Name: development of an in vitro assay to evaluate contractile function of mesenchymal cells that underwent epithelial-mesenchymal transition
Uploaded: 2016-06-10T14:00:00
Description: Watch this Scientific Journal Video about Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition at JoVE.com

We thank Dr. Tadashi Koyama for technical help. This work was supported in part by JSPS KAKENHI Grant Numbers 23249045, 15K09211, 15K19172; a grant to the Respiratory Failure Research Group from the Ministry of Health, Labour and Welfare, Japan; a grant for research on allergic disease and immunology, Japan.

1. الاستعدادات والثقافة من خلايا الرئة طلائي <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ثقافة A549 الخلايا الظهارية الرئة البشرية (خط خلية ملتصقة) في Dulbecco لتعديل النسر متوسطة (DMEM) تستكمل مع 10٪ مصل بقري جنيني (FBS)، و 100 وحدة دولية / مل البنسلين، و 100 ميكروغرام / مل الست...

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ويضم البروتوكول وضعت في هذه الدراسة خطوتين. يتم تنفيذ الخطوة الأولى للحث على EMT، في حين أن الخطوة الثانية هي فحص هلام الانكماش. وبما أنه من المهم للتأكد من أن الخلايا خضعت EMT الخطوة 2 توفر عنصرا مكملا ممتازا للتغيرات التعبير المورفولوجية والجينات. وأظهرت دراسات سابقة ?...

وتشارك التليف في التسبب في أمراض مختلفة التقدمية المزمنة، مثل مرض الرئة الخلالي، تليف القلب، وتليف الكبد والفشل الكلوي المحطة، التصلب الجهازي، وأمراض المناعة الذاتية 1. بين أمراض الرئة الخلالي، والتليف الرئوي مجهول السبب (IPF) هو مرض التدريجي المزمن ويظهر سوء أحوال الطقس. السمة المميزة المرضية للفريق الحكومي الدولي هو تطوير بؤر أرومي ليفي التي تتكون من الخلايا الليفية وmyofibroblasts تنشيط التي ترتبط مع التكهن. واقترح أصول هذه الخلايا الليفية الرئوية التي يمكن جنيها من عدة خلايا اللحمة المتوسطة، بما في ذلك الخلايا الليفية الرئوية المقيمين أصلا وتعميم الخلايا الليفية من نخاع العظام. في الآونة الأخيرة، وقد اقترح الانتقالية الظهارية الوسيطة (EMT) لتترافق مع تشكيل خلايا اللحمة المتوسطة 2، والمساهمة في التسبب في اضطرابات متليفة. ويعتقد أن EMتي يلعب دورا هاما في عملية نمو الجنين، التئام الجروح، وتطور السرطان، بما في ذلك غزو الورم والانبثاث 3. بعد عملية EMT، الخلايا الظهارية الحصول على قدرة خلايا اللحمة المتوسطة التي كتبها ضياع معالم الظهارية، مثل E-كادهيرين، والتعبير عن علامات الوسيطة، مثل فيمنتين، وα العضلات الملساء الأكتين (SMA) 4،5. وأظهرت دراسات سابقة الدليل على أن عملية EMT ارتبط تطور التليف الأنسجة في الكلى 6 و 7 الرئة. بالإضافة إلى ذلك، والتهاب مزمن يعزز المرض متليف 8؛ علاوة على ذلك، فإن هذه السيتوكينات الالتهابية مثل عامل نخر الورم أعضاء الفصيلة 14 (TNFSF14؛ ضوء)، عامل نخر الورم (TNF) -α، وانترلوكين 1β، وقد ثبت لتعزيز EMT 9-12. جل الكولاجين فحص الانكماش، انكماش الخلايا الفحص المعتمدة على الكولاجين التي هي جزء لا يتجزأ الليفية في النوع الأولالكولاجين جل ثلاثة الأبعاد، هو المثل الأعلى في نموذج المختبر لتقييم انقباض. انقباض هي واحدة من وظائف مميزة من الخلايا الليفية، وتسهم في ترميم الجروح العادية وتليف 13. في هذا الاختبار، ويعتقد أن المرفق من الخلايا الليفية إلى النوع الأول الكولاجين من خلال الآليات التي تعتمد على إنتغرين من المفترض أن تنتج التوتر الميكانيكي تحت بعض الظروف، وبالتالي يؤدي إلى تقلص الأنسجة. هنا، يتم الإبلاغ عن تطوير فحص هلام انكماش يمكن تكييفها لتقييم الاستحواذ على وظيفة مقلص في الخلايا التي خضع EMT. يوضح هذا التقرير أن هذا الاختبار تعديل هو مناسبة لتقييم انقباض في خلايا اللحمة المتوسطة التي خضعت EMT.

