Name: a protocol to acquire the degenerative tenocyte from humans
Uploaded: 2018-06-09T14:30:00
Description: Watch this Scientific Journal Video about A Protocol to Acquire the Degenerative Tenocyte from Humans at JoVE.com

这项研究得到了韩国卫生技术研发 & 发展研究所 (KHIDI) 的资助, 该项目由大韩民国卫生部 & 福利部提供资金 (赠款号: HI16C1559)。

该议定书是根据《赫尔辛基宣言》进行的, 《议定书》是在茶 Bundang 医疗中心的机构审查委员会批准的。
1. 退行性肌腱组织从患者中收获
<ol><li>诊断侧髁的病史和使用体检结果: 压痛在腱的起源接近侧髁;疼痛通过腕关节的伸展而诱发。</li>
	<li>使用以下纳入标准:18 岁以上;侧髁的历史至少六月;非手术治疗顽固;自注射皮质类固醇、富含血小板血浆或肉毒杆菌毒素以来, 至少有?...

<ol>
	<li>Ackermann, P. W., Renstrom, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tendinopathy+in+sport.">Tendinopathy in sport.</a> Sports Health. 4 (3), 193-201 (2012).</li><li>Maffulli, N., Wong, J., Almekinders, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Types+and+epidemiology+of+tendinopathy.">Types and epidemiology of tendinopathy.</a> Clinical Sports Medicine. 22 (4), 675-692 (2003).</li><li>Lui, P. P., Chan, L. S., Fu, S. C., Chan, K. M. <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+sensory+neuropeptides+in+tendon+is+associated+with+failed+healing+and+activity-related+tendon+pain+in+collagenase-induced+tendon+injury.">Expression of sensory neuropeptides in tendon is associated with failed healing and activity-related tendon pain in collagenase-induced tendon injury.</a> American Journal of Sports Medicine. 38 (4), 757-764 (2010).</li><li>Han, S. H., 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=Effects+of+corticosteroid+on+the+expressions+of+neuropeptide+and+cytokine+mRNA+and+on+tenocyte+viability+in+lateral+epicondylitis.">Effects of corticosteroid on the expressions of neuropeptide and cytokine mRNA and on tenocyte viability in lateral epicondylitis.</a> Journal of Inflammation (London). 9 (1), 40 (2012).</li><li>Uchio, Y., 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+neuropeptides+and+cytokines+at+the+extensor+carpi+radialis+brevis+muscle+origin.">Expression of neuropeptides and cytokines at the extensor carpi radialis brevis muscle origin.</a> Journal of Shoulder and Elbow Surgery. 11 (6), 570-575 (2002).</li><li>Lieber, R. L., Loren, G. J., Friden, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+measurement+of+human+wrist+extensor+muscle+sarcomere+length+changes.">In vivo measurement of human wrist extensor muscle sarcomere length changes.</a> Journal of Neurophysiology. 71 (3), 874-881 (1994).</li><li>Regan, W., Wold, L. E., Coonrad, R., Morrey, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscopic+histopathology+of+chronic+refractory+lateral+epicondylitis.">Microscopic histopathology of chronic refractory lateral epicondylitis.</a> American Journal of Sports Medicine. 20 (6), 746-749 (1992).</li><li>Sharma, P., Maffulli, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tendon+injury+and+tendinopathy:+healing+and+repair.">Tendon injury and tendinopathy: healing and repair.</a> Journal of Bone and Joint Surgery American volume. 87 (1), 187-202 (2005).</li><li>Lui, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+technology+for+tendon+regeneration:+current+status,+challenges,+and+future+research+directions.">Stem cell technology for tendon regeneration: current status, challenges, and future research directions.</a> Stem Cells Cloning. 8, 163-174 (2015).</li><li>Dahlgren, L. A., van der Meulen, M. C., Bertram, J. E., Starrak, G. S., Nixon, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin-like+growth+factor-I+improves+cellular+and+molecular+aspects+of+healing+in+a+collagenase-induced+model+of+flexor+tendinitis.">Insulin-like growth factor-I improves cellular and molecular aspects of healing in a collagenase-induced model of flexor tendinitis.</a> Journal of Orthopedic Research. 20 (5), 910-919 (2002).</li><li>Schnabel, L. V., 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=Mesenchymal+stem+cells+and+insulin-like+growth+factor-I+gene-enhanced+mesenchymal+stem+cells+improve+structural+aspects+of+healing+in+equine+flexor+digitorum+superficialis+tendons.">Mesenchymal stem cells and insulin-like growth factor-I gene-enhanced mesenchymal stem cells improve structural aspects of healing in equine flexor digitorum superficialis tendons.</a> Journal of Orthopedic Research. 27 (10), 1392-1398 (2009).</li><li>Durgam, S. S., Stewart, A. A., Sivaguru, M., Wagoner Johnson, A. J., Stewart, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tendon-derived+progenitor+cells+improve+healing+of+collagenase-induced+flexor+tendinitis.">Tendon-derived progenitor cells improve healing of collagenase-induced flexor tendinitis.</a> Journal of Orthopedic Research. 34 (12), 2162-2171 (2016).</li><li>Titan, A., Andarawis-Puri, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tendinopathy:+Investigating+the+Intersection+of+Clinical+and+Animal+Research+to+Identify+Progress+and+Hurdles+in+the+Field.">Tendinopathy: Investigating the Intersection of Clinical and Animal Research to Identify Progress and Hurdles in the Field.</a> Journal of Bone and Joint Surgery Review. 4 (10), (2016).