加味四君子汤含药血清对胃癌细胞SGC-7901凋亡相关因子表达的影响
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摘要
目的:研究加味四君子汤含药血清对胃癌细胞SGC-7901凋亡相关因子表达的影响,进一步探讨其具体抗肿瘤作用机制。方法:40只SD大鼠随机分为加味四君子汤低、中、高剂量组(0.213,0.426,0.853 g·kg~(-1)),空白组,每组10只,空白组给予等体积的生理盐水灌胃,各组连续灌胃10 d,末次给药1.5 h后,水合氯醛腹腔麻醉,心脏采血,分离血清,56℃灭菌,0.22μm过滤,制备加味四君子汤高、中、低剂量组含药血清,各剂量组含药血清孵育胃癌细胞SGC-7901,运用流式细胞仪检测细胞早期和晚期凋亡情况,运用实时荧光定量聚合酶链式反应(Real-time PCR)技术检测肿瘤抑制基因p53,c-核蛋白类基因(c-Myc),半胱氨酸天冬氨酸蛋白酶-3(Caspase-3),凋亡相关基因B细胞淋巴瘤-2(Bcl-2)mRNA的表达,运用细胞免疫荧光技术检测p53,c-Myc,Caspase-3,Bcl-2蛋白的相对表达量情况。结果:与空白组比较,加味四君子汤含药血清高剂量组细胞凋亡比率显著升高(P<0.01),且可使胃癌细胞SGC-7901早期+晚期凋亡率达到22.58%(P<0.01)。Real-time PCR结果显示加味四君子汤中剂量组能显著促进Caspase-3 mRNA表达(P<0.01),加味四君子汤高剂量组能显著上调p53及c-Myc mRNA表达量(P<0.01),加味四君子汤高剂量组显著抑制Bcl-2 mRNA表达(P<0.01)。免疫荧光结果显示加味加味四君子汤高剂量组能使c-Myc,Caspase-3,p53蛋白表达水平显著升高(P<0.01),而Bcl-2蛋白表达水平显著降低(P<0.05)。结论:加味四君子汤含药血清能抑制抗凋亡蛋白Bcl-2的表达,促进凋亡相关分子p53,c-Myc,Caspase-3的表达,发挥抗肿瘤作用。
Objective: To explore the effect of modified Si Junzitang( MSJZT) drug serum on the expression of apoptosis-related molecules of gastric cancer cell SGC-7901 and further its anti-tumor mechanism.Method: A total of 40 SD rats were randomly divided into four groups: low-dose,middle-dose,high-dose MSJZT( 0. 213,0. 426,0. 853 g·kg~(-1)) groups and normal group( n = 10). The treatment groups were administrated through gastric perfusion,and the normal group was given the equivalent volume of normal saline for 10 days.1. 5 h after the last treatment,chloral hydrate peritoneal anesthesia was performed,blood was collected from heart,and different doses of serum were separated to prepare drug-containing serum of low-dose,middle-dose,high-dose MSJZT groups,in order to incubate SGC-7901 gastric cancer cell. Early and late apoptosis rates were detected with flow cytometry. Afterwards,the tumor suppressor gene p53,c-nucleoprotein gene( c-Myc),cysteine-aspartic acid protease-3( Caspase-3), B-cell lymphoma-2( Bcl-2) mRNA expressions were confirmed by fluorescence quantitative polymerase chain reaction( Real-time PCR). The protein expressions of p53,c-Myc,Caspase-3,Bcl-2 were detected by immunofluorescence. Result: Compared with the normal group,the high-dose MSJZT group could obviously increase the apoptosis rate to 22. 58%( P < 0. 01). The results of Real-time PCR showed that the middle-dose MSJZT group significantly promoted the mRNA expression of Caspase-3( P < 0. 01),the medium-dose group significantly increased the mRNA expressions of p53 and c-Myc( P < 0. 01),and the high-dose group significantly inhibited the mRNA expression of Bcl-2( P < 0. 01). The immunofluorescence results showed that the high-dose MSJZT group could significantly increase the protein expressions of c-Myc, Caspase-3 and p53( P < 0. 01),while the protein expression of Bcl-2 was significantly reduced( P < 0. 05). Conclusion: MSJZT drug serum could exert an anti-tumor effect by inhibiting the expression of the anti-apoptotic protein Bcl-2,and promoting the expressions of pro-apoptotic-related molecules p53,c-Myc,Caspase-3.
