论文标题:松果菊苷对大鼠肝癌细胞GJIC影响的研究
Study on the Effect of Echinacoside on GJIC in Rat Hepatocarcinoma Cell
论文作者
论文导师 李杰芬,论文学位 博士,论文专业 中西医结合基础
论文单位 广州中医药大学,点击次数 731,论文页数 116页File Size12091K
2008-04-01论文网 http://www.lw23.com/lunwen_96712/
Echinacoside;; rat hepatocarcinoma cell CBRH7919;; suicide gene;; Gap Junctional Intercellular Communication;; connexin
一、研究目的及意义: 通过检测吴茱萸碱、松果菊苷、淫羊藿苷及葫芦巴碱对大鼠肝癌细胞CBRH7919GJIC功能的影响,以筛选出能促进CBRH7919细胞GJIC功能的中药活性成分;并进一步定量检测筛选出的活性成分对CBRH7919细胞GJIC功能的影响;在此基础上再检测该活性成分对CBRH7919细胞HSV-tk/GCV系统旁观者效应的影响,以了解该活性成分对GJIC功能是否有促进作用;并进一步检测该活性成分对CBRH7919细胞Cx26和Cx32表达的影响,以期探讨该活性成分影响CBRH7919细胞GJIC功能的机制,为临床综合治疗肿瘤和拓展中药的新用途提供实验依据。 二、研究方法: 1.检测吴茱萸碱、松果菊苷、淫羊藿苷及葫芦巴碱对CBRH7919细胞的作用:分别用终浓度为OμM、0.078125μM、0.15625μM、0.3125μM、0.625μM、1.25μM、2.5μM的吴茱萸碱和终浓度为0μM、12.5μM、25μM、50gM、100μM、200μM、400μM的松果菊苷及淫羊藿苷以及终浓度为0μM、25μM、50μM、100μM、200μM、400μM、800μM葫芦巴碱作用CBRH7919细胞48hrs。倒置显微镜下观察药物作用前后各组细胞的数目和形态;MTT法检测各组细胞存活率。 2.检测吴茱萸碱、松果菊苷、淫羊藿苷及葫芦巴碱对CBRH7919细胞GJIC功能的影响:分别用终浓度为0.25gM吴茱萸碱和50gM松果菊苷及淫羊藿苷以及100μM葫芦巴碱作用CBRH7919细胞48hrs,然后采用划痕标记染料示踪技术(scrape-loading dyetransfer assay,SLDT)检测各组细胞GJIC功能。 3.检测松果菊苷对CBRH7919细胞GJIC功能的影响:分别用终浓度为25μM、50μM、100μM的松果菊苷作用CBRH7919细胞24hrs和48hrs,然后采用预标记双染料传输技术(preloading and dye transefer)及流式细胞术检测各组细胞GJIC功能。 4.检测松果菊苷对CBRH7919细胞HSV-tk/GCV系统旁观者效应的影响:用终浓度为25μM、50μM、100μM的松果菊苷与终浓度为15.7μM的GCV分别和联合作用CBRH7919Rk细胞以及含10%CBRH7919/tk~+细胞的CBRH7919/tk~±混合细胞48hrs。倒置显微镜下观察药物作用前后各组细胞的数目和形态;MTT法检测各组细胞抑制率,两两比较分析各组细胞抑制率的差异和旁观者效应大小,并用金正钧Q值分析松果菊苷与HSV-tk/GCV系统联合的相互作用是否具有协同性。 5.检测松果菊苷对CBRH7919细胞Cx26和Cx32表达的影响:分别用终浓度为25μM、50μM、100μM的松果菊苷作用CBRH7919细胞24hrs和48hrs,然后采用FITC间接免疫荧光染色,倒置显微镜观察和流式细胞仪检测各组细胞表达Cx26和Cx32的阳性细胞数及荧光强度;采用Western-blot技术检测各组细胞Cx26和Cx32的表达量。 三、研究结果: 1.吴茱萸碱、松果菊苷、淫羊藿苷及葫芦巴碱对CBRH7919细胞的作用:镜下观察可见,空白对照组细胞生长状况良好,贴壁很稳,密度高,细胞呈梭状、菱形、三角形或多边形;与对照组细胞相比,吴吴茱萸碱0.625μM及以上浓度组、松果菊苷200μM及以上浓度组、淫羊藿苷200μM及以上浓度组、葫芦巴碱400μM及以上浓度组均出现细胞数目减少、贴壁的细胞非常少、细胞碎片很多、细胞大部分死亡(细胞变圆、脱落),吴茱萸碱各浓度组细胞存活率分别为:107.81±0.01%、111.25±0.01%、103.26±0.01%、87.00±0.02%、71.91±0.01%、64.81±0.02%;松果菊苷各浓度组细胞存活率分别为:104.90±0.03%、112.26±0.02%、112.79±0.01%、112.31±0.01%、91.44±0.02%、60.79±0.01%;淫羊藿苷各浓度组细胞存活率分别为:102.89±0.01%、106.49±0.01%、113.64±0.03%、115.12±0.02%、109.19±0.01%、99.97±0.02%;葫芦巴碱各浓度组细胞存活率分别为:99.84±0.02%、98.76±0.02%、97.06±0.02%、87.27±0.00.02%、85.31±0.04%、84.72±0.04%。 2.吴茱萸碱、松果菊苷、淫羊藿苷及葫芦巴碱对CBRH7919细胞GJIC功能的影响:空白对照组细胞的荧光染料主要出现在划痕旁1列细胞中,半定量为(±),荧光亮度低,细胞间染料传输功能差;与空白对照组细胞相比,细胞经0.25μM吴茱萸碱作用48hrs后的CBRH7919细胞中,荧光染料出现在划痕附近至划痕旁的2～3列细胞内,半定量为(++),荧光亮度增加,细胞间染料传输功能增强;细胞经50μM松果菊苷作用48hrs后的CBRH7919细胞中,荧光染料出现在划痕的附近细胞内至划痕以外的5～6列甚至7～8列细胞内,半定量为(++++),形成连片状的荧光染色,荧光亮度明显增加,细胞间染料传输功能显著增强;细胞经50μM淫羊藿苷及100μM葫芦巴碱作用48hrs后的CBRH7919细胞中,荧光染料出现在划痕附近至划痕旁的1～2列细胞内,半定量为(+),荧光亮度稍增加,细胞间染料传输轻微增强。 3.松果菊苷对CBRH7919细胞GJIC功能的影响:流式细胞仪测定结果显示,与空白对照组相比,经松果菊苷作用24hrs后,松果菊苷25μM组、50μM组、100μM组的红色和绿色荧光皆阳性的细胞数占总细胞数的比例增加,并且随着松果菊苷浓度的增加,红色和绿色荧光皆阳性的细胞数占总细胞数的比例逐渐增加,但增加的幅度不大;经松果菊苷作用48hrs后,松果菊苷25μM组、50μM组、100μM组的红色和绿色荧光皆阳性的细胞数占总细胞数的比例进一步增加,与空白对照组相比,增加的幅度明显增大。但松果菊苷终浓度100μM组与终浓度50μM组相比,红色和绿色荧光皆阳性的细胞数占总细胞数的比例不再上升,比终浓度50μM组略低。 4.