【作者】 李姵萱；

【导师】 王新华；

【作者基本信息】 广州中医药大学 ， 中医临床基础， 2011， 博士

【摘要】 1.目的：疏肝清毒汤是对癌症具有比较客观疗效的—自拟方,本文以其来处理肝癌细胞株HepG-2,以初步探讨疏肝清毒汤体外抗肝癌的作用机制。2.方法：2.1细胞的培养：用0.06%胰蛋白酶将HepG-2细胞分散成单个细胞悬液,用含10%胎牛血清的DMEM培养液配成浓度为3×105个/ml的细胞悬液,按0.1ml/孔分种于96孔板,置37℃、饱和湿度、5%CO2培养箱内培养。2.2疏肝清毒汤对HepG-2细胞的毒性作用实验：将疏肝清毒汤原液作系列倍比稀释成以下八个浓度100、50、25、12.5、6.25、3.13、1.56、0.78g/L。用0.06%胰蛋白酶将HepG-2细胞分散成单个细胞悬液,用含10%胎牛血清的DMEM培养液配成浓度为3×105个/ml的细胞悬液,按0.1ml/孔分种于96孔板,置37℃、饱和湿度、5%CO2培养箱内培养,培养24小时细胞贴壁,2d后换含药培养液0.1ml/孔,每个浓度2孔,并设不加药物的对照。置37℃、饱和湿度、5%CO2培养箱内继续培养,12d后去掉上清,将所余细胞用MTT法测药物细胞毒性。2.3疏肝清毒汤对HepG-2细胞生长曲线的影响：取对数生长期细胞用10%胎牛血清的DMEM培养液配成细胞数为1×104/ml的单细胞悬液,在24孔培养板中接种细胞,每孔1ml。培养24小时细胞贴壁后,用完全培养基将疏肝清毒汤稀释至浓度为60g/L和30g/L,对照组用2%DMEM取代疏肝清毒汤。37℃、5%CO2、饱和湿度条件下于CO2培养箱中培养。在培养后即刻及1、2、3、4、5、6、7天,各取样4孔,消化后各孔取40μl,加0.4%台盼蓝染液10μl,置血球计数板计数拒染的活细胞数,并与培养时间作图。2.4疏肝清毒汤对HepG-2细胞AFP分泌的影响：将疏肝清毒汤原液作系列倍比稀释成以下八个浓度60、30、15、7.5、3.751、1.875、0.938、0.469g/L。用0.06%胰蛋白酶将HepG-2细胞分散成单个细胞悬液,用含10%胎牛血清的DMEM培养液配成浓度为3×105个/ml的细胞悬液,按0.1