Objective: To explore the effect of modified Si Junzitang( MSJZT) drug serum on the expression of apoptosis-related molecules of gastric cancer cell SGC-7901 and further its anti-tumor mechanism.Method: A total of 40 SD rats were randomly divided into four groups: low-dose,middle-dose,high-dose MSJZT( 0. 213,0. 426,0. 853 g·kg~(-1)) groups and normal group( n = 10). The treatment groups were administrated through gastric perfusion,and the normal group was given the equivalent volume of normal saline for 10 days.1. 5 h after the last treatment,chloral hydrate peritoneal anesthesia was performed,blood was collected from heart,and different doses of serum were separated to prepare drug-containing serum of low-dose,middle-dose,high-dose MSJZT groups,in order to incubate SGC-7901 gastric cancer cell. Early and late apoptosis rates were detected with flow cytometry. Afterwards,the tumor suppressor gene p53,c-nucleoprotein gene( c-Myc),cysteine-aspartic acid protease-3( Caspase-3), B-cell lymphoma-2( Bcl-2) mRNA expressions were confirmed by fluorescence quantitative polymerase chain reaction( Real-time PCR). The protein expressions of p53,c-Myc,Caspase-3,Bcl-2 were detected by immunofluorescence. Result: Compared with the normal group,the high-dose MSJZT group could obviously increase the apoptosis rate to 22. 58%( P < 0. 01). The results of Real-time PCR showed that the middle-dose MSJZT group significantly promoted the mRNA expression of Caspase-3( P < 0. 01),the medium-dose group significantly increased the mRNA expressions of p53 and c-Myc( P < 0. 01),and the high-dose group significantly inhibited the mRNA expression of Bcl-2( P < 0. 01). The immunofluorescence results showed that the high-dose MSJZT group could significantly increase the protein expressions of c-Myc, Caspase-3 and p53( P < 0. 01),while the protein expression of Bcl-2 was significantly reduced( P < 0. 05). Conclusion: MSJZT drug serum could exert an anti-tumor effect by inhibiting the expression of the anti-apoptotic protein Bcl-2,and promoting the expressions of pro-apoptotic-related molecules p53,c-Myc,Caspase-3.
引文
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[20]李雪莉,郝远瑞,邹建湘,等.C-myc,Bcl-2与胃癌生物学行为和细胞凋亡[J].新消化病学杂志,1997,5(12):28-29.
[21]Um H D.Bcl-2 family proteins as regulators of cancer cell invasion and metastasis:a review focusing on mitochondrial respiration and reactive oxygen species[J].Oncotarget,2016,7(5):5193-5203.
[22]陈伟,马磊,杨立山.甘草次酸对哮喘大鼠气道重塑及肺组织Casepase-3、Bax、Bcl-2表达的影响[J].中药药理与临床,2016,32(4):16-19.
[23]于曼,胡图强.姜黄素通过P53通路诱导人舌鳞癌细胞的凋亡[J].湖北医药学院学报,2018,37(2):132-135.
[2]Mocellin S,Verdi D,Pooley K A,et al.Genetic variation and gastric cancer risk:a field synopsis and Metaanalysis[J].Gut,2015,64(8):1209-1219.
[3]Iveson T,Donehower R C,Davidenko I,et al.Rilotumumab in combination with epirubicin,cisplatin,and capecitabine as first-line treatment for gastric or oesophagogastric junction adenocarcinoma:an openlabel,dose de-escalation phase 1b study and a doubleblind,randomised phase 2 study[J].Lancet Oncol,2014,15(9):1007-1018.
