Name: creation and transplantation of an adipose-derived stem cell (asc) sheet in a diabetic wound-healing model
Uploaded: 2017-08-04T12:30:00
Description: Watch this Scientific Journal Video about Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model at JoVE.com

作者感谢塑料部和整形外科手术，东京大学医学院博士由纪子古贺提供实用的建议。我们还感谢先生秀和村田的糖尿病中心的东京妇女医疗大学医学院为优异的技术支持。这项研究被支持由创新中心创造先进的跨学科研究领域项目的发展中国家创新系统"单元格表组织工程中心 (CSTEC)"项目从部的教育、 文化、 体育、 科学和技术 （文部科学省） 的日本。

All experimental protocols presented below were approved by the Animal Welfare Committee of Tokyo Women&#39;s Medical University School of Medicine and abided by all requirements of the Guidelines for Proper Conduct of Animal Experiments.
1. Preparation of Animals, Instruments, Culture Media, and Dishes
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	<li>Prepare complete culture medium using minimum essential medium alpha containing 20% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Store this for several months at 4 &#176...

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成功地培养区域审计事务中心表最关键的步骤如下： 1) 温度必须在温度反应性培养皿培养过程维持在大约 37 ° C。在区域审计事务中心表的创建，过程中每道工序进行 37 ° C 的热板，和每个试剂被加热到 37 ° C，以防止细胞自发地分离从菜31。2) 收件人 ZDF 大鼠必须进行监测，以防止被删除的非胶粘剂的酱汁，是区域审计事务中心床单移植成功的关键。如果删除了更衣室，必须应?...

糖尿病患者的人口世界范围内增加，达到 4 亿 2015年1;估计有 15-25%的糖尿病患者处于危险从下肢糖尿病溃疡2的进展。糖尿病下肢溃疡是棘手的可能需要长期治疗与康复训练后完全恢复。长治疗期间经常导致显著降低患者的生活质量。因此，治疗糖尿病创面必须开发新的治疗方法，减少或防止恶化。评价糖尿病创面愈合，我们优化糖尿病溃疡创面愈合大鼠模型，模仿实际临床情况，并评估是否移植脂肪源性干细胞 (ASC) 床单使用单元格表工程加速伤口愈合。
骨髓间质细胞 (MSCs) 表现出极大的潜力，加速创面愈合其自我更新的能力，他们的免疫调节作用，它们能够分化成多种细胞谱系3。陶瓷是一种类型的 MSC 来源于脂肪组织，和他们表现出骨髓间充质干细胞来自其他组织，包括其潜在的血管生成和旁分泌活动4，5的几个优势。脂肪组织是较为丰富的人体，并其辅助功能允许收集使用微创手术。因此，陶瓷已用于实验伤口愈合应用6，7。
以前的报道表明，单个单元格 MSC 悬浮液直接注入伤口周围地区可以加快伤口愈合8，9。然而，尽管报告的加速伤口愈合的糖尿病溃疡模型的单细胞悬液注射后，在伤口部位移植细胞的存活时间尚不清楚。
在此研究中，我们应用使用温度反应性培养皿的细胞板工程。这些菜有温度响应聚合物N-异丙基丙烯共价键到10他们表面上。接枝的聚合物层允许温度控制的细胞粘附或脱离表面的培养皿。这道菜的表面变得疏水在 37 ° C，让细胞粘附和增殖，而细胞自发地从表面分离的时候就亲水在温度低于 32 ° c。体外培养的细胞可以收获作为完整细胞到细胞连接与细胞外基质 (ECMs) 的连续单元格表只是通过降低的温度;因此，蛋白水解酶，破坏 ECM，如胰蛋白酶，并非需要的11。因此，细胞板工程可以保护细胞间连接和改善细胞移植的疗效。
此外，细胞板移植增加细胞存活率时相比，细胞注射12。在此协议中，作为延迟的伤口愈合 2 型糖尿病与肥胖模型选取 Zucker 糖尿病脂肪肝 (ZDF) 鼠。在大约 4 个星期中 ZDF 大鼠自发形成肥胖。他们然后患 2 型糖尿病与肥胖之间 8 和 12 周龄，此时他们表现出高血糖与胰岛素抵抗、 血脂异常和甘油13相关联。延迟的伤口愈合，血流量减少外围及血管中，糖尿病肾病也观察到14，，1516。此外，ZDF 大鼠可能适当模型研究顽固性皮肤溃疡，例如糖尿病溃疡的愈合。
人类和啮齿类动物在创面愈合的机制上的差异是与皮肤的解剖学差异相关联。正常大鼠愈合的伤口基于伤口收缩而创面愈合的人类基于重新上皮和肉芽组织的形成。通常情况下，伤口小夹板固定在啮齿动物模型中使用有助于尽量减少伤口收缩并允许逐步形成的肉芽组织17，虽然非糖尿病大鼠伤口已经几乎完全闭合的收缩。然而，糖尿病创面收缩 ZDF 在大鼠是受损，并且伤口愈合主要发生通过重新上皮和肉芽组织的形成;因此，这一进程是更类似于人类的伤口愈合14。
糖尿病伴骨外露创面后清创术临床上经常遇到。先前的研究审查了直径为 12 毫米全层皮肤伤口的裸鼠裸鼠18，19背上和背上的正常小鼠20直径为 10 毫米全层皮肤伤口。发展为严重的糖尿病创面的临床模型，较大的 (15 x 10 mm2) 全层皮肤缺损暴露骨并没有骨膜被创造了，作为先前描述21，大鼠 2 型糖尿病和肥胖。
通过同种异体移植的 ASC 床单创造了大鼠 ASC （区域审计事务中心） 床单从正常 Lewis 大鼠的陶瓷。在临床实践中，自体移植是行不通的因为溃疡的糖尿病患者常表现出严重的糖尿病并发症，如不加控制高血糖和高身体质量指数，这些并发症原因伤口愈合障碍，增加了从这些患者获得脂肪组织的难度。此外，从动物与糖尿病展览体干改变属性和受损功能22。因此，议定书 》 在这里提出了一种描述区域审计事务中心床单从正常大鼠同种异体移植和人工皮肤对糖尿病大鼠的应用。
在本协议中使用的双层人工皮肤防止伤口的自发性收缩、 促进合成的一种新的结缔组织矩阵，和类似于真正的真皮23。在此协议中，人工皮肤是放置区域审计事务中心工作表上，用尼龙线，防止伤口收缩或扩大造成的松散大鼠皮肤固定。此外，人造皮肤提供一种三维框架为 ASC 床单、 保持潮湿的环境下的移植的 ASC 床单和伤口，和保护伤口免受感染和外部力量。最后，非胶粘剂敷料被放置在伤口保护它免受外部冲击、 保持潮湿的伤口环境，和吸收渗出物。
一个区域审计事务中心表是薄薄的、 灵活的和可变形和可以坚持移动收件人的网站，例如24跳动的心。单元格表工程已被用于各种组织重建和可以生成疗效25，26.表现出临床治疗潜力的 ASC 床单可能加速许多类型的伤口的愈合。此外，ASC 床单，结合人工皮肤，使用同种异体移植可能适用于治疗难治性溃疡或烧伤，例如那些观察外周动脉疾病或胶原病，或者他们可能注射到患者营养不良或使用类固醇。这种方法提高移栽体干的效率。伤口愈合 ZDF 大鼠模型产生严重的伤口情况，类似于人类的伤口愈合过程，模仿在小型实验动物临床条件。

