肝癌细胞来源的外泌体对其增殖、凋亡及自噬的生物学行为影响

高洁; 李江艳; 黄桦; 张伦军; 邓蓉

doi:10.16766/j.cnki.issn.1674-4152.003538

肝癌细胞来源的外泌体对其增殖、凋亡及自噬的生物学行为影响

doi: 10.16766/j.cnki.issn.1674-4152.003538

高洁¹,
李江艳^1, ,,
黄桦²,
张伦军¹,
邓蓉¹

1.
蚌埠医科大学第一附属医院检验科，安徽蚌埠 233004
2.
蚌埠医科大学检验医学院，安徽蚌埠 233030

基金项目:

国家自然科学基金项目 82200661

蚌埠医学院重点项目 2020byzd059

蚌埠医学院重点项目 2020byzd029

详细信息

通讯作者:
李江艳，E-mail: 1258813867@qq.com

中图分类号: R735.7 R730.23
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- 文章访问数: 347
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- PDF下载量: 14
- 被引次数: 0
出版历程
- 收稿日期: 2024-03-04
- 网络出版日期: 2024-07-22

Effects of exosomes derived from hepatocellular carcinoma cells on their biological behaviors of proliferation, apoptosis and autophagy

1.
Department of Clinical Laboratory, the First Affiliated Hospital of Bengbu Medical University, Bengbu, Anhui 233004, China

摘要

摘要: 目的外泌体在肝癌的发生发展中发挥重要作用，本研究旨在探讨HepG₂肝癌细胞增殖、凋亡及自噬与外泌体释放的影响及相关机制。方法通过超速离心法提取HepG₂细胞外泌体，采用透射电镜、纳米颗粒跟踪、Western blotting分析HepG₂细胞中外泌体形态、径粒分布及标志蛋白表达；将外泌体与HepG₂细胞共同培养以后，采用CCK-8检测HepG₂细胞增殖变化、流式细胞术检测HepG₂细胞凋亡率，Western blotting检测凋亡相关蛋白(Bax、Bcl-2)和自噬相关蛋白(LC3-Ⅱ/LC3-Ⅰ、p62、Beclin-1)表达。结果提取的HepG₂细胞外泌体形态符合外泌体特征，直径在30~150 nm之间，标志蛋白Alix和CD63表达明显。CCK-8结果显示，24 h外泌体组与对照组吸光度相比差异无统计学意义(P＞0.05)，48 h及72 h外泌体组吸光度均高于对照组(P＜0.05)。流式细胞术检测结果显示，对照组细胞凋亡率为(14.96±0.28)%，外泌体组细胞凋亡率为(11.16±0.50)%，细胞凋亡率下降(t=11.485，P＜0.001)。Western blotting结果显示，与对照组比较，外泌体组凋亡相关蛋白Bcl-2表达上调(P＜0.01)、Bax表达下调(P＜0.01)，自噬相关蛋白LC3-Ⅱ/LC3-Ⅰ表达上调(P＜0.01)、Beclin-1表达上调(P＜0.05)、p62表达下调(P＜0.01)。结论 HepG₂细胞来源的外泌体可以促进细胞增殖，通过诱导自噬上调而抑制细胞凋亡。
- 肝癌细胞 /
- 外泌体 /
- 增殖 /
- 凋亡 /
- 自噬
Abstract: Objective Exosomes play an important role in the occurrence and development of hepatocellular carcinoma. This study aims to investigate the effects of exosomes on the proliferation, apoptosis, autophagy, and exosome release of HepG₂ hepatocellular carcinoma cells and the related mechanisms. Methods Exosomes of HepG₂ cells were extracted by ultrafast centrifugation. Exosome morphology, diameter distribution and marker protein expression of HepG₂ cells were analyzed by transmission electron microscopy, nanoparticle tracking and Western blotting analysis. After the co-culture of exosomes and HepG₂ cells, a CCK-8 kit was used to detect the proliferation of HepG₂ cells, the apoptosis of HepG₂ cells was detected by flow cytometry, and the expressions of apoptosis-related proteins (Bax, Bcl-2) and autophagy-related proteins (LC3-Ⅱ/LC3-Ⅰ, p62, Beclin-1) were detected by Western blotting. Results The morphology of exosomes extracted from HepG₂ cells was consistent with the characteristics of exosomes, and the diameter between 30 and 150 nm. The marker proteins Alix and CD63 were significantly expressed. The results of CCK-8 showed that the absorbance of HepG₂ cells in the 24 h exosome group was not significantly different from that in the control group(P > 0.05), but the absorbance of the exosome group at 48 hours and 72 hours were higher than that of the control group (P < 0.05). Flow cytometry showed that the apoptosis rate was (14.96±0.28) % in the control group and (11.16±0.50) % in the exosome group, and the apoptosis rate decreased (t=11.485, P < 0.001). Western blotting results showed that compared with the control group, the expression of apoptosis-related protein Bcl-2 in exosome group was up-regulated (P < 0.01), and the expression of Bax was down-regulated (P < 0.01). Autophagy associated protein LC3-Ⅱ/LC3-Ⅰ expression was up-regulated (P < 0.01), Beclin-1 expression was up-regulated (P < 0.05), and p62 expression was down-regulated (P < 0.01). Conclusion Exosomes derived from HepG₂ cells can promote cell proliferation and inhibit apoptosis by inducing up-regulation of autophagy.
- Hepatocellular carcinoma cells /
- Exosome /
- Proliferation /
- Apoptosis /
- Autophagy

HTML全文

图 1 外泌体鉴定

注：A为透射电镜观察HepG₂外泌体形态；B为纳米颗粒跟踪分析检测HepG₂外泌体的径粒分布；C为Western blotting检测HepG₂外泌体的蛋白标志物。

Figure 1. Identification of exosomes

下载: 全尺寸图片幻灯片

图 2 流式细胞术检测细胞凋亡率

注：^aP＜0.01。

Figure 2. Apoptosis rate was detected by flow cytometry

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图 3 Western blotting检测凋亡相关蛋白表达

注：1为对照组，2为外泌体组；^aP＜0.01。

Figure 3. The expression of apoptosis-related proteins was detected by Western blot

下载: 全尺寸图片幻灯片

图 4 Western blotting检测自噬相关蛋白表达

注：1为对照组，2为外泌体组；^aP＜0.01，^bP＜0.05。

Figure 4. The expression of autophagy related proteins was detected by Western blot

下载: 全尺寸图片幻灯片

表 1 CCK-8法检测不同时间点细胞吸光度值(x±s)

Table 1. Absorbance of cells at different time measured by CCK-8 method(x±s)

组别	孔数	24 h	48 h	72 h
对照组	3	1.72±0.06	1.94±0.06	2.30±0.02
外泌体组	3	1.75±0.05	2.12±0.06	2.69±0.12
F值		0.601	14.394	33.483
P值		0.481	0.019	0.004
注：F_时间=199.236，P_时间＜0.001；F_组间=40.178，P_组间=0.003；F_交互=11.407，P_交互=0.005。

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