摘要
目的探讨溴隐亭对垂体泌乳素腺瘤血管形成的影响及其分子作用机制。方法 2014年1月—2015年6月,体外培养MMQ细胞株并进行MTT比色实验,共设置6个实验组、1个对照组和1个空白组,实验组在培养基和细胞悬液中添加不同浓度(0.125、0.250、0.500、1.000、2.000、4.000μg/ml)溴隐亭,分别记为0.125μg/ml组、0.250μg/ml组、0.500μg/ml组、1.000μg/ml组、2.000μg/ml组、4.000μg/ml组,对照组加入等体积的培养基及细胞悬液,空白组仅加入等体积的培养基;根据半数抑制浓度(IC50)筛选最适浓度溴隐亭进行后续试验。再采用最适浓度的溴隐亭处理MMQ细胞48 h,制备条件培养液(CM),各实验组在培养基和细胞悬液基础上分别加入最适溶度的溴隐亭和CM,对照组加入等体积的培养基和细胞悬液,采用酶联免疫吸附试验(ELISA)检测泌乳素(PRL)浓度及其变化率。用携带GFP基因的慢病毒(LV-GFP)感染人脐静脉血管内皮细胞(HUVEC)制备HUVEC/LV-GFP,用CM孵育HUVEC/LV-GFP细胞,各实验组在培养基和细胞悬液基础上分别加入最适浓度的溴隐亭和CM,CM组在培养基和细胞悬液基础上仅加入CM,1.000μg/ml组在培养基和细胞悬液基础上仅加入1.000μg/ml溴隐亭,对照组加入等体积培养基和细胞悬液;24 h后在荧光显微镜下观察血管样结构形成情况并计数。采用Western blotting法检测对照组与最适浓度溴隐亭组垂体瘤转化基因(PTTG)和血管内皮生长因子(VEGF)表达情况。结果 6个实验组MMQ细胞增殖抑制率时间与组间存在交互作用(P<0.05);培养48 h、72 h 0.125μg/ml组、0.250μg/ml组、0.500μg/ml组、1.000μg/ml组、2.000μg/ml组、4.000μg/ml组MMQ细胞增殖抑制率高于培养24 h,培养72 h 0.125μg/ml组、0.250μg/ml组、0.500μg/ml组、1.000μg/ml组、2.000μg/ml组MMQ细胞增殖抑制率高于培养48 h(P<0.05);而培养48 h与培养72 h 4.000μg/ml组MMQ细胞增殖抑制率比较,差异无统计学意义(P>0.05)。通过软件计算抑制MMQ细胞增殖的IC50接近0.500μg/ml,遂选用0.250、0.500、1.000μg/ml溴隐亭进行后续实验。1.000μg/ml+CM组PRL浓度及变化率均低于0.500μg/ml+CM组、0.250μg/ml+CM组和对照组;0.500μg/ml+CM组PRL浓度及变化率均低于0.250μg/ml+CM组和对照组;0.250μg/ml+CM组PRL浓度及变化率均低于对照组(P<0.05)。CM组MMQ细胞外血管样结构计数多于0.250μg/ml+CM组、0.500μg/ml+CM组、1.000μg/ml+CM组、1.000μg/ml组、对照组;0.250μg/ml+CM组MMQ细胞外血管样结构计数多于0.500μg/ml+CM组、1.000μg/ml+CM组、1.000μg/ml组、对照组;0.500μg/ml+CM组MMQ细胞外血管样结构计数多于1.000μg/ml+CM组、1.000μg/ml组、对照组;1.000μg/ml+CM组MMQ细胞外血管样结构计数多于1.000μg/ml组、对照组;1.000μg/ml组与对照组MMQ细胞外血管样结构计数比较,差异无统计学意义(P>0.05)。对照组PTTG、VEGF表达水平高于0.250μg/ml组、0.500μg/ml组、1.000μg/ml组,0.250μg/ml组PTTG、VEGF表达水平高于0.500μg/ml组、1.000μg/ml组,0.500μg/ml组PTTG、VEGF表达水平高于1.000μg/ml组(P<0.05)。结论溴隐亭可通过抑制垂体泌乳素腺瘤的血管形成而抑制其生长与侵袭,而这种抑制作用与下调PTTG/VEGF信号通路有关。
Objective To investigate the impact of bromocriptine on vascularization of prolactinoma and the molecular mechanism. Methods From January 2014 to June 2015,MMQ cell strains were cultured in vitro,MTT colorimetric assay was used to find the best concentration of bromocriptine. A group added 0. 125 μg / ml of bromocriptine,B group added 0. 250 μg / ml of bromocriptine,C group added 0. 500 μg / ml of bromocriptine,D group added 1. 000 μg / ml of bromocriptine,E group added2. 000 μg / ml of bromocriptine,F group added 4. 000 μg / ml of bromocriptine,control group added isovolumetric culture medium and cell suspension,blank control group added isovolumetric culture medium. IC50 was calculated to find the best concentration of bromocriptine. After that, MMQ cell strains were cultured by the best concentration of bromocriptine for 48 hours, and conditioned medium（ CM） was prepared at the same time,then B group added with CM served as B1 group,C group added with CM served as C1 group,D group added with CM served as D1 group,control group added isovolumetric culture medium and cell suspension as before,and ELISA method was used to detect the PRL concentration and the change rate. Lentivirus with GFP gene was used to infect the HUVEC and prepared for HUVEC / LV-GFP cells,then B group hatched by CM served as B2 group,C group hatched by CM served as C2 group,D group hatched by CM served as D2 group,control group add with CM served as control- CM group; after 24 hours of culture,fluorescence microscope was used to observe the formation and counts of capillary structure. Western blotting method was used to detect the expressions of PTTG and VEGF of control group, of B group, of C group,of D group. Results There was interaction of inhibition ratio of cell multiplication between time and group among A group,B group,C group,D group,E group and F group（ P 〈 0. 