Chinese Journal of Tissue Engineering Research ›› 2026, Vol. 30 ›› Issue (1): 130-138.doi: 10.12307/2026.540
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Zhao Yang1, Li Jialin2, Wu Xiao1, Zou Yourui3, Liu Yang3, Ma Hui3
Received:
2024-11-18
Accepted:
2025-01-20
Online:
2026-01-08
Published:
2025-07-02
Contact:
Ma Hui, PhD, Professor, General Hospital of Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China
About author:
Zhao Yang, Master candidate, Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China
Supported by:
CLC Number:
Zhao Yang, Li Jialin, Wu Xiao, Zou Yourui, Liu Yang, Ma Hui. Choline kinase alpha silencing affects proliferation and apoptosis in glioma cells by inducing mitochondrial dysfunction[J]. Chinese Journal of Tissue Engineering Research, 2026, 30(1): 130-138.
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2.2 CHKA沉默对U87MG和U251细胞线粒体结构和形态的影响 透射电子显微镜观察结果显示,与shNC组U87MG和U251细胞比较,shCHKA组U87MG和U251细胞线粒体结构紊乱,出现线粒体肿胀、线粒体空泡化(图2A);Western blot实验结果显示(图2B,C),shCHKA组U87MG和U251细胞中ACO2、TOM20和ATP5A蛋白表达水平均较shNC组显著降低(P < 0.01,P < 0.001),提示CHKA沉默导致线粒体结构破坏和功能减弱[20-22];而SOD2蛋白表达水平显著升高(P < 0.01,P < 0.000 1),可能是细胞为应对线粒体功能障碍引发的活性氧积累而激活的代偿性抗氧化反应[23]。 "
2.3 CHKA沉默对U87MG和U251细胞线粒体功能的影响 为了进一步明确CHKA沉默对线粒体功能的影响,分别检测了CHKA沉默前后U87MG和U251细胞活性氧、线粒体膜电位和细胞ATP含量的变化。利用荧光显微镜对各组细胞的活性氧水平进行检测,结果显示shCHKA组U87MG和U251细胞内活性氧荧光强度较shNC组显著升高(P < 0.01,图3A,D)。利用聚集体与单体的JC-1荧光比值来评估各组细胞的线粒体膜电位,荧光图像中红色荧光表示JC-1聚集体(高膜电位),绿色荧光表示JC-1单体(低膜电位),结果显示shCHKA 组JC-1聚集体/单体荧光比值较低,这表明CHKA沉默后U87MG和U251细胞的线粒体膜电位水平显著降低(P < 0.01,P < 0.001,图3B,E)。利用化学发光法检测各组细胞内ATP水平的变化,结果以纳米摩尔(nmol)表达,结果显示shCHKA组U87MG和U251细胞内ATP水平较shNC组显著降低(P < 0.01,图3C)。 "
2.4 CHKA沉默对U87MG和U251细胞增殖和凋亡的影响 该研究验证了CHKA沉默会引起U87MG和U251细胞线粒体结构和功能的一系列损伤,同时活性氧增加等线粒体异常现象是细胞早期凋亡的标志事件之一[24-25],因此猜测CHKA沉默可以引起U87MG和U251细胞增殖能力和凋亡水平的变化。利用CCK-8实验检测各组U87MG和U251细胞的增殖能力,结果显示shCHKA组U87MG和U251细胞的增殖能力较shNC组显著减弱(P < 0.01,图4A)。利用流式细胞术检测各组U87MG和U251细胞的凋亡率,结果显示shCHKA组U87MG和U251细胞的凋亡率较shNC组显著升高(P < 0.001,图4B)。综上所述,通过在胶质瘤细胞中沉默CHKA,验证了CHKA沉默后胶质瘤细胞的线粒体发生了功能障碍,并可能进一步导致细胞增殖能力减弱和凋亡水平增加,影响了胶质瘤细胞的生长和恶性表型。 "
2.5 Mdivi-1对植入CHKA沉默U87MG细胞的裸鼠皮下肿瘤生长的影响 为体内体外共同验证以上结论,皮下植入U87MG胶质瘤细胞,记录肿瘤0-25 d体积变化,25 d切除肿瘤并记录肿瘤大小和肿瘤质量,结果显示:shCHKA组的裸鼠瘤体质量较shNC组明显减轻,生长速度显著降低(P < 0.000 1);同时为了明确CHKA沉默是通过改变线粒体功能从而导致胶质瘤的抑制作用,在CHKA沉默的基础上腹腔注射线粒体分裂抑制剂Mdivi-1,保护线粒体功能,反向验证CHKA沉默后对胶质瘤的抑制作用。结果显示:shCHKA+ Mdivi-1组的裸鼠瘤体质量较shCHKA组显著增加,生长速度显著变快(P < 0.000 1,图5)。 2.6 Mdivi-1对CHKA沉默的U87MG和U251细胞线粒体功能的影响 shCHKA+Mdivi-1组U87MG和U251细胞内活性氧荧光强度较shCHKA组显著降低(P < 0.05,P < 0.01,图6A,D)。shCHKA+Mdivi-1组U87MG和U251细胞线粒体膜电位较shCHKA组显著升高(P < 0.01,图6B,E)。shCHKA+Mdivi-1组U87MG和U251细胞内ATP水平较shCHKA组显著升高(P < 0.01,P < 0.001,图6C)。 "
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