<table border="0" cellpadding="0" cellspacing="0" width="598">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM</td>
			<td style="width:96px;">sigma aldrich</td>
			<td style="width:119px;">11965-092</td>
			<td style="width:167px;">For A549 medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS</td>
			<td style="width:96px;">GIBCO</td>
			<td style="width:119px;">10437</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Transforming Growth Factor-&#946;1, Human, recombinant</td>
			<td style="width:96px;">Wako Laboratory chemicals</td>
			<td style="width:119px;">209-16544</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Recombinant Human TNF-&#945;</td>
			<td style="width:96px;">R&#38;D systems</td>
			<td style="width:119px;">210-TA/CF</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">E-Cadherin (24E10) Rabbit mAb</td>
			<td style="width:96px;">Cell Signaling Technology</td>
			<td style="width:119px;">#3195</td>
			<td>1:3000 dilution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Vimentin (D21H3) Rabbit mAb</td>
			<td style="width:96px;">Cell Signaling Technology</td>
			<td style="width:119px;">#5741</td>
			<td>1:3000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-&#945;-Tubulin antibody</td>
			<td style="width:96px;">sigma aldrich</td>
			<td style="width:119px;">T9026</td>
			<td>1:10000 dilution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Monoclonal Anti-Actin, &#945;-Smooth Muscle antibody&#160;</td>
			<td style="width:96px;">sigma aldrich</td>
			<td style="width:119px;">A5228</td>
			<td>1:10000 dilution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Anti-N-cadherin antibody</td>
			<td style="width:96px;">BD Transduction Laboratories</td>
			<td style="width:119px;">#610920</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Anti-Mouse IgG, HRP-Linked Whole Ab Sheep (secondary antibody)</td>
			<td style="width:96px;">GE Healthcare</td>
			<td style="width:119px;">NA931-100UL</td>
			<td>1:20000 dilution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Anti-Rabbit IgG, HRP-Linked Whole Ab Donkey (secondary antibody)</td>
			<td style="width:96px;">GE Healthcare</td>
			<td style="width:119px;">NA934-100UL</td>
			<td>1:20000 dilution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">blocking reagent</td>
			<td style="width:96px;">GE Healthcare</td>
			<td style="width:119px;">RPN418</td>
			<td>2% in TBS-T</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">6 Well Clear Flat Bottom TC-Treated Multiwell Cell Culture Plate, with Lid</td>
			<td style="width:96px;">corning</td>
			<td style="width:119px;">#353046</td>
			<td></td>
		</tr>
		<tr height="21">
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</table>

development of an in vitro assay to evaluate contractile function of mesenchymal cells that underwent epithelial-mesenchymal transition

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is the formation of fibroblastic foci, which is associated with the disease severity. Mesenchymal cells consisting of the fibroblastic foci are proposed to be derived from several cell sources, including originally resident intrapulmonary fibroblasts and circulating fibrocytes from bone marrow. Recently, mesenchymal cells that underwent epithelial-mesenchymal transition (EMT) have been also supposed to contribute to the pathogenesis of fibrosis. In addition, EMT can be induced by transforming growth factor &#946;, and EMT can be enhanced by pro-inflammatory cytokines like tumor necrosis factor &#945;. The gel contraction assay is an ideal in vitro model for the evaluation of contractility, which is one of the characteristic functions of fibroblasts and contributes to wound repair and fibrosis. Here, the development of a gel contraction assay is demonstrated for evaluating contractile ability of mesenchymal cells that underwent EMT.

خلال EMT، الخلايا الظهارية تفقد علامات الظهارية، مثل E-كادهيرين، واكتساب التعبير عن علامات الوسيطة، مثل فيمنتين وα العضلات الملساء الأكتين 4،5. حضانة الخلايا الظهارية A549 الإنسان الرئة مع TGF-β1 وTNF-α تحرض EMT. ظهور خلايا A549 العادية بمثابة حجر كوبلي ?...

Watch this Scientific Journal Video about Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition at JoVE.com

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Here, we describe the development and application of a gel contraction assay for evaluating contractile function in mesenchymal cells that underwent epithelial-mesenchymal transition.

تطوير<em&gt; في المختبر</em&gt; الفحص لتقييم مقلص وظيفة الخلايا الجذعية الوسيطة التي خضعت طلائي-الوسيطة الانتقالية

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is ...

Research

JoVE Journal

Developmental Biology

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples

على الطريقة الأنزيمية لانقاذ الوسيطة الخلايا الجذعية من عينات متخدر نخاع العظم

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade ...

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

في المختبر وفي النماذج الحية لدراسة الانتقال القرنية غشائي-الوسيطة

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema ...

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

عزل ماوس بطانة الرحم طلائي واللحمية خلايا ل<em&gt; في المختبر</em&gt; Decidualization

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. ...

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

في المختبر نمو بصيلات بريانترال الماوس تحت ظروف محاكاة الجاذبية الصغرية

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We ...

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

في المختبر تمايز الخلايا الجذعية البشرية الوسيطة إلى الفنية كارديوميوسيتي مثل الخلايا

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

والرزن ثقافة المشارك خاتم الابهري: أداة ملائمة تقييم إمكانات الأوعية الوسيطة Stromal الخلايا في المختبر

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature ...

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells (HSPCs) in Developing Zebrafish Embryos

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos

تطوير التحليل في المختبر كوانتيتاتي الجذعية المكونة للدم والسلف الخلايا (هسبكس) في تطوير الأجنة الزرد

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

التحليل الحسابي ل Germline ايليجانس كاينورهابديتيس دراسة توزيع الأنوية، والبروتينات، وسيتوسكيليتون

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

في المختبر توليد المشتقات سميط من البشرية التي يسببها الخلايا الجذعية Pluripotent

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

تقييم الضرر التأكسدي في الخلايا السطحية الرئيسية للفأرة /الخلايا الجذعية استجابة للأضرار فوق البنفسجية-C (UV-C)

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...