</li><li>Dirks, R. C., Warden, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+for+the+study+of+tendinopathy.">Models for the study of tendinopathy.</a> Journal of Musculoskeletal Neuronal Interaction. 11 (2), 141-149 (2011).</li><li>de Mos, M., 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=Can+platelet-rich+plasma+enhance+tendon+repair?+A+cell+culture+study.">Can platelet-rich plasma enhance tendon repair? A cell culture study.</a> American Journal of Sports Medicine. 36 (6), 1171-1178 (2008).</li><li>Scherb, M. B., Han, S. H., Courneya, J. P., Guyton, G. P., Schon, L. C. <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+bupivacaine+on+cultured+tenocytes.">Effect of bupivacaine on cultured tenocytes.</a> Orthopedics. 32 (1), 26 (2009).</li><li>Wong, M. 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=Effect+of+dexamethasone+on+cultured+human+tenocytes+and+its+reversibility+by+platelet-derived+growth+factor.">Effect of dexamethasone on cultured human tenocytes and its reversibility by platelet-derived growth factor.</a> Journal of Bone and Joint Surgery American Volume. 85 (10), 1914-1920 (2003).</li><li>Tempfer, H., 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=Effects+of+crystalline+glucocorticoid+triamcinolone+acetonide+on+cultered+human+supraspinatus+tendon+cells.">Effects of crystalline glucocorticoid triamcinolone acetonide on cultered human supraspinatus tendon cells.</a> Acta Orthopaedics. 80 (3), 357-362 (2009).</li><li>Menon, A., 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=New+insights+in+extracellular+matrix+remodeling+and+collagen+turnover+related+pathways+in+cultured+human+tenocytes+after+ciprofloxacin+administration.">New insights in extracellular matrix remodeling and collagen turnover related pathways in cultured human tenocytes after ciprofloxacin administration.</a> Muscles Ligaments Tendons J. 3 (3), 122-131 (2013).</li><li>Backman, L. J., Fong, G., Andersson, G., Scott, A., Danielson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substance+P+is+a+mechanoresponsive,+autocrine+regulator+of+human+tenocyte+proliferation.">Substance P is a mechanoresponsive, autocrine regulator of human tenocyte proliferation.</a> PLoS One. 6 (11), 27209 (2011).</li><li>Backman, L. J., Eriksson, D. E., Danielson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substance+P+reduces+TNF-alpha-induced+apoptosis+in+human+tenocytes+through+NK-1+receptor+stimulation.">Substance P reduces TNF-alpha-induced apoptosis in human tenocytes through NK-1 receptor stimulation.</a> Britich Journal of Sports Medicine. 48 (19), 1414-1420 (2014).</li><li>Rolf, C. G., Fu, B. S., Pau, A., Wang, W., Chan, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+cell+proliferation+and+associated+expression+of+PDGFRbeta+causing+hypercellularity+in+patellar+tendinosis.">Increased cell proliferation and associated expression of PDGFRbeta causing hypercellularity in patellar tendinosis.</a> Rheumatology (Oxford). 40 (3), 256-261 (2001).</li><li>Hoppe, 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=Tenocytes+of+chronic+rotator+cuff+tendon+tears+can+be+stimulated+by+platelet-released+growth+factors.">Tenocytes of chronic rotator cuff tendon tears can be stimulated by platelet-released growth factors.</a> Journal of Shoulder and Elbow Surgery. 22 (3), 340-349 (2013).</li><li>Schipper, O. N., Dunn, J. H., Ochiai, D. H., Donovan, J. S., Nirschl, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nirschl+surgical+technique+for+concomitant+lateral+and+medial+elbow+tendinosis:+a+retrospective+review+of+53+elbows+with+a+mean+follow-up+of+11.7+years.">Nirschl surgical technique for concomitant lateral and medial elbow tendinosis: a retrospective review of 53 elbows with a mean follow-up of 11.7 years.</a> American Journal of Sports Medicine. 39 (5), 972-976 (2011).</li><li>Cook, J. L., Feller, J. A., Bonar, S. F., Khan, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+tenocyte+morphology+is+more+prevalent+than+collagen+disruption+in+asymptomatic+athletes'+patellar+tendons.">Abnormal tenocyte morphology is more prevalent than collagen disruption in asymptomatic athletes' patellar tendons.</a> Journal of Orthopedic Research. 22 (2), 334-338 (2004).</li><li>Yuan, T., 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=Creating+an+Animal+Model+of+Tendinopathy+by+Inducing+Chondrogenic+Differentiation+with+Kartogenin.">Creating an Animal Model of Tendinopathy by Inducing Chondrogenic Differentiation with Kartogenin.</a> PLoS One. 11 (2), 0148557 (2016).</li><li>Lui, P. P., Maffulli, N., Rolf, C., Smith, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+are+the+validated+animal+models+for+tendinopathy.">What are the validated animal models for tendinopathy.</a> Scandinavian Journal of Medical Scientific Sports. 21 (1), 3-17 (2011).</li><li>Wilhelm, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lateral+epicondylitis+review+and+current+concepts.">Lateral epicondylitis review and current concepts.</a> Journal of Hand Surgery American Volume. 34 (7), 1359-1360 (2009).</li></ol>

