乳岩宁方诱导MCF-7细胞凋亡及抑制荷瘤裸鼠微血管生成作用的实验研究

【作者】 邹阳；

【导师】 殷东风；

【作者基本信息】 辽宁中医药大学 ， 中医内科学， 2010， 博士

【摘要】 目的:MCF-7是人源乳腺癌细胞, ER、PR、VEGF均阳性表达,本实验分为两部分,通过体外实验观察乳岩宁方对MCF-7细胞凋亡的影响,通过体内实验观察乳岩宁方对MCF-7荷瘤裸鼠乳腺癌的抑瘤作用、抑制MCF-7荷瘤裸鼠肿瘤血管生成及对磷酸化Akt蛋白表达水平的影响,来探讨乳岩宁方的作用机制,可能与PI3K/Akt信号转导通路相关。材料与方法:实验细胞株:MCF-7购于湖南湘雅医学院中心实验室细胞库(美国标准生物品收藏中心ATCC制备)。实验动物:SPF级小鼠(4-6周龄)50只雌雄各半,体重20±2 g ;雌性BALB/c-nu/nu裸鼠(4-6周龄)30只,体重20±2 g购于大连医科大学实验动物中心实验动物质量合证:SCXKGGD2004-0017。实验方法:一、含乳岩宁方SPF小鼠血清制备:50只SPF级小鼠适应性喂养3天,随机分为对照组、用药组,用药组按人与小鼠体表面积比值折算成相当于人临床剂量10倍量给药,对照组灌服等体积生理盐水,于每天上、下午相同时间(每次间隔12小时)灌胃,连续灌胃7天,后禁食不禁水,于末次给药2次,中间间隔lh,于最后一次给药后l小时,无菌操作下取血。血样常温放置4小时后4℃过夜,离心2000rpm×15min,0.22um微孔滤膜过滤分装于EP管中,-20℃保存,使用前56℃水浴灭活30min。二、MTT比色法测定乳岩宁含药血清对MCF-7细胞增殖的影响实验步骤:1将对数生长期的细胞以0.25%的消化胰酶/EDTA 800ul消化,用10%FBS的DMEM制成单细胞悬液;2调整细胞浓度为2×105/ml,接种于3块96孔板中,每孔200ul,于37℃,5%CO2饱和湿度条件培养24 h; 3换用0.5%FBS的DMEM培养基,继续于37℃,5%CO2饱和湿度条件培养24 h,同步细胞于G0/G1期;4根据DMEM培养基中血清来源不同将细胞随机分为6组,即5%正常血清组、10%正常血清组、20%正常血清组及相同体积分数的5%中药血清组、10%中药血清组、20%中药血清组,每组设10个复孔;5每孔分别添加各组不同体积分数血清及DMEM培养基,每孔终体积为200ul,继续分别培养24 h、48 h、72 h;6在不同的终点时间均换成无血清DMEM100ul,并加入MTT (5mg/ml) 20uL,孵育4 h后,吸弃培养液,然后加入二甲基亚砜( DMSO) 150uL/孔,放于水平摇床轻微震荡10 min;7用酶标仪在490nm波长处测定吸光度(OD值),结果取均值;计算细胞增殖抑制率。细胞增殖抑制率=(对照组OD值-实验组OD值)/对照组OD值×100%。三、AO/EB荧光染色法观察乳岩宁对MCF-7细胞凋亡的影响取对数生长期MCF-7细胞,PBS液洗涤1次,0.25%胰酶/EDTA 800ul消化后,用含10%胎牛血清的低糖DMEM培养液配成104个/ml的细胞悬液。以每孔2ml体积接种于6孔板,孔内放玻片。细胞培养生长抑制2天后,分为乳岩宁含药血清浓度为5%、10%、20%、及对照组共4组,每组3个复孔,每组给药24h。1天后,吸去培养液,取出6孔板的玻片,PBS洗3次后,10%甲醛固定30分钟, AO/EB染色,碳酸盐缓冲甘油封片,荧光显微镜观察,激发波长为405 nm。每组分别计数3次,每次计数细胞300个,计算凋亡率,用SPSS10.0软件X2检验进行统计分析,确定影响凋亡的含药血清浓度。含药血清浓度为20%组,凋亡率最高,因此以下实验选择用20%浓度含药血清进行实验。四、流式细胞仪检测MCF-7细胞周期。五、乳岩宁对MCF-7细胞BAX/BCL2/ Caspase3蛋白表达的影响取对数生长期的MCF-7细胞,用0.25%胰酶/EDTA 800ul消化后,配成104个/ml的细胞悬液,以每瓶5ml体积接种于培养瓶中,每2天换液一次。在含有10%胎牛血清低糖DMEM培养基中培养,待其生长至70%融合时,换用0.5%FBS的DMEM培养基继续培养24 h,同步细胞于G0/G1期。将细胞分为对照组(含20%空白血清)和中药血清组(含20%中药血清),培养24 h,1天后,倒去培养液。将细胞培养液吸弃,预冷PBS洗细胞2次,倒出PBS,加入预冷的细胞裂解液,置冰上裂解细胞;细胞样品完全破碎后,用细胞刮刮下细胞,移至准备好的1.5ml的EP管中。冷冻高速离心机中离心,4℃1500转/min离心20分钟,取上清,即是蛋白。用Western blot的方法进行检测。六、乳岩宁含药血清诱导MCF-7细胞凋亡相关信号转导机制Western blot检测P-AKT蛋白表达取对数生长期平滑肌细胞,用PBS液洗涤1次,用0.25%胰酶/EDTA 800ul消化后,制成单细胞悬液,以每瓶5ml体积接种于培养瓶中,于37℃,5%CO2条件下培养;待其生长至70%融合时,换用0.5%FBS的DMEM培养基继续培养24 h,同步细胞于G0/G1期;将细胞分为正常血清组(含20%空白血清)和中药血清组(含20%中药血清),培养24 h;在终点时间,更换含有PDGF-BB(5ng/ml)的培养基继续孵育30 min;分别于5min、10min、20min、30min将细胞培养基吸弃,细胞刮收集细胞,后续Western blot检测。七、以及MCF-7荷瘤裸鼠病理切片制备、透射电镜观察细胞凋亡微观状态、免疫组化检测细胞凋亡原位杂交(TUNEL)法、MVD表达、ELISA方法检测MCF-7荷瘤裸鼠血清中EGF/VEGF表达,以及Western blot法对PI3K-Akt蛋白检测等。结果:1. MTT检测提示乳岩宁20%含药血清干预24小时对MCF-7有明显抑制作用。流式细胞仪凋亡检测试剂盒(AnnexinⅤ-FITC)检测提示乳岩宁含药血清可诱导MCF-7细胞凋亡。2.流式细胞仪细胞周期检测提示乳岩宁含药血清诱导MCF-7细胞停滞于G0/G1-S期,且呈量效关系。3.蛋白检测乳岩宁含药血清可下调BCL-2蛋白表达,提高Bax及Caspase3蛋白表达,降低Bcl-2/Bax比值,下调P-Akt表达,说明乳岩宁含药血清诱导MCF-7细胞凋亡机制之一是通过影响PI3K/Akt通路中磷酸化Akt蛋白表达水平,进而干预细胞周期相关调控蛋白的表达实现。4. MCF-7荷瘤裸鼠在体实验病理、电镜形态学观察细胞凋亡微观状态,免疫组化检测细胞凋亡原位杂交(TUNEL)法、蛋白检测P-Akt弱表达,印证乳岩宁方对MCF-7荷瘤裸鼠亦具有诱导细胞凋亡作用。5.乳岩宁方分组干预后免疫组化检测MCF-7荷瘤裸鼠MVD下降、ELISA方法检测MCF-7荷瘤裸鼠血清中EGF/VEGF表达下调,说明乳岩宁方具有抑制乳腺癌血管生成作用,对MCF-7荷瘤裸鼠乳腺癌具有多靶点治疗作用。结论:1.乳岩宁方对MCF-7细胞及MCF-7荷瘤裸鼠乳腺癌具有诱导细胞凋亡作用。诱导肿瘤细胞凋亡的作用机制与PI3K/Akt信号转导通路相关。2.乳岩宁方具有抗MCF-7荷瘤裸鼠乳腺癌血管生成作用。更多还原

