中药组方黄芩汤抑制胃癌细胞增殖的实验研究

doi:10.3877/cma.j.issn.2095-1221.2019.06.004

中华细胞与干细胞杂志(电子版) ›› 2019, Vol. 09 ›› Issue (06) : 340 -344. doi: 10.3877/cma.j.issn.2095-1221.2019.06.004

所属专题：文献；

论著

中药组方黄芩汤抑制胃癌细胞增殖的实验研究

林少琴¹, 张绍琴², 陈思玉¹, 陈舒怡¹, 陈雄³, 欧阳学农³^,^†(

)

^1. 350025　福州，联勤保障部队第九〇〇医院肿瘤科；350122　福州，福建中医药大学药学院
^2. 350003　福州，福建省肿瘤医院中西医结合科
^3. 350025　福州，联勤保障部队第九〇〇医院肿瘤科

收稿日期:2019-10-12 出版日期:2019-12-01
通信作者: 欧阳学农
基金资助:
国家自然科学基金(81274002); 福建省自然科学基金(2016J01586)

Experimental study of traditional Chinese medicine Huangqin Decoction in inhibition of gastric cancer cell proliferation

Shaoqin Lin¹, Shaoqin Zhang², Siyu Chen¹, Shuyi Chen¹, Xiong Chen³, Xuenong Ouyang³^,^†()

^1. Department of Oncology, 900 Hospital of the Joint Logistics Team, Fuzhou 350025, China; College of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou 350025, China
^2. Department of Integrated Traditional Chinese and Western Medicine, Fujian Cancer Hospital, Fuzhou 350025, China
^3. Department of Oncology, 900 Hospital of the Joint Logistics Team, Fuzhou 350025, China

Received:2019-10-12 Published:2019-12-01
Corresponding author: Xuenong Ouyang
About author:
Corresponding author: Ouyang Xuenong, Email:oyxnong@163.com

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林少琴, 张绍琴, 陈思玉, 陈舒怡, 陈雄, 欧阳学农. 中药组方黄芩汤抑制胃癌细胞增殖的实验研究[J/OL]. 中华细胞与干细胞杂志(电子版), 2019, 09(06): 340-344.

Shaoqin Lin, Shaoqin Zhang, Siyu Chen, Shuyi Chen, Xiong Chen, Xuenong Ouyang. Experimental study of traditional Chinese medicine Huangqin Decoction in inhibition of gastric cancer cell proliferation[J/OL]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2019, 09(06): 340-344.

目的

探讨黄芩汤对胃癌细胞生长增殖及干细胞标志物表达的影响。

方法

采用体外优化培养的胃癌细胞株SGC-7901模型，分别加入生理盐水、5-FU、黄岑汤和5-FU+黄芪汤干预，MTT法检测胃癌细胞增殖情况，Real-time PCR检测胃癌干细胞相关表面标志物CD44、EpCAM、CD90的表达情况；并进一步利用胃癌细胞裸鼠荷瘤模型，观察黄岑汤协同5-FU对体内肿瘤生长的抑制效果。细胞增殖曲线多组间数据比较采用重复测量数据方差分析，单时间点多组数据采用单因素方差分析，组间比较采用Tukey法。

结果

优化培养的胃癌细胞SGC- 7901呈球形生长，细胞活性染色显示良好的活性状态；MTT结果显示，黄岑汤组（0.44±0.04）能够抑制胃癌细胞的增殖，从第4天起与对照组（0.59±0.02）相比，差异具有统计学意义（F = 39.550，P < 0.01）；黄岑汤联合5-FU组（0.36±0.04）加强5-FU对肿瘤细胞的抑制作用，从第3天起与对照组（0.50±0.01）相比，差异具有统计学意义（F = 10.670，P < 0.01），与5-FU组（0.42±0.03）对比，差异具有统计学意义（F = 10.670，P < 0.05）；Real-time PCR结果显示黄岑汤联合5-FU可抑制胃癌细胞中肿瘤干细胞相关表面标志物CD44、EpCAM、CD90表达（P < 0.01）；移植瘤生长抑制实验发现黄芪汤组对荷瘤鼠移植瘤的生长具有抑制作用，与对照组相比移植瘤体积减小，差异具有统计学意义（P < 0.01），黄岑汤联合5-FU组与5-FU组相比对移植瘤抑制效果更明显，差异具有统计学意义（P < 0.01）。

结论

黄芪汤能抑制胃癌细胞的生长和增殖，并能协同5-FU产生抗肿瘤作用。

关键词: 胃癌细胞, 黄芩汤, 细胞增殖, 5-氟尿嘧啶

Objective

To investigate the effect of Huangqin Decoction on the growth and proliferation of gastric cancer cells and the expression of stem cell markers.

