支持这项工作是由德意志研究联合会（DFG; KL 1415/4-2 1415/3-2和KL）。

1。准备<ol><li>准备培养皿<ol><li>解冻胶原蛋白​​IV型O / N在4°C和0.1％无菌醋酸溶解。使用量取决于接种细胞的类型和数量的菜肴。使用2微克/厘米2。 </li><li>孵育至少1小时，于RT菜肴，并用蒸馏水（A. tridest）洗两次。 </li><li>允许菜肴正常干燥。 </li></ol></li><li>补充介质<ol><li>提高到500毫升培养基中添加1.75克葡萄糖，4.5 g / L的葡​​萄糖水平。为了调节介质600毫渗透摩尔，添加100mM NaCl的100mM的尿素，分别由200毫渗透摩尔，100毫渗透摩尔，增加渗透压。 </li><li>到600 mosmol DMEM培养基中添加1％非必需氨基酸，1％ultroser，500μM的二丁酰环腺苷酸（dbcAMP），20 U / ml的制霉菌素和0.25微克/毫升庆大霉素（1:200稀释的原液的浓度为50微克/毫升）新鲜的一天的准备。 </li></ol></LI&gt; <li>酶溶液<ol><li>加入1毫克/毫升透明质酸酶和胶原酶2.2毫克/毫升的DPBS含0.25微克/毫升的庆大霉素和制霉菌素。准备1毫升这种酶溶液消化2内部肾髓质。该解决方案必须通过过滤灭菌，并存储在50ml Falcon管中。 </li></ol></li></ol> 2。培养原代大鼠内髓集合管（IMCD）细胞所有动物的处理程序进行符合地方和联邦立法。 <ol><li>麻醉大鼠在CO 2（在一个盒子里饱和CO 2）或异氟醚（U-400麻醉机，有限公司，马耳他Univentor;空气流量350-400毫升/分钟2-2.5％异氟醚）。杀头动物出血发白内部较深的外髓之间的边界，允许精确检测。此外，出血孤立的红细胞数量减少到最小。移除肾脏和补充用庆大霉素和制霉菌素（见上文）在冰上的无菌DPBS中把它们洗干净。所有进一步的步骤是无菌洁净工作台条件下进行。 </li><li> DPBS溶液肾脏转移到一个无菌的压缩，以除去过多的液体。取出脂肪肾胶囊用无菌镊子和剪刀。 </li><li>经肾仍然固定在压缩，稍微切的外侧（皮层）的纵向轴线的旁边。确保不完全划破，但把它留在一块，在肾盂连接。 </li><li>用弯剪刀仔细解剖发白的内髓质，被包围玫瑰波光粼粼的外髓。收集他们在DPBS，冰60毫米的培养皿中含有庆大霉素和制霉菌素。 </li><li>一旦所有的髓质分离和收集的冰，减小体积的DPBS的水平，他们只是布满缓冲。使用一个无菌的的剃刀刃切髓质切块53毫米。 </li><li>简单火焰熔化和圆一个无菌巴斯德吸管尖底。确保移液器冷却，然后再继续。 </li><li>组织悬液转移到50毫升猎鹰管含有新鲜配制的无菌酶溶液用透明质酸酶和胶原酶（见1.3.1）用巴斯德吸管圆润。紧紧关上盖子，并在37°C下连续搅拌（220转）一个普通的桌面水浴1.5 - 2小时。 </li><li>研磨的悬浮液，吸液管的混合物向下几次与轮巴斯德吸管（见上文），直到变得均匀混浊的悬浮液。 </li><li>离心悬浮在300 XG 5分钟和16°C。弃上清，悬浮颗粒在DPBS彻底，含有庆大霉素和制霉菌素再次离心。重复此洗涤步骤一次。 </li><li>重悬细胞在完全培养基和种子到c的ulture菜和/或微滴度孔板。 2髓质（1只）的编制将产生足够一个60毫米培养皿中的细胞。 </li><li> 24小时后，洗细胞两次与600 mosmol DMEM，并添加新鲜培养基。在接下来的一周，应清洗细胞的2-3倍（ 图1）。播种后6-8天，髓集合管的细胞，可用于实验。请记住他们在实验开始前24小时，二丁酰环腺苷酸（dbcAMP）和制霉菌素未经孵化。否则AQP2将位于只在质膜。 </li></ol>