松果菊苷对CBRH7919细胞HSV-tk/GCV系统旁观者效应的影响:镜下观察发现,空白对照组和松果菊苷组细胞生长状况良好,细胞贴壁很稳,细胞密度高,细胞呈梭状、菱形、三角形或多边形;GCV组细胞数目减少,细胞密度减低,小部分细胞死亡(细胞变圆、脱落);松果菊苷联合GCV组均出现细胞数目减少、细胞密度减低、贴壁的细胞减少、细胞死亡(细胞变圆、脱落)数增加;用25μM、50μM、100μM浓度的松果菊苷联合10%tk~+/GCV组的细胞抑制率(%)分别为16.74±0.05、17.16±0.06、18.88±0.07,与单纯10%tk~+/GCV组(7.31±0.08)和相应的各浓度单纯松果菊苷组(2.31±0.07、3.11±0.05、4.62±0.04)的抑制率相比明显要高。各组细胞实际抑制率和理论抑制率的两两比较及金正钧Q值计算结果显示,25μM、50μM、100μM浓度下的松果菊苷联合10%tk~+/GCV组的实际抑制率(分别为16.74、17.16、18.88)显著高于理论抑制率(分别为9.45、10.19、11.59),金正钧Q值分别为1.77、1.68、1.62,均大于1.15,说明其增效作用为协同作用。 5.松果菊苷对CBRH7919细胞Cx26和Cx32表达的影响:用倒置荧光显微镜观察和流式细胞仪分析结果显示,与空白对照组相比,经松果菊苷作用后,CBRH7919细胞表达Cx26和Cx32的阳性细胞数增多、荧光强度增强。Western-blot测定结果显示,与空白对照组相比,经松果菊苷作用后,CBRH7919细胞Cx26和Cx32的表达量增加,并呈一定的剂量效应依赖关系。 四、结论: 1.吴茱萸碱对细胞对大鼠肝癌细胞CBRH7919有较强的细胞毒性;松果菊苷、淫羊藿苷及葫芦巴碱对CBRH7919细胞的细胞毒性较弱。 2.在筛选的四种中药活性成分中,松果菊苷能显著促进和恢复CBRH7919细胞GJIC的功能,故选取松果菊苷作为进一步观察影响CBRH7919细胞GJIC功能的药物。今后在研究药物影响细胞GJIC的实验中,划痕标记染料示踪实验可以作为初步筛选待选药物的指标。 3.松果菊苷具有上调大鼠肝癌细胞CBRH7919 GJIC功能的作用,并呈一定的剂量效应效应依赖关系。 4.松果菊苷联合HSV-tk/GCV系统能显著地提高tk~+、tk~-混合细胞对GCV的敏感性,具有明显增强HSV-tk/GCV系统的杀伤效应的作用,而且松果菊苷对HSV-tk/GCV系统杀伤肿瘤细胞的增效作用是一种协同性相互作用。松果菊苷可能通过提高HSV-tk/GCV系统旁观者效应来实现这种协同性增效作用,其机制可能主要是松果菊苷促进和恢复了CBRH7919细胞GJIC的功能。 5.松果菊苷对大鼠肝癌细胞CBRH7919 Cx26和Cx32的表达有促进作用,并呈一定的剂量效应依赖关系,而且可能促进Cx26和Cx32的靶向输送,使表达的Cx26和Cx32主要分布在细胞膜上,有利于细胞间通讯。 6.松果菊苷可能通过促进大鼠肝癌细胞CBRH7919 Cx表达、成熟与定位分布,在一定程度上恢复了Cx介导的GJIC功能,重新建立细胞间连接,进而提高HSV-tk/GCV系统旁观者效应来实现与HSV-tk/GCV系统协同性增效作用。
Objective: By investigating the effect of evodiamine,echinacoside,icariin and trignelline on rat hepatocarcinoma cells CBRH7919,to clarify the active components which can accelerate the function of GJIC and then examine the effect of these active components on cells CBRH7919.Base on the facts,to examine the effect of these active components on the bystander effect of HSV-tk/GCV system in CBRH7919 to research whether these active components can accelerate the function of GJIC or not.Furthermore,by investigating their effect on the expression of Connexin 26 and Connexin 32 in CBRH7919,to explore the effect mechanism of these active components on the function of GJIC in CBRH7919 for constructing the foundation of clinical comprehensive tumor therapy and exploring new applicaions of Chinese medicine. Methods: 1.To examine the effect of evodiamine,echinacoside,icariin and trignelline on rat hepatocarcinoma cells CBRH7919:The cells CBRH7919 were treated seperately by evodiamine with final concentration at 0μM,0.078125μM,0.15625μM,0.3125μM, 0.625μM,1.25μM and 2.5μM,echinacoside and icariin each with final concentration at 0μM,12.5μM,25μM,50μM,100μM,200μM and 400μM,trignelline with final concentration at 0μM,25μM,50μM,100μM,200μM,400μM and 800μM for 48hrs.The quantity and the form of cells in each group were observed under inverted phase contrast microscope.The viability of cell in each group was detected by MTT assay. 