骨髓间充质干细胞及补脑Ⅰ号处理后血清对小鼠海马神经元缺氧缺糖模型ICAM-1、NF200表达的影响

doi:10.3877/cma.j.issn.1674-0785.2022.01.017

中华临床医师杂志(电子版) ›› 2022, Vol. 16 ›› Issue (01) : 100 -106. doi: 10.3877/cma.j.issn.1674-0785.2022.01.017

基础研究

骨髓间充质干细胞及补脑Ⅰ号处理后血清对小鼠海马神经元缺氧缺糖模型ICAM-1、NF200表达的影响

杨辉¹^,^†(

), 鲁利香², 易健³, 易旭⁴

^1. 550001　贵阳，贵州中医药大学第二临床医学院神经内科
^2. 550001　贵阳，贵州中医药大学研究生院
^3. 410007　长沙，湖南中医药大学第一附属医院中心实验室
^4. 550001　贵阳，贵州中医药大学第二附属医院中心实验室

收稿日期:2021-08-15 出版日期:2022-01-15
通信作者: 杨辉
基金资助:
国家自然科学基金项目(81260601)

Effects of BMSCs and Bunao No. 1 medicated serum on expression of ICAM-1 and NF200 in mouse hippocampal neurons with oxygen-glucose deprivation

Hui Yang¹^,^†(), Lixiang Lu², Jian Yi³, Xu Yi⁴

^1. Department of Neurology, the Second Clinical Medical College, Guizhou University of Traditional Chinese Medicine, Guiyang 550001, China
^2. Graduate School of Guizhou University of Traditional Chinese Medicine, Guiyang 550001, China
^3. Central Laboratory, the First Affiliated Hospital of Hunan University of Traditional Chinese Medicine, Changsha 410007, China
^4. Central Laboratory, the Second Affiliated Hospital of Guizhou University of Traditional Chinese Medicine, Guiyang 550001, China

Received:2021-08-15 Published:2022-01-15
Corresponding author: Hui Yang

全文 ( ) 下载PDF ( ) 导出引用

引用本文：

杨辉, 鲁利香, 易健, 易旭. 骨髓间充质干细胞及补脑Ⅰ号处理后血清对小鼠海马神经元缺氧缺糖模型ICAM-1、NF200表达的影响[J]. 中华临床医师杂志(电子版), 2022, 16(01): 100-106.

Hui Yang, Lixiang Lu, Jian Yi, Xu Yi. Effects of BMSCs and Bunao No. 1 medicated serum on expression of ICAM-1 and NF200 in mouse hippocampal neurons with oxygen-glucose deprivation[J]. Chinese Journal of Clinicians(Electronic Edition), 2022, 16(01): 100-106.

目的

观察骨髓间充质干细胞（BMSCs）、补脑Ⅰ号血清对小鼠海马神经元缺氧缺糖（OGD）模型神经中丝200（NF200）、细胞间黏附因子-1（ICAM-1）的影响。

方法

分离培养BMSCs、海马神经元，对海马神经元进行OGD造模。制备补脑Ⅰ号血清与正常血清。经MTT法筛选补脑Ⅰ号血清对海马神经元OGD模型干预的最佳浓度为10%，时间为72 h。实验分为正常组、模型组、BMSCs组、正常血清组、中药血清组、BMSCs＋正常血清组、BMSCs＋中药血清组。qRT-PCR检测细胞NF200、ICAM-1 mRNA表达；Western Blot检测细胞NF200、ICAM-1蛋白表达。

结果

与正常组比较，模型组、BMSCs组、正常血清组、BMSCs＋正常血清组、中药血清组、BMSCs＋中药血清组ICAM-1、NF200 mRNA及蛋白表达升高（P＜0.05）；与模型组比较，BMSCs组、BMSCs＋正常血清组、中药血清组、BMSCs＋中药血清组NF200 mRNA及蛋白表达升高，ICAM-1 mRNA及蛋白表达降低，差异有统计学意义（P＜0.05）；BMSCs＋中药血清组较BMSCs组、正常血清组、中药血清组及BMSCs＋正常血清组的NF200 mRNA及蛋白表达升高，ICAM-1 mRNA及蛋白表达减少，差异有统计学意义（P＜0.05）。

结论

BMSCs和补脑Ⅰ号血清可能通过抑制炎症、促进神经再生及双重作用修复OGD损伤的海马神经元，两者联合应用效果优于单独使用。

关键词: 补脑Ⅰ号, 骨髓间充质干细胞, 缺氧缺糖, 海马神经元, 神经中丝200, 细胞间黏附因子-1

Objective

To observe the effects of Bunao No.1 medicated serum and bone marrow mesenchymal stem cells (BMSCs) on neurofilament 200 (NF200) and intercellular adhesion factor-1 (ICAM-1) expression in hippocampal neurons with oxygen-glucose deprivation (OGD).

Methods

BMSCs and hippocampal neurons were isolated and cultured, and hippocampal neurons were induced by OGD. Bunao No. 1 medicated serum and normal serum were prepared. MTT assay showed that the optimal concentration of Bunao No. 1 medicated serum for the intervention of hippocampal neurons with OGD was 10%, and the best duration was 72 hours. The cells was divided into a normal group, model group, BMSCs group, normal serum group, Bunao No. 1 medicated serum group, BMSCs＋normal serum group, and BMSCs＋Bunao No. 1 medicated serum group. The mRNA and protein expression of NF200 and ICAM-1 were detected by qRT-PCR and Western Blot, respectively.

Results

Compared with the normal group, the mRNA and protein expression of ICAM-1 and NF200 in the model group, BMSCs group, normal serum group, BMSCs＋normal serum group, Bunao No.1 medicated serum group, and BMSCs＋Bunao No.1 medicated serum group increased significantly (P＜0.05). Compared with the model group, the mRNA and protein expression of NF200 significantly increased and those of ICAM-1 significantly decreased in the BMSCs group, BMSCs＋normal serum group, Bunao No. 1 medicated serum group, and BMSCs＋Bunao No.1 medicated serum group (P＜0.05). Compared with the BMSCs group, normal serum group, Bunao No.1 medicated serum group, and BMSCs＋normal serum group, the mRNA and protein expression of NF200 significantly increased and those of ICAM-1 significantly decreased in the BMSCs＋Bunao No.1 medicated serum group (P＜0.05).

Conclusion

BMSCs and Bunao No.1 medicated serum can inhibit inflammation, promote nerve regeneration, and repair the hippocampal neurons damaged by OGD. Combined use of BMSCs and Bunao No.1 medicated serum is better than their single use.

Key words: Bunao No.1, Bone marrow mesenchymal stem cells, Hypoxia and glucose deficiency, Hippocampal neurons, Neurofilament 200, Intercellular adhesion

图1 骨髓间充质干细胞（BMSCs）原代培养（×100）。图1a为BMSCs原代培养48 h见细胞贴壁；图1b为BMSCs原代培养72 h细胞伸展生长，呈多边形、三角形、短梭形等；图1c为BMSCs原代培养第10天，呈伸展生长

图2 骨髓间充质干细胞（BMSCs）表面标记情况。图2a为CD44阳性率为80.1%；图2b为CD45阴性率为85.6%；图2c为CD90阳性率为84.0%注：Count为

图3 P3代骨髓间充质干细胞（BMSCs）的生长曲线注：OD为

图4 神经元特异性烯醇化酶（NSE）免疫组化鉴定（×100）

图5 海马神经元缺氧缺糖（OGD）造模（×100）

图6 各浓度补脑Ⅰ号含药血清对骨髓间充质干细胞（BMSCs）增殖的影响注：OD为

表1 各组小鼠海马神经元细胞ICAM-1、NF200 mRNA表达量比较（

x ˉ

±s，n=3）

组别	ICAM-1	NF200
正常组	0.25±0.09	0.32±0.11
模型组	0.97±0.18^a	0.61±0.63^a
BMSCs组	0.66±0.12^abcd	1.96±0.58^abcd
正常血清组	0.99±0.14^ad	0.71±0.26^ad
BMSCs＋正常血清组	0.69±0.13^abcd	1.92±0.50^abcd
中药血清组	0.48±0.11^abc	2.86±0.62^abc
BMSCs＋中药血清组	0.36±0.07^abc	4.61±1.16^abc

表1 各组小鼠海马神经元细胞ICAM-1、NF200 mRNA表达量比较（

x ˉ

±s，n=3）

组别	ICAM-1	NF200
正常组	0.25±0.09	0.32±0.11
模型组	0.97±0.18^a	0.61±0.63^a
BMSCs组	0.66±0.12^abcd	1.96±0.58^abcd
正常血清组	0.99±0.14^ad	0.71±0.26^ad
BMSCs＋正常血清组	0.69±0.13^abcd	1.92±0.50^abcd
中药血清组	0.48±0.11^abc	2.86±0.62^abc
BMSCs＋中药血清组	0.36±0.07^abc	4.61±1.16^abc

图7 各组海马神经元细胞ICAM-1、NF200蛋白表达情况注：A为正常组；B为模型组；C为BMSCs组；D为正常血清组；E为BMSCs＋正常血清组；F为中药血清组；G为BMSCs＋中药血清组；NF200为神经丝蛋白200；ICAM-1为细胞间黏附因子-1；BMSCs为骨髄间充质干细胞

表2 各组小鼠海马神经元细胞ICAM-1、NF200蛋白表达量比较（

x ˉ

±s，n=3）

组别	ICAM-1	NF200
正常组	0.028±0.016	0.029±0.014
模型组	0.694±0.338^a	0.072±0.015^a
BMSCs组	0.605±0.285^abcd	0.373±0.017^abcd
正常血清组	0.682±0.323^abd	0.087±0.021^abd
BMSCs＋正常血清组	0.595±0.285^abcd	0.365±0.012^abcd
中药血清组	0.478±0.225^abc	0.510±0.024^abc
BMSCs＋中药血清组	0.320±0.151^abc	0.620±0.021^abc

表2 各组小鼠海马神经元细胞ICAM-1、NF200蛋白表达量比较（

x ˉ

±s，n=3）

组别	ICAM-1	NF200
正常组	0.028±0.016	0.029±0.014
模型组	0.694±0.338^a	0.072±0.015^a
BMSCs组	0.605±0.285^abcd	0.373±0.017^abcd
正常血清组	0.682±0.323^abd	0.087±0.021^abd
BMSCs＋正常血清组	0.595±0.285^abcd	0.365±0.012^abcd
中药血清组	0.478±0.225^abc	0.510±0.024^abc
BMSCs＋中药血清组	0.320±0.151^abc	0.620±0.021^abc

1	BartoLome F, de La Cueva M, PascuaL C, et al. Amyloid β-induced impairments on mitochondrial dynamics, hippocampal neurogenesis, and memory are restored by phosphodiesterase 7 inhibition [J]. ALzheimers Res Ther, 2018, 10(1): 24.
2	Scher AI, Xu Y, Korf ES, et al. Hippocampal shape analysis in Alzheimer's disease: a population-based study [J]. Neuroimage, 2007, 36(1): 8-18.
3	Iturria-Medina Y, Sotero RC, Toussaint PJ, et al. Alzheimer's disease neuroimaging initiative. Early role of vascular dysregulation on late-onset Alzheimer's disease based on multifactorial data-driven analysis [J]. Nat Commun, 2016, 7: 11934.
4	Arvanitakis Z, Capuano AW, Leurgans SE, et al. Relation of cerebral vessel disease to Alzheimer's disease dementia and cognitive function in elderly people:a cross-sectional study [J]. Lancet Neurol, 2016, 15(9): 934-943.
5	况时祥, 杨辉, 刘琛, 等. 补脑Ⅰ号治疗老年人轻度认知功能障碍临床观察 [J]. 新中医, 2011, 43(8): 31-32.
6	赵芝兰, 况时祥, 张树森, 等. 补脑Ⅰ号对复合Alzheimer病模型大鼠学习记忆障碍的改善及其作用机制实验研究 [J]. 新中医, 2013, 45(7): 161-163.
7	赵芝兰, 况时祥, 薛红, 等. 补脑Ⅰ号对阿尔茨海默病模型大鼠早期淀粉样蛋白和突触结构与功能的影响 [J]. 时珍国医国药, 2013, 24(6): 1393-1394.
8	杨辉, 贾桃, 况时祥, 等. BMSCs移植对AD大鼠海马组织SOD、AchE活性与MDA、Ach含量的影响及补脑Ⅰ号的干预作用 [J]. 贵阳中医学院学报, 2019, 41(1): 17-20.
9	屈相玲, 韩云霞, 周训蓉, 等. 参蓉胶囊对痴呆小鼠学习记忆能力及海马CA1区NF-κB、IκB-α、Caspase-3蛋白表达的影响 [J]. 时珍国医国药, 2019, 30(12): 2890-2892.
10	杨辉, 鲁利香, 肖政华, 等. 缺氧缺糖海马神经元模型最佳造模时间的选择 [J]. 贵州医科大学学报, 2017, 42(7): 778-782.
11	汪晖. 药理学实验 [M]. 武汉: 湖北科学技术出版社, 2007: 20.
12	Gao JJ, Hu YW, Wang YC, et al. ApoM suppresses TNF-а-induced expression of ICAM-1 and VCAM-1 through inhibiting the activity of NF-κB [J]. DNA Cell Biol, 2015, 34(8): 550-556.
13	Yusuf-Makagiansar H, Anderson ME, Yakovleva TV, et al. Inhibition of LFA-1/ICAM-1 and VLA-4/VCAM-1 as a therapeutic approach to inflammation and autoimmune disease [J]. Med Res Rev, 2002, 22(2): 146-167.
14	刑娟, 范崇桂, 沈雷, 等. 芹黄素对缺血缺氧性脑损伤大鼠的神经保护作用 [J]. 神经解剖学杂志, 2019, 35(5): 515-520.
15	巩敏杰, 安佳琪, 吴锋, 等. 褪黑素通过 Nrf2/HO-1信号通路减轻大鼠脑缺血再灌注损伤 [J]. 西安交通大学学报(医学版), 2019, 40(6): 857-863, 876.

[1]	于秀章, 郄明蓉, 侯敏敏. 子宫内膜异位症与干细胞研究现状[J]. 中华妇幼临床医学杂志(电子版), 2021, 17(02): 132-137.
[2]	宋斯迪, 鲁曦, 金发光, 梁源, 鱼高乐. SDF-1α/CXCR4信号促进骨髓间充质干细胞对海水吸入性肺损伤的治疗作用[J]. 中华肺部疾病杂志(电子版), 2019, 12(01): 28-33.
[3]	周艳群, 陈鹏, 刘增慧, 毛晶晶, 黎耀和. 多发性骨髓瘤患者骨髓间充质干细胞衰老关键基因和通路的生物信息学分析与验证[J]. 中华细胞与干细胞杂志(电子版), 2022, 12(05): 274-281.
[4]	夏杰, 唐紫萌, 吴向未. E-钙黏蛋白对血管内皮祖细胞和骨髓间充质干细胞之间的黏附作用研究[J]. 中华细胞与干细胞杂志(电子版), 2021, 11(06): 343-350.
[5]	刘一栋, 贾玉凤, 王燕, 刘扬, 宁金月. 大豆异黄酮上调Wnt-1基因表达促进骨髓间充质干细胞增殖和成骨分化的研究[J]. 中华细胞与干细胞杂志(电子版), 2021, 11(05): 292-297.
[6]	李应明, 陈华, 伍燕. K562来源的外泌体对骨髓间充质干细胞造血和成骨基因表达的影响[J]. 中华细胞与干细胞杂志(电子版), 2021, 11(04): 207-214.
[7]	钱道海, 宋国栋, 张洲, 马志龙, 王冠男, 于文建, 陈鹏, 王小明. 骨髓间充质干细胞对重症急性胰腺炎胰腺组织修复的促进作用研究[J]. 中华细胞与干细胞杂志(电子版), 2019, 09(06): 351-357.
[8]	朱聪, 黄国锋, 江惠祥, 吴本文, 林剑彪, 林伟斌, 高明明, 丁真奇. 间歇式轴向压应力对组织工程骨种子细胞的黏附增殖与成骨分化促进作用的研究[J]. 中华细胞与干细胞杂志(电子版), 2018, 08(06): 334-342.
[9]	张睿, 崔乐. IL-12重组质粒联合骨髓间充质干细胞对MCF-7人乳腺癌细胞侵袭及裸鼠移植瘤生长的影响分析[J]. 中华细胞与干细胞杂志(电子版), 2018, 08(03): 140-145.
[10]	谢林岑, 陈月秋, 沈振亚. 高表达microRNA-155的骨髓间充质干细胞对免疫调节的影响[J]. 中华细胞与干细胞杂志(电子版), 2018, 08(02): 88-94.
[11]	冉凤英, 陈龙, 张斌强, 尚兵, 余飞, 陈炜, 陈琴华. TLRs信号通路激活的MSCs外泌体对巨噬细胞极化的影响[J]. 中华细胞与干细胞杂志(电子版), 2018, 08(02): 65-71.
[12]	刘然然, 方倩倩, 唐泽文. 周围神经损伤对骨髓间充质干细胞增殖及成骨分化影响的研究[J]. 中华神经创伤外科电子杂志, 2023, 09(01): 7-11.
[13]	张伟丽, 罗敏, 彭江, 赵斌. BMP-2/Smads信号通路促进骨代谢失衡大鼠骨髓间充质干细胞的成骨分化[J]. 中华老年骨科与康复电子杂志, 2021, 07(02): 65-72.
[14]	张威, 魏雅楠, 韩娜. 骨髓间充质干细胞来源外泌体对促进大鼠坐骨神经钳夹伤的修复作用[J]. 中华临床医师杂志(电子版), 2021, 15(04): 265-271.
[15]	周敏, 武东辉, 吴亚霖, 庞静雯, 庄秀妹. 亲水性钛表面微纳米形貌对大鼠骨髓间充质干细胞增殖与成骨分化的影响[J]. 中华临床医师杂志(电子版), 2018, 12(02): 98-102.

阅读次数

全文

摘要

选择文件类型/文献管理软件名称

选择包含的内容

Effects of BMSCs and Bunao No. 1 medicated serum on expression of ICAM-1 and NF200 in mouse hippocampal neurons with oxygen-glucose deprivation

模态框（Modal）标题

选择文件类型/文献管理软件名称

选择包含的内容

Effects of BMSCs and Bunao No. 1 medicated serum on expression of ICAM-1 and NF200 in mouse hippocampal neurons with oxygen-glucose deprivation