Name: primary culture of rat adrenocortical cells and assays of steroidogenic functions
Uploaded: 2019-03-12T12:30:00
Description: Watch this Scientific Journal Video about Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions at JoVE.com

这项工作得到了台湾国家科学委员会的资助 (nsc102-2320-b-07-008-my3) 和国立城公大学医院研究补助金 (nckuh-10102045) 的支持。

所有的程序, 包括动物科目已批准高雄医科大学动物护理和使用委员会。
1. 实验程序
<ol><li>用co2安乐死产出成年 (8至12周) 女性 sd 大鼠。</li>
	<li>应用70% 乙醇, 让它被吸收到皮肤, 然后用纸巾擦拭皮肤。</li>
	<li>把老鼠放在一个干净的塑料板上仰面位置。</li>
	<li>在中线用 "外部" 弯曲钳提起皮肤, 并通过创建 "y" 形状 (从大腿的水平到肋下的角度) 来执行钝?...

<ol>
	<li>Frith, C. H., Jones, T. C., Mohr, U., Hunt, R. D., Capen, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histology,+Adrenal+Gland,+Mouse.">Histology, Adrenal Gland, Mouse.</a> Endocrine System. Monographs on Pathology of Laboratory Animals. , 8-12 (1983).</li><li>Keegan, C. E., Hammer, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+insights+into+organogenesis+of+the+adrenal+cortex.">Recent insights into organogenesis of the adrenal cortex.</a> Trends in Endocrinology Metabolism. 13 (5), 200-208 (2002).</li><li>Marieb, E., Wilhelm, P. B., Mallatt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chapter 17: The Endocrine System. , 558-581 (2017).</li><li>Chrousos, G. 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(1-2), 135-141 (2004).</li><li>Bielohuby, 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=Growth+analysis+of+the+mouse+adrenal+gland+from+weaning+to+adulthood:+time-+and+gender-dependent+alterations+of+cell+size+and+number+in+the+cortical+compartment.">Growth analysis of the mouse adrenal gland from weaning to adulthood: time- and gender-dependent alterations of cell size and number in the cortical compartment.</a> American Journal of Physiology-Endocrinology Metabolism. 293 (1), E139-E146 (2007).</li><li>van Weerden, W. M., Bierings, H. G., van Steenbrugge, G. J., de Jong, F. H., Schroder, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adrenal+glands+of+mouse+and+rat+do+not+synthesize+androgens.">Adrenal glands of mouse and rat do not synthesize androgens.</a> Life Sciences. 50 (12), 857-861 (1992).</li><li>Wang, T., Rainey, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+adrenocortical+carcinoma+cell+lines.">Human adrenocortical carcinoma cell lines.</a> Molecular and Cellular Endocrinology. 351 (1), 58-65 (2012).</li><li>Payne, A. H., Hales, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+steroidogenic+enzymes+in+the+pathway+from+cholesterol+to+active+steroid+hormones.">Overview of steroidogenic enzymes in the pathway from cholesterol to active steroid hormones.</a> Endocrine Review. 25 (6), 947-970 (2004).</li><li>Ruggiero, C., Lalli, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+ACTH+Signaling+on+Transcriptional+Regulation+of+Steroidogenic+Genes.">Impact of ACTH Signaling on Transcriptional Regulation of Steroidogenic Genes.</a> Frontiers in Endocrinology. 7, 24 (2016).</li><li>Chen, Y. C., Liang, Y. L., Huang, Y. L., Huang, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+Toona+sinensis-stimulated+adrenal+steroidogenesis+in+primary+rat+adrenal+cells.">Mechanism of Toona sinensis-stimulated adrenal steroidogenesis in primary rat adrenal cells.</a> Journal of Functional Foods. 14 (2015), 318-323 (2015).</li><li>Rybak, S. M., Ramachandran, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+culture+of+normal+rat+adrenocortical+cells.+I.+Culture+conditions+for+optimal+growth+and+function.">Primary culture of normal rat adrenocortical cells. I. Culture conditions for optimal growth and function.</a> In Vitro. 17 (7), 599-604 (1981).</li><li>Rybak, S. M., Ramachandran, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+culture+of+normal+rat+adrenocortical+cells.+II.+Quantitation+of+steroid+dehydrogenase+stain.">Primary culture of normal rat adrenocortical cells. II. Quantitation of steroid dehydrogenase stain.</a> In Vitro. 17 (7), 605-611 (1981).</li><li>O'Hare, M. J., Neville, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Steroid+metabolism+by+adult+rat+adrenocortical+cells+in+monolayer+culture.">Steroid metabolism by adult rat adrenocortical cells in monolayer culture.</a> Journal of Endocrinology. 58 (3), 447-462 (1973).</li><li>Di Blasio, A. M., Fujii, D. K., Yamamoto, M., Martin, M. C., Jaffe, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+cell+proliferation+and+steroidogenesis+in+cultured+human+fetal+adrenal+cells+chronically+exposed+to+adrenocorticotropic+hormone:+rationalization+of+in+vitro+and+in+vivo+findings.">Maintenance of cell proliferation and steroidogenesis in cultured human fetal adrenal cells chronically exposed to adrenocorticotropic hormone: rationalization of in vitro and in vivo findings.</a> Biology of Reproduction. 42 (4), 683-691 (1990).</li><li>Brasaemle, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thematic+review+series:+adipocyte+biology.+The+perilipin+family+of+structural+lipid+droplet+proteins:+stabilization+of+lipid+droplets+and+control+of+lipolysis.">Thematic review series: adipocyte biology. The perilipin family of structural lipid droplet proteins: stabilization of lipid droplets and control of lipolysis.</a> Journal of Lipid Research. 48 (12), 2547-2559 (2007).</li><li>Hoeflich, A., Bielohuby, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+adrenal+gland+growth:+signal+integration+by+extracellular+signal+regulated+kinases1/2.">Mechanisms of adrenal gland growth: signal integration by extracellular signal regulated kinases1/2.</a> Journal of Molecular Endocrinology. 42 (3), 191-203 (2009).</li><li>O'Hare, M. J., Neville, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+responses+to+corticotrophin+and+cyclic+AMP+by+adult+rat+adrenocortical+cells+in+monolayer+culture.">Morphological responses to corticotrophin and cyclic AMP by adult rat adrenocortical cells in monolayer culture.</a> Journal of Endocrinology. 56 (3), 529-536 (1973).</li></ol>