以前的一些研究报告了如何使用不同的程序, 如胶原酶或 kartogenin 注射, 跑步机跑步, 以及更多的26,27建立慢性 tendinopathic 动物模型。虽然许多研究显示有希望的治疗效果的基础上这些动物模型, 使用人类退行性 tenocyte 的实验将是至关重要的肌腱领域, 以重现疗效的治疗。在这篇文章中, 我们建立了一个成功地收获和文化的人类退化肌腱细胞一致的协议。?...

肌腱是一种慢性退行性肌肉骨骼的情况, 发展在身体的各个部分。最近, 由于越来越多地参与娱乐运动和提高预期寿命1、2, 发达世界的肌腱病例数量大大增加。肌腱的病因被认为是多因素的, 这些原因包括缺血、氧自由基损伤、收缩与血管扩张 innervations 的不平衡、内微泪和神经调节的变化3 ,4,5,6,7,8。大多数治疗肌腱只会缓解症状。此外, 没有组织再生的治疗需要很长的时间来恢复和获得有限的反应肌腱, 这给医生9的临床挑战。
目前治疗方案的无能以及缺乏退行性肌腱的自愈能力, 使研究人员对探索替代治疗策略感兴趣。最近, 新的研究报告了许多有希望的结果, 以提高肌腱肌腱的愈合效果, 使用生长因子, 干细胞为主的治疗, 和基因治疗10,11,12。
通过文献综述, 我们发现, 所涉及的研究可分为两类, 根据其分析材料: 动物模型, 如鼠, 一只老鼠, 或兔子;和人类模型。关于动物模型, 目前有两种常用的技术来产生肌腱: 化学诱导损伤或机械超载的模型。然而, 每个动物模型是有限的复制复杂的人类肌腱病理13,14。
大多数使用人体标本的论文都是在组织学上分析的, 或者是以健康的人类 tenocyte 为基础进行体外实验, 而不是变性 tenocyte15、16、17、18,19,20,21. 只有少数论文报告说, 他们使用了人类退化 tenocyte, 但他们没有详细描述用于从人类22、23获得变性 tenocyte 的协议。在这方面, 应该指出, 动物模型或健康组织/tenocyte 的成功结果不一定能预测人的疗效或有效剂量, 因为肌腱变性是一个复杂的过程, 而发病机制是仍未完全理解。
总的来说, 有必要描述从人体组织获得退行性 tenocyte 的标准协议, 而不会对捐献者造成不良影响。本文介绍了如何获得人类退行性 tenocyte 的协议。为验证该协议, 对所采集的组织进行组织学分析。然后, 采用免疫细胞化学 (ICC)、定量的实时聚合酶链反应 (qRT PCR) 和活性测定方法, 证实了 tenocyte 的退化性。