05）; after 48 hours,72 hours of culture,inhibition ratio of cell multiplication of A group,of B group,of C group,of D group,of E group,of F group was statistically significantly higher than that after 24 hours of culture,respectively; after 72 hours of culture,inhibition ratio of cell multiplication of A group,of B group,of C group,of D group,of E group was statistically significantly higher than that after 48 hours of culture,respectively（ P 〈 0. 05）,while no statistically significant differences of inhibition ratio of cell multiplication of F group was found compared to that after 48 hours of culture（ P 〉 0. 05）. The calculation showed that,IC50 of inhibition of cell multiplication was close to0. 500 μg / ml. The PRL concentration and the change rate of D1 group were statistically significantly lower those of C1 group,of B1 group,of control group,PRL concentration and the change rate of C1 group were statistically significantly lower than those of B1 group,of control group,PRL concentration and the change rate of B1 group were statistically significantly lower than those of control group（ P 〈 0. 05）. Capillary structure count of control- CM group was statistically significant more than that of B2 group,of C2 group, of D2 group, of D group, of control group, respectively; capillary structure count of B2 group was statistically significant more than that of C2 group,of D2 group,of D group,of control group,respectively; capillary structure count of C2 group was statistically significant more than that of D2 group,of D group,of control group,respectively; capillary structure count of D2 group was statistically significant more than that of D group,of control group,respectively（ P 〈 0. 05）;while no statistically significant differences of capillary structure count was found between D group and control group（ P 〉 0. 05）.Expressions of PTTG and VEGF of control group were statistically significantly higher than those of B group,of C group,of D group; expressions of PTTG and VEGF of B group were statistically significantly higher than those of C group, of D group;expressions of PTTG and VEGF of C group were statistically significantly higher than those of D group（ P 〈 0. 05）. Conclusion Bromocriptine can inhibit development and invasion of prolactinoma by inhibiting the vascularization,the molecular mechanism is possibly correlated with the down- regulation PTTG / VEGF signaling pathway.
出处
《实用心脑肺血管病杂志》
2016年第3期43-48,共6页
Practical Journal of Cardiac Cerebral Pneumal and Vascular Disease
基金
广西壮族自治区卫生厅自筹经费科研课题(z2013624)
广西壮族自治区临床重点专科建设项目
关键词
催乳素瘤
溴隐亭
血管内皮生长因子类
Prolactinoma
Bromocriptine
Vascular endothelial growth factors