肾上腺在环境适应中起着关键作用3。肾上腺皮质分泌的激素可以调节和调节生理功能和稳态3。体内模型反映了人体的真正生理效应。然而, 它仍然受到许多复杂因素的影响, 在实验中造成不稳定的影响。相比之下, 体外细胞模型具有优势, 与动物模型相比是无与伦比的。首先, 环境条件在体外系统中易于控制。激素很可能受到任何刺激的影响。体内实验通常会产?...

内分泌系统负责调节生理活动和稳态1。肾上腺位于肾脏的颅极, 是分泌矿物皮质激素、糖皮质激素和雄激素2, 3 的主要内分泌器官之一。肾上腺有两个不同的部分: 皮层和髓质。肾上腺皮质由三层组成: 外肾小球、中间小泡和内网状 3.肾小球带是醛固酮的原始分泌位点, 醛固酮是一种矿物质皮质激素, 它有助于在肾脏中重新吸收钠离子和水 3.在正常生理条件下, 小束带主要产生糖皮质激素的基础水平3。啮齿类动物中的皮质类固醇和人类的皮质醇通过调节血糖来帮助身体应对压力.在一定程度上, 他们可以抑制炎症反应和调节免疫系统4,5。与其他哺乳动物不同的是, 由于肾上腺缺乏17α-羟化酶表达, 小鼠和大鼠没有功能性网状带.因此, 小鼠和大鼠的肾上腺缺乏肾上腺 c-19 类固醇 (皮质醇和肾上腺雄激素) 的分泌。
原代培养的肾上腺皮质细胞已被证明是有用的研究机制控制肾上腺生理 8.像其他类固醇生成组织, 每个肾上腺区域合成其类固醇从游离胆固醇9。在肾上腺皮质激素 (acth) 刺激下, 游离胆固醇从破裂的脂滴中释放, 从而提高了小泡和网状10中类固醇的产生。
许多群体试图从肾上腺皮质癌中建立稳定的细胞系。然而, 一些担忧限制了肾上腺皮质细胞系的使用, 就像体外模型 8一样。细胞系的类固醇反应可能因通道、血清批号、血清质量等而异。此外, 商业肾上腺细胞系 (y1 和 sw13) 不分泌皮质酮, 使研究肾上腺类固醇发生变得更加困难 8.使用牛和马肾上腺作为体外模型可能是一个很好的选择, 尽管来自这些市场的组织可能掩盖了一些未知的风险。与它们相比, 大鼠肾上腺的管理更容易, 来源相对较稳定, 无病原菌。总之, 本文所述的方法可用于研究大鼠肾上腺束细胞中皮质酮合成的相关机制。