【Abstract】 Purpose:MCF-7 is from human breast cancer, ER,PR and VEGF are all positive expression。This experimental research was consist of two parts , Accoring to observe the influence to the apoptosis of MCF-7 cell by Ruyanning decoction in vitro ,and According to the experiments in vivo about tumor-inhibitition effect, inhabiting angiogenesis of breast cancer ,the influence to the expression of phosphorylatal Akt protein on MCF-7 tumor bearing nude ,to investigate the mechanisms of Ruyanning decoction were related to signal transduction passageway of PI3K/Akt .Material and method:PartⅠThe preparation of mice’blood serum of Ruyanning Decoction. Choosing fifty BALB/C Nude mice of SPF rank, weight between 18 and 22 gram, age between 6 and 8 weeks, rats were randomly assigned into two groups with 25 in each group （the normal blood serum group, the herbal blood serum group）, the herbal blood serum group:Taking in Ruyanning Decoction whose dose was converted according to ratio of body surface area with mice , amount to ten times of clinical dose , intragastric administration was forty gram every kilogram everyday, while giving the same dose normal saline to the control group, the frequency of intragastric administration was twice a day at the same time everyday, 12 hours interval,congtinuing 7days. Ruyanning Decoction was boiled in routine, At last time,mice weren’t given the food , given the herb two times as original dose,the interval of the two times was one hour ,after 1 hour at the last time, then took blood in a sterile operation manner 1 hour before gave herb.Mice were anesthesed by chloral hydrate.The sample of blood was laid up in a four centigrade degree refrigerator till the next day after placed in normal temperature 4 hours. 2000 rpm in the centrifuge for 15 minutes, then filtrated though 0.22um filter membrane, and packed the blood serum respectively in EP pipe, conserved in negative 80 centigrade degree in refrigerator, inactivated it in 56 centigrade degree water for 30 minutes before blood serum being used.PartⅡThe influence of blood serum of Ruyanning Decoction to proliferation of human breast cancer cell of MCF-7 were detected using MTT color metric assay. Experimental process:1. Took cell of ogarithm period of growth,then were digested by pancreatic enzymes /EDTA of 0.25 percentage and made into single cell fluid with Dulbecco’s modified Eagle’s medium（DMEM） composed by 10% fetal bovine serum 100 units/ml penicillin, and 100 ug/ml streptomycin. 2.Adjusting the density of MCF-7 cell to 2×10⁵/ml ,then inoculated 200ul in the three boards with ninty-six holes each, Then the cells were maintained in 37 centigrade degree ,a humidified incubator with 5% CO2 and grown on 100-mm culture dishes in Dulbecco’s modified Eagle’s medium（DMEM） with low glucose for 24H.3. After 24 hours exchanged Dulbecco’s modified Eagle’s medium with 5 percentage fetal bovine serum,continued to cultivate in 37 centigrade, a