miR-15b基因干扰在脑缺血再灌注损伤中的作用及机制研究

doi:10.11958/20220433

摘要/Abstract

摘要：

目的探讨微小核糖核酸-15b（miR-15b）基因干扰对脑缺血再灌注损伤的影响及其作用机制。方法取新生24 h内Wistar乳鼠的大脑皮层星形胶质细胞传代培养并鉴定，氧糖剥夺/再恢复法处理模拟体内脑缺血再灌注损伤（模型组）;无特殊处理的大脑皮层星形胶质细胞为对照组。构建miR-15b干扰腺病毒载体及阴性对照载体，分别转染上述模型组细胞，记为过表达组、沉默组、过表达对照组、沉默对照组，另设置空白组。24 h后，倒置相差显微镜观察各组细胞形态学改变;氮兰四唑盐（MTS）法检测细胞存活率，乳酸脱氢酶（LDH）漏出率实验检测细胞活力;流式细胞术检测细胞凋亡率;实时荧光定量聚合酶链反应检测各组细胞miR-15b以及B淋巴细胞瘤-2（Bcl-2）、胱天蛋白酶-3（Caspase-3）、Caspase-9 mRNA表达;Western blot检测各组细胞Bcl-2、Caspase-3、Caspase-9蛋白表达;荧光素酶报告基因实验验证miR-15b是否靶向调控Bcl-2。结果与对照组比，空白组、过表达对照组和沉默对照组贴壁细胞减少，细胞缩小变圆，分布松散，细胞存活率和Bcl-2 mRNA及蛋白表达降低，细胞活力、凋亡率、miR-15b表达、Caspase-3和Caspase-9 mRNA及蛋白表达升高（P<0.05）;与空白组和过表达对照组比，过表达组贴壁细胞减少，分布更加稀疏，可见细胞漂浮于培养液中，细胞存活率及Bcl-2 mRNA和蛋白表达降低，细胞活力、凋亡率、miR-15b表达、Caspase-3和Caspase-9 mRNA和蛋白表达升高（P<0.05）;与空白组和沉默对照组比，沉默组贴壁细胞增加，但仍低于对照组，细胞有轻微缩小，细胞存活率和Bcl-2 mRNA及蛋白表达升高，细胞活力、凋亡率、miR-15b表达、Caspase-3和Caspase-9 mRNA及蛋白表达降低（P<0.05）。荧光素酶报告基因实验结果证实miR-15b可靶向调控Bcl-2。结论 miR-15b过表达可通过线粒体途径诱导受损的神经细胞形变、凋亡，加重脑缺血再灌注损伤。

关键词: 再灌注损伤, 缺氧缺血，脑, 神经元, RNA干扰疗法, 微小核糖核酸-15b, B淋巴细胞瘤-2, 半胱氨酸蛋白酶-3, 半胱氨酸蛋白酶-9

Abstract:

Objective To investigate the effect of microRNA-15b (miR-15b) gene interference on cerebral ischemia-reperfusion injury and its mechanism. Methods Astrocytes from cerebral cortex of newborn Wistar rats within 24 hours of birth were cultured and identified. Oxygen glucose deprivation/recovery method was used to treat the simulated cerebral ischemia-reperfusion injury in vivo, which was recorded as the model group. miR-15b interfering adenovirus vector and negative control vector were constructed and transfected into cells of the above model group respectively, which were recorded as the overexpression group, the silence group, the overexpression control group and the silence control group. Astrocytes from the cerebral cortex without special treatment were used as the blank control group. After 24 hours, the morphological changes of cells were observed by inverted phase contrast microscope in each group. The cell viability was detected by MTT assay, and the cell viability was detected by lactate dehydrogenase (LDH) leakage rate. The apoptosis rate was detected by flow cytometry. Real-time quantitative polymerase chain reaction was used to detect the expression levels of miR-15b, blymphoma-2 (Bcl-2), cysteine protease-3 (Caspase-3) and cysteine protease-9 (Caspase-9) messenger RNA (mRNA). The expressions of Bcl-2, Caspase-3 and Caspase-9 protein levels were detected by Western blot assay. Luciferase reporter gene experiment verified that miR-15b could target and regulate Bcl-2. Results Compared with the control group, the adherent cells decreased in the blank group, the overexpression control group and the silence control group, cells shrunk and became round, and the distribution was loose, cell viability and Bcl-2 mRNA and protein expression decreased, cell viability and apoptosis rate, and the expression levels of miR-15b, Caspase-3, Caspase-9 mRNA and protein increased (P<0.05). Compared with the blank group and the silencing control group, the number of adherent cells in the overexpression group decreased and was more sparsely distributed. It could be seen that cells floated in the culture medium, the cell survival rate and the expression of Bcl-2 mRNA and protein in the overexpression group decreased, the cell viability and apoptosis rate, and the expression of miR-15b, the expressions of Caspase-3, Caspase-9 mRNA and protein increased (P<0.05). Compared with the blank group and the silenced control group, the adherent cells in the silenced group increased, but still lower than the control group. The cells shrank slightly, the cell survival rate and the expression of Bcl-2 mRNA and protein increased in the silencing group, the cell viability and apoptosis rate, and the expression of miR-15b, the expressions of Caspase-3, Caspase-9 mRNA and protein decreased (P<0.05). Results of luciferase reporter gene experiments confirmed that miR-15b can target and regulate Bcl-2. Conclusion Overexpression of miR-15b can aggravate cerebral ischemia-reperfusion injury, presumably through the mitochondrial pathway to induce the deformation and apoptosis of damaged nerve cells.

