番茄来源胞外囊泡样颗粒对口腔鳞状细胞癌的作用效果研究

doi:10.11958/20252768

摘要/Abstract

摘要：

目的探讨番茄来源胞外囊泡样颗粒（TDNVs）对口腔鳞状细胞癌（OSCC）恶性生物学行为的影响及其潜在分子机制。方法采用优化的超速离心法及蔗糖密度梯度离心法提取、纯化番茄果实中的TDNVs，经透射电子显微镜（TEM）、纳米颗粒追踪分析（NTA）实验鉴定其形态及粒径，考马斯亮蓝染色检测TDNVs蛋白含量。通过细胞增殖及毒性检测、流式细胞术实验分别检测TDNVs对WSU-HN6和SCC-15细胞增殖、周期及凋亡能力的影响。Real-time PCR及Western blot实验检测细胞周期及凋亡相关基因的表达情况。结果成功分离并鉴定TDNVs，TDNVs的平均粒径为167.8 nm，颗粒浓度约为1.3×10¹¹ 颗粒/mL，且TDNVs具有丰富的蛋白质组分。体外实验表明，TDNVs能够显著抑制OSCC细胞增殖能力（P<0.05），诱导细胞周期阻滞在G₂/M期并促进细胞凋亡（P<0.05）。同时，TDNVs处理导致OSCC细胞中周期蛋白依赖性激酶1（CDK1）、细胞周期蛋白B1（Cyclin B1）、B淋巴细胞瘤-2基因（Bcl-2）mRNA及蛋白表达水平降低（P<0.05），而Bcl-2相关X蛋白（Bax）mRNA及蛋白表达水平升高（P<0.05）。结论 TDNVs能够通过诱导细胞周期阻滞并促进细胞凋亡，从而抑制OSCC细胞的恶性进展。

关键词: 口腔肿瘤, 癌, 鳞状细胞, 番茄, 细胞外囊泡, 细胞增殖, 细胞凋亡, 细胞周期

Abstract:

Objective To explore the effect of tomato derived nanovesicles (TDNVs) on the malignant biological behavior of oral squamous cell carcinoma (OSCC) and its potential molecular mechanism. Methods TDNVs in tomato fruits were extracted and purified by optimizing ultracentrifugation and sucrose density gradient centrifugation. The morphology and particle size were identified by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) experiments. Coomassie Brilliant Blue staining was employed to detect the TDNVs protein content. The effects of TDNVs on the proliferation, cell cycle and apoptosis abilities of WSU-HN6 and SCC-15 cells were detected by cell counting kit 8 (CCK-8) and flow cytometry, respectively. The expression of genes related to cell cycle and apoptosis was detected by Real-time PCR and Western blot experiment. Results TDNVs were successfully isolated and identified, and the average particle size of TDNVs was 167.8 nm. The particle concentration is approximately 1.3×10¹¹ particles/mL. In vitro experiments demonstrated that TDNVs could significantly inhibit the proliferation ability of OSCC cells (P<0.05), induced cell cycle arrest at the G₂/M phase and promoted cell apoptosis (P<0.05). Meanwhile, TDNVs treatment led to a decrease in the mRNA expression levels of cyclin-dependent kinase 1 (CDK1), Cyclin B1 and B-lymphoblastoma-2 gene (Bcl-2) in OSCC cells (P<0.05). The mRNA expression level of Bcl-2 associated X protein (Bax) increased (P<0.05). Conclusion TDNVs can inhibit the malignant progression of OSCC cells by inducing cell cycle arrest and promoting apoptosis.

Key words: mouth neoplasms, carcinoma, squamous cell, lycopersicon esculentum, extracellular vesicles, cell proliferation, apoptosis, cell cycle

中图分类号:

R739.81

图/表 13

表1 引物序列

Tab.1 Primer sequence

基因	引物序列（5′→3′）	产物大小/bp
GAPDH	上游：TCAGCAATGCCTCCTGCAC	117
	下游：TCTGGGTGGCAGTGATGGC	117
CDK1	上游：AGTCTACGGGCTACCCGATT	706
	下游：CCACTCTGCCCTAGGCTTTC	706
Cyclin B1	上游：TGGTGAATGGACTGTCAAGAACA	117
	下游：TACACCTTTGCCACAGCCTT	117
Bcl-2	上游：AAAAATACAACATCACAGAGGAAGT	334
	下游：TCCCGGTTATCGTACCCTGT	334
Bax	上游：TGATGGACGGGTCCGGG	422
	下游：TGTCCAGCCCATGATGGTTC	422

图1 TDNVs的鉴定 A：TDNVs的电镜图片，标尺=100 nm；B：TDNVs的粒径大小和浓度；C：考马斯亮蓝染色显示TDNVs蛋白质分布，n=3。

Fig.1 The identification of TDNVs

图2 不同浓度TDNVs处理WSU-HN6和SCC-15细胞增殖曲线 WSU-HN6细胞：F时间=44.556，F浓度=34.241；SCC-15细胞：F时间=49.301，F浓度=48.808。均P<0.01。

Fig.2 Proliferation curves of WSU-HN6 and SCC-15 cells treated with different concentrations of TDNVs

