这项研究是由医学，韩国首尔，蔚山大学学院的学生研究资助（13-14），并从峨山生命科学研究所，韩国首尔赠款（2014-464）的支持。

1.细胞系<ol><li>培养10 6端粒酶永生化人角膜上皮在一个6孔板的细胞（HCECs）:在37℃培养箱中使用支气管上皮细胞生长培养基（BEGM）于5％CO 2气氛（密度1039.9细胞/毫米2），直到他们达到95％汇合。 </li></ol> 2. Western印迹分析创建上皮擦伤伤口后， <ol><li>横跨汇合培养HCECs的阱的表面上的条纹无菌200μl的枪头每个培养皿四次在生物安全柜（II级，A2型）和在37℃培养箱用5％ 的 CO 2气氛中分别孵育HCECs 1，5，10，30，60，和120分钟。 </li><li>根据角膜上皮创伤后的孵育时间用一个6孔板为六个不同的样品。 </li><li>洗负伤HCEC单层三次用1×PBS，然后加入2.0毫升的BEGM到每个孔中。 </li><li>分离使用2 HCECs毫升0.25％胰蛋白酶 - 乙二胺四乙酸（EDTA），每孔5分钟，离心，在900×g离心在一个15毫升管5分钟，并吸出胰蛋白酶-EDTA使用1毫升吸移管。 </li><li>暂停HCECs用1ml 1×PBS中，并将它们传送到1.5ml管中。 </li><li>以10,000×g离心15秒离心机HCECs和吸出1×PBS中使用1毫升吸管。 </li><li>悬浮HCECs在100μl冰冷的裂解缓冲液（10mM的Tris，10 mM氯化钠，2mM EDTA的，25毫氟化钠，2毫的Na 3 VO 4，1mM的苯甲基磺酰氟[PMSF]，蛋白酶抑制剂[1μM的胃酶抑素A， 1μM的亮肽素，和0.1μM的抑肽酶]和0.5％的Triton X-100 [pH为7]），并将它们混合均匀。 </li><li>孵育细胞30分钟，在冰上以诱导细胞裂解。 </li><li>离心裂解物在10,000×g离心15分钟，4℃，然后上清液转移至新的1.5毫升管（90微升等份），并将它们存储在-80℃。 </li><li>确定使用Bradford PR细胞裂解物的总蛋白浓度otein检测。9 </li><li>负载30微克总细胞蛋白质成10％或12％丙烯酰胺凝胶进行十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS-PAGE），然后电泳转移分离蛋白带向硝酸纤维素滤膜用200毫安的电流1小时，在4℃ C到免疫印迹试验使用。 </li><li>块硝酸纤维素滤膜，用5％脱脂在Tris缓冲盐水乳用吐温20（TBST）1小时后，添加初级兔多克隆抗体对非磷酸化的HSP27（1:1000稀释）或抗磷酸HSP27初级兔多克隆抗体（1 :1,000稀释度），在5％牛血清白蛋白（BSA），并在4℃在摇床上过夜孵育该膜。 </li><li>用TBST洗涤后5％BSA的10分钟3次:检测用辣根过氧化物酶缀合的山羊抗 - 兔抗体免疫反应条带（10,000稀释1）。 </li><li>在免疫印迹鲁米诺试剂孵化膜（6-7米升每10cm×5厘米膜），在室温下1分钟。 </li><li>移除试剂溶液的膜，除去多余的液体，具有吸收巾，并将其放置在一个塑料片保护。 </li><li>在黑暗的房间提供一个安全的工作灯，将覆盖膜在胶卷暗盒与蛋白质的一面朝上。 </li><li>放置在膜顶部X射线胶片和暴露1分钟。 </li></ol> 3. siRNA转染分析10 <ol><li>在5×10 5细胞培养HCECs /孔在37℃孵化用BEGM直到他们达到95％汇合于5％CO 2气氛中的6孔板中。 </li><li>稀释转染试剂（2.5或7.5微升）与转（稀释倍数为41或14.3）100微升减少血清中和溶解HSP27特异性的，并在100微升炒对照siRNA降低血清中创建的10或50纳米HSP27特异性和炒对照siRNA。 </li><li>混合100μ;用100μl稀释转染试剂升siRNA溶液（1:1的比例），并孵育混合物在室温下15分钟。 </li><li>所述siRNA脂质复合物添加到细胞中。然后经过4小时改变介质在37℃下完成BEGM孵育细胞2天。 </li><li>通过Western印迹分析转染细胞从4.1节所述4.7。 </li></ol> 4. Western blot检测用于siRNA转染细胞11 <ol><li>提取HSP27特异性和使用100μl的冰冷的裂解缓冲液（10mM的Tris，10 mM氯化钠，2mM的乙二胺四乙酸（EDTA），25mM的氟化钠，2毫的Na 3 VO加扰对照siRNA转染HCECs在生物安全柜4，1mM的苯甲基磺酰氟（PMSF），蛋白酶抑制剂，和0.5％的Triton X-100，pH 7）中。 </li><li>孵育细胞30分钟，在冰上以诱导细胞裂解。 </li><li>以10,000 xg离心沉淀裂解物15分钟，然后上清转移至新的1.5mL桶ES（90微升等份），并将它们存储在-80℃。 </li><li>确定使用Bradford蛋白测定的细胞裂解物的蛋白浓度。9 </li><li>与在10％或12％丙烯酰胺凝胶等量总细胞蛋白的负荷样品，若使凝胶进行SDS-PAGE，并电泳以200毫安的电流，在4℃下传送所分离的蛋白带向硝酸纤维素滤膜1小时至在免疫印迹试验使用。 </li><li>块硝酸纤维素滤膜，用5％脱脂在Tris缓冲盐水乳用吐温20（TBST）1小时后，添加抗磷酸和未磷酸化HSP27初级抗体（1:1000稀释度），磷酸化Akt（1:1,000稀释度），非磷酸化的Akt（1:1000稀释，用作细胞存活标记物），Bcl-2的相关X蛋白（1:1000稀释，用作促凋亡蛋白），和甘油醛-3-磷酸脱氢酶（GAPDH; 1 1:200稀释，用作上样对照）的5％牛血清白蛋白（BSA），并过夜孵育所述膜于4℃在振荡器上。 </li><li>检测用辣根过氧化物酶缀合的山羊抗 - 兔抗体免疫反应条带（1:10,000稀释），在5％的BSA洗涤后用TBST 3次，每次10分钟洗涤。 </li><li>在室温下1分钟孵育的印迹鲁米诺试剂膜（6-7毫升每10cm×5厘米膜）。 </li><li>移除试剂溶液的膜，除去多余的液体，具有吸收巾，并将其放置在一个塑料片保护。 </li><li>在黑暗的房间提供一个安全的工作灯，将覆盖膜与蛋白质面朝上胶片盒。 </li><li>放置在膜的顶部X射线胶片和暴露1分钟。 </li></ol> 5.划痕引起的定向伤人分析评估细胞迁移12 <ol><li>在生物安全柜，通过在表面上拖动的汇合培养物的孔中的一个无菌枪头使伤口HSP27特异性siRNA转染或炒对照siRNA转染HCECs。 </li><li>伤人后立即，洗细胞两次用1×磷酸盐缓冲盐水（PBS），并保持它们在BEGM培养在37℃培养箱用5％ 的 CO 2气氛中24小时伤人后。 </li><li>照片使用正置显微镜放大100倍的图像HCEC伤后24小时，进行使用过滤背景压扁 在图像分析软件命令。 </li><li>使用选择测量命令，确定利息（AOI）与同尺寸的多边形形状，可以从一端垂直覆盖，结束初期伤口的区域，并在每个样品的受伤面积确定三个不同的AOI。 </li><li>自动计数使用计算/尺寸测量菜单选项在各个领域的手机号码。 </li></ol>凋亡6.流式细胞仪分析<ol><li>文化HSP27特异性siRNA转染和CONTROL S的iRNA转染的含有各10nM的siRNA的HCECs在/孔在6孔板5×10 5个细胞的浓度，直到细胞达到在37℃培养箱中，在BEGM一个5％CO 2气氛中95％汇合。 </li><li>使用1毫升吸管分离使用每孔2mL 0.25％胰蛋白酶-EDTA HCECs 5分钟，离心，在900×g离心在一个15毫升管5分钟，并吸出胰蛋白酶-EDTA。 </li><li>用冷PBS洗涤细胞两次，然后以10 6个细胞/ ml的浓度重悬在1×细胞结合缓冲液（0.1M HEPES /氢氧化钠[pH为7.4]，1.4 M氯化钠，和25mM 氯化钙 ）。 </li><li>转移100μl的细胞悬浮液（1×10 5个细胞）到5ml的培养管中。 </li><li>添加5μl的异硫氰酸荧光素缀合的膜联蛋白V和5μl碘化丙啶。 </li><li>轻轻涡旋细胞，并培育他们在黑暗中室温下15分钟。 </li><li>通过流动Ç加入200μl1X结合缓冲液中向每个管和分析细胞1小时内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>

笔者在本研究中提及的任何材料或方法没有财务或专有权益。

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...