humidified incubator with 5% CO₂ for 24H,in correspondence with the cell stage of G0/G1 .4.There are six groups in all according to different proportion blood serum of Ruyanning Decoction,the concentration of herbal blood serum was five percentage, ten percentage and twenty percentage, corresponding to the normal blood serum.There were ten repeated holes in each group.5. Added different proportion blood serum and culture dishes into each hole ,the volume of each hole was 200ul,the five percentage blood serum and ten percentage blood serum were consisited of twenty percentage blood serum and the normal blood serum,every hole kept concentration as twenty percentage blood serum . Cultivated for 24 hours,48 hours,72 hours respectively.6. Then exchanged Dulbecco’s modified Eagle’s medium with no fetal bovine serum at different time , then added MTT （5mg/ml ） 20ul,continued cultured for 4 hours,throwed the nutrient solution away ,detected the each time point value of OD by Microplate reader device, Data are presented as mean±S.E.M. of the number of independent experiments shown in each Figure,then caculated the inhibition rate of cell proliferation.PartⅢTo observe the influence of blood serum of Ruyanning Decoction to the apoptosis of MCF-7 cell in the way of AO/EB fluorescent staining. Took cell of ogarithm period of growth,washed by PBS once, then were digested by pancreatic enzymes /EDTA of 0.25 percentage and made into single cell fluid with Dulbecco’s modified Eagle’s medium（DMEM） composed by 10% fetal bovine serum 100 units/ml penicillin, and 100 ug/ml streptomycin. Adjusting the density of MCF-7 cell to 1×10⁴/ml ,then inoculated 2ml in the boards with six holes each. After two days ,cell was divided into four groups with normal blood serum and herbal blood serum, acted on the MCF-7 cell for 24 hours repectively, then droped the nutrient solution, fixed by 10 % formaldehyde for 30 minutes ,AO/EB fluorescent staining , carbonate buffer glycerine seal ,cell was observed by fluorescen- ce microscope with 405nm wavelength.Each group was calculated for three times with 300 cell each time ,then calculated the apoptosis rate. Data are presented as mean±S.E.M. of the number of independent experiments shown in each Figure by SPSS 10.0,determining the optimal experimental concentration.PartⅣAccording to the result of MTT, choosed the optimal concentration of herb serum, cells was divided into normal blood serum and herbal blood serum. The media were then removed and fresh growth media with drugs were added, acted on the cell for 12hours and 24hours respectively. The medium was carefully removed and combined with a 1ml phosphate-buffered saline （PBS） rinse to constitute the“detached sample”.The adherent cells were trypsinized and suspended in growth medium. Samples of the adherent and detached suspensions were counted and asses -sed for viability by exclusion of trypan blue. The remaining cell suspensions were washed 1×with PBS and then suspended in