 우리는 스크래치에 의한 방향 상처 분석, 웨스턴 블로 팅, siRNA를 형질 분석, 면역 형광 분석을 포함하여이 분석에 사용되는 방법을 설명하고, 유동 세포 계측법.

<table><tbody><tr><td>Biological safety cabinet</td><td>CHC LAB Co.Ltd,&amp;nbsp; Daejeon, Republic of Korea&amp;nbsp;</td><td>CHC-777A2-06</td><td>Class II, Type A2&amp;nbsp;</td></tr><tr><td>Stealth RNAi&amp;trade; siRNA</td><td>Thermo Fisher Scientific, Inc., Waltham, MA</td><td></td><td>RNAi siRNA; scrambled control-siRNA and HSP27-specific siRNA</td></tr><tr><td>BEGM&lt;sup&gt;TM&lt;/sup&gt;</td><td>Lonza, Inc., Walkersville, MD</td><td>CC-3171, CC4175</td><td>Bronchial epithelium growth medium&amp;nbsp;</td></tr><tr><td>Protease inhibitor&amp;nbsp;</td><td>Sigma-Aldrich, Inc., St. Louis, MO</td><td>P8340 ,P7626</td><td>1 &amp;#956;M Pepstatin A, 1 &amp;#956;M Leupeptin, 0.1 &amp;#956;M Aprotinin</td></tr><tr><td>Bradford protein assay&amp;nbsp;</td><td>Bio-Rad Laboratories, Hercules, CA</td><td>#500-0001</td><td>Bradford protein assay&amp;nbsp;</td></tr><tr><td>Nitrocellulose filters&amp;nbsp;</td><td>Amersham, Little Chalfont, UK</td><td>RPN3032D</td><td>Western blotting membrane</td></tr><tr><td>Non-phosphorylated HSP27&amp;nbsp;</td><td>Abcam Inc., Cambridge, MA</td><td>ab12351</td><td>1:1,000 dilution (Total HSP27)</td></tr><tr><td>Phosphorylated HSP27 (Ser85)</td><td>Abcam Inc., Cambridge, MA</td><td>ab5594</td><td>1:1,000 dilution HSP27 was phosphorylated at Ser85</td></tr><tr><td>Lipofectamine&lt;sup&gt;&amp;reg; &lt;/sup&gt;RNAiMAX reagent&amp;nbsp;</td><td>Invitrogen, Carlsbad, CA</td><td>13-778-075</td><td>Transfection reagent</td></tr><tr><td>Phosphorylated Akt (Ser473)</td><td>Cell Signaling Technology, Danvers, MA</td><td>No. 4060</td><td>1:1,000 dilution Akt was phosphorylated at Ser473 (cell survival marker)</td></tr><tr><td>Non-phosphorylated Akt&amp;nbsp;</td><td>Cell Signaling Technology, Danvers, MA</td><td>No. 4061</td><td>1:1,000 dilution (Total Akt)</td></tr><tr><td>Bcl-2-associated X protein&amp;nbsp;</td><td>Cell Signaling Technology, Danvers, MA</td><td>No. 4062</td><td>1:1,000 (anti-apoptotic protein marker)</td></tr><tr><td>GAPDH</td><td>Santa Cruz Biotechnology, Santa Cruz, CA</td><td>No. 4063</td><td>1:1,000 loading control marker (house keeping gene)</td></tr><tr><td>Horseradish peroxidase-conjugated goat anti-rabbit antibodies</td><td>Thermo Fisher Scientific, Inc., Waltham, MA</td><td>NCI1460KR</td><td>1:10,000 dilution</td></tr><tr><td>OPTI-MEM</td><td>Invitrogen, Carlsbad, CA</td><td>31985</td><td>reduced serum medium for transfection</td></tr><tr><td>Image analysis software</td><td>Olympus, Inc., Tokyo, Japan</td><td>Image-Pro Plus 5.0</td><td></td></tr><tr><td>Skimed milk powder&amp;nbsp;</td><td>Carl Roth GmbH + Co. KG, Karlstruhe, Germany</td><td>T145.2</td><td></td></tr><tr><td>Tris&amp;nbsp;</td><td>Amresco LCC, Inc. Solon, OH</td><td>No-0497</td><td></td></tr><tr><td>Sodium Chloride&amp;nbsp;</td><td>Amresco LCC, Inc. Solon, OH</td><td>No-0241</td><td></td></tr><tr><td>Six well culture plate</td><td>Thermo Fisher Scientific, Inc., Waltham, MA</td><td>140675</td><td>35.00 mm diameter / well</td></tr><tr><td>24-well culuture dish</td><td>Thermo Fisher Scientific, Inc., Waltham, MA</td><td>142475</td><td></td></tr><tr><td>Orbital shaker</td><td>N-Bioteck, Inc., Seoul, South Korea</td><td>NB1015</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Santa Cruz Biotechnology, Santa Cruz, CA</td><td>sc-2323&amp;nbsp;</td><td></td></tr><tr><td>BDFACSCanto&lt;sup&gt;TM&lt;/sup&gt; II</td><td>BD Biosciences, Franklin Lakes, NJ</td><td></td><td>Flow cytometry</td></tr><tr><td>X-Ray Film</td><td>Kodak, Rochester, NY</td><td></td><td>Medical X-Ray Cassette with Green 400 Screen&amp;nbsp;</td></tr><tr><td>western blotting luminol reagent</td><td>Santa Cruz Biotechnology, Santa Cruz, CA</td><td>sc-2048&amp;nbsp;</td><td></td></tr><tr><td>FITC Annexin V Apoptosis Detection Kit I</td><td>BD Biosciences, Franklin Lakes, NJ</td><td>556547</td><td></td></tr></tbody></table>