2.To examine the effect of evodiamine,echinacoside,icariin and trignelline on the function of GJIC in rat hepatocarcinoma cells CBRH7919:The cells CBRH7919 were treated by evodiamine with final concentration at 0.25μM and echinacoside,icariin with final concentration at 50μM and trignelline with final concentration at 100μM for 48hrs.The function of GJIC in each group was detected by scrape-loading dye transfer assay. 3.To examine the bystander effect of echinacoside on the function of GJIC in cell CBRH7919:The cells CBRH7919 were treated by echinacoside with final concentration at 25μM,50μM and 100μM for 24hrs and 48hrs.The function of GJIC in each group was detected by preloading and dye transefer assay and flow cytometry. 4.To examine the influence of echinacoside on the bystander effect on HSV-tk/GCV mechanism in cells CBRH7919:the cells CBRH7919,and the mixed cells with 10%tk~+and tk~- were treated seperately by echinacoside with final concentration at 25μM,50μM and 100μM and GCV with final concentration at 15.7μM,or treated by echinacoside plus GCV with the same concentration,for 48hrs,then the quantity and form of the cells in each group were observed under inverted phase contrast microscope before and after the treatment.The inhibition rate of cells in each group was detected by MTT assay.The Q-value was used to test the synergistic effects on echinacoside combined with the HSV-tk/GCV system. 5.To examine the effect of echinacoside on the expression of Connexin 26 and Connexin 32 in cells CBRH7919:The cells CBRH7919 were treated by echinacoside with final concentration at 25μM,50μM and 100μM for 24hrs and 48hrs.The quantity of positive cells and fluorescence intensity of the expression of Connexin 26 and Connexin 32 in the cells of each group was detected by FITC indireect immunno-flurescent assay and flow cytometry,observed under inverted phase contrast microscope.Western-blot assay was used to examine the expression level of the Connexin 26 and Connexin 32 of cells in each group. Results: 1.The effect of evodiamine,echinacoside,icariin and trignelline on rat hepatocarcinoma cell CBRH7919:Observed under inverted phase contrast microscope,the cells in the blank control group were found in good state with enhanced adherence,high density,displaying spindle,diamond,triangle or polygon in shape.Compared with the cells in the control group,the quantity of cells in the treated group decreased and less cells adhered. Furthermore,a great deal of cell debrus was seen and most cells were found dead(the cells turned round and dropped).The viability of cell in the evodiamine group was 107.81±0.01%,111.25±0.01%,103.26±0.01%,87.00±0.02%,71.91±0.01%,64.81±0.02% respectively.The viability of cell in the echinacoside group was 104.90±0.03%, 112.26±0.02%,112.79±0.01%,112.31±0.01%,91.44±0.02%,60.79±0.01%respectively. The Viability of cell in the icariin group was 102.89±0.01%,106.49±0.01%,113.64±0.03%, 115.12±0.02%,109.19±0.01%,99.97±0.02%respectively.The viability of cell in the trignelline group was 99.84±0.02%,98.76±0.02%,97.06±0.02%,87.27±0.00.02%, 85.31±0.04%,84.72±0.04%respectively. 