<ol>
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作者宣称，他们有没有竞争经济利益。

我们提出了一个详细的协议的准备和培养原代大鼠IMCD细胞。该方法产生高达21平方厘米的细胞从一个大鼠20。实验要求标准的细胞培养设备，并可以在大约6小时内由一个人进行。因此，这种做法是适合作为一个标准的实验室方法。 原代大鼠的IMCD细胞可以接种在培养皿不同大小，范围从96孔板到60毫米的菜肴。然而，环比增长384格式的程序需要优化。我们避免?...

A correction was made to <a href="http://www.jove.com/video/50366">Culturing Primary Rat Inner Medullary Collecting Duct Cells</a>. There was an error with an author's name. The author's last name had a typo and was corrected to: Enno Klussmann instead of: Enno Klussman

A correction to author list has been made for article Culturing Primary Rat Inner Medullary Collecting Duct Cells.

Erratum: Culturing Primary Rat Inner Medullary Collecting Duct Cells

在肾集合管主细胞，精氨酸加压素（AVP）控制水的重吸收，通过刺激插入水通道蛋白2（AQP2）到质膜。 AVP结合G蛋白偶联后叶加压素2型受体（V2R），刺激腺苷酸环化酶，从而cAMP的形成。启动该信号级联激活蛋白激酶A（PKA）。蛋白激酶A磷酸化水通道在丝氨酸256（S256），这是关键的触发再分配到质膜的细胞内小泡。膜插入沿渗透压梯度和微调体内水分的动态平衡，促进水的重吸收。 失调的AVP-介导的水的重吸收，由于像差的AVP分泌或AVP-激活的的信令原因或伴有严重的疾病。水的重吸收减少V2R或AQP2突变造成的，而导致肾性尿崩症海拔血液ATED血浆AVP水平与心血管疾病，如慢性心脏衰竭及抗利尿激素分泌综合征（SIADH）水的重吸收过多。 AVP-介导的水的重吸收的意义，不仅是因为它蕴涵在疾病。 AVP诱导易位AQP2息囊泡与细胞膜的融合代表了严格cAMP依赖的胞吐过程，这是目前不是很了解。为cAMP依赖的胞吐作用的其他例子是在肾脏和H +在胃中分泌的肾素分泌。因此，阐明AQP2易位肾主细胞的分子机制，不仅有助于理解相似的分子过程，在其他类型的细胞，但也可能与AVP-介导的水的重吸收障碍有关的疾病的治疗新疗法铺平道路。 êlucidating控制机制A​​QP2需要收集管主细胞模型。对于这样一些不同的哺乳动物肾细胞系都可用。然而，这些模型有几个缺点。在许多细胞系统中的水通道蛋白水平低（ 表1，M1，HEK293，COS-7，MDCK细胞，LLC-PK1）1-10，其他异位过度表达人类（MCD4，WT10）11,12或大鼠（CD8） 13 AQP2。在MCD4和COS-7细胞中的V2受体不表达。据我们所知，没有永久的细胞株作为一种模式，这是来自肾脏内髓集合管，肾AQP2表达最丰富的那部分，但是从皮质集合管1,11,13-16 。这种细胞模型，例如广泛使用的永生mpkCCD（ 例如 17），或最近建立的mTERT-CCD 14个细胞系。两种细胞系内源性表达AQP2和V2R但因为它们是来自最有可能含有皮质集合管内髓集合管（IMCD）细胞相比，不同的蛋白质组。例如，他们必须表达不同的Na +的运输系统。 在这里，我们提出了一个协议文化主髓集合管细胞表达V2R和AQP2内源性。这一模式，因此，代表着最密切的生理情况在肾集合管。建立文化，只需要标准的实验室设备其他实验室可以很容易地采用这种方式。