rna interference-based investigation of the function of heat shock protein 27 during corneal epithelial wound healing

Small interfering RNA (siRNA) is among the most widely used RNA interference methods for the short-term silencing of protein-coding genes. siRNA is a synthetic RNA duplex created to specifically target a mRNA transcript to induce its degradation and it has been used to identify novel pathways in various cellular processes. Few reports exist regarding the role of phosphorylated heat shock protein 27 (HSP27) in corneal epithelial wound healing. Herein, cultured human corneal epithelial cells were divided into a scrambled control-siRNA transfected group and a HSP27-specific siRNA-transfected group. Scratch-induced directional wounding assays, and western blotting, and flow cytometry were then performed. We conclude that HSP27 has roles in corneal epithelial wound healing that may involve epithelial cell apoptosis and migration. Here, step-by-step descriptions of sample preparation and the study protocol are provided.

인산화 HSP27의 발현은 크게 5, 10로 증가하고, 처음 30 분 후에는 13 unwounded 인체 각막 상피 세포에 비하여 부상. 웨스턴 블롯 분석 Bax의 발현이 크게 HSP27 특정 siRNA를 형질 인체 각막 상피 세포 (그림 1A-E)에서 증가하는 반면 인산화 HSP27과 인산화 된 Akt의 발현이 모두 유의하게 감소 된 것으로 나타났습니다. 인산화 HSP27의 발현을 30 % 40 10 뉴 멕시코 % 및 HSP...

Watch this Scientific Journal Video about RNA Interference-based Investigation of the Function of Heat Shock Protein 27 during Corneal Epithelial Wound Healing at JoVE.com

RNA Interference-based Investigation of the Function of Heat Shock Protein 27 during Corneal Epithelial Wound Healing

Herein, we present a protocol to use heat shock protein 27 (HSP27)-specific small interfering RNA to assess the function of HSP27 during corneal epithelial wound healing. RNA interference is the best method for effectively knocking-down gene expression to investigate protein function in various cell types.

각막 상피 ​​창상 치유 기간 동안 열 충격 단백질 27의 기능의 RNA 간섭 기반 조사

Small interfering RNA (siRNA) is among the most widely used RNA interference methods for the short-term silencing of protein-coding genes. siRNA is a ...

rna-interference-based-investigation-function-heat-shock-protein-27

Universidad de Cartagena UDEC

Research

JoVE Journal

Genetics

7.4K 조회수.  Saevit Eye Hospital. Herein, we present a protocol to use heat shock protein 27 (HSP27)-specific small interfering RNA to assess the function of HSP27 during corneal epithelial wound healing. RNA interference is the best method for effectively knocking-down gene expression to investigate protein function in various cell types.