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	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Scalpel</td>
			<td style="width:199px;">Kisanbio</td>
			<td style="width:119px;">KS-Q0306-15</td>
			<td style="width:167px;">No. 15</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Mini-blade</td>
			<td style="width:199px;">Beaver</td>
			<td style="width:119px;">374769</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:235px;">Dulbecco&#39;s modified Eagle&#39;s medium (DMEM)</td>
			<td style="width:199px;">Gibco</td>
			<td style="width:119px;">11995065</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Collagenase &#8545;</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:119px;">C6885</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">PBS</td>
			<td>Gibco</td>
			<td style="width:119px;">14190250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">fetal bovine serum (FBS)</td>
			<td>Gibco</td>
			<td style="width:119px;">16000044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 mM ascorbic acid-2-phosphate</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:119px;">A5960</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Antibiotic-Antimycotic solution</td>
			<td>Gibco</td>
			<td style="width:119px;">15240062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">4% formaldehyde</td>
			<td style="width:199px;">Bio-solution</td>
			<td style="width:119px;">BP031</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Triton X-100&#160;</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:119px;">X100-100ml</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">BSA</td>
			<td style="width:199px;">Rdtech</td>
			<td style="width:119px;">C0082</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">TWEEN 20</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:119px;">P9416-100ml</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">MKX (C-5)</td>
			<td style="width:199px;">Santa cruz biotechnology</td>
			<td style="width:119px;">sc-515878</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Tenomodulin (N-14)</td>
			<td style="width:199px;">Santa cruz biotechnology</td>
			<td style="width:119px;">sc-49325</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Fluorescence Mounting Medium</td>
			<td style="width:199px;">DAKO</td>
			<td style="width:119px;">S3023</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:235px;">DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td style="width:199px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">D1306</td>
			<td></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:235px;">WST-1</td>
			<td style="width:199px;">Dojindo Molecular Technologies</td>
			<td style="width:119px;">CK04</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">BrdU Cell Proliferation Assay Kit</td>
			<td style="width:199px;">Cell Signaling Technology</td>
			<td style="width:119px;">#6813</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:235px;">TRIzol Reagent</td>
			<td style="width:199px;">Invitrogen</td>
			<td style="width:119px;">15596018</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:235px;">iScript cDNA Synthesis Kit&#160;</td>
			<td style="width:199px;">Bio-Rad</td>
			<td style="width:119px;">170-8891</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:235px;">TaqMan Gene Expression Master Mix</td>
			<td style="width:199px;">Applied Biosystems</td>
			<td style="width:119px;">4369016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">GAPDH</td>
			<td style="width:199px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">Hs02786624_g1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">COL3A1</td>
			<td style="width:199px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">Hs00943809_m1</td>
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a protocol to acquire the degenerative tenocyte from humans