Methods

The gastric cancer cell line SGC-7901, which was optimally cultivated in vitro, was added with physiological saline, 5-FU, Huangqin Decoction, and 5-FU + Huangqin Decoction for intervention. The proliferation of gastric cancer cells was detected by MTT assay, and the surface markers associated with gastric cancer stem cells, CD44, EpCAM and CD90, were detected by Real-time PCR. Furthermore, a nude mouse model bearing gastric cancer cells was used to observe the inhibitory effect of Huangqin Decoction in combination with 5-FU on tumor growth in vivo. Cell proliferation data were analyzed by repeated measures multivariate analysis of variance, data comparison between multiple groups was made by using one-way ANOVA, and comparison between groups by using Tukey's method.

Results

The optimally cultured gastric cancer cells SGC-7901 showed a spherical growth, and the cell viability staining showed a good active state. The MTT results showed that the Huangqin Decoction group (0.44±0.04) could significantly inhibit the proliferation of gastric cancer cells, compared with the control group (0.59±0.02) from the fourth day There was a statistical difference (P < 0.01) ; Huangqin Decoction combined with 5-FU group (0.36±0.04) could significantly enhance the inhibitory effect of 5-FU on tumor cells, and there was a statistical difference from the control group (0.50±0.01) (P < 0.01) and the 5-FU group (0.42±0.03) on day 3 (P < 0.05) ; Real-time PCR results showed that Huangqin Decoction combined with 5-FU could inhibit tumor stem cell-related surface markers CD44, EpCAM, and CD90 in gastric cancer cells. Expression (P < 0.01) ; the tumor growth inhibition experiment found that the Huangqin Decoction group had a significant inhibitory effect on the growth of tumor-bearing mice, and the tumor volume was decreased significantly compared with the control group (P < 0.01) , Huangqin Decoction combined with 5-FU has a more significant inhibitory effect on transplanted tumors compared with the 5-FU group (P < 0.01) .

Conclusion

Huangqin Decoction can inhibit the growth and proliferation of gastric cancer cells, and exert an antitumor effect when combined with 5-FU.

Key words: Gastric cancer cells, Huangqin Decoction, Cell proliferation, 5-Fluorouracil

表1 实时荧光定量PCR引物序列

基因	Forward primer	Reverse primer
CD44	GCGTAACGCGTCCCGGACACCA	GCGAGGCTTTCAACACAACC
EpCAM	CGCAGCTCAGGAAGAATGTG	TGAAGTACACTGGCATTGACGA
CD90	CCGACTTCCGAAGCACACAGT	GATGCACAAGGAGTGCGGGATCC
GAPDH	CAAGGTCATCCATGACAACTTTG	GTCCACCACCCTGTTGCTGTAG

表1 实时荧光定量PCR引物序列

基因	Forward primer	Reverse primer
CD44	GCGTAACGCGTCCCGGACACCA	GCGAGGCTTTCAACACAACC
EpCAM	CGCAGCTCAGGAAGAATGTG	TGAAGTACACTGGCATTGACGA
CD90	CCGACTTCCGAAGCACACAGT	GATGCACAAGGAGTGCGGGATCC
GAPDH	CAAGGTCATCCATGACAACTTTG	GTCCACCACCCTGTTGCTGTAG

