腺病毒介导的SPRY1过表达对肝细胞癌凋亡和自噬的影响

doi:10.11958/20252541

天津医药 ›› 2026, Vol. 54 ›› Issue (4): 337-343.doi: 10.11958/20252541

• 细胞与分子生物学 • 下一篇

腺病毒介导的SPRY1过表达对肝细胞癌凋亡和自噬的影响

刘瑢(), 项艳曾, 马丽娟, 张鹏^△()

宁夏医科大学总医院药剂科（邮编750004）

收稿日期:2025-07-23 修回日期:2025-11-20 出版日期:2026-04-15 发布日期:2026-04-14
通讯作者: ^△E-mail：zhp435656@163.com
作者简介:刘瑢（1978），女，副主任药师，主要从事肝癌作用机制及药物治疗方面研究。E-mail：Liurong17795163761@163.com
基金资助:
宁夏自然科学基金项目(2022AAC03484)

The effect of adenovirus-mediated SPRY1 overexpression on apoptosis and autophagy in hepatocellular carcinoma

LIU Rong(), XIANG Yanzeng, MA Lijuan, ZHANG Peng^△()

Department of Pharmacy, General Hospital of Ningxia Medical University, Yinchuan 750004, China

Received:2025-07-23 Revised:2025-11-20 Published:2026-04-15 Online:2026-04-14
Contact: ^△E-mail：zhp435656@163.com

刘瑢, 项艳曾, 马丽娟, 张鹏. 腺病毒介导的SPRY1过表达对肝细胞癌凋亡和自噬的影响[J]. 天津医药, 2026, 54(4): 337-343.

LIU Rong, XIANG Yanzeng, MA Lijuan, ZHANG Peng. The effect of adenovirus-mediated SPRY1 overexpression on apoptosis and autophagy in hepatocellular carcinoma[J]. Tianjin Medical Journal, 2026, 54(4): 337-343.

摘要/Abstract

摘要：

目的探讨腺病毒介导的斑点蛋白酪氨酸激酶底物（SPRY）1过表达对人肝癌细胞增殖、凋亡及自噬的影响及其作用机制。方法构建携带SPRY1的腺病毒载体Ad5-SPRY1，感染人肝癌细胞系Huh7和Hep3B，实验分为对照组（空白对照）、Ad5-GFP组（感染空载腺病毒Ad5-GFP）和Ad5-SPRY1组（感染重组腺病毒Ad5-SPRY1）。采用qPCR检测SPRY1 mRNA表达水平；Western blot检测SPRY1、细胞-髓细胞瘤病毒癌基因同源物（c-MYC）、B细胞淋巴瘤/白血病-2基因（BCL2）及自噬标志蛋白微管相关蛋白1A/1B轻链3（LC3）Ⅰ/Ⅱ的表达；流式细胞术检测细胞凋亡率；透射电子显微镜观察自噬体超微结构；Ad-mCherry-GFP-LC3B荧光报告系统检测自噬流变化。结果 Ad5-SPRY1感染Huh7、Hep3B细胞48 h后，SPRY1 mRNA和蛋白表达水平高于对照组和Ad5-GFP组（P<0.05）。流式细胞术检测显示，感染Huh7、Hep3B细胞24 h后，Ad5-SPRY1组细胞凋亡率低于对照组和Ad5-GFP组（P<0.05）。Western blot结果显示，Ad5-SPRY1组c-MYC和BCL2蛋白表达水平高于对照组和Ad5-GFP组（P<0.05）。透射电镜观察显示，Ad5-SPRY1组细胞质中可见大量双层膜自噬体结构；Western blot结果显示，Ad5-SPRY1组LC3Ⅱ蛋白表达水平高于对照组和Ad5-GFP组（P<0.05）。Ad-mCherry-GFP-LC3B荧光检测显示，Ad5-SPRY1组黄色斑点数量多于对照组和pcDNA3.1组（P<0.05）。结论腺病毒介导的SPRY1过表达可显著抑制肝癌细胞凋亡并诱导自噬，其作用机制可能与上调c-MYC、BCL2表达及增强LC3介导的自噬流有关；SPRY1可能成为肝癌治疗的潜在新靶点。

关键词: 癌, 肝细胞, 细胞凋亡, 自噬, 斑点蛋白酪氨酸激酶底物1, 原癌基因蛋白质c-myc, 原癌基因蛋白质bcl-2

Abstract:

Objective To investigate the effect and mechanism of adenovirus-mediated overexpression of Sprouty RTK signaling antagonist 1 (SPRY1) on proliferation, apoptosis and autophagy in human hepatocellular carcinoma (HCC) cells. Methods The recombinant adenovirus vector Ad5-SPRY1 was constructed and used to infect human HCC cell lines Huh7 and Hep3B. Cells were divided into the control group (infected with Ad5-GFP) and the SPRY1 overexpression group (infected with Ad5-SPRY1). SPRY1 mRNA expression was detected by qPCR. Western blot assay was used to analyze the protein levels of SPRY1, c-MYC, BCL2 and autophagy-related proteins LC3Ⅰ/Ⅱ. Cell apoptosis was measured by flow cytometry with Annexin V-PE/7-AAD double staining. Autophagosome formation was observed by transmission electron microscopy (TEM), and autophagic flux was assessed using the Ad-mCherry-GFP-LC3B reporter system. Results After 48 hours of Ad5-SPRY1 infection in Huh7 and Hep3B cells, the expression levels of SPRY1 mRNA and protein were higher than those in the control group and the Ad5-GFP group (P<0.05). Flow cytometry analysis showed that after 24 hours of infection in Huh7 and Hep3B cells, the apoptosis rate in the Ad5-SPRY1 group was lower than that in the control group and the Ad5-GFP group (P<0.05). Western blot results showed that the expression levels of c-MYC and BCL2 proteins in the Ad5-SPRY1 group were higher than those in the control group and the Ad5-GFP group (P<0.05). Transmission electron microscopy observation revealed a large number of double-membrane autophagosome structures in the cytoplasm of the Ad5-SPRY1 group. Western blot results indicated that the expression level of LC3Ⅱ protein in the Ad5-SPRY1 group was higher than that in the control group and the Ad5-GFP group (P<0.05). Ad-mCherry-GFP-LC3B fluorescence assay showed that the number of yellow spots in the Ad5-SPRY1 group was higher than that in the control group and pcDNA3.1 group (P<0.05). Conclusion Adenovirus-mediated SPRY1 overexpression inhibits apoptosis and promotes autophagy in HCC cells, possibly through upregulation of c-MYC and BCL2 and enhancement of LC3-mediated autophagic flux. SPRY1 may represent a novel therapeutic target for hepatocellular carcinoma.

Key words: carcinoma, hepatocellular, apoptosis, autophagy, SPla and RyR domain-containing protein 1, proto-oncogene proteins c-myc, proto-oncogene proteins bcl-2

中图分类号:

R735.7

图/表 11

表1 SPRY1检测相关的PCR序列

Tab.1 Sequences of SPRY1 for PCR

基因名称	引物序列（5′→3′）	产物大小/bp
SPRY1	上游：TCCCTGGTCATAGGTCTGAAAG	187
SPRY1	下游：TGCCGGTTACAGGCCAAAC	187
GAPDH	上游：GGAGCGAGATCCCTCCAAAAT	197
GAPDH	下游：GGCTGTTGTCATACTTCTCATGG	197

图1 腺病毒的产生及对肝癌细胞的感染效果检测 A：Ad5-SPRY1转染HEK293细胞10 d后的CPE，箭头示变圆脱落的细胞；标尺为500 μm；B：Ad5-SPRY1感染Huh7细胞24 h后的GFP表达情况；左侧标尺为500 μm；右侧标尺为200 μm。

Fig.1 Detection of adenovirus production and infection efficiency in Huh7 cells

图2 Western blot检测SPRY1蛋白在Huh7、Hep3B细胞中的表达 A：对照组；B：Ad5-GFP组；C：Ad5-SPRY1组。

Fig.2 Detection of SPRY1 protein expression in Huh7 and Hep3B cells by Western blot assay

表2 各组Huh7细胞和Hep3B细胞中SPRY1 mRNA和蛋白表达水平比较（n=3，$\bar{x}±s$）

Tab.2 Comparison of SPRY1 mRNA and protein expression levels between Huh7 cells and Hep3B cells of each group