비디오: RNA Interference-based Investigation of the Function of Heat Shock Protein 27 during Corneal Epithelial Wound Healing

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to follow and manipulate the wound response ex vivo in an environment that closely mimics that of epithelial tissue injury in vivo. This issue was addressed by creating a clinically relevant epithelial ex vivo injury-repair model based on cataract surgery. In this culture model, the response of the lens epithelium to wounding can be followed live in the cells&#8217; native microenvironment, and the molecular mediators of wound repair easily manipulated during the repair process. To prepare the cultures, lenses are removed from the eye and a small incision is made in the anterior of the lens from which the inner mass of lens fiber cells is removed. This procedure creates a circular wound on the posterior lens capsule, the thick basement membrane that surrounds the lens. This wound area where the fiber cells were attached is located just adjacent to a continuous monolayer of lens epithelial cells that remains linked to the lens capsule during the surgical procedure. The wounded epithelium, the cell type from which fiber cells are derived during development, responds to the injury of fiber cell removal by moving collectively across the wound area, led by a population of vimentin-rich repair cells whose mesenchymal progenitors are endogenous to the lens1. These properties are typical of a normal epithelial wound healing response. In this model, as in vivo, wound repair is dependent on signals supplied by the endogenous environment that is uniquely maintained in this ex vivo culture system, providing an ideal opportunity for discovery of the mechanisms that regulate repair of an epithelium following wounding.

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

Described here is the establishment of a clinically relevant ex vivo mock cataract surgery model that can be used to investigate mechanisms of the injury response of epithelial tissues within their native microenvironment.

임상 적으로 구축<em&gt; 전의 VIVO</em기본 미세 환경에서 상피 상처 복구 조사를위한&gt; 모의 백내장 수술 모델

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to ...

연구

발생학

Live-Cell Imaging of Intact Ex Vivo Globes Using a Novel 3D Printed Holder

Corneal epithelial wound healing is a migratory process initiated by the activation of purinergic receptors expressed on epithelial cells. This activation results in calcium mobilization events that propagate from cell to cell, which are essential for initiating cellular motility into the wound bed, promoting efficient wound healing. The Trinkaus-Randall lab has developed a methodology for imaging the corneal wound healing response in ex vivo murine globes in real time. This approach involves enucleating an intact globe from a mouse that has been euthanized per established protocols and immediately incubating the globe with a calcium indicator dye. A counterstain that stains other features of the cell can be applied at this stage to assist with imaging and show cellular landmarks. The protocol worked well with several different live cell dyes used for counterstaining, including SiR actin to stain actin and deep red plasma membrane stain to stain the cell membrane. To examine the response to a wound, the corneal epithelium is injured using a 25 G needle, and the globes are placed in a 3D printed holder. The dimensions of the 3D printed holder are calibrated to ensure immobilization of the globe throughout the duration of the experiment and can be modified to accommodate eyes of different sizes. Live cell imaging of the wound response is performed continuously at various depths throughout the tissue over time using confocal microscopy. This protocol allows us to generate high-resolution, publication-quality images using a 20x air objective on a confocal microscope. Other objectives can also be used for this protocol. It represents a significant improvement in the quality of live cell imaging in ex vivo murine globes and permits the identification of nerves and epithelium.

Live-Cell Imaging of Intact Ex Vivo Globes Using a Novel 3D Printed Holder

The present work describes a novel experimental protocol that utilizes a 3D printed holder to enable high-resolution live cell imaging of enucleated globes. Through this protocol, the cellular calcium signaling activity in wounded corneal epithelium from ex vivo globes can be observed in real time.

새로운 3D 프린팅 홀더를 사용한 온전한 생체 외 구체의 라이브 셀 이미징

Corneal epithelial wound healing is a migratory process initiated by the activation of purinergic receptors expressed on epithelial cells. This activation ...

생화학

Investigating Scarless Tissue Regeneration in Embryonic Wounded Chick Corneas

Chick embryonic corneal wounds display a remarkable capacity to fully and rapidly regenerate, whereas adult wounded corneas experience a loss of transparency due to fibrotic scarring. The tissue integrity of injured embryonic corneas is intrinsically restored with no detectable scar formation. Given its accessibility and ease of manipulation, the chick embryo is an ideal model for studying scarless corneal wound repair. This protocol demonstrates the different steps involved in wounding the cornea of an embryonic chick in ovo. First, eggs are windowed at early embryonic ages to access the eye. Second, a series of in ovo physical manipulations to the extraembryonic membranes are conducted to ensure access to the eye is maintained through later stages of development, corresponding to when the three cellular layers of the cornea are formed. Third, linear cornea wounds that penetrate the outer epithelial layer and the anterior stroma are made using a microsurgical knife. The regeneration process or fully restored corneas can be analyzed for regenerative potential using various cellular and molecular techniques following the wounding procedure. Studies to date using this model have revealed that wounded embryonic corneas display activation of keratocyte differentiation, undergo coordinated remodeling of ECM proteins to their native three-dimensional macrostructure, and become adequately re-innervated by corneal sensory nerves. In the future, the potential impact of endogenous or exogenous factors on the regenerative process could be analyzed in healing corneas by using developmental biology techniques, such as tissue grafting, electroporation, retroviral infection, or bead implantation. The current strategy identifies the embryonic chick as a crucial experimental paradigm for elucidating the molecular and cellular factors coordinating scarless corneal wound healing.

