大鼠肾上腺皮质细胞的原代培养及非甾体功能的测定
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| female/ male, SD/Wistar rats (8-12 wk) | BioLASCO | male or femal rats are suitable for experiments. Female's adrenal glands are larger than male rats, however, female rats own the oestrous cycles that may influence the experiments | |
| collagenase, type II | Sigma | C-6885 | isolation of adrenal cells |
| DMEM | ThermoFisher | 12800-017 | powder media for adrenal cells |
| Ham's F12 | ThermoFisher | 21700-075 | powder media for adrenal cells |
| HEPES | JT.baker | 4018 | buffer (powder) |
| NaHCO3 | JT.baker | 3506-1 | |
| Horse serum | Hyclone | 16050014 | |
| Fetal bovine serum | SAFC | 12103C | |
| penicillin (10,000 U)-streptomycin (10,000 ug/ml) | Corning | 30-002-Cl | antibiotics |
| curved forceps | BRAUN | BD343R | |
| forceps | BRAUN | BD331R | |
| scissor | BRAUN | BC224R | cut rat skin and muscle |
| delicate scissor | BRAUN | BC101R | cut adrenal glands |
| adipose-differentiation related protein | Abcam | ab108323 | antibody for lipid droplet associated protein |
| corticosterone ELISA kit | cayman | 500655 | assay for corticosterone synthesis |
| inverted light microscopy | Leica | ||
| fluorescence microscope | Nikon | ||
| Triton X-100 | JT.baker | X198-07 | for immunofluorescence staining |
| goat anti-rabbit IgG Alexa 488 Fluor secondary antibody | ThermoFisher | A-11034 | for immunofluorescence staining |
| anti-ADFP | Abcam | 108323 | for immunofluorescence staining |
| ProLong Gold antifade mountant | ThermoFisher | P36935 | for immunofluorescence staining |
References
- Frith, C. H., Jones, T. C., Mohr, U., Hunt, R. D., Capen, C. C. Histology, Adrenal Gland, Mouse. Endocrine System. Monographs on Pathology of Laboratory Animals. , 8-12 (1983).
- Keegan, C. E., Hammer, G. D. Recent insights into organogenesis of the adrenal cortex. Trends in Endocrinology Metabolism. 13 (5), 200-208 (2002).
- Marieb, E., Wilhelm, P. B., Mallatt, J. . Chapter 17: The Endocrine System. , 558-581 (2017).
- Chrousos, G. P. The hypothalamic-pituitary-adrenal axis and immune-mediated inflammation. New England Journal Medicine. 332 (20), 1351-1362 (1995).
- Bornstein, S. R., Rutkowski, H., Vrezas, I. Cytokines and steroidogenesis. Molecular and Cellular Endocrinology. (1-2), 135-141 (2004).
- Bielohuby, M., et al. 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. American Journal of Physiology-Endocrinology Metabolism. 293 (1), E139-E146 (2007).
- van Weerden, W. M., Bierings, H. G., van Steenbrugge, G. J., de Jong, F. H., Schroder, F. H. Adrenal glands of mouse and rat do not synthesize androgens. Life Sciences. 50 (12), 857-861 (1992).
- Wang, T., Rainey, W. E. Human adrenocortical carcinoma cell lines. Molecular and Cellular Endocrinology. 351 (1), 58-65 (2012).
- Payne, A. H., Hales, D. B. Overview of steroidogenic enzymes in the pathway from cholesterol to active steroid hormones. Endocrine Review. 25 (6), 947-970 (2004).
- Ruggiero, C., Lalli, E. Impact of ACTH Signaling on Transcriptional Regulation of Steroidogenic Genes. Frontiers in Endocrinology. 7, 24 (2016).
- Chen, Y. C., Liang, Y. L., Huang, Y. L., Huang, B. M. Mechanism of Toona sinensis-stimulated adrenal steroidogenesis in primary rat adrenal cells. Journal of Functional Foods. 14 (2015), 318-323 (2015).
- Rybak, S. M., Ramachandran, J. Primary culture of normal rat adrenocortical cells. I. Culture conditions for optimal growth and function. In Vitro. 17 (7), 599-604 (1981).
- Rybak, S. M., Ramachandran, J. Primary culture of normal rat adrenocortical cells. II. Quantitation of steroid dehydrogenase stain. In Vitro. 17 (7), 605-611 (1981).
- O’Hare, M. J., Neville, A. M. Steroid metabolism by adult rat adrenocortical cells in monolayer culture. Journal of Endocrinology. 58 (3), 447-462 (1973).
- Di Blasio, A. M., Fujii, D. K., Yamamoto, M., Martin, M. C., Jaffe, R. B. 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. Biology of Reproduction. 42 (4), 683-691 (1990).
- Brasaemle, D. L. Thematic review series: adipocyte biology. The perilipin family of structural lipid droplet proteins: stabilization of lipid droplets and control of lipolysis. Journal of Lipid Research. 48 (12), 2547-2559 (2007).
- Hoeflich, A., Bielohuby, M. Mechanisms of adrenal gland growth: signal integration by extracellular signal regulated kinases1/2. Journal of Molecular Endocrinology. 42 (3), 191-203 (2009).
- O’Hare, M. J., Neville, A. M. Morphological responses to corticotrophin and cyclic AMP by adult rat adrenocortical cells in monolayer culture. Journal of Endocrinology. 56 (3), 529-536 (1973).