图3 TDNVs对WSU-HN6和SCC-15细胞周期分布的影响

Fig.3 Effects of TDNVs on the cell cycle distribution of WSU-HN6 and SCC-15 cells

表2 各组细胞周期分布比较（n=3，%，$\bar{x}±s$）

Tab.2 Comparison of cell cycle distribution between the three groups

组别	WSU-HN6			SCC-15
组别	G₁	S	G₂/M	G₁	S	G₂/M
Blank组	65.38±2.98	16.45±2.15	18.16±1.65	56.30±3.49	28.10±1.81	15.60±2.01
PBS组	63.09±3.97	16.09±3.54	20.82±1.42	55.52±2.99	26.80±3.07	17.69±2.32
5 mg/L组	34.64±2.22^ab	9.70±0.66^ab	55.66±2.06^ab	40.17±1.69^ab	25.57±2.20	34.26±0.56^ab
F	89.061^＊＊	7.376^＊	439.508^＊＊	31.068^＊＊	0.817	96.625^＊＊

图4 TDNVs对WSU-HN6和SCC-15细胞CDK1和Cyclin B1蛋白表达的影响 A、B、C依次为WSU-HN6细胞Blank组、PBS组、5 mg/L组；D、E、F依次为SCC-15细胞Blank组、PBS组、5 mg/L组。图6同。

Fig.4 Effect of TDNVs on CDK1 and Cyclin B1 protein expression in WSU-HN6 and SCC-15 cells

表3 各组细胞CDK1、Cyclin B1 mRNA表达水平比较（n=3，%，$\bar{x}±s$）

Tab.3 Comparison of CDK1 and Cyclin B1 mRNA levels between the three groups of cells

组别	WSU-HN6		SCC-15
组别	CDK1	Cyclin B1	CDK1	Cyclin B1
Blank组	1.00±0.06	1.00±0.02	1.00±0.04	1.00±0.01
PBS组	0.97±0.08	1.02±0.16	1.06±0.07	1.04±0.23
5 mg/L组	0.61±0.02^ab	0.45±0.04^ab	0.73±0.03^ab	0.64±0.04^ab
F	82.974^＊＊	40.112^＊＊	36.553^＊＊	8.088^＊

表4 各组细胞CDK1、Cyclin B1蛋白表达水平比较（n=3，%，$\bar{x}±s$）

Tab.4 Comparison of CDK1 and Cyclin B1 protein levels between the three groups of cells

组别	WSU-HN6		SCC-15
组别	CDK1	Cyclin B1	CDK1	Cyclin B1
Blank组	1.00±0.02	1.00±0.02	1.00±0.01	1.00±0.06
PBS组	1.08±0.09	1.12±0.08	0.94±0.04	1.04±0.06
5 mg/L组	0.29±0.05^ab	0.21±0.06^ab	0.68±0.63^ab	0.29±0.25^ab
F	160.195^＊＊	227.726^＊＊	43.739^＊＊	409.108^＊＊

图5 TDNVs对WSU-HN6和SCC-15细胞凋亡的影响

Fig.5 Effects of TDNVs on apoptosis of WSU-HN6 and SCC-15 cells

表5 各组细胞凋亡比例比较（n=3，%，$\bar{x}±s$）

Tab.5 Comparison of apoptosis ratios between the three groups

组别	WSU-HN6	SCC-15
Blank组	5.90±0.30	3.43±1.27
PBS组	6.12±0.28	3.50±0.78
5 mg/L组	20.17±1.15^ab	18.38±1.99^ab
F	400.604^＊＊	107.332^＊＊

图6 TDNVs对WSU-HN6和SCC-15细胞Bcl-2和Bax蛋白表达的影响

Fig.6 Effect of TDNVs on Bcl-2 and Bax protein expression in WSU-HN6 and SCC-15 cells

表6 各组细胞 Bcl-2、Bax mRNA表达水平比较（n=3，%，$\bar{x}±s$）

Tab.6 Comparison of Bcl-2 and Bax mRNA levels between the three groups of cells

组别	WSU-HN6		SCC-15
组别	Bcl-2	Bax	Bcl-2	Bax
Blank组	1.00±0.08	1.00±0.02	1.00±0.12	1.00±0.03
PBS组	1.00±0.14	1.02±0.24	0.90±0.07	1.02±0.20
5 mg/L组	0.58±0.04^ab	1.81±0.02^ab	0.47±0.05^ab	1.56±0.04^ab
F	18.776^＊＊	32.658^＊＊	29.482^＊＊	22.367^＊＊

表7 各组细胞 Bcl-2、Bax 蛋白表达水平比较（n=3，%，$\bar{x}±s$）

Tab.7 Comparison of Bcl-2 and Bax protein levels between the three groups of cells

组别	WSU-HN6		SCC-15
组别	Bcl-2	Bax	Bcl-2	Bax
Blank组	1.00±0.02	1.00±0.02	1.00±0.01	1.00±0.01
PBS组	1.04±0.04	1.09±0.11	0.92±0.16	1.09±0.30
5 mg/L组	0.54±0.05^ab	2.21±0.53^ab	0.25±0.04^ab	2.74±0.13^ab
F	146.687^＊＊	13.852^＊＊	55.052^＊＊	157.001^＊＊

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