PBS before being fixed and processed for flow cytometric analyses of DNA content as described previously. Percentages of proliferation cells and cells in the G1, S, and G2/M stages of the cell cycle were determined with a DNA histogram-fitting program. Attempts were made to collect a minimum of 104events/sample for subse quent analyses.Choosed the optimal concentration of herb serum, cell was divided to normal blood serum and herbal blood serum, collected cell after blood serum acted on the cell for 6 hours,12 hours,18 hours,24 hours respectively .Then MCF-7 cell were starved as described above for 24 hours and then treated with the various agents indicated in the text. They were then washed twice with phosphate-buffered saline （PBS） and lysed in ice cold lysis buffer. Cells were detected by FCM. Then carried out statistical analysis on the result. Differences from control were assessed by analysis of variance with ANOVA tests when multiple comparisons were required.PartⅤTo observe the influence of blood serum of Ruyanning Decoction to the expression of BAX/BCL2, Caspase3 , P-Akt protein of MCF-7 cell. Took cell of ogarithm period of growth,then were digested by pancreatic enzymes /EDTA of 0.25 percentage and made into single cell fluid with Dulbecco’s modified Eagle’s medium（DMEM） composed by 10% fetal bovine serum 100 units/ml penicillin, and 100 ug/ml streptomycin. Adjusting the density of MCF-7 cell to 1×104/ml ,then inoculated 5ml in the culture bottle, Then the cells were maintained in 37 centigrade degree ,a humidified incubator with 5% CO2 and grown on 100mm culture dishes in Dulbecco’s modified Eagle’s medium（DMEM） with low glucose for 24H. After 24 hours exchanged Dulbecco’s modified Eagle’s medium with 5 percentage fetal bovine serum,continued to cultivate in 37 centigrade, a humidified incubator with 5% CO2 for 24H,in correspondence with the cell stage of G0/G1.Choosing the optimal experimental concentration, divided into normal blood serum and herbal blood serum, acted on the MCF-7 cell for 24 hours repectively, then collected cell.MCF-7 were starved as described above for 24 hours and then treated with the various agents indicated in the text. They were then washed twice with phosphate-buffered saline（PBS） and lysed in ice-cold lysis buffer.To detected the expression of BAX/BCL2, Caspase3 , P-Akt protein of MCF-7 cell in the way of Western blot.PartⅥTo observe the morphological change of MCF-7 cell by transmission electron microscope,to detect the apoptosis by TUNEL in the way of immunohistochemistry , the expression of MMP-2/MMP-9、MVD was detected by immunohistochemistry, the expression of EGF/VEGF was detected by ELISA.Results:1.The result of MTT color metric assay revealed that there was no significant difference between the five percentage herbal blood serum at different time with 24 hours, 48 hours, 72 hours and the normal blood serum. AS 5 percentage of blood serum was concerned, there was no significant difference between