老年大鼠神经组织星形胶质细胞的分选培养与炎性特征分析*

doi:10.12047/j.cjap.6275.2022.071

中国应用生理学杂志 ›› 2022, Vol. 38 ›› Issue (4): 379-384.doi: 10.12047/j.cjap.6275.2022.071

• 技术方法 • 上一篇

老年大鼠神经组织星形胶质细胞的分选培养与炎性特征分析^*

岳夏雅, 赵欣^△, 张雨彤, 郝娜, 郭霞, 张宇

细胞生理学教育部重点实验室, 山西医科大学生理学系, 太原 030001

收稿日期:2022-02-17 修回日期:2022-07-07 出版日期:2022-07-28 发布日期:2022-11-23
通讯作者: ^△Tel: (0351) 3985163; E-mail: zhyucnm@163.com, xinzhao2006@163.com
基金资助:
^*国家自然科学基金项目(81371254); 山西省‘1331工程’重点学科建设计划经费(XK201708); 山西省自然科学基金(201801D121318); 山西省省筹资金资助回国留学人员科研项目(2021-080); 山西省研究生创新项目(2021Y406)

Isolation, culture and inflammatory characteristics of astrocytes in aged rats nervous tissue

YUE Xia-ya, ZHAO Xin^△, ZHANG Yu-tong, HAO Na, GUO Xia, ZHANG Yu

Key Laboratory of Cellular Physiology, Ministry of Education; Department of Physiology, Shanxi Medical University, Taiyuan 030001, China

Received:2022-02-17 Revised:2022-07-07 Online:2022-07-28 Published:2022-11-23

摘要/Abstract

摘要： 目的: 建立优化的年轻与老年大鼠神经组织星形胶质细胞的分离纯化方法,比较年轻大鼠与老年大鼠星形胶质细胞的形态、功能差异,探讨老化后星形胶质细胞的功能改变及其在衰老过程中发挥的可能机制。方法: 采用50%-35%的percoll密度梯度离心法分选年轻(2月龄)和老年(20月龄)SD大鼠的大脑与脊髓星形胶质细胞;每组细胞设置3个复孔,培养72 h后,采用免疫荧光检测星形胶质细胞特异性标志物胶质纤维酸性蛋白(GFAP),观察不同年龄阶段星形胶质细胞的形态特征;qPCR检测衰老标志(p16、p21)的表达,β-半乳糖苷酶染色检测星形胶质细胞的衰老情况;qPCR检测促炎因子(IL-1β、TNF-α)与抗炎因子(IL-10)的表达水平。结果: 采用50%-35%的percoll梯度分选得到的星形胶质细胞的数量多、活性好、纯度高达95%以上,可用于后续实验。与年轻大鼠神经组织的星形胶质细胞相比,分选自老年大鼠神经组织的星形胶质细胞在细胞形态上偏向激活态,突起较少;星形胶质细胞β-半乳糖苷酶染色阳性率升高,p16、p21表达也明显增多(P<0.01);老年大鼠神经组织的星形胶质细胞的促炎因子(IL-1β、TNF-α)表达升高(P<0.05),抗炎因子(IL-10)表达有所降低(P<0.05)。结论: 50%-35%的percoll梯度可以作为大鼠神经组织星形胶质细胞的分选纯化、原代培养的方法;随着年龄的增加,星形胶质细胞发生细胞老化,表现出促炎症表型,促进神经系统的炎性衰老,可能是神经系统老化及神经退行性疾病的机制之一。

关键词: 星形胶质细胞, 神经组织, 老化, 炎性因子, 细胞培养, 大鼠

Abstract: Objective: To establish an optimized method for the isolation and purification of astrocytes from the neural tissues of young and aged rats. Then, the morphological and functional differences of astrocytes between young and aged rats were compared to explore the functional changes of astrocytes after aging and its possible mechanism in the aging process. Methods: Young (2 months old) and aged (20 months old) SD rats were used. Astrocytes in brain and spinal cord tissue were purified by 50% - 35% percoll density gradient centrifugation. Each group of cells was set up with three duplicate wells. After 72 h of culture, Glial fibrillary acidic protein (GFAP) which was astrocyte specific marker were detected by immunofluorescence to evaluate the morphological characteristics. Cell senescence markers (p16 and p21) and β- Galactosidase were detected by qPCR and staining respectively. The expressions of pro-inflammatory cytokines (IL-1β, TNF-α) and anti-inflammatory cytokines were detected by qPCR. Results: Using 50%-35% percoll gradient separation, astrocytes were obtained with large number, good activity and purity of more than 95%, which could be used in subsequent experiments. Compared with the astrocytes in the nerve tissue of young rats, the astrocytes in the nervous tissue of the aged rats had fewer protrusions and tended to be activated in cell morphology; the positive rate of β -galactosidase staining was increased significantly and the expressions of p16 and p21 were increased (P<0.01). The expressions of pro-inflammatory cytokines (IL-1β, TNF-α) were increased (P<0.05), and the expression of anti-inflammatory cytokine (IL-10) was decreased (P<0.05) in astrocytes of the aged rats nervous tissue. Conclusion: The percoll gradient of 50% - 35% could be used as a method for separation, purification and primary culture of astrocytes. With the increase of age, astrocytes undergo cellular senescence, showing a pro-inflammatory phenotype, promoting inflammaging of the nervous system, which may be one of the mechanisms of nervous system aging and neurodegenerative diseases.

Key words: astrocytes, nervous tissue, aging, inflammatory cytokines, cell culture, rats

中图分类号:

R338.8

岳夏雅, 赵欣, 张雨彤, 郝娜, 郭霞, 张宇. 老年大鼠神经组织星形胶质细胞的分选培养与炎性特征分析^*[J]. 中国应用生理学杂志, 2022, 38(4): 379-384.

YUE Xia-ya, ZHAO Xin, ZHANG Yu-tong, HAO Na, GUO Xia, ZHANG Yu. Isolation, culture and inflammatory characteristics of astrocytes in aged rats nervous tissue[J]. CJAP, 2022, 38(4): 379-384.

参考文献

[1] 宋博雅, 郝选明. 运动干预对衰老模型大鼠血清炎性因子水平的影响[J]. 中国应用生理学杂志, 2020, 36(1): 42-45.
[2] Franceschi C, Capri M, Monti D, et al. Inflammaging and anti-inflammaging: a systemic perspective on aging and longevity emerged from studies in humans[J]. Mech Ageing Dev, 2007, 1281(1): 92-105.
[3] Walsh ME, Sloane LB, Fischer KE, et al. Use of nerve conduction velocity to assess peripheral nerve health in aging mice[J]. J Gerontol A Biol Sci Med Sci, 2015, 70(11): 1312-1319.
[4] Linnerbauer M, Wheeler MA, Quintana FJ.Astrocyte crosstalk in CNS inflammation[J]. Neuron, 2020, 108(4): 608-622.
[5] Han RT, Kim RD, Molofsky AV, et al. Astrocyte-immune cell interactions in physiology and pathology[J]. Immunity, 2021, 54(2): 211-224.
[6] Rodriguez-Arellano JJ, Parpura V, Zorec R, et al. Astrocytes in physiological aging and Alzheimer's disease[J]. Neuroscience, 2016, 323(5): 170-182.
[7] Li Y, Ning L, Yin Y, et al. Age-related shifts in gut microbiota contribute to cognitive decline in agedrats[J]. Aging (Albany NY), 2020, 12(9): 7801-7817.
[8] Verkhratsky A, Nedergaard M.Physiology of astroglia[J]. Physiol Rev, 2018, 98(1): 239-389.
[9] Sikora E, Bielak-Zmijewska A, Mosieniak G.What is and what is not cell senescence[J]. Postepy Biochem, 2018, 64(2): 110-118.
[10] Vazquez-Villasenor I, Garwood CJ, Heath PR, et al. Expression of p16 and p21 in the frontal association cortex of ALS/MND brains suggests neuronal cell cycle dysregulation and astrocyte senescence in early stages of thedisease[J]. Neuropathol Appl Neurobiol, 2020, 46(2): 171-185.
[11] Clarke LE, Liddelow SA, Chakraborty C, et al. Normal aging induces A1-like astrocyte reactivity[J]. Proc Natl Acad Sci U S A, 2018, 115(8): E1896-E1905.
[12] Giovannoni F, Quintana FJ.The role of astrocytes in CNS inflammation[J]. Trends Immunol, 2020, 41(9): 805-819.
[13] 康文博, 李晓红, 陈翀, 等. 亚低温对脊髓损伤后反应性星形胶质细胞增生的影响[J]. 中国应用生理学杂志, 2016, 32(4): 289-292.
[14] Chai B, Zheng ZH, Liao X, et al. The protective role of omentin-1 in IL-1beta-induced chondrocyte senescence[J]. Artif Cells Nanomed Biotechnol, 2020, 48(1): 8-14.
[15] Juric DM, Carmankrzan M.Interleukin-1 beta, but not IL-1 alpha, mediates nerve growth factor secretion from rat astrocytes via type I IL-1 receptor[J]. Int J Dev Neurosci, 2001, 19(7): 675-683.
[16] Dvoriantchikova G, Ivanov D.Tumor necrosis factor-alpha mediates activation of NF-κB and JNK signaling cascades in retinal ganglion cells and astrocytes in opposite ways[J]. Eur J Neurosci, 2014, 40(8): 3171-3178.
[17] He P, Liu Q, Wu J, et al. Genetic deletion of TNF receptor suppresses excitatory synaptic transmission via reducing AMPA receptor synaptic localization in corticalneurons[J]. FASEB J, 2012, 26(1): 334-345.
[18] Iyer SS, Cheng G.Role of interleukin 10 transcriptional regulation in inflammation and autoimmune disease[J]. Crit Rev Immunol, 2012, 32(1): 23-63.
[19] Ferrucci L, Fabbri E.Inflammageing: chronic inflammation in ageing, cardiovascular disease, and frailty[J]. Nat Rev Cardiol, 2018, 159: 505-522.
[20] vonLeden RE, Khayrullina G, Moritz KE, et al. Age exacerbates microglial activation, oxidative stress, inflammatory and NOX2 gene expression, and delays functional recovery in a middle-aged rodent model of spinal cord injury[J]. J Neuroinflamm, 2017, 14(1): 161-176.

老年大鼠神经组织星形胶质细胞的分选培养与炎性特征分析^*

Isolation, culture and inflammatory characteristics of astrocytes in aged rats nervous tissue

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