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			<td>Name </td>
			<td>Company Catalog</td>
			<td>Number</td>
		</tr>
		<tr>
			<td>Collagen Type IV Mouse</td>
			<td>BD Biosciences</td>
			<td>356233</td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>SIGMA</td>
			<td>H6254</td>
		</tr>
		<tr>
			<td>Collagenase type CLS-II</td>
			<td>Biochrom AG</td>
			<td>C2-22</td>
		</tr>
		<tr>
			<td>DMEM + GlutMAX</td>
			<td>Invitrogen GIBCO</td>
			<td>21885108</td>
		</tr>
		<tr>
			<td>Nystatin</td>
			<td>SIGMA</td>
			<td>N4014</td>
		</tr>
		<tr>
			<td>Ultroser G</td>
			<td>Cytogen</td>
			<td>15950-017</td>
		</tr>
		<tr>
			<td>Non essential amino acids (NEA)</td>
			<td>Biochrom AG</td>
			<td>K0293</td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Invitrogen GIBCO</td>
			<td>15710</td>
		</tr>
		<tr>
			<td>DBcAMP</td>
			<td>BIOLOG</td>
			<td>D009</td>
		</tr>
	</tbody>
</table>

culturing primary rat inner medullary collecting duct cells

精氨酸加压素（AVP）肾集合管主细胞，从而微调体内水分的动态平衡，促进水的重吸收。 AVP的后叶加压素V2受体（V2R）的表面上的细胞结合，从而诱导的cAMP合成。这刺激细胞信号传导过程中的水通道蛋白2（AQP2）的磷酸化的变化。蛋白激酶A phoshorylates AQP2，从而触发细胞内的囊泡AQP2易位到质膜促进水的重吸收原尿。从主细胞垂体AVP激活的信号畸变AVP释放可引起中央或肾性尿崩症，分别升高血浆AVP水平与心血管疾病，如慢性心脏衰竭及抗利尿激素分泌综合征。 在这里，我们提出了一个协议cultivati对原代大鼠内髓集合管（IMCD）细胞，快递V2R和AQP2内源性的。这些细胞是适用于阐明的分子机制，水通道的控制，从而发现新AVP-介导的水的重吸收的失调相关的疾病的治疗的药物靶点。髓集合管细胞是从大鼠肾脏内髓质和六到八天播种后用于实验。髓集合管细胞可以定期细胞培养皿，培养瓶和不同格式的微滴定板中培养，该过程只需要几个小时，适合标准的细胞培养实验室。

原代大鼠IMCD细胞培育成功，将导致在一个融合的单层播种后6-8天（ 图2）。每60毫米培养皿中有大约6×10 6细胞。这些细胞紧紧坚持培养皿，这些被涂上IV型胶原，基底膜成分18。因此，IMCD细胞将无法分离，即使在一些彻底的洗涤程序。高达80％的培养细胞表达出来的内源性和AQP2。这是主要的细胞，，而AQP2细胞缺乏被认为是闰细胞，非髓集合管细胞是来自于薄的四肢，?...

Watch this Scientific Journal Video about Culturing Primary Rat Inner Medullary Collecting Duct Cells at JoVE.com

Culturing Primary Rat Inner Medullary Collecting Duct Cells

精氨酸加压素（AVP）控制体内水分的动态平衡微调肾主细胞，通过促进水的重吸收。在这里，我们提出了一个协议的初级大鼠内髓集合管细胞AVP-介导的水的重吸收的分子机制的阐明适合种植。

原代培养大鼠内髓集合管细胞

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP ...

culturing-primary-rat-inner-medullary-collecting-duct-cells

Research

JoVE Journal

Biology

14.7K 次观看.  Max-Delbrück-Center for Molecular Medicine. 精氨酸加压素（AVP）控制体内水分的动态平衡微调肾主细胞，通过促进水的重吸收。在这里，我们提出了一个协议的初级大鼠内髓集合管细胞AVP-介导的水的重吸收的分子机制的阐明适合种植。