组别	Huh7细胞		Hep3B细胞
组别	mRNA	蛋白	mRNA	蛋白
对照组	1.00±0.12	1.00±0.08	1.00±0.03	1.00±0.03
Ad5-GFP组	1.00±0.07	1.00±0.07	1.00±0.06	1.00±0.04
Ad5-SPRY1组	81.89±4.07^ab	3.31±0.19^ab	69.19±3.07^ab	2.61±0.11^ab
F	1 180.719^＊	320.415^＊	1 477.800^＊	541.412^＊

表3 各组Huh7细胞和Hep3B细胞凋亡情况比较（n=3，$\bar{x}±s$）

Tab.3 Comparison of apoptosis status between two groups of Huh7 cells and Hep3B cells

组别	Huh7细胞
组别	细胞凋亡率/%			c-MYC蛋白		BCL2蛋白
对照组	32.14±0.78			1.00±0.03		1.00±0.13
Ad5-GFP组	33.14±1.05			1.00±0.07		1.00±0.04
Ad5-SPRY1组	17.02±1.58^ab			2.30±0.14^ab		1.94±0.11^ab
F	181.832^＊			216.545^＊		85.748^＊
组别		Hep3B细胞
组别		细胞凋亡率/%	c-MYC蛋白		BCL2蛋白
对照组		27.51±2.90	1.00±0.07		1.00±0.02
Ad5-GFP组		28.84±2.35	1.00±0.10		1.00±0.10
Ad5-SPRY1组		3.53±0.63^ab	2.94±0.07^ab		1.74±0.10^ab
F		127.296^＊	638.889^＊		115.649^＊

图3 流式细胞术检测腺病毒对肝癌细胞凋亡的影响 Annexin V-PE/7-AAD双染法，右下象限为早期凋亡细胞，右上象限为晚期凋亡细胞；A：对照组；B：Ad5-GFP组；C：Ad5-SPRY1组。

Fig.3 Effect of Ad5-SPRY1 on apoptosis of hepatocellular carcinoma cells detected by flow cytometry

图4 Western blot检测腺病毒对肝癌细胞凋亡的影响 A：对照组；B：Ad5-GFP组；C：Ad5-SPRY1组。

Fig.4 Effect of Ad5-SPRY1 on apoptosis of hepatocellular carcinoma cells detected by Western blot assay

图5 透射电镜检测腺病毒Ad5-SPRY1对肝癌细胞自噬的影响透射电镜观察自噬体结构，箭头示双层膜自噬体；上层10 000倍标尺为500 μm；下层25 000倍标尺为250 μm。

Fig.5 Effect of Ad5-SPRY1 on autophagy in hepatocellular carcinoma cells detected by transmission electron microscope

图6 Western blot检测腺病毒Ad5-SPRY1对肝癌细胞自噬相关蛋白表达的影响 A：对照组；B：Ad5-GFP组；C：Ad5-SPRY1组。

Fig.6 Effect of Ad5-SPRY1 on autophagy related protein expression in hepatocellular carcinoma cells detected by Western blot assay

图7 激光共聚焦检测腺病毒Ad5-SPRY1对肝癌细胞自噬及相关蛋白表达的影响 A：对照组；B：pcDNA3.1组；C：pcDNA3.1-SPRY1组；标尺为10 μm。

Fig.7 Effect of Ad5-SPRY1 on autophagy in hepatocellular carcinoma cells detected by laser scanning confocal microscope

表4 各组Huh7细胞和Hep3B细胞中蛋白表达水平和自噬颗粒的比较（n=3，%，$\bar{x}±s$）

Tab.4 Comparison of LC3Ⅱ protein expression levels and autophagic granules between Huh7 cells and Hep3B cells of each group

组别	Huh7细胞		Hep3B细胞
组别	LC3Ⅰ/Ⅱ蛋白	黄色斑点/个	黄色斑点/个
对照组	1.00±0.06	8.67±2.52	8.33±2.52
pcDNA3.1组	1.00±0.07	8.33±1.53	7.67±4.04
pcDNA3.1-SPRY1组	1.78±0.10^ab	25.33±3.21^ab	16.67±3.21^ab
F	110.120^＊	44.754^＊	6.859^＊

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腺病毒介导的SPRY1过表达对肝细胞癌凋亡和自噬的影响

The effect of adenovirus-mediated SPRY1 overexpression on apoptosis and autophagy in hepatocellular carcinoma

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