<table>
	<tbody>
		<tr>
			<td>female/ male, SD/Wistar rats (8-12 wk)</td>
			<td>BioLASCO</td>
			<td></td>
			<td>male or femal rats are suitable for experiments. Female&#39;s adrenal glands are larger than male rats, however, female rats own the oestrous cycles that may influence the experiments</td>
		</tr>
		<tr>
			<td>collagenase, type II</td>
			<td>Sigma</td>
			<td>C-6885</td>
			<td>isolation of adrenal cells</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>ThermoFisher</td>
			<td>12800-017</td>
			<td>powder media for adrenal cells</td>
		</tr>
		<tr>
			<td>Ham&#39;s F12</td>
			<td>ThermoFisher</td>
			<td>21700-075</td>
			<td>powder media for adrenal cells</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>JT.baker</td>
			<td>4018</td>
			<td>buffer (powder)</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>JT.baker</td>
			<td>3506-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>Hyclone</td>
			<td>16050014</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>SAFC</td>
			<td>12103C</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin (10,000 U)-streptomycin (10,000 ug/ml)</td>
			<td>Corning</td>
			<td>30-002-Cl</td>
			<td>antibiotics</td>
		</tr>
		<tr>
			<td>curved forceps</td>
			<td>BRAUN</td>
			<td>BD343R</td>
			<td></td>
		</tr>
		<tr>
			<td>forceps</td>
			<td>BRAUN</td>
			<td>BD331R</td>
			<td></td>
		</tr>
		<tr>
			<td>scissor</td>
			<td>BRAUN</td>
			<td>BC224R</td>
			<td>cut rat skin and muscle</td>
		</tr>
		<tr>
			<td>delicate scissor</td>
			<td>BRAUN</td>
			<td>BC101R</td>
			<td>cut adrenal glands</td>
		</tr>
		<tr>
			<td>adipose-differentiation related protein</td>
			<td>Abcam</td>
			<td>ab108323</td>
			<td>antibody for lipid droplet associated protein</td>
		</tr>
		<tr>
			<td>corticosterone ELISA kit</td>
			<td>cayman</td>
			<td>500655</td>
			<td>assay for corticosterone synthesis</td>
		</tr>
		<tr>
			<td>inverted light microscopy</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>fluorescence microscope</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>JT.baker</td>
			<td>X198-07</td>
			<td>for immunofluorescence staining</td>
		</tr>
		<tr>
			<td>goat anti-rabbit IgG Alexa 488 Fluor secondary antibody</td>
			<td>ThermoFisher</td>
			<td>A-11034</td>
			<td>for immunofluorescence staining</td>
		</tr>
		<tr>
			<td>anti-ADFP</td>
			<td>Abcam</td>
			<td>108323</td>
			<td>for immunofluorescence staining</td>
		</tr>
		<tr>
			<td>ProLong Gold antifade mountant</td>
			<td>ThermoFisher</td>
			<td>P36935</td>
			<td>for immunofluorescence staining</td>
		</tr>
	</tbody>
</table>

primary culture of rat adrenocortical cells and assays of steroidogenic functions

肾上腺皮质分泌的激素对动物对抗压力和疾病至关重要。本文介绍的方法是原代培养大鼠肾上腺细胞的程序和相关功能检测 (脂滴表面蛋白的免疫荧光染色, 以及皮质酮分析)。与体内模型不同的是, 肾上腺单层培养物中的间实验变化较小, 实验条件易于控制。此外, 老鼠的来源也比其他动物更稳定, 比如牛的动物。还有几种人肾上腺细胞系 (nci-h295、nci-h295r、sw13 等) 可用于肾上腺研究。然而, 这些系的类固醇生产仍将受到多种因素的影响, 其中包括血清批号、传道数、不同基因的突变/丢失等。大鼠肾上腺皮质细胞的原代培养除了缺乏17α-羟化酶外, 是研究肾上腺生理的一种较好、更简便的方法。总之, 原发性大鼠肾上腺培养可以作为一个很好的体外平台, 研究肾上腺系统中有关试剂的机制。

使用这里描述的程序, 原代培养的肾上腺细胞可以在相反的对比显微镜下进行区分 (图 1a)。为了进一步证实细胞质内的囊泡是脂滴, 可以在第3天培养物上进行脂肪分化相关蛋白 (adrp) 的免疫荧光染色 (图 1b)。玻璃被单上长出了细胞。用磷酸盐缓冲盐水 (pbs) 对细胞进行三次清洗, 并用4% 的甲醛固定。用 pbs 清洗三次后, 细胞被阻断, ...

