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浙江大学学报（农业与生命科学版）
浙江大学学报（农业与生命科学版）  2019, Vol. 45 Issue (5): 533-541    DOI: 10.3785/j.issn.1008-9209.2019.01.221
生物科学与技术     
蜂王浆蛋白体外消化产物对胃癌细胞SGC-7901增殖和凋亡的影响及其可能机制
王天石1(),王馨梦2,付莉1()
1. 锦州医科大学食品科学与工程学院，辽宁 锦州 121000
2. 锦州医科大学生物化学与分子生物学教研室，辽宁 锦州 121000
Effects of royal jelly protein in vitro digestion products on proliferation and apoptosis of gastric cancer cell SGC-7901 and its possible mechanism
Tianshi WANG1(),Xinmeng WANG2,Li FU1()
1. College of Food Science and Engineering, Jinzhou Medical University, Jinzhou 121000, Liaoning, China
2. Department of Biochemistry and Molecular Biology, Jinzhou Medical University, Jinzhou 121000, Liaoning, China
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摘要：

通过研究蜂王浆蛋白体外消化产物对胃癌细胞SGC-7901增殖、凋亡的影响，并探讨其可能的作用机制，为蜂王浆蛋白辅助治疗胃癌奠定实验基础。采用碱提酸沉法提取蜂王浆蛋白，并通过体外模拟消化实验得到蜂王浆蛋白体外消化产物，然后用不同质量浓度的蜂王浆蛋白体外消化物诱导体外培养的胃癌细胞SGC-7901，通过倒置显微镜观察其对胃癌细胞形态的影响，并通过集落形成实验检测诱导后胃癌细胞的集落形成能力；采用3-(4，5-二甲基噻唑-2)-2，5-二苯基四氮唑溴盐（MTT）法检测其对胃癌细胞增殖的抑制作用，通过流式细胞术检测细胞周期及凋亡，并通过蛋白质印迹法检测其诱导后胃癌细胞中p53和PARP-1蛋白的表达情况。结果表明：蜂王浆蛋白体外消化产物使胃癌细胞形态发生皱缩，细胞密度降低，集落形成能力降低（P＜0.05），对胃癌细胞SGC-7901的增殖具有抑制作用。0.2 mg/mL体外消化产物经24 h和48 h培养后对胃癌细胞抑制率分别达到（57.58±3.48）%和（62.84±1.98）%（P＜0.05）。与空白对照组相比，蜂王浆蛋白体外消化产物对SGC-7901细胞增殖的抑制作用呈剂量依赖性，S期细胞减少，从而引起细胞凋亡；且经蜂王浆蛋白体外消化产物诱导后，胃癌细胞中p53蛋白表达量增加（P＜0.05），PARP-1蛋白表达量减少（P＜0.05）。说明蜂王浆蛋白体外消化产物能抑制胃癌细胞SGC-7901的增殖并诱导其发生凋亡，其机制可能与p53表达上调和PARP-1表达下调有关。
关键词： 蜂王浆蛋白体外消化产物;  胃癌;  细胞增殖;  细胞周期;  细胞凋亡    
Abstract:

To study the effects of in vitro digestion products of royal jelly protein on the proliferation and apoptosis of gastric cancer cell SGC-7901 and to explore its possible mechanism so as to provide an experimental basis for the royal jelly protein in the treatment of gastric cancer, the royal jelly protein was extracted by alkali extraction and acid precipitation. The in vitro digestion products of royal jelly protein were obtained by in vitro simulated digestion experiments. The gastric cancer cells SGC-7901 were cultured in vitro with different concentrations of royal jelly protein in vitro digestion products, and the influence on gastric cancer cells were observed by an inverted microscope. The colony formation ability of gastric cancer cells after induction was tested. The 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay was used to detect the inhibition against proliferation of the gastric cancer cells; the cell cycle and apoptosis were detected by flow cytometry; and the expression levels of p53 and PARP-1 proteins in the gastric cancer cells were detected by Western blotting. The results showed that the in vitro digestion products of royal jelly protein leaded to the shrinkage of gastric cancer cells, decreased cell density and colony formation ability (P＜0.05), and inhibited the proliferation of gastric cancer cell SGC-7901. When the in vitro digestion product concentration was 0.2 mg/mL, the inhibition rates at 24 h and 48 h were (57.58±3.48)% and (62.84±1.98)%, respectively (P＜0.05). Compared with the blank control group, the in vitro digestion products of royal jelly protein inhibited the proliferation of SGC-7901 cells in a dose-dependent manner, and the S phase cells decreased, which leading to the apoptosis. After induction by in vitro digestion products of royal jelly protein, the expression of p53 increased in the gastric cancer cells (P＜0.05), and the expression level of PARP-1 protein decreased (P＜0.05). The above results show that the in vitro digestion products of royal jelly protein can inhibit the proliferation of gastric cancer cell SGC-7901 and induce its apoptosis. The mechanism may be related to the up-regulation of p53 expression and the down-regulation of PARP-1 expression.
Key words: royal jelly protein in vitro digestion products    gastric cancer    cell proliferation    cell cycle    apoptosis
收稿日期: 2019-01-22 出版日期: 2019-12-05
CLC:  TS 201.1  
基金资助: 辽宁省自然科学基金(201801675)
通讯作者: 付莉     E-mail: a297248362@163.com;fuli1979119@126.com
作者简介: 王天石（https://orcid.org/0000-0002-7056-3023），E-mail：a297248362@163.com
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引用本文:

王天石,王馨梦,付莉. 蜂王浆蛋白体外消化产物对胃癌细胞SGC-7901增殖和凋亡的影响及其可能机制[J]. 浙江大学学报（农业与生命科学版）, 2019, 45(5): 533-541.

Tianshi WANG,Xinmeng WANG,Li FU. Effects of royal jelly protein in vitro digestion products on proliferation and apoptosis of gastric cancer cell SGC-7901 and its possible mechanism. Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(5): 533-541.

链接本文:

http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2019.01.221    或    http://www.zjujournals.com/agr/CN/Y2019/V45/I5/533

图1  不同质量浓度蜂王浆蛋白体外消化产物对细胞形态的影响A0. 293T细胞以0.2 mg/mL蛋白体外消化产物处理的对照组；A. SGC-7901细胞以DMSO处理的对照组；B. SGC-7901细胞以0.05 mg/mL蛋白体外消化产物处理的实验组；C. SGC-7901细胞以0.1 mg/mL蛋白体外消化产物处理的实验组；D. SGC-7901细胞以0.2 mg/mL蛋白体外消化产物处理的实验组。
图2  不同质量浓度蜂王浆蛋白体外消化产物对细胞集落形成能力的影响A0～D对应的处理详见图1注。
图3  不同质量浓度蜂王浆蛋白体外消化产物对细胞集落形成数目的影响A0～D对应的处理详见图1注。*表示与DMSO对照组相比在P＜0.05水平差异有统计学意义，**表示与DMSO对照组相比在 P＜0.01水平差异有高度统计学意义；n=3。

处理

Treatment

蜂王浆蛋白体外消化产物质量浓度

Concentration of royal jelly protein

in vitro digestion products/(mg/mL)

增殖抑制率

Proliferation inhibition rate/%

24 h48 h

DMSO对照组

Control group

000

实验组

Experimental group

0.0530.84±1.99*42.28±4.63*
0.145.10±2.70*51.47±2.04**
0.257.58±3.48**62.84±1.98**
表1  不同质量浓度蜂王浆蛋白体外消化产物对胃癌细胞增殖抑制率的影响
图4  不同质量浓度蜂王浆蛋白体外消化产物诱导48 h后胃癌细胞的流式细胞周期图A. SGC-7901细胞以DMSO处理的对照组；B. SGC-7901细胞以0.05 mg/mL蛋白体外消化产物处理的实验组；C. SGC-7901细胞以 0.1 mg/mL蛋白体外消化产物处理的实验组；D. SGC-7901细胞以0.2 mg/mL蛋白体外消化产物处理的实验组。PI-A：经碘化丙啶染色的荧光强度值。

处理

Treatment

蜂王浆蛋白体外消化产物质量浓度

Concentration of royal jelly protein

in vitro digestion products/(mg/mL)

细胞占比

Cell ratio/%

G0-G1期

G0-G1 phase

S期

S phase

G2-M期

G2-M phase

DMSO对照组

Control group

042.71±1.3349.78±0.967.51±1.58

实验组

Experimental group

0.0559.11±2.37*38.05±1.83*2.84±1.36
0.173.19±0.94**24.11±0.45**2.70±1.25
0.278.64±0.79**18.26±0.34**2.10±0.78
表2  不同质量浓度蜂王浆蛋白体外消化产物诱导48 h后对胃癌细胞周期的影响
图5  不同质量浓度蜂王浆蛋白体外消化产物诱导胃癌细胞48 h后的流式细胞凋亡图A～D对应的处理详见图4注。Q1：坏死细胞；Q2：晚期凋亡细胞；Q3：活细胞；Q4：早期凋亡细胞。7-AAD-A：经7-氨基放线菌素D染色的荧光强度值；PE-A：经膜联蛋白V-PE染色的荧光强度值。*表示与DMSO对照组相比在P＜0.05水平差异有统计学意义；n=3。
图6  不同质量浓度蜂王浆蛋白体外消化产物对胃癌细胞中p53和PARP-1表达的影响A～D对应的处理详见图4注。*表示与DMSO对照组相比在 P＜0.05水平差异有统计学意义；n=3。
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