Name: rna interference-based investigation of the function of heat shock protein 27 during corneal epithelial wound healing
Uploaded: 2016-09-27T12:30:00
Description: 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

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?...

<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>

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。

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			<td height="38" style="height:38px;width:212px;">Biological safety cabinet</td>
			<td style="width:247px;">CHC LAB Co.Ltd,&#160; Daejeon, Republic of Korea&#160;</td>
			<td style="width:109px;">CHC-777A2-06</td>
			<td style="width:199px;">Class II, Type A2&#160;</td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;width:212px;">Stealth RNAi&#8482; siRNA</td>
			<td style="width:247px;">Thermo Fisher Scientific, Inc., Waltham, MA</td>
			<td style="width:109px;"></td>
			<td style="width:199px;">RNAi siRNA; scrambled control-siRNA and HSP27-specific siRNA</td>
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		<tr height="38">
			<td height="38" style="height:38px;width:212px;">BEGMTM</td>
			<td style="width:247px;">Lonza, Inc., Walkersville, MD</td>
			<td style="width:109px;">CC-3171, CC4175</td>
			<td style="width:199px;">Bronchial epithelium growth medium&#160;</td>
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			<td height="38" style="height:38px;width:212px;">Protease inhibitor&#160;</td>
			<td style="width:247px;">Sigma-Aldrich, Inc., St. Louis, MO</td>
			<td style="width:109px;">P8340 ,P7626</td>
			<td style="width:199px;">1 uM Pepstatin A, 1 uM Leupetin, 0.1 uM Aprotin</td>
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			<td height="20" style="height:20px;width:212px;">Bradford protein assay&#160;</td>
			<td style="width:247px;">Bio-Rad Laboratories, Hercules, CA</td>
			<td style="width:109px;">#500-0001</td>
			<td style="width:199px;">Bradford protein assay&#160;</td>
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			<td height="20" style="height:20px;width:212px;">Nitrocellulose filters&#160;</td>
			<td style="width:247px;">Amersham, Little Chalfont, UK</td>
			<td style="width:109px;">RPN3032D</td>
			<td style="width:199px;">Western blotting membrane</td>
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			<td height="38" style="height:38px;width:212px;">Non-phosphorylated HSP27&#160;</td>
			<td style="width:247px;">Abcam Inc., Cambridge, MA</td>
			<td style="width:109px;">ab12351</td>
			<td style="width:199px;">1:1000 dilution (Total HSP27)</td>
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			<td height="38" style="height:38px;width:212px;">Phosphorylated HSP27 (Ser85)</td>
			<td style="width:247px;">Abcam Inc., Cambridge, MA</td>
			<td style="width:109px;">ab5594</td>
			<td style="width:199px;">1: 1000 dilution HSP27 was phosphorylated at Ser85</td>
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			<td height="42" style="height:42px;width:212px;">Lipofectamine&#174; RNAiMAX reagent&#160;</td>
			<td style="width:247px;">Invitrogen, Carlsbad, CA</td>
			<td style="width:109px;">13-778-075</td>
			<td style="width:199px;">Transfection reagent</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:212px;">Phosphorylated Akt (Ser473)</td>
			<td style="width:247px;">Cell Signaling Technology, Danvers, MA</td>
			<td style="width:109px;">No. 4060</td>
			<td style="width:199px;">1: 1000 dilution Akt was phosphorylated at Ser473 (cell survival marker)</td>
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			<td height="38" style="height:38px;width:212px;">Non-phosphorylated Akt&#160;</td>
			<td style="width:247px;">Cell Signaling Technology, Danvers, MA</td>
			<td style="width:109px;">No. 4061</td>
			<td style="width:199px;">1:1000 dilution (Total Akt)</td>
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			<td height="38" style="height:38px;width:212px;">Bcl-2-associated X protein&#160;</td>
			<td style="width:247px;">Cell Signaling Technology, Danvers, MA</td>
			<td style="width:109px;">No. 4062</td>
			<td style="width:199px;">1: 1000 (anti-apoptotic protein marker)</td>