2.The effect of evodiamine,echinacoside,icariin and trignelline on the function of GJIC on CBRH7919:The fluorescent dyes in the blank control group were seen mainly transfering to the first row of cells,with semi-quantitative(±),low fluoscence brightness and weak intercellular transfer ability.Compared with the cells in the blank control group,the fluorescence dyes in the group treated by evodiamine at 0.25μM appeared neighboring the second and third row,with semi-quantitative(++),enhanced fluorescence brightness and reinforced intercellular transfer ability,while the fluorescence dyes in the group treated by echinacoside at 100μM appeared besides or beyond the fifth and sixth row or even seven and eighth row,with semi-quantitative(+++),increased apparently fluorescence brightness, remarkably reinforced intercellular transfer ability.The fluorescence dyes were seen connecting into sheets,while the fluorescence dyes in the the cells treated by icariin at 50μM and trignelline at 100μM were found appeared besides the first and second row,with semi-quantitative(+),slightly enhanced fluorescence brightness and intercellular transfer ability. 3.The influence of echinacoside on the function of GJIC in CBRH7919:the results measured by flow cytometry showed that compared with the blank control group,after treated by echinacoside with the concentration at 25μM,50μM and 100μM,for 24hrs,the ratio of positive cells in the red-green fluorescence occupying in the total cells increased with the increasing of the concentration,while the ratio of positive cells in the green fluorescence occupied in the total cells increased,but only slightly.After being treated for 48hrs,the ratio of positive cells in the red-green fluorescence occupying in the total cells increased more than that in the blank control group.However,compared with the treated group at 50μM,the treated group at 100μM was found that the ratio of the positive cells in the red-green fluorescence occupying in the total cells stopped increasing and was less than that in the treated group at 50μM. 4.The influence of echinacoside on the bystander effect on HSV-tk/GCV in CBRH7919: Observed under inverted phase contrast microscope,the cells in the blank control group were found in good state with enhanced adherence,high density,displaying spindle, diamond,triangle or polygon in shape.Compared with the cells in the control group,the quantity of cells in the treated group decreased,less cells adhered,a great deal of cell debrus was seen and most cells were found dead(the cells turned round and dropped). When combining the echinacoside at 25μM,50μM and 100μM with 10%tk~+/GCV,the inhibition rate was 16.74±0.05,17.16±0.06,18.88±0.07 respectively,which was higher remarkably than that when only treated by 10%tk~+/GCV(7.31±0.08)and the echinacoside at 25μM,50μM and 100μM respectively(2.31±0.07,3.11±0.05,4.62±0.04).When comparing the actual inhibition rate with the theoretical one,the Q-value analysis indicated that when combining the echinacoside with concentration at 25μM,50μM and 100μM with 10%tk~+/GCV,the actual inhibition rate was 16.74,17.16,18.88 respectively,which were remarkable higher than the theoretical ones(9.45,10.19,11.59 respectively),and the Q-value was 1.77,1.68,1.62 respectively.All were higher than 1.15,which indicated synergistic effect. 