复方藤梨汤含药血清对人脑胶质瘤U251细胞增殖、凋亡及上皮间质转化的影响

doi:10.11958/20221840

天津医药 ›› 2023, Vol. 51 ›› Issue (8): 841-846.doi: 10.11958/20221840

复方藤梨汤含药血清对人脑胶质瘤U251细胞增殖、凋亡及上皮间质转化的影响

武海博(), 梁燕, 沈雷, 范展, 傅国惠^△()

南阳市中心医院神经内科（邮编473000）

收稿日期:2022-11-09 修回日期:2023-02-05 出版日期:2023-08-15 发布日期:2023-08-10
通讯作者: ^△E-mail：fgh377818@sina.com
作者简介:武海博（1988），男，主治医师，主要从事胶质瘤方面研究。E-mail：wuhaibo0929@126.com
基金资助:
河南省医学科技攻关计划联合共建项目(LHGJ20191452);河南省科技厅科技攻关项目(202102310408)

Effects of Fufang Tengli Decoction drug-containing serum on proliferation, apoptosis and epithelial-mesenchymal transition of human glioma U251 cells

WU Haibo(), LIANG Yan, SHEN Lei, FAN Zhan, FU Guohui^△()

Department of Neurology, Nanyang Central Hospital, Nanyang 473000, China

Received:2022-11-09 Revised:2023-02-05 Published:2023-08-15 Online:2023-08-10
Contact: ^△E-mail: fgh377818@sina.com

武海博, 梁燕, 沈雷, 范展, 傅国惠. 复方藤梨汤含药血清对人脑胶质瘤U251细胞增殖、凋亡及上皮间质转化的影响[J]. 天津医药, 2023, 51(8): 841-846.

WU Haibo, LIANG Yan, SHEN Lei, FAN Zhan, FU Guohui. Effects of Fufang Tengli Decoction drug-containing serum on proliferation, apoptosis and epithelial-mesenchymal transition of human glioma U251 cells[J]. Tianjin Medical Journal, 2023, 51(8): 841-846.

摘要/Abstract

摘要：

目的探究复方藤梨汤含药血清对人脑胶质瘤U251细胞增殖、凋亡及上皮间质转化的影响。方法将24只雄性SD大鼠分为对照组和低、中、高剂量组[复方藤梨汤6、12、24 g/（kg·d）]，每组6只，制备含药血清处理人脑胶质瘤U251细胞。采用CCK-8法检测细胞活力，集落形成实验检测细胞克隆形成能力，流式细胞术检测细胞周期分布与细胞凋亡，定量聚合酶链反应检测原癌基因C-myc、细胞周期蛋白D1（CyclinD1）基因表达，Western blot法检测细胞凋亡蛋白[B细胞淋巴瘤-2（Bcl-2）、Bcl-2相关X蛋白（Bax）、胱天蛋白酶（Caspase）-3、Caspase-8]以及β-连环蛋白（β-catenin）、上皮性黏附蛋白（E-cadherin）及波形蛋白（Vimentin）表达。结果选取体积分数10%的含药血清为最终剂量。与对照组比较，低、中、高剂量组细胞活力、克隆形成数、C-myc mRNA、CyclinD1 mRNA、Vimentin表达水平降低，处于S期细胞比例、Bax/Bcl-2比值、Caspase-3表达水平升高（P<0.05）；低、高剂量组G1期细胞比例降低（P<0.05）；中、高剂量组G2期细胞比例、β-catenin表达水平降低，Caspase-8和E-cadherin表达水平升高（P<0.05）；高剂量组细胞凋亡率升高（P<0.05）。结论复方藤梨汤含药血清可促使人脑胶质瘤U251细胞阻滞于S期，抑制细胞增殖，诱导细胞凋亡，通过调节β-catenin、E-cadherin和Vimentin表达抑制上皮间质转化。

关键词: 神经胶质瘤, 上皮-间质转化, 细胞增殖, 细胞凋亡, 细胞增殖抑制药（中药）, 复方藤梨汤含药血清, 人脑胶质瘤U251细胞

Abstract:

Objective To explore effects of Fufang Tengli Decoction drug-containing serum on the proliferation, apoptosis and epithelial-mesenchymal transition (EMT) of human glioma U251 cells. Methods A total of 24 male SD rats were divided into the control group, the low-dose, medium-dose and high-dose Fufang Tengli Decoction groups [6, 12, 24 g/(kg·d)], 6 rats in each group. After 10 d of continuous intragastric administration, blood samples was collected to prepare drug-containing serum, and human glioma U251 cells were treated for 24 h. The cells activity was detected by Cell Counting Kit-8. The cell cloning was detected by colony formation assay. The distribution of cells cycles and apoptosis were detected by flow cytometry. The expressions of proto-oncogene C-myc and cyclin D1 (CyclinD1) genes were detected by quantitative polymerase chain reaction. The expressions of B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax), cysteine aspartase-3 (caspase-3), caspase-8, β-catenin, epithelial cadherin (E-cadherin) and vimentin (Vimentin) were detected by Western blot assay. Results The drug-containing serum with 10% volume fraction was selected as the final dose. Compared with the control group, cells activity, number of clone formation and expression levels of C-myc, CyclinD1 mRNA and Vimentin were decreased, while numbers of cells in S phase and expression levels of Bax/Bcl-2 and Caspase-3 were increased in the low-dose, medium-dose and high-dose Fufang Tengli Decoction groups (P<0.05). The proportion of cells in G2 phase and expression level of β-catenin were decreased, while expression levels of Caspase-8 and E-cadherin were increased in the medium-dose and high-dose Fufang Tengli Decoction groups (P<0.05). Cell apoptosis rate was increased in the high-dose Fufang Tengli Decoction group (P<0.05). Conclusion Fufang Tengli Decoction drug-containing serum can block human glioma U251 cells in S phase, inhibit cell proliferation, induce apoptosis, regulate expressions of β-catenin, E-cadherin and Vimentin, and inhibit EMT.