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			<td height="57" style="height:57px;width:212px;">GAPDH</td>
			<td style="width:247px;">Santa Cruz Biotechnology, Santa Cruz, CA</td>
			<td style="width:109px;">No. 4063</td>
			<td style="width:199px;">1:1000 loading control&#160; marker (house keeping gene)</td>
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			<td height="43" style="height:43px;width:212px;">Horseradish peroxidase-conjugated goat anti-rabbit antibodies</td>
			<td style="width:247px;">Thermo Fisher Scientific, Inc., Waltham, MA</td>
			<td style="width:109px;">NCI1460KR</td>
			<td style="width:199px;">1:10000 dilution</td>
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			<td height="38" style="height:38px;width:212px;">OPTI-MEM</td>
			<td style="width:247px;">Invitrogen, Carlsbad, CA</td>
			<td style="width:109px;">31985</td>
			<td style="width:199px;">reduced serum medium for transfection</td>
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			<td height="40" style="height:40px;width:212px;">Image analysis software</td>
			<td style="width:247px;">Olympus, Inc., Tokyo, Japan</td>
			<td style="width:109px;">Image-Pro Plus 5.0</td>
			<td style="width:199px;"></td>
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			<td height="44" style="height:44px;width:212px;">Skimed milk powder&#160;</td>
			<td style="width:247px;">Carl Roth GmbH + Co. KG, Karlstruhe, Germany</td>
			<td style="width:109px;">T145.2</td>
			<td style="width:199px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:212px;">Tris&#160;</td>
			<td style="width:247px;">Amresco LCC, Inc. Solon, OH</td>
			<td style="width:109px;">No-0497</td>
			<td style="width:199px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:212px;">Sodium Chloride&#160;</td>
			<td style="width:247px;">Amresco LCC, Inc. Solon, OH</td>
			<td style="width:109px;">No-0241</td>
			<td style="width:199px;"></td>
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		<tr height="38">
			<td height="38" style="height:38px;width:212px;">Six well culture plate</td>
			<td style="width:247px;">Thermo Fisher Scientific, Inc., Waltham, MA</td>
			<td style="width:109px;">140675</td>
			<td style="width:199px;">35.00 mm diameter / well</td>
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		<tr height="38">
			<td height="38" style="height:38px;width:212px;">24-well culuture dish</td>
			<td style="width:247px;">Thermo Fisher Scientific, Inc., Waltham, MA</td>
			<td style="width:109px;">142475</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:212px;">Orbital shaker</td>
			<td style="width:247px;">N-Bioteck, Inc., Seoul, South Korea</td>
			<td style="width:109px;">NB1015</td>
			<td style="width:199px;"></td>
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		<tr height="38">
			<td height="38" style="height:38px;width:212px;">Bovine serum albumin</td>
			<td style="width:247px;">Santa Cruz Biotechnology, Santa Cruz, CA</td>
			<td style="width:109px;">sc-2323&#160;</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:212px;">BDFACSCantoTM II</td>
			<td style="width:247px;">BD Biosciences, Franklin Lakes, NJ</td>
			<td style="width:109px;"></td>
			<td style="width:199px;">Flow cytometry</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:212px;">X-Ray Film</td>
			<td style="width:247px;">Kodak, Rochester, NY</td>
			<td style="width:109px;"></td>
			<td style="width:199px;">Medical X-Ray Cassette with Green 400 Screen&#160;</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:212px;">western blotting luminol reagent</td>
			<td style="width:247px;">Santa Cruz Biotechnology, Santa Cruz, CA</td>
			<td style="width:109px;">sc-2048&#160;</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:212px;">FITC Annexin V Apoptosis Detection Kit I</td>
			<td style="width:247px;">BD Biosciences, Franklin Lakes, NJ</td>
			<td style="width:109px;">556547</td>
			<td style="width:199px;"></td>
		</tr>
	</tbody>
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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 ...

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