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目的 探究细粒棘球蚴囊液中外泌体的存在并探讨其是否通过JAK2/STAT3信号通路调控小鼠肝癌细胞Hepa1-6的增殖、凋亡。方法 使用囊液直接干预Hepa1-6细胞，CCK8、EdU检测囊液对其增殖能力的影响；使用超速离心法对细粒棘球蚴囊液外泌体进行提取，并使用透射电镜、Western-Blotting、粒径分析对其进行验证；通过外泌体-PKH67吞噬试验验证外泌体进入细胞情况；通过CCK8、EDU等检测细粒棘球蚴囊液外泌体对Hepa1-6细胞增殖的作用；使用Western-Blotting检测外泌体对Hepa1-6细胞凋亡的作用；通过Western-Blotting检测JAK2/STAT3信号通路相关蛋白的表达情况并通过相关性研究探讨此通路与肝癌细胞增殖的关系。结果 1.细粒棘球蚴囊液促进Hepa1-6增殖；2.Western-Blotting结果显示细粒棘球蚴囊液外泌体表面标志物CD63,CD9,TSG101表达阳性；3.透射电镜可见外泌体形态为双层膜结构的盘状囊泡，粒径分析显示囊泡直径分布区间为159.2±81.1 nm; 4.外泌体24 h可进入细胞内；5.CCK8、EdU结果显示细粒棘球蚴囊液外泌体在24、48 h均可促进Hepa1-6细胞的增殖(P<0.05);6. Western-Blotting结果显示细粒棘球蚴囊液外泌体在干预Hepa1-6 24 h、48 h时均促进Bcl-2的表达，且随着浓度增加促进作用增加(P<0.05),Bax、caspase-3、caspase-9表达量降低，随着浓度增加抑制作用增强；7. Western-Blotting结果显示细粒棘球蚴囊液外泌体干预Hepa1-6 24h后t-JAK2、t-STAT3表达量无明显变化，p-JAK2、p-STAT3蛋白表达随着外泌体浓度增加而增加，且与肝癌细胞增殖存在正相关。结论 细粒棘球蚴囊液及其外泌体可以通过JAK2/STAT3信号通路促进Hepa1-6增殖，同时抑制其凋亡。
Abstract：Objective To investigate the existence of exosomes in the hydatid cyst fluid and whether they regulate the proliferation and apoptosis of mouse hepatocellular carcinoma cells Hepa1-6 through JAK2/STAT3 signaling pathway. Methods Hepa1-6 cells were directly interfered with Echinococcus granulosus cyst fluid. CCK8 and EdU were used to detect the effect of Echinococcus granulosus cyst fluid on the proliferation of Hepa1-6 cells. Ultrafast centrifugation method was used to extract exosomes from the hydatid cyst fluid, and the exosomes were verified by transmission electron microscopy, Western-Blotting and particle size analysis. The entry of exosomes into cells was verified by exosom-PKH67 phagocytosis test; The effect of exosomes in the hydatid cyst fluid on the proliferation of Hepa1-6 cells was detected by CCK8 and EDU. The effect of exosomes on apoptosis of Hepa1-6 cells was detected by Western-Blotting. The expression of JAK2/STAT3 signaling pathway related proteins was detected by Western-Blotting, and the relationship between this pathway and the proliferation of hepatoma cells was explored through correlation studies. Results 1. The hydatid fluid promoted the proliferation of Hepa1-6; 2.Western-Blotting results showed positive expressions of CD63,CD9,TSG101 in the exosome surface markers of echinococcus granulosus cyst fluid. 3. Transmission electron microscopy showed that the exosomes were disk-like vesicles with double-layer membrane structure, and particle size analysis showed that the vesicles were distributed in a diameter range of 159.2±81.1 nm; 4. Exosomes can enter cells within 24 hours; 5.CCK8 and EdU results showed that the exosomes of echinococcus granulosus cyst fluid could promote the proliferation of Hepa1-6 cells at both 24h and 48h(P<0.05). 6. Western-Blotting results showed that the exosomes of fine echinococcus hydatid fluid promoted the expression of Bcl-2 at 24h and 48h after Hepa1-6 intervention, and the promoting effect increased with the increase of concentration(P<0.05). The expressions of Bax, caspase-3 and caspase-9 decreased, and the inhibitory effect increased with the increase of concentration. 7. Western-Blotting results showed that the expression levels of t-JAK2 and t-STAT3 did not change significantly after the intervention of Hepa1-6 by exosomes in the fine hydatid cyst fluid for 24 h, and the protein expressions of p-JAK2 and p-Stat3 increased with the increase of exosomes concentration, and they were positively correlated with the proliferation of liver cancer cells. Conclusion Echinococcus granulosus cyst fluid and its exosomes can promote the proliferation of Hepa1-6 and inhibit its apoptosis through JAK2/STAT3 signaling pathway.
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基本信息:
DOI：10.13880/j.cnki.65-1174/n.2024.22.032
中图分类号:R735.7
引用信息:
[1]伍希雅,崔杰,林芷伊,等.细粒棘球蚴囊液外泌体通过JAK2/STAT3信号通路调控小鼠肝癌细胞Hepa1-6增殖与凋亡[J].石河子大学学报(自然科学版),2024,42(06):742-749.DOI:10.13880/j.cnki.65-1174/n.2024.22.032.
基金信息:
国家科技计划项目(S2022EB5188); 国家卫生健康委中亚高发病防治重点实验室开放基金(KF202105); 新疆生产建设兵团南疆重点项目(S2021AB3280)
2024-03-24
2024
2024-06-25
2024
1
2024-10-09
2024-10-09
2024-10-09