normal blood serum and herbal, while on the percentage of 20 herbal blood serum, there was significant difference about the value of OD comparing to normal blood serum, illuminated that 20 herbal blood serum had obviously effect on the proliferation of MCF-7 cell. The herbal blood serum inhibited the proliferation of rat aortic vascular smooth muscle cell in a concentration-dependent and time-dependent manner.2.The analytical result of Annexin V-FITC/PI double staining detected via Flow cytometry （FCM） showed that:Herbal blood serum of Ruyanning Decoction inhibited the transformation from G0/G1 phase to S phase in a definite range. Comparing to the control group , herbal blood serum delayed the process of cell cycle obviously,the proportion that on G0/G1 phase was more than normal blood serum, while on the S phase the proportion went down sharply, cell blocked on the G1 phase. The herbal blood serum inhibited the proliferation of MCF-7 cell in a concentration-dependent and time-dependent manner.3. The result of Western blot revealed that Ruyanning Decoction serum could down-regulate the expression of Bcl-2 protein and phosphorylatal Akt protein, up-regulate Bax and Caspase3,decreased the ratio of Bcl-2/Bax,in the herbal blood serum, and there was significant difference comparing to normal blood serum.The result show that blood serum of Ruyanning Decoction could induce apoptosis of MCF-7 cell,one of possible mechanism of which effected the expression of phosphorylatal Akt protein in the passageway of PI3K/Akt,then meddled related regulated protein of cell cycle.4.The result of transmission electron microscope revealed that: the morphology of cell in normal blood serum was in a average state, the nucleolus was clear; while in the herbal blood serum the morphology of cell manifested typical apoptosis, for example, cell nucleus was irregular, concentrated cytoplasm, the nucleolus of the cell was disappear, the pyknosis of chromatin, gathered under the caryotheca aside, the swelling of endoplasmic reticulum etc. The result of TUNEL in the way of immunohistochemistry also revealed that Ruyanning Decoction could induce apoptosis of MCF-7 cell. 5.The result of the expression of MMP-2/MMP-9、MVD detected by immunohistochemistry revealed that MVD of MCF-7 mammary cancer loaded on Nude mice falled ,improved the inhibition rate of tumor ,down-regulated the expression of EGF/VEGF of blood serum of micedetected by ELISA ,the expression of MMP-2/MMP-9 reduced ,illuminated Ruyanning Decoction had the effect to inhibite angiogenesis of mammary cancer,had multi-target curable effect.Conclusion:1. The prescription of Ruyanning has the effect of inducing the apoptosis of the cell of MCF-7 breast cancer and MCF-7 tumor bearing nude . and MCF-7 tumor bearing nude by the prescription of Ruyanning are related to signal transduction passageway of PI3K/Akt .2. Angiogenesis of MCF-7 breast cancer on tumor bearing nude can be inhabited by the prescription of Ruyanning.更多还原