The present protocol demonstrates the different steps involved in wounding the cornea of an embryonic chick in ovo. The regenerating or fully restored corneas can be analyzed for regenerative potential using various cellular and molecular techniques following the wounding procedure.&#160;

배아 상처 병아리 각막의 흉터없는 조직 재생 조사

Chick embryonic corneal wounds display a remarkable capacity to fully and rapidly regenerate, whereas adult wounded corneas experience a loss of ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

각 막에 대 한 모델로 눈 버와 각 막 상피 마모 상처 치유

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

의학

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.

A protocol for an ex vivo corneal organ culture model useful for wound healing studies is described. This model system can be used to assess the effects of agents to promote regenerative healing or drug toxicity in an organized 3D multicellular environment.

Ex Vivo 막 기관 문화 모델에 대 한 상처 치유 연구

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two ...

An Epithelial Abrasion Model for Studying Corneal Wound Healing

The cornea is critical for vision, accounting for about two-thirds of the refractive power of the eye. Crucial to the role of the cornea in vision is its transparency. However, due to its external position, the cornea is highly susceptible to a wide variety of injuries that can lead to the loss of corneal transparency and eventual blindness. Efficient corneal wound healing in response to these injuries is pivotal for maintaining corneal homeostasis and preservation of corneal transparency and refractive capabilities. In events of compromised corneal wound healing, the cornea becomes vulnerable to infections, ulcerations, and scarring. Given the fundamental importance of corneal wound healing to the preservation of corneal transparency and vision, a better understanding of the normal corneal wound healing process is a prerequisite to understanding impaired corneal wound healing associated with infection and disease. Toward this goal, murine models of corneal wounding have proven useful in furthering our understanding of the corneal wound healing mechanisms operating under normal physiological conditions. Here, a protocol for creating a central corneal epithelial abrasion in mouse using a trephine and a blunt golf club spud is described. In this model, a 2 mm diameter circular trephine, centered over the cornea, is used to demarcate the wound area. The golf club spud is used with care to debride the epithelium and create a circular wound without damaging the corneal epithelial basement membrane. The resulting inflammatory response proceeds as a well-characterized cascade of cellular and molecular events that are critical for efficient wound healing. This simple corneal wound healing model is highly reproducible and well-published and is now being used to evaluate compromised corneal wound healing in the context of disease.

Here, a protocol for creating a central corneal epithelial abrasion wound in the mouse using a trephine and a blunt golf club spud is described. This corneal wound healing model is highly reproducible and is now being used to evaluate compromised corneal wound healing in the context of diseases.

각막 상처 치유를 연구하기위한 상피 마모 모델

The cornea is critical for vision, accounting for about two-thirds of the refractive power of the eye. Crucial to the role of the cornea in vision is its ...

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, and result in a large, persistent corneal epithelial defect. Epithelial defect with the following corneal opacity and peripheral neovascularization result in irreversible visual impairment and hinder future management, especially keratoplasty. Since the animal model can be used as an effective drug development platform, models of corneal injury to the mouse and alkali burn to rabbit corneal epithelium are developed here. New Zealand white rabbit is used in the alkali burn model. Different concentrations of sodium hydroxide can be applied onto the central circular area of the cornea for 30 s under intramuscular and topical anesthesia. After copious isotonic normal saline irrigation, residual loose corneal epithelium was removed with corneal burr deep down to the Bowman's layer within this circular area. Wound healing was documented by fluorescein staining under Cobalt blue light. C57BL/6 mice were used in the traumatic model of murine corneal epithelium. The murine central cornea was marked using a skin punch, 2 mm in diameter, and then debrided by a corneal rust ring remover with a 0.5 mm burr under a stereomicroscope. These models can be prospectively used to validate the therapeutic effect of eye drops or mixed agents such as stem cells, which potentially facilitate corneal epithelial regeneration. By observing corneal opacity, peripheral neovascularization, and conjunctival congestion with stereomicroscope and imaging software, therapeutic effects in these animal models can be monitored.