5.The influence of echinacoside on the expression of Connexin 26 and Connexin 32 in CBRH7919:the results,observed under inverted phase contrast microscope and measured by flow cytometry showed that when comparing with the blank control group,the quantity and fluorescent intensity of positive cells in the expression of Connexin 26 and Connexin 32 in CBRH7919 were found increased and reinforced.Detected by western-blot assay and compared with blank control group,the result showed the expression level increased in Connexin 26 and Connexin 32,after treated by echinacoside,which indicated the dose dependent manner. Conclusion: 1.Evodiamine has stronger cytotoxicity on CBRH7919,and the toxicity from echinacoside, icariin and trignelline on CBRH7919 is weak. 2.Among the four kinds of active components that have been selected,echinacoside could be sorted out for the medicine to further explore its effect on the function of CBRH7919. When conducting the experiment for the medical effect on GJIC in future, scaraping-loading dye transfer assay could be the initial index to screen the medicine candidate. 3.Echinacoside has the ability of up-regulating the function of GJIC on CBRH7919 and indicates dose dependent manner to some extent. 4.When combined with HSV-tk/GCV,echinacoside could remarkably increase the sensitivity of mixed cells tk~+,tk~- on GCV,and has distinct killing effect on the HSV-tk/GCV system.The synergistic effect of echinacoside on the killing ability to tumor cells by HSV-tk/GCV system is cooperative interaction,rather than addictive action.Echinacoside may probably fulfill its cooperative interaction by enhancing the bystander effect of HSV-tk/GCV system.The mechanism might be that echinacoside has the ability to accelerate or recover the function of GJIC in CBRH7919. 5.Echinacoside has stimulatory effects on the expression of Connexin 26 and Connexin 32, and indicates dose dependent manner to some extent.Moreoever,it might stimulate the targeted drug delivery on Connexin 26 and Connexin 32,and force the expression of Connexin 26 and Connexin 32 to locate mainly on the membrane,which facilitates the intercellular communication. 6.By inducing the expression of Connexin,maturation and localization of CBRH7919, echinacoside might recover the GJIC function mediated by connexin to some extent,and rebuilt the GJIC to increase the bystander effect of HSV-tk/GCV system and thus to fulfill the synergistic action with HSV-tk/GCV system.
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