角膜上皮细胞（CEC上）不断脱落到泪膜，而他们同时由角膜缘和角膜上皮基底层细胞所替代。1各种外在压力能促使中欧国家的凋亡和脱屑。2热休克蛋白（HSP）是高度保守的，并且可以根据分子大小被分为两个家族。3最大HSP家族包括HSP90，HSP70和HSP60，而较小的家族包括HSP27 4 HSP27的磷酸化是已知在细胞存活中起重要作用和所需的细胞迁移，因为这种蛋白在肌动蛋白重塑的作用。5-7。因此，我们试图测试上皮伤口愈合的体外模型HSP27磷酸化在CEC迁移和凋亡的潜在作用。 RNA干扰或者使用小或短干扰RNA（RNAi）技术（siRNA）的有GE在基础和应用生物学nerated兴趣，因为它可能允许击倒任何感兴趣的基因的表达。8在此，我们使用的特定HSP27-siRNA转以评估HSP27的给CEC伤口愈合和细胞凋亡的贡献。传统的RNAi方法在细胞中的基因敲除使用合成的RNA双链体，包括可组装到创建的siRNAs 2未修饰21聚体寡核苷酸。所述RNAi的siRNA，我们在此本研究中使用的是转染细胞的简单高效的方法，以及该试剂可与各种永生化细胞系。在此研究中，我们证明用于该分析的方法，包括一个刮诱导定向伤口测定法，western印迹，siRNA转染测定法，免疫荧光测定法，和流式细胞术。

<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.

在5，10磷酸化HSP27的表达显著上升，划痕后30分钟，未受伤的HCECs 13伤人比较。免疫印迹分析表明，磷酸HSP27和磷酸化Akt的表达均被显著降低，而Bax的表达在特定HSP27-siRNA转染HCECs（ 图1A-E）中的显著增加。减少了30％，在10nM的40％和HSP27特异性为50nM的siRNA转染的细胞，分别与对照siRNA转染的细胞，但磷酸化HSP27表达相比没有减少（ 图1A-B中的磷?...

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.

角膜上皮伤口愈合过程中的热休克蛋白27的功能为基础的RNA干扰研究

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 次观看.  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.

视频: 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.

建立临床相关<em&gt;离体</em&gt;调查上皮创伤修复的微环境本地人模拟白内障手术模型

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

科研

发育生物学

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.

使用新型3D打印支架对完整的 离体 地球仪进行活细胞成像

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

生物化学

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;

研究胚胎损伤雏鸡角膜的无疤痕组织再生

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.

眼毛刺角膜上皮磨损与角膜创面愈合的模型研究

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 ...

医学

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.

创面愈合研究的前活体角膜器官培养模型

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.

研究角膜伤口愈合的上皮擦伤模型

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.

角膜上皮机械和化学损伤的离体和体内动物模型

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.

高通量 SiRNA 筛选氯化苦和氟化氢引起的角膜上皮细胞损伤

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

生物学

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.

斑马鱼角膜伤口愈合:从磨损到伤口闭合成像分析

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.

角膜上皮伤口愈合过程中的热休克蛋白27的功能为基础的RNA干扰研究

摘要

探索更多视频

此视频中的章节

建立临床相关

使用新型3D打印支架对完整的离体地球仪进行活细胞成像

研究胚胎损伤雏鸡角膜的无疤痕组织再生

眼毛刺角膜上皮磨损与角膜创面愈合的模型研究

创面愈合研究的前活体角膜器官培养模型

研究角膜伤口愈合的上皮擦伤模型

角膜上皮机械和化学损伤的离体和体内动物模型

高通量 SiRNA 筛选氯化苦和氟化氢引起的角膜上皮细胞损伤

斑马鱼角膜伤口愈合:从磨损到伤口闭合成像分析

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

角膜上皮伤口愈合过程中的热休克蛋白27的功能为基础的RNA干扰研究

摘要

探索更多视频

此视频中的章节

建立临床相关

使用新型3D打印支架对完整的 离体 地球仪进行活细胞成像

研究胚胎损伤雏鸡角膜的无疤痕组织再生

眼毛刺角膜上皮磨损与角膜创面愈合的模型研究

创面愈合研究的前活体角膜器官培养模型

研究角膜伤口愈合的上皮擦伤模型

角膜上皮机械和化学损伤的离体和体内动物模型

高通量 SiRNA 筛选氯化苦和氟化氢引起的角膜上皮细胞损伤

斑马鱼角膜伤口愈合:从磨损到伤口闭合成像分析

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

使用新型3D打印支架对完整的离体地球仪进行活细胞成像