<table border="0" cellpadding="0" cellspacing="0" width="678">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">&#945;-MEM glutamax</td>
			<td style="width:169px;">Invitrogen</td>
			<td style="width:119px;">32571-036</td>
			<td style="width:167px;">Carlsbad, CA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum (FBS)</td>
			<td>Japan Bioserum Co Ltd.</td>
			<td style="width:119px;">S1650-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/streptomycin</td>
			<td>Life Technologies</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase A</td>
			<td>Roche Diagnostics</td>
			<td style="width:119px;">10 103 578 001</td>
			<td>Mannheim, Germany</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">60-cm2 Primaria tissue culture dish</td>
			<td>BD Biosciences</td>
			<td>353803</td>
			<td>Franklin Lakes, NJ</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s Phosphate Buffer Saline (PBS)</td>
			<td>Life Technologies</td>
			<td style="width:119px;">1490-144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.25% Trypsin-ethylenediamine tetraacetic acid (EDTA)</td>
			<td>Life Technologies</td>
			<td style="width:119px;">25200-056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-ascorbic acid phosphate magnesium salt n-hydrate</td>
			<td style="width:169px;">Wako</td>
			<td>013-19641</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">35-mm temperature-responsive culture dish (UpcellTM)</td>
			<td>CellSeed</td>
			<td>NUNC-174904</td>
			<td>Tokyo, Japan</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Microwarm plate (MP-1000)</td>
			<td style="width:169px;">Kitazato Science Co., Ltd.</td>
			<td style="width:119px;">1111</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rodent mechanical ventilator</td>
			<td>Stoelting</td>
			<td>#50206</td>
			<td>Wood Dale, IL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4% isoflurane</td>
			<td>Pfizer Japan</td>
			<td>114-13340-3</td>
			<td>Tokyo, Japan</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Artificial skin (Pelnac&#174;)</td>
			<td>Smith &#38; Nephew</td>
			<td>PN-R40060</td>
			<td>&#160;Tokyo, Japan</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Non-adhesive dressing (Hydrosite plus&#174;)</td>
			<td>Smith &#38; Nephew</td>
			<td align="right">66800679</td>
			<td>Known as Allevyn non-adhessing&#174; in the United State</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">5-0 nylon suture</td>
			<td>Alfresa</td>
			<td style="width:119px;">EP1105NB45-KF2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20 CELLSTAR TUBES</td>
			<td>greiner bio-one</td>
			<td style="width:119px;">227 261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">15mL Centrifuge Tube</td>
			<td>Corning Incorporated</td>
			<td align="right" style="width:119px;">430791</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">14 GOLDMAN-FOX PERIOSTEAL</td>
			<td>Hu-Friedy</td>
			<td style="width:119px;">P14</td>
			<td>Chicago, IL</td>
		</tr>
	</tbody>
</table>

