国际麻醉学与复苏杂志   2022, Issue (2): 0-0
    
PTPIP51调节线粒体‑内质网结构偶联在N2a细胞氧糖剥夺/复糖复氧损伤中的作用
于群, 赵慧, 王炳琪, 陈怀龙, 张文博, 王海鹏, 王明山, 时飞1()
1.潍坊医学院麻醉学院
The role of protein tyrosine phosphatase interacting protein 51 in oxygen glucose deprivation/reoxygenation injury by regulating the mitochondria‑associated endoplasmic reticulum membranes in N2a cells
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摘要：

目的 观察蛋白酪氨酸磷酸酶相互作用蛋白（protein tyrosine phosphatase interacting protein, PTPIP）51对线粒体‑内质网结构偶联（the mitochondria‑associated endoplasmic reticulum membranes, MAMs）的影响，探讨其在小鼠神经母细胞瘤（mouse neuro‑blastoma N2a, N2a）细胞氧糖剥夺/复糖复氧（oxygen glucose deprivation/reoxygenation, OGD/R）损伤中的作用。 方法 将指数生长的N2a细胞株置于37 ℃、5%CO2、95%空气的细胞培养箱内培养至细胞密度达70%，按照随机数字表法分为4组：对照组（C组）、氧糖剥夺/复糖复氧组（OGD/R组）、干扰小RNA（small interfering RNA, siRNA）‑PTPIP51转染组（siRNA组）、siRNA‑NC转染组（NC组）。OGD/R模型制备：将N2a细胞高糖培养液换成低糖培养液，置于37 ℃、5%CO2、95% N2缺氧环境中培养3 h后重新置于高糖培养液中正常条件下培养。C组继续在正常条件下培养；OGD/R组仅制备OGD/R模型；siRNA组与NC组在OGD/R模型制备前48 h分别转染siRNA‑PTPIP51及siRNA‑NC，余同OGD/R组。各组于复糖复氧12 h、24 h后收集细胞检测。细胞计数试剂盒（cell counting kit‑8, CCK‑8）法检测细胞存活率，流式细胞术检测细胞凋亡率，实时荧光定量PCR（real‑time fluorescent quantitative PCR, RT‑qPCR）检测PTPIP51 mRNA表达水平，Western blot检测PTPIP51蛋白水平，共聚焦显微镜观察线粒体‑内质网共定位水平。 结果 与C组比较，其他3组细胞存活率降低（P<0.05），细胞凋亡率、线粒体‑内质网共定位水平、PTPIP51 mRNA及PTPIP51蛋白水平均升高（P<0.05）；与OGD/R组比较，siRNA组细胞存活率升高 (P<0.05），细胞凋亡率、线粒体‑内质网共定位水平、PTPIP51 mRNA及PTPIP51蛋白水平均降低（P<0.05）。 结论 PTPIP51表达上调介导的线粒体‑内质网共定位水平提高是N2a细胞OGD/R损伤的机制之一。
关键词： 线粒体‑内质网结构偶联； 氧糖剥夺； 蛋白酪氨酸磷酸酶相互作用蛋白51；
Abstract:

Objective To observe the effect of protein tyrosine phosphatase interacting protein (PTPIP) 51 on the mitochondria‑associated endoplasmic reticulum membranes (MAMs), and explore its role in oxygen glucose deprivation/reoxygenation (OGD/R) injury in mouse neuro‑blastoma N2a (N2a) cells. Methods The cells at the exponential phrase were cultured in an incubator at 37 °C, 5% CO2, and 95% air and allowed to grow to 70% confluence. According to the random number table method, they were divided into four groups: a control group (group C), an oxygen‑glucose deprivation and reoxygenation group (group OGD/R), a small interfering RNA (siRNA)‑PTPIP51 transfection group (group siRNA), and a siRNA‑NC transfection group (group NC). The OGD/R model was established as follows: high‑sugar medium used for N2a cell cultivation was replaced by low‑sugar medium, and the cells were cultured at 37 ℃ in 5% CO2 and 95% N2 hypoxia environment for 3 h, and then re‑cultured in high‑sugar medium under normal conditions. Group C continued to grow under normal conditions. The OGD/R model was established in group OGD/R. Group siRNA and group NC were transfected with siRNA‑PTPIP51 or siRNA‑NC respectively 48 h before the OGD/R model was established, and other treatments were the same as group OGD/R. After 12 h and 24 h of re‑glucose and re‑oxygenation, the cell survival rate was measured by the cell counting kit‑8 (CCK‑8) assay. The apoptotic rate was determined by flow cytometry. The expression of PTPIP51 mRNA and protein was detected by real‑time fluorescent quantitative PCR (RT‑qPCR) or Western blot. The co‑localization of mitochondrial‑endoplasmic reticulum was observed under a confocal microscope. Results Compared with group C, the cell viability rate decreased in the other three groups (P<0.05). The apoptosis rate, the co‑localization of mitochondrial‑endoplasmic reticulum, and the expression of PTPIP51 mRNA and protein increased in the other three groups (P<0.05). Compared with group OGD/R, the cell viability rate increased in group siRNA (P<0.05), while the apoptosis rate, the co‑location of mitochondrial‑endoplasmic reticulum, and the expression of PTPIP51 mRNA and protein decreased in group siRNA (P<0.05). Conclusion The increase of co‑localization of mitochondrial‑endoplasmic reticulum mediated by PTPIP51 up‑regulation is one of the mechanisms of OGD/R injury in N2a cells.
Key words: Mitochondria‑associated endoplasmic reticulum membranes; Oxygen‑glucose deprivation; Protein tyrosine phosphatase interacting protein 51; Calcium overload