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目的 探讨敲低锌转运蛋白ZIP7对小鼠睾丸支持细胞增殖的影响及其可能的作用机制。方法 使用小鼠睾丸支持细胞系(TM4细胞)进行实验，TM4细胞培养后采用细胞免疫荧光染色法观察ZIP7在TM4细胞中的亚细胞定位；将TM4细胞分为对照组和ZIP7 siRNA组(采用siRNA转染敲低TM4细胞中ZIP7表达),采用CCK8法检测两组细胞的增殖能力，使用DHE荧光探针检测两组细胞内活性氧(ROS)的水平，实时荧光定量PCR(qPCR)检测两组细胞内ZIP7 mRNA的相对表达量，Western blot法检测两组细胞内ZIP7、c-Jun氨基末端激酶(JNK)、磷酸化JNK(p-JNK)、细胞外信号调节激酶(ERK)及磷酸化ERK(p-ERK)蛋白表达水平。结果 细胞免疫荧光染色法结果显示ZIP7定位于TM4细胞的内质网上。siRNA干扰使得ZIP7 siRNA组细胞中ZIP7的mRNA和蛋白表达显著低于对照组(P<0.05);siRNA转染敲低TM4细胞内ZIP7的表达后，ZIP7 siRNA组细胞增殖能力显著低于对照组(P<0.001),细胞内ROS水平显著高于对照组(P<0.001)。与对照组比较，ZIP7 siRNA组细胞的p-ERK/ERK蛋白表达无显著差异(P>0.05),而p-JNK/JNK蛋白表达显著增加(P<0.05)。结论 敲低ZIP7可以明显抑制TM4细胞增殖，该过程可能通过细胞内ROS水平的升高及JNK磷酸化的激活介导。
Abstract：Objective:To investigate the effects and its possible mechanism of knocking down the zinc transporter protein ZIP7 on the proliferation of mouse testicular sertoli cells.Methods:Experiments were carried out using a mouse testicular sertoli cell line(TM4 cells). The subcellular localization of ZIP7 in TM4 cells was observed by cellular immunofluorescence staining after the culture of TM4 cells. TM4 cells were divided into the control group and the ZIP7 siRNA group(siRNA transfection was used to knock down the expression of ZIP7 in TM4 cells). The proliferation viability of the two groups were detected by CCK8 assay, and the levels of reactive oxygen species(ROS) in the two groups were detected using a DHE fluorescent probe. The protein expression levels of ZIP7,c-Jun amino-terminal kinase(JNK),phosphorylated JNK(p-JNK),extracellular signal-regulated kinase(ERK) and phosphorylated ERK(p-ERK) in the cells of both groups were detected by Western blot.Results:The results of cellular immunofluorescence staining showed that ZIP7 was located in the endoplasmic reticulum of TM4 cells. The siRNA interference resulted in significantly lower mRNA and protein expressions of ZIP7 in ZIP7 siRNA group than those in the control group(P<0.05). After transfection with siRNA and knockdown of ZIP7 expression in TM4 cells, the proliferative viability of cells in the ZIP7 siRNA group was significantly lower than that in the control group, while the intracellular ROS level was significantly higher than that of the control group(P<0.001). Compared with the control group, there was no significant difference in the expression of p-ERK/ERK protein in the cells of the ZIP7 siRNA group(P>0.05),while the expression of p-JNK/JNK protein was significantly increased(P<0.05).Conclusions:Knockdown of ZIP7 can significantly inhibited the proliferation of TM4 cells. The process may be mediated through elevated ROS levels and activation of JNK phosphorylation.
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基本信息:
中图分类号:R-332
引用信息:
[1]特力格尔,刘璇,李媛静,等.敲低锌转运蛋白ZIP7抑制小鼠睾丸支持细胞增殖的研究[J].生殖医学杂志,2024,33(02):201-207.
基金信息:
河北省省级科技计划资助项目(226Z7722G); 河北省自然科学基金(H2022314001,H2021314001)
2023-08-14
2023
2023-10-30
2024-01-12
2024
1
2024-02-15
2024-02-15