Key words: glioma, epithelial-mesenchymal transition, cell proliferation, apoptosis, cell proliferation inhibitors (TCD), Fufang Tengli Decoction, human glioma U251 cells

中图分类号:

R739.41

图/表 9

图1 复方藤梨汤含药血清对U251细胞克隆形成能力的影响

Fig.1 Effects of Fufang Tengli Decoction drug-containing serum on clone formation ability of U251 cells

图2 复方藤梨汤含药血清对U251细胞周期分布的影响

Fig.2 Effects of Fufang Tengli Decoction drug-containing serum on distribution of U251 cell cycles

表1 各组细胞周期分布情况比较（n=3，%，$\bar{x}±s$）

Tab.1 Comparison on distribution of cell cycles between the four groups

组别	G1期	S期	G2期
对照组	37.82±0.29	43.16±0.88	19.05±0.60
低剂量组	36.12±0.25^a	46.33±0.33^a	17.54±0.10
中剂量组	38.27±0.73^b	47.57±0.74^a	14.16±1.03^ab
高剂量组	36.58±0.65^ac	50.39±1.63^abc	13.03±2.13^ab
F	11.230^＊＊	26.304^＊＊	15.896^＊＊

表2 各组细胞中C-myc和CyclinD1 mRNA相对表达水平比较（n=3，$\bar{x}±s$）

Tab.2 Comparison on relative expression levels of C-myc and CyclinD1 mRNA between the four groups

组别	C-myc	CyclinD1
对照组	1.00±0.00	1.00±0.00
低剂量组	0.83±0.08^a	0.77±0.05^a
中剂量组	0.69±0.12^a	0.59±0.11^ab
高剂量组	0.54±0.09^ab	0.50±0.11^ab
F	16.339^＊＊	22.510^＊＊

表3 各组细胞凋亡率及凋亡相关蛋白表达水平比较（n=3，$\bar{x}±s$）

Tab.3 Comparison of apoptosis rate and expression levels of apoptosis-related proteins between the four groups

组别	细胞凋亡率/%	Bax/Bcl-2	Caspase-3	Caspase-8
对照组	6.90±0.51	0.36±0.09	0.78±0.15	0.26±0.05
低剂量组	7.28±0.25	1.97±0.36^a	1.46±0.25^a	0.33±0.07
中剂量组	7.57±0.28	2.83±0.24^ab	1.53±0.14^a	0.45±0.09^a
高剂量组	11.06±0.37^abc	2.92±0.18^ab	1.89±0.17^abc	0.78±0.09^abc
F	83.244^＊＊	73.297^＊＊	19.353^＊＊	27.266^＊＊

图3 Western blot法检测各组U251细胞中凋亡相关蛋白表达结果 1：对照组；2：低剂量组；3：中剂量组；4：高剂量组。

Fig.3 Expressions of apoptosis-related proteins in U251 cells detected by Western blot assay

图4 复方藤梨汤含药血清对U251细胞凋亡的影响

Fig.4 Effects of Fufang Tengli Decoction drug-containing serum on apoptosis of U251 cells

表4 各组细胞中上皮间质转化标记物表达水平比较（n=3，$\bar{x}±s$）

Tab.4 Comparison on expression levels of EMT markers between the four groups

组别	β-catenin	E-cadherin	Vimentin
对照组	0.87±0.10	0.59±0.09	1.18±0.12
低剂量组	0.79±0.09	0.71±0.13	0.72±0.07^a
中剂量组	0.56±0.11^ab	0.94±0.10^a	0.43±0.11^ab
高剂量组	0.35±0.07^abc	1.20±0.16^abc	0.39±0.08^ab
F	20.546^＊＊	14.633^＊＊	43.713^＊＊

图5 Western blot法检测各组U251细胞中上皮间质转化标记物蛋白表达结果 1：对照组；2：低剂量组；3：中剂量组；4：高剂量组。

Fig.5 Expressions of EMT markers in U251 cells detected by Western blot assay

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复方藤梨汤含药血清对人脑胶质瘤U251细胞增殖、凋亡及上皮间质转化的影响

Effects of Fufang Tengli Decoction drug-containing serum on proliferation, apoptosis and epithelial-mesenchymal transition of human glioma U251 cells

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