图1 倒置荧光显微镜下观察不同培养时间点胃癌细胞形态变化（FDA/PI双荧光染色，×100）

表2 黄芩汤抑制胃癌细胞增殖情况比较（±s，n = 3）

分组	第1天	第2天	第3天	第4天	第5天	第6天	第7天
对照组	0.41±0.01	0.43±0.02	0.50±0.01	0.59±0.02	0.74±0.03	0.79±0.02	0.82±0.04
黄岑汤组	0.41±0.04	0.41±0.02	0.45±0.03	0.44±0.04^a	0.45±0.04^a	0.53±0.01^a	0.54±0.03^a
5-FU组	0.41±0.03	0.40±0.01	0.42±0.03^a	0.44±0.03^a	0.49±0.03^a	0.49±0.02^a	0.50±0.03^a
黄岑汤+5-FU组	0.41±0.04	0.40±0.03	0.36±0.04^a	0.33±0.02^ab	0.30±0.04^ab	0.29±0.03^ab	0.28±0.03^ab
F值	0.030	0.992	10.670	39.550	75.940	253.200	128.400
P值	0.992	0.444	< 0.01	< 0.01	< 0.01	< 0.01	< 0.01

表2 黄芩汤抑制胃癌细胞增殖情况比较（±s，n = 3）

分组	第1天	第2天	第3天	第4天	第5天	第6天	第7天
对照组	0.41±0.01	0.43±0.02	0.50±0.01	0.59±0.02	0.74±0.03	0.79±0.02	0.82±0.04
黄岑汤组	0.41±0.04	0.41±0.02	0.45±0.03	0.44±0.04^a	0.45±0.04^a	0.53±0.01^a	0.54±0.03^a
5-FU组	0.41±0.03	0.40±0.01	0.42±0.03^a	0.44±0.03^a	0.49±0.03^a	0.49±0.02^a	0.50±0.03^a
黄岑汤+5-FU组	0.41±0.04	0.40±0.03	0.36±0.04^a	0.33±0.02^ab	0.30±0.04^ab	0.29±0.03^ab	0.28±0.03^ab
F值	0.030	0.992	10.670	39.550	75.940	253.200	128.400
P值	0.992	0.444	< 0.01	< 0.01	< 0.01	< 0.01	< 0.01

表3 黄岑汤抑制胃癌细胞肿瘤干细胞表面标志物表达（±s，n = 3）

分组	CD44	EpCAM	CD90
对照组	1.00±0.02	1.00±0.03	1.00±0.02
黄岑汤组	0.95±0.02	0.80±0.03^a	0.85±0.02^a
5-FU组	0.86±0.03^a	0.74±0.02^a	0.82±0.03^a
黄岑汤+5FU组	0.45±0.03^ab	0.36±0.02^ab	0.43±0.02^ab
F值	276.300	315.000	412.200
P值	< 0.01	< 0.01	< 0.01

表3 黄岑汤抑制胃癌细胞肿瘤干细胞表面标志物表达（±s，n = 3）

分组	CD44	EpCAM	CD90
对照组	1.00±0.02	1.00±0.03	1.00±0.02
黄岑汤组	0.95±0.02	0.80±0.03^a	0.85±0.02^a
5-FU组	0.86±0.03^a	0.74±0.02^a	0.82±0.03^a
黄岑汤+5FU组	0.45±0.03^ab	0.36±0.02^ab	0.43±0.02^ab
F值	276.300	315.000	412.200
P值	< 0.01	< 0.01	< 0.01

表4 各组荷瘤鼠移植瘤的体积（mm³，±s）

分组	动物数（只）	第0天	第15天
对照组	5	149.52±63.97	506.73±17.26
5-FU组	5	142.68±35.72	380.70±24.63^a
黄芪汤组	5	144.70±41.06	407.33±18.29^a
5-FU+黄芪汤组	5	146.29±28.82	301.84±19.74^ab
F值	?	0.008	92.250
P值	?	0.998	< 0.01

表4 各组荷瘤鼠移植瘤的体积（mm³，±s）

分组	动物数（只）	第0天	第15天
对照组	5	149.52±63.97	506.73±17.26
5-FU组	5	142.68±35.72	380.70±24.63^a
黄芪汤组	5	144.70±41.06	407.33±18.29^a
5-FU+黄芪汤组	5	146.29±28.82	301.84±19.74^ab
F值	?	0.008	92.250
P值	?	0.998	< 0.01

图2 荷瘤裸鼠体内移植瘤大小

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