[4]QI F,LI A,Inagaki Y,et al.Chinese herbal medicines as adjuvant treatment during chemo-or radio-therapy for cancer[J].Biosci Trends,2011,4(6):297-307.
[5]Pedersen B,Koktved D P,Nielsen L L.Living with side effects from cancer treatment-a challenge to target information[J].Scand J Caring Sci,2013,27(3):715-723.
[6]高启龙,朱亚楠,石变,等.青龙衣含药血清抑制胃癌SGC-7901细胞生长和诱导凋亡[J].中国实验方剂学杂志,2017,23(13):111-115.
[7]邹超,杨宇飞.四君子汤加减降低胃肠肿瘤患者术后复发转移的研究进展[J].世界中医药,2015,10(2):297-300,303.
[8]张俊杰.四君子汤加味辅助治疗胃癌53例疗效观察[J].国医论坛,2015,30(6):48-49.
[9]WU B,XUAN Z R.Progress in research on applying Sijunzi decoction in treating digestive malignant tumor[J].Chin J Integr Med,2007,13(2):156-159.
[10]JIE Y,HE W,YANG X,et al.Kruppel-like factor 4 acts as a potential therapeutic target of Sijunzi decoction for treatment of colorectal cancer[J].Cancer Gene Ther,2017,24(9):361-366.
[11]QIAN J,XIE H,GUO C X,et al.Si Jun Zi decoction demolition parties inhibit proliferation and induce apoptosis of human gastric cancer BGC823 side population[J].Afr J Tradit Complement Altern Med,2015,12(6):77-89.
[12]魏伟,吴希美,李元建.药理实验方法学[M].4版.北京:人民卫生出版社,2010:70-73.
[13]陈皎皎,胡陵静,张国铎,等.四君子汤加味对肺癌化疗骨髓抑制的作用机制[J].中国实验方剂学杂志,2018,24(2):180-185.
[14]Kim E,Moon A.Ursolic acid inhibits the invasive phenotype of SNU-484 human gastric cancer cells[J].Oncol Let,2015,9(2):897-902.
[15]张志敏,秦传蓉,章必成,等.小檗碱对肝癌Hep G2细胞增殖和凋亡的调节作用[J].医药导报,2018,37(5):512-518.
[16]刘倩,傅缨,资晓飞,等.小檗碱对高糖环境下HCCLM3细胞凋亡与自噬的影响[J].中国实验方剂学杂志,2019,25(2):68-73.
[17]丁大伟,章永红.以细胞凋亡通路为靶点的抗肿瘤中药研究进展[J].中国老年学杂志,2018,38(1):239-241.
[18]白阳,叶健,王敬泽.c-Myc功能及其下游靶点[J].细胞生物学杂志,2007,29(2):191-196.
[19]徐媛辉,唐丹凤,潘小玲,等.原癌基因c-Myc的生物学功能及在卵泡发育中的作用[J].南昌大学学报:医学版,2010,50(11):97-101.
[20]李雪莉,郝远瑞,邹建湘,等.C-myc,Bcl-2与胃癌生物学行为和细胞凋亡[J].新消化病学杂志,1997,5(12):28-29.
[21]Um H D.Bcl-2 family proteins as regulators of cancer cell invasion and metastasis:a review focusing on mitochondrial respiration and reactive oxygen species[J].Oncotarget,2016,7(5):5193-5203.
[22]陈伟,马磊,杨立山.甘草次酸对哮喘大鼠气道重塑及肺组织Casepase-3、Bax、Bcl-2表达的影响[J].中药药理与临床,2016,32(4):16-19.
[23]于曼,胡图强.姜黄素通过P53通路诱导人舌鳞癌细胞的凋亡[J].湖北医药学院学报,2018,37(2):132-135.
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