肌腱, 一种因肌腱变性而发展起来的痛苦状态, 由于体力活动的增加和预期寿命的延长, 在发达世界中的上升。尽管患病率不断上升, 但潜在的发病机制仍不清楚, 治疗一般是有症状的。最近, 研究了多种治疗方法, 包括生长因子、干细胞和基因治疗, 以期提高退行性肌腱的愈合效力。然而, 这些研究中的大多数只对动物模型或健康的人类肌腱细胞进行。尽管一些研究使用病理学肌腱细胞, 我们最好的知识目前还没有描述如何获得人类退行性肌腱细胞的协议。本研究的目的是描述获取人类退行性肌腱细胞的标准协议。最初, 肌腱组织是在手术期间从侧髁的病人身上收获的。然后从腕桡肌肌腱中取出活检标本, 并与手术时观察到的结构变化有关。所有收获的肌腱看起来都是钝的、灰色的、易碎的和水肿的, 这使得它们在视觉上与健康的不同。肌腱细胞被培养并用于实验。同时, 在组织学上分析了一半的收获组织, 并表明它们具有相同的肌腱 (angiofibroblastic 发育不良或增生) 的主要特征。免疫细胞化学的二次分析证实, 培养的干细胞是肌腱细胞与大多数细胞有阳性染色的莫霍克和 tenomodulin 蛋白。用增殖试验或 qRT PCR 方法对细胞与健康对照进行比较, 确定肌腱细胞退行性的质量。退行性 tenocyte 显示出较高的增殖率和类似基因表达模式的肌腱, 匹配以前的报告。总的来说, 这一新的议定书可能为今后研究肌腱提供了有用的工具。

组织学分析表明, 从侧髁的收获组织具有 tendinopathic 肌腱的特征。肌腱退行性肌腱的 H & E 段揭示了一种无条理的胶原束, 极性和细直、强包装的平行纤维结构。在样品中常见的组织学征象如高细胞构成、大核无典型纺锤形等。此外, 退化肌腱的胶原束是由 H & E 染色的相对较弱的阿利新, 但呈阳性染色, 表明软骨和多糖物质增加。
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Watch this Scientific Journal Video about A Protocol to Acquire the Degenerative Tenocyte from Humans at JoVE.com

A Protocol to Acquire the Degenerative Tenocyte from Humans

体外应用退行性肌腱细胞是必要的, 当调查的效果, 新的治疗肌腱。然而, 大多数研究研究只使用动物模型或健康的 tenocyte。我们提出了以下的协议, 以隔离人类退行性肌腱细胞在手术期间。

从人类获得变性 Tenocyte 的一种协议

Tendinopathy, a painful condition that develops in response to tendon degeneration, is on the rise in the developed world due to increasing physical ...

This method can help answer key questions about human degenerative tenocyte biology as only few studies have described human degenerative tenocyte isolation and culture in detail. The main advantage of this technique is that we can obtain human degenerative tenocyte without additional patient injury because the degenerative tissue is acquired during standard epicondylitis surgery. Demonstrating the procedure will be Dr.Soo-Hong Han, Minjung Baek, and Hyung Kyung Kim from my Level Three.To acquire a degenerative tendocyte tissue sample, use a number 15 surgical scalpel to make a three to five centimeter skin incision just anterior medial to the lateral epicondyle, and proximal to the level of the joint to expose the lateral elbow and visually locate the extensor carpi radialis longus in the extensor aponeurosis interface. Use the scalpel to make a splitting incision two to three millimeters in depth between the extensor carpi radialis longus and the extensor aponeurosis at the interface. After anterior retraction of the extensor carpi radialis longus, locate the pathologic degenerative tissue by its characteristic dull grayish color and typically edematous and friable tissue, and use the scalpel and mini blades to carefully, but sharply, resect all of the degenerative tissue.When all the tissue has been harvested, close the wound with nonabsorbable suture material according to standard protocols and carefully remove all of the surrounding tissue from an approximately one cubic centimeter piece of tissue sample, placing the clean tendon tissue into 10 milliliters of PBS as soon as it has been trimmed. Then fix about half of the harvested tissue in neutral buffered 10%formalin in a chemical fume hood for 12 hours. To set up a tenocyte culture, transfer the other half of the harvested control and degenerated tissue samples into individual tissue culture dishes containing fresh PBS and mince the tissues into one cubic meter or smaller pieces, digest the tissue fragments in 30 milliliters of DMEM supplemented with 30 milligrams per milliliter of collagenase two per tissue group at 37 degrees Celsius for one hour.At the end of the digestion, filter the cell solutions through individual 100 micron cell strainers and deactivate the collagenase with one milliliter of FBS per tube. Collect the tenocytes by centrifugation and re-suspend the pellets at three times 10 to the fifth cells per 10 milliliters of DMEM supplemented with 10%FBS and 1%antibiotic, antimycotic solution concentration. Then, seed the cells into one 100 centimeter culture dish per condition, and culture the tenocytes in a 37 degree Celsius and 5%carbon dioxide incubator for one week, changing the culture medium every three days until an 80%confluence has been reached.For Immunocytochemical analysis, transfer two times 10 to the fifth cells in 200 microliters of fresh medium to each well of one eight-well chamber slide per experimental group and grow the cells to confluence for 24 hours. The next day, wash the cells with PBS, followed by fixation with 500 microliters of 4%formaldehyde per well, and permeabilization with 0.5%triton X 100 in PBS for 10 minutes. To block any non-specific binding, incubate the cells with 1%bovine serum albumin, and 0.05%Tween 20 in PBS, as experimentally appropriate, followed by incubation with the flourescently-tagged primary antibodies of interest at four degrees Celsius overnight protected from light.The next morning, label the cells with the appropriate secondary antibodies for one hour at room temperature protected from light, and mount the samples with 0.5 microliters of an appropriate fluorescence mounting medium per chamber. Then image the samples on a fluorescence microscope under a 400 times objective. For pathological analysis of the tissue, embed the formal and fixed control and degenerated tendon tissue samples in four to five milliliters of paraffin and acquire four to five micron sections from each paraffin block.For hematoxylin in eosin, or H&E staining, deparaffinize the samples with three five-minute immersions in xylene followed by hydration with graded ethanol immersions and a two-minute tap water wash. Next, label the samples with hematoxylin for five minutes, followed by another two-minute water wash. Dip the rinsed samples in 1%hydrochloric acid and 70%ethanol for three seconds, followed by a three-minute water wash.Neutralize the acid with a 10 second dip in 0.5%ammonia, followed by another three-minute water wash. Label the neutralized samples with the eosin for five minutes, and dehydrate the H&E stained samples with graded ethanol immersions. After three xylene immersions, mount the samples with an appropriate mounting medium and a cover slide.To perform immunohistochemical analysis of the tissue samples, use the bond polymer refined detection kit according to the manufacturer's instructions with the following minor modifications. After deparaffinization with xylene as demonstrated, immerse the samples in bond epitope retrieval solution for 30 minutes at 100 degrees Celsius for antigen retrieval, followed by a five-minute incubation in hydrogen peroxide to quench any endogenous peroxidase. Next, label the sections for fifteen minutes at room temperature with the primary antibodies of interest.At the end of the incubation, label the samples with the appropriate biotin-free polymeric horseradish peroxidase linker secondary antibody conjugate system for 50 minutes at room temperature. Then mount the sections with a cover slip in the appropriate mounting medium and image the samples on a light microscope at a 400 times magnification. H&E analyses reveals that tissue harvested from the degenerated lateral epicondylitis demonstrates disorganized collagen bundles with a loss of polarity and fine, straight, strongly packed parallel fiber structures.Indeed, the collagen bundles are positive for Alcian Blue staining in the degenerated tissue sections indicating an increased proteoglycan and glycosaminoglycan presence within the damaged tissue. Immunohistochemical analysis of the degenerated tendons also indicates a cytoplasmic granular positive reaction to vascular endothelial growth factor that is not observed in control, healthy tendon tissue samples. Both types of cultured tissue sample cells can be confirmed as tenocytes by immunofluorachemistry with most of the cultured cells expressing representative tenocyte markers such as the mohawk and tenomodulin proteins and demonstrating an elongated appearance.Of note, the degenerative tenocytes also demonstrate a higher rate of proliferation in culture compared to tenocytes harvested from healthy controls as well as a higher expression of multiple genes known to be related to the development of tendinopathy. In conclusion, this validated protocol using degenerative tenocyte paves the way for future investigations in tendon biology and for testing new interventions.

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