外源性IL-33介导BV2细胞调控PC12细胞生长的实验研究

Experimental study on the regulation of PC12 cell growth by activated BV2 cells via IL-33

  • 摘要: 背景 小胶质细胞-神经元互作对脊髓损伤后的神经修复至关重要。白细胞介素-33(interleukin-33,IL-33)是来自白细胞介素-1 家族的多功能细胞因子,其在脊髓损伤后调节小胶质细胞-神经元互作中起关键作用。目的 探究活化BV2 细胞对PC12 细胞生长的调控作用。方法 使用细胞计数试剂盒-8(cell counting kit-8,CCK8) 检测不同浓度脂多糖(lipopolysaccharide,LPS)干预BV2 细胞后BV2 细胞活化个数及BV2 细胞存活率,筛选影响其活化的最适浓度。利用免疫荧光染色检测BV2 细胞突起长度及分支变化情况。将普通培养基培养的BV2 细胞与含LPS 培养基培养的BV2 细胞分别与PC12 细胞共培养,利用免疫荧光染色检测PC12 细胞突起长度及分支变化情况。利用实时荧光定量 PCR(real-time quantitative PCR,RT-qPCR)检测白细胞介素6(interleukin-6,IL-6),肿瘤坏死因子α(tumor necrosis factor-α,TNF-α),白细胞介素4(interleukin-4,IL-4),白细胞介素10(interleukin-10,IL-10)基因相对表达量;使用10 ng/mL IL-33 干预活化的BV2细胞,并将其与PC12 细胞共培养,利用免疫荧光染色检测PC12 细胞突起长度及分支变化情况。结果 CCK8 结果显示,当LPS浓度小于1 μg/mL 时,细胞呈明显活化形态的个数较少,当LPS浓度大于1 μg/mL 时,细胞存活率出现下降,所以1 μg/mL 为影响BV2 细胞活化的最适浓度;光学显微镜观察及免疫荧光染色实验结果显示,相比于静息态BV2 细胞,活化BV2 细胞突起变短,胞体近端分支变多,形态呈现阿米巴样结构,RT-qPCR 结果显示,促炎因子IL-6,TNF-α 表达水平上调(P<0.01),而抗炎因子IL-4,IL-10 的表达差异无统计学意义(P>0.05);光学显微镜观察及免疫荧光染色结果显示,与活化BV2 细胞共培养后,PC12 细胞突起变短,分支变少;IL-33 的干预减轻了这一现象。结论 IL-33 可以调控活化BV2 细胞,减少其对PC12细胞生长的抑制作用。

     

    Abstract: Background Microglia-neuron interactions are crucial for neural repair after spinal cord injury. Interleukin-33 (IL-33) is a multifunctional cytokine from the interleukin-1 family, which plays a key role in regulating microglia-neuron interactions following spinal cord injury.Object Investigate the regulatory effect of activated BV2 cells on the growth of PC12 cells.Methods Using the cell counting kit-8 (CCK8) to detect the number of activated BV2 cells and BV2 cell viability after intervention with different concentrations of lipopolysaccharide (LPS), to screen for the optimal concentration affecting their activation. Immunofluorescence staining was used to observe changes in BV2 cell process length and branching. BV2 cells cultured in regular medium and BV2 cells cultured in LPS-containing medium were co-cultured with PC12 cells, respectively, and immunofluorescence staining was used to detect changes in PC12 cell process length and branching. Real-time quantitative PCR (RT-qPCR) was used to detect the relative expression levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin-4 (IL-4), and interleukin-10 (IL-10) genes. Activated BV2 cells were treated with 10 ng/mL IL-33 and co-cultured with PC12 cells, and immunofluorescence staining was used to detect changes in PC12 cell process length and branching..Results CCK8 results showed that when the LPS concentration was less than 1 μg/mL, the number of cells exhibiting obvious activated morphology was low, while when the LPS concentration was greater than 1 μg/mL, cell viability decreased. Therefore, 1μg/mL is the optimal concentration affecting BV2 cell activation. Observations under an optical microscope and results from immunofluorescence staining experiments showed that, compared to resting BV2 cells, activated BV2 cells had shorter processes and more proximal branching of the cell body, exhibiting an amoeboid morphology. RT-qPCR results showed that the expression levels of pro-inflammatory factors IL-6 and TNF-α were upregulated (P<0.01), while the expression of anti-inflammatory factors IL-4 and IL-10 showed no significant difference (P>0.05). Optical microscopy and immunofluorescence staining results indicated that after co-culture with activated BV2 cells, PC12 cells exhibited shorter processes and fewer branches; IL-33 intervention alleviated this phenomenon. Conclusion IL-33 can regulate activated BV2 cells and reduce their inhibitory effect on PC12 cell growth.

     

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