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Here, animal models based on mouse and rabbit are developed for mechanical and chemical injury of corneal epithelium to screen new therapeutics and the underlying mechanism.

각막 상피의 기계적 및 화학적 손상에 대한 Ex Vivo 및 In Vivo 동물 모델

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, ...

High Throughput SiRNA Screening for Chloropicrin and Hydrogen Fluoride-Induced Cornea Epithelial Cell Injury

Toxicant-induced ocular injury is a true ocular emergency because chemicals have the potential to rapidly inflict significant tissue damage. Treatments for toxicant-induced corneal injury are generally supportive as no specific therapeutics exist to treat these injuries. In the efforts to develop treatments and therapeutics to care for exposure, it can be important to understand the molecular and cellular mechanisms of these injuries. We propose that utilization of high throughput small inhibitory RNA (siRNA) screening can be an important tool that could help to more rapidly elucidate the molecular mechanisms of chemical cornea epithelial injury. siRNA are double stranded RNA molecules that are 19-25 nucleotides long and utilize the post-transcriptional gene silencing pathway to degrade mRNA which have homology to the siRNA. The resulting reduction of expression of the specific gene can then be studied in toxicant exposed cells to ascertain the function of that gene in the cellular response to the toxicant. The development and validation of in vitro exposure models and methods for the high throughput screening (HTS) of hydrogen fluoride- (HF) and chloropicrin- (CP) induced ocular injury are presented in this article. Although we selected these two toxicants, our methods are applicable to the study of other toxicants with minor modifications to the toxicant exposure protocol. The SV40 large T antigen immortalized human corneal epithelial cell line SV40-HCEC was selected for study. Cell viability and IL-8 production were selected as endpoints in the screening protocol. Several challenges associated with the development of toxicant exposure and cell culture methods suitable for HTS studies are presented. The establishment of HTS models for these toxicants allows for further studies to better understand the mechanism of injury and to screen for potential therapeutics for chemical ocular injury.

High throughput small inhibitory RNA screening is an important tool that could help to more rapidly elucidate the molecular mechanisms of chemical cornea epithelial injury. Herein, we present the development and validation of exposure models and methods for the high throughput screening of hydrogen fluoride- and chloropicrin-induced cornea epithelial injury.

Chloropicrin 및 수소 불 화물 유발 각 막 상피 세포 상해에 대 한 높은 처리량 SiRNA 심사

Toxicant-induced ocular injury is a true ocular emergency because chemicals have the potential to rapidly inflict significant tissue damage. Treatments ...

생물학

Zebrafish Corneal Wound Healing: From Abrasion to Wound Closure Imaging Analysis

As the transparent surface of the eye, the cornea is instrumental for clear sight. Due to its location, this tissue is prone to environmental insults. Indeed, the eye injuries most frequently encountered clinically are those to the cornea. While corneal wound healing has been extensively studied in small mammals (i.e., mice, rats, and rabbits), corneal physiology studies have neglected other species, including zebrafish, despite zebrafish being a classic research model.
This report describes a method of performing a corneal abrasion on zebrafish. The wound is performed in vivo on anesthetized fish using an ocular burr. This method allows for a reproducible epithelial wound, leaving the rest of the eye intact. After abrasion, wound closure is monitored over the course of 3 h, after which the wound is reepithelialized. By using scanning electron microscopy, followed by image processing, the epithelial cell shape, and apical protrusions can be investigated to study the various steps during corneal epithelial wound closure.
The characteristics of the zebrafish model permit study of the epithelial tissue physiology and the collective behavior of the epithelial cells when the tissue is challenged. Furthermore, the use of a model deprived of the influence of the tear film can produce&#160;new answers regarding corneal response to stress. Finally, this model also allows the delineation of the cellular and molecular events involved in any epithelial tissue subjected to a physical wound. This method can be applied to the evaluation of drug effectiveness in preclinical testing.

This protocol focuses on damaging the ocular surface of zebrafish through abrasion to assess the subsequent wound closure at the cellular level. This approach exploits an ocular burr to partly remove the corneal epithelium and uses scanning electron microscopy to track changes in cell morphology during wound closure.

