粉防己碱对人视网膜母细胞瘤细胞株S0-Rb50增殖凋亡影响的可能机制研究  

Possible Mechanism of Tetrandrine on Proliferation and Apoptosis of Human Retinoblastoma Cell Line S0-Rb50

作　　者：马高恩 焦云娟[2] 贺琳[3] 刘向玲[1] Ma Gaoen;Jiao Yunjuan;He Lin(Department of Ophthalmology,Third Affiliated Hospital of Xinxiang Medical College,Henan 453003,China)

机构地区：[1]新乡医学院第三附属医院眼一科,453003 [2]新乡医学院病理教研室,453003 [3]新乡医学院眼科医院眼科,453003

出　　处：《医学研究杂志》2020年第4期117-122,99,共7页Journal of Medical Research

基　　金：河南省医学科技攻关计划(201602159)。

摘　　要：目的观察粉防己碱(tetrandrine,Tet)通过磷脂酰肌醇-3激酶(PI 3K)/蛋白激酶B(Akt)通路对人视网膜母细胞瘤细胞株S0-Rb50增殖及凋亡的影响。方法对数期生长S0-Rb50细胞分为4组,即对照组、Tet低、中、高剂量组,分别用0、2.5、5.0、10.0μmol/L Tet处理,观察干预24h细胞形态学变化;MTT法检测干预24、48、72h时各组吸光度(A)值;流式细胞术检测干预24h各组细胞周期及细胞凋亡率;实时荧光定量PCR及Western blot法检测干预24h各组细胞PI 3K、Akt、B淋巴细胞瘤-2基因(Bcl-2)、Bcl-2相关X蛋白(Bax)mRNA和蛋白表达量及裂解的含半胱氨酸天冬氨酸蛋白水解酶3(cleaved caspase-3)蛋白表达量。结果倒置相差显微镜观察发现,对照组细胞形态规则、融合成片,不同干预组细胞随Tet浓度升高细胞体积逐渐减小、核质比例减小,不能融合成片,视野内漂浮细胞或细胞崩解碎片增多。与对照组比较,Tet高剂量组干预24h时A值较低,差异有统计学意义(P<0.05),Tet各剂量组干预48、72h时A值均较低,且随干预剂量升高呈降低趋势,差异均有统计学意义(P<0.05)。对照组MTT试验A值随干预时间的延长呈升高趋势(P<0.05),Tet高剂量组MTT试验A值随干预时间的延长无明显变化(P>0.05)。与对照组比较,Tet各剂量组G 0/G 1、S期细胞占比、PI 3K、Bcl-2 mRNA和蛋白相对表达量及pAkt/Akt较低,且随干预剂量升高呈降低趋势,差异均有统计学意义(P<0.05);与对照组比较,Tet各剂量组G 2/M期细胞占比、凋亡率、Bax mRNA和蛋白相对表达量及cleaved caspase-3蛋白相对表达量较高,且随干预剂量升高呈降低趋势,差异均有统计学意义(P<0.05)。结论Tet可抑制人视网膜母细胞瘤细胞株S0-Rb50增殖、促进凋亡,可能与抑制PI 3K/Akt信号通路、下调Bcl-2 mRNA和蛋白、上调Bax mRNA和蛋白及cleaved caspase-3蛋白表达相关。Objective To observe the effect of tetrandrine(Tet)on the proliferation and apoptosis of human retinoblastoma cell line S0-Rb50 through phosphatidylinositol 3-kinase(PI 3K)/protein kinase B(Akt)pathway.Methods Logarithmic growth S0-Rb50 cells were divided into four groups:control group,Tet low,medium and high dose groups,treated with 0,2.5,5.0 and 10.0mmol/L Tet respectively.Morphological changes of S0-Rb50 cells were observed after 24 hours of intervention.The optical density(A)of each group was measured by MTT method at 24,48 and 72 hours after intervention.Detection of cell cyclee and apoptotic rate were made by flow cytometry after 24 hours of intervention.The mRNA and protein expressions of PI 3K,Akt,B lymphoma-2 gene(Bcl-2),Bcl-2 related X protein(Bax)and the protein expression of cleavage of cleaved caspase-3 were detected by real-time fluorescence quantitative PCR and Western blot.Results Inverted phase contrast microscopy showed that the cells in the control group were regular in shape and fused into fragments.With the increase of Tet concentration,the cell volume and the ratio of nuclear plasma in the different intervention groups decreased gradually,and the cells could not fuse into fragments.Floating cells or cell disintegration fragments increased in the visual field.Compared with the control group,the A value of Tet high dose group was lower at 24 hours after intervention(P<0.05),and the A values of Tet groups were lower at 48 hours and 72 hours after intervention,and decreased with the increase of intervention dose(P<0.05).The A value of MTT test in the control group increased with the prolongation of the intervention time(P<0.05),while the A value of MTT test in the high dose Tet group did not change significantly with the prolongation of the intervention time(P>0.05).Compared with the control group,the proportion of G 0/G 1 and S phase cells,the relative expressions of PI 3K,Bcl-2 mRNA and protein and pAkt/Akt in Tet groups were lower,and decreased with the increase of intervention dose(P<0.05).C

关 键 词：人视网膜母细胞瘤 粉防己碱 增殖 凋亡 

分 类 号：R739.74[医药卫生—肿瘤]