Watch this Scientific Journal Video about Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions at JoVE.com

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

大鼠肾上腺皮质细胞的原代培养及非甾体功能的测定

肾上腺皮质分泌的激素对动物对抗压力和疾病至关重要。在这里, 我们提出了一个方案, 培养原发性大鼠肾上腺细胞。它可以作为研究肾上腺类固醇发生和脂质生物合成中感兴趣的试剂机制的一个很好的体外平台。

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary ...

提出的协议是非常容易的，它将帮助研究人员准备初级肾上腺培养。它提供了一个简单的程序，开始肾上腺类固醇发生。该技术可用于筛查与压力相关的疾病，所有代理抑制炎症反应的潜力。演示这个程序的将是我实验室的硕士生李伟昌。首先使用70%乙醇对安乐死SD大鼠进行消毒，让它被吸收到皮肤中。用纸巾擦拭皮肤后，将大鼠放在干净的塑料板上。接下来，使用外部弯曲钳子在中线抬起皮肤。使用剪刀从大腿水平到子成本角创建 Y 形，对皮肤进行钝解剖。用70%乙醇冲洗剪刀后，切肌肉，也形成Y形。在肝脏后面的右胃后找到左肾上腺。用一对标准钳子和一对弯曲的钳子，抬起胃和肝脏，然后用另一个弯曲的钳子分离肾上腺，并把它们放入无菌盘中。使用精细钳子小心地将肾上腺从每个腺体中移出。现在，用一把精致的剪刀将肿瘤切成小块，用足够量的无血清DMEM F-12介质覆盖组织。准备三个不同浇注尺寸的巴氏移液器，通过加热燃烧器。使用第一个具有最大开口的移液器，将所有肾上腺碎片转移到含有胶原蛋白酶 II 的 50mL 锥形管中。然后在37摄氏度的水浴中孵育管子20分钟，每5分钟摇动一次，总共4次。在酶消化过程中正确执行组织分散是非常关键的。使用第二个，然后最小的倒大小移液器重复移液和孵育，直到所有组织件变小。然后在四摄氏度的冷介质中加入六倍体积的啤酒，以停止酶反应。然后在 800 次 G 下中分 10 分钟，并丢弃两次上一液。添加适当的介质体积，然后重新挂起单元格。在95%的湿度和5%的二氧化碳中，将细胞种植在所需的盘子或碗中，在37摄氏度下过夜。第二天，用无血清、温暖的DMEM F-12基基仔细清洗细胞两次。加入新鲜生长介质，将细胞保持37摄氏度、95%的湿度和5%的二氧化碳，直到第3天。利用此协议，获得了原培养大鼠肾上腺细胞。第3天肾上腺细胞在相位对比显微镜下得到确认。第3天培养物进行的脂肪分化相关蛋白质的免疫荧光染色证实，细胞质内的囊泡是脂质液滴。在大鼠肾上腺细胞的肾上腺素激素刺激后，通过皮质酮测定细胞的激素生成能力，显示皮质酮释放增加，ACTH浓度较高。该方法可应用于其他初级培养系统，如原发性心脏培养、肾上腺内腺培养等。这种技术还可能有助于研究肾上腺皮质细胞和色度芬细胞之间的相互作用。

Research

JoVE Journal

Biology

Primary Culture of Adult Rat Heart Myocytes

成年大鼠心脏心肌细胞的原代培养

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

科研

生物学

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

原代培养及质粒电穿孔尔蒂小鼠器官。

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

前初级人类输卵管上皮细胞的体外培养

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

Isolation and Primary Culture of Rat Hepatic Cells

大鼠肝细胞的分离和小学文化

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Culturing Primary Rat Inner Medullary Collecting Duct Cells

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 ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

小鼠原代胰腺腺泡细胞的分离和培养

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

Viability Assays for Cells in Culture

活性检测在培养细胞

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

隔离和鼠标主动脉内皮细胞原代培养

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

A Tissue Culture Model of Estrogen-producing Primary Bovine Granulosa Cells

雌激素原代牛颗粒细胞的组织培养模型

Ovarian granulosa cells (GC) are the major source of estradiol synthesis. Induced by the preovulatory luteinizing hormone (LH) surge, cells of the theca ...

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

微型猪原主主动脉内皮细胞的隔离与培养

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a ...