제브라피쉬 각막 상처 치유: 마모에서 상처 폐쇄 영상 분석까지

As the transparent surface of the eye, the cornea is instrumental for clear sight. Due to its location, this tissue is prone to environmental insults. ...

Establishing a Severe Corneal Inflammation Model in Rats Based on Corneal Epithelium Curettage Combined with Corneal Sutures

Corneal inflammation, especially severe corneal inflammation, plays a significant role in the development of corneal limbal stem cell dysfunction. Constructing appropriate animal models can help us focus on the effects of severe inflammation on corneal limbal stem cells. A 2 mm rust remover&#160;was&#160;used to remove the central corneal epithelium of Sprague Dawley (SD) rats to create an injury. Then, the central stroma of the cornea was sutured with nylon sutures to induce persistent inflammation. In this way, a corneal inflammation model with central corneal epithelium abrasion and central stroma suturing was constructed, which induced severe corneal inflammation. The changes in corneal inflammation and the condition of the limbal stem cells at 1, 3, and 7 days post-modeling were observed. On the 3rd day after modeling, the rats&#39; corneal limbus was severely edematous, with obvious neovascularization and local hyperplasia, which are typical signs of limbal stem cell deficiency. By the 7th day, the corneal edema gradually worsened, and the neovascularization continued to increase. Through quantitative reverse transcription polymerase chain reaction (RT-qPCR) and immunofluorescence staining, we found that the corneal epithelial inflammatory factors were significantly upregulated, the corneal epithelial differentiation was abnormal, the corneal epithelial stem cells were significantly reduced, and the cell proliferation and stemness had also decreased. Therefore, this model demonstrates that severe inflammation can induce limbal stem cell damage without directly damaging the limbal stem cells. The model is beneficial for observing the effects of severe inflammation on the biological mechanisms of stem cells and provides an ideal platform for studying the mechanisms of corneal epithelial stem cell dysfunction induced by inflammation.

We developed a rat model of severe corneal inflammation through corneal epithelium curettage combined with corneal sutures. The study evaluated corneal inflammation patterns, epithelial proliferation, and changes in limbal stem cells under inflammatory conditions.

각막 상피 창상 치유 기간 동안 열 충격 단백질 27의 기능의 RNA 간섭 기반 조사

요약

더 많은 비디오 탐색

이 비디오의 챕터

임상 적으로 구축

새로운 3D 프린팅 홀더를 사용한 온전한 생체 외 구체의 라이브 셀 이미징

배아 상처 병아리 각막의 흉터없는 조직 재생 조사

각 막에 대 한 모델로 눈 버와 각 막 상피 마모 상처 치유

Ex Vivo 막 기관 문화 모델에 대 한 상처 치유 연구

각막 상처 치유를 연구하기위한 상피 마모 모델

각막 상피의 기계적 및 화학적 손상에 대한 Ex Vivo 및 In Vivo 동물 모델

Chloropicrin 및 수소 불 화물 유발 각 막 상피 세포 상해에 대 한 높은 처리량 SiRNA 심사

제브라피쉬 각막 상처 치유: 마모에서 상처 폐쇄 영상 분석까지

Establishing a Severe Corneal Inflammation Model in Rats Based on Corneal Epithelium Curettage Combined with Corneal Sutures

각막 상피 ​​창상 치유 기간 동안 열 충격 단백질 27의 기능의 RNA 간섭 기반 조사

요약

더 많은 비디오 탐색

이 비디오의 챕터

임상 적으로 구축

새로운 3D 프린팅 홀더를 사용한 온전한 생체 외 구체의 라이브 셀 이미징

배아 상처 병아리 각막의 흉터없는 조직 재생 조사

각 막에 대 한 모델로 눈 버와 각 막 상피 마모 상처 치유

Ex Vivo 막 기관 문화 모델에 대 한 상처 치유 연구

각막 상처 치유를 연구하기위한 상피 마모 모델

각막 상피의 기계적 및 화학적 손상에 대한 Ex Vivo 및 In Vivo 동물 모델

Chloropicrin 및 수소 불 화물 유발 각 막 상피 세포 상해에 대 한 높은 처리량 SiRNA 심사

제브라피쉬 각막 상처 치유: 마모에서 상처 폐쇄 영상 분석까지

Establishing a Severe Corneal Inflammation Model in Rats Based on Corneal Epithelium Curettage Combined with Corneal Sutures

각막 상피 창상 치유 기간 동안 열 충격 단백질 27의 기능의 RNA 간섭 기반 조사