creation and transplantation of an adipose-derived stem cell (asc) sheet in a diabetic wound-healing model

人工皮肤取得了相当大的治疗效果，在临床实践中。然而，可能会延长人工皮肤治疗糖尿病患者与阻碍的血流或大的伤口的伤口。作为一种新技术治疗糖尿病溃疡，出现了基于细胞疗法及细胞板工程已改善细胞移植的疗效。大量的报告建议脂肪源性干细胞 （陶瓷），一种间充质基质细胞 (MSC)，表现出由于他们在脂肪组织和其可访问性集合时相比骨髓间充质干细胞从其他组织中的相对丰度的治疗潜力。因此，陶瓷似乎是一个良好的来源的干细胞用于治疗目的。在这项研究，ASC 床单从正常 Lewis 大鼠附睾脂肪已成功创建使用温度反应性培养皿和正常培养基含有抗坏血酸。ASC 床单被移植进 Zucker 糖尿病脂肪酸 (ZDF) 大鼠，大鼠模型的 2 型糖尿病、 肥胖等，表现出减少创面愈合。伤口被创建后颅骨表面、 ASC 床单被移植到伤口，和双层人工皮肤被用来支付床单。收到 ASC 床单的 ZDF 大鼠有更好地愈合比没有移植的 ASC 床单 ZDF 老鼠。这种方法是有限的因为 ASC 床单是敏感的干燥条件，需要一个潮湿的伤口环境的维护。因此，人工皮肤用于封面 ASC，防止干燥。同种异体移植的 ASC 床单结合人工皮肤也可能适用于其他难治性溃疡或烧伤，例如那些观察外周动脉疾病与胶原病和可能给予营养不良或使用类固醇的患者。因此，这种治疗可能改善糖尿病创面愈合的治疗选项的第一步。

本议定书试图建立一种新的基于单元格的难治性糖尿病伤口疗法。简要 （如图 1所示），同种异体区域审计事务中心床单从正常大鼠使用单元格表工程被创造了，然后移植对糖尿病大鼠使用双层人工皮肤全层皮肤缺损。光镜图像的区域审计事务中心表 (图 2A) 的一个好例子和一个坏的榜样的区域审计事务中心表 (图 2B) 如图 2</stron...

Watch this Scientific Journal Video about Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model at JoVE.com

Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model

脂肪干细胞 （陶瓷） 容易孤立和收获从正常大鼠脂肪。ASC 床单可以使用单元格表工程创建，可以移植到 Zucker 糖尿病脂肪肝大鼠表现出全层皮肤缺损伴骨外露，然后用双层的人工皮肤覆盖。

创作和移植的糖尿病伤口愈合模型中脂肪源性干细胞 (ASC) 表

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients ...

Research

JoVE Journal

Medicine

Creation and Transplantation of an Adipose-derived Stem Cell (ASC) Sheet in a Diabetic Wound-healing Model

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

主动脉原位移植大鼠模型，研究慢性血管病变的发展

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

科研

医学

A Mouse Model of in Utero Transplantation

A Mouse Model of in Utero Transplantation

一个小鼠模型在子宫内移植

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

在干细胞移植在体外模拟缺血/再灌注模型

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

采用实时定量PCR检测供体细胞植入一个具有竞争力的小鼠骨髓移植模型

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Murine Model of Wound Healing

伤口愈合的小鼠模型

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

在脊髓损伤的实验挫伤型号神经干细胞移植

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

Transplantation of Adipose Tissue-Derived Stem Cell Sheet to Reduce Leakage After Partial Colectomy in A Rat Model

脂肪组织干细胞移植对大鼠部分结肠切除术后渗漏的减少

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of ...

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

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 ...

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

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 ...

Targeting Gray Rami Communicantes in Selective Chemical Lumbar Sympathectomy

选择性化学腰椎交感神经切除术中灰色拉米关节的靶向性

Chemical lumbar sympathectomy (CLS) is a commonly used, minimally invasive procedure for the treatment of conditions including ischemic diseases of the ...