Key words: reperfusion injury, hypoxia-ischemia, brain, neurons, RNAi therapeutics, microRNA-15b, B-lymphoma-2, Caspase-3, Caspase-9

中图分类号:

R329.21

图/表 8

图1 大鼠大脑皮层星形胶质细胞鉴定（免疫化学染色，×200）

Fig.1 Identification of astrocytes in rat cerebral cortex (immunochemical staining，×200)

图2 各组细胞形态学及转染效率（×100）

Fig.2 Cell morphology and transfection efficiency of each group (×100)

图3 各组细胞凋亡率分析

Fig.3 Analysis of apoptosis rate in each group

表1 各组细胞的存活率、细胞活力及凋亡率比较（n=3，%，$\bar{x}±s$）

Tab.1 Analysis of cell survival rate, cell viability and apoptosis rate in each group

组别	存活率	细胞活力	凋亡率
对照组	95.62±6.39	1.85±0.33	4.55±0.81
空白组	29.51±5.84^a	52.27±9.02^a	40.26±7.04^a
过表达对照组	28.94±5.73^a	49.58±9.23^a	42.55±7.30^a
沉默对照组	26.45±6.86^a	51.14±9.55^a	41.93±7.55^a
过表达组	9.12±1.02^abc	77.25±11.78^abc	64.54±10.06^abc
沉默组	55.23±8.04^abd	27.36±5.15^abd	21.18±2.03^abd
F	76.586^＊＊	28.127^＊＊	28.700^＊＊

表2 各组细胞的miR-15b以及Bcl-2、Caspase-3、Caspase-9 mRNA相对表达量比较（n=3，$\bar{x}±s$）

Tab.2 Analysis of relative expression levels of miR-15b, Bcl-2, Caspase-3 and Caspase-9 mRNA in cells of each group

组别	miR-15b	Bcl-2	Caspase-3	Caspase-9
对照组	1.03±0.18	1.01±0.20	0.98±0.17	1.00±0.19
空白组	1.78±0.31^a	0.52±0.09^a	2.05±0.38^a	1.62±0.28^a
过表达对照组	1.85±0.34^a	0.50±0.09^a	2.01±0.35^a	1.59±0.26^a
沉默对照组	1.82±0.33^a	0.47±0.08^a	1.99±0.36^a	1.65±0.30^a
过表达组	2.98±0.52^abc	0.18±0.03^abc	3.26±0.57^abc	2.74±0.51^abc
沉默组	1.11±0.19^bd	0.87±0.16^abd	1.24±0.21^bd	1.07±0.16^bd
F	16.952^＊＊	30.145^＊＊	21.377^＊＊	14.108^＊＊

图4 各组细胞Bcl-2、Caspase-3、Caspase-9蛋白表达 A：对照组;B：空白组;C：过表达对照组;D：沉默对照组;E：过表达组;F：沉默组。

Fig.4 The expression levels of Bcl-2, Caspase-3 and Caspase-9 protein in cells of each group

表3 各组细胞的Bcl-2、Caspase-3、Caspase-9蛋白相对表达量分析

Tab.3 Analysis of relative expression levels of Bcl-2, Caspase-3 and Caspase-9 proteins in cells of each group

组别	Bcl-2	Caspase-3	Caspase-9
对照组	1.71±0.27	0.52±0.08	0.48±0.07
空白组	0.79±0.12^a	1.51±0.25^a	1.15±0.21^a
过表达对照组	0.76±0.12^a	1.55±0.28^a	1.22±0.22^a
沉默对照组	0.80±0.13^a	1.46±0.24^a	1.06±0.18^a
过表达组	0.30±0.08^abc	2.31±0.37^abc	1.85±0.31^abc
沉默组	1.26±0.18^abd	0.78±0.14^bd	0.51±0.12^bd
F	26.973^＊＊	20.052^＊＊	19.406^＊＊

图5 miR-15b在Bcl-2的结合位点图

Fig.5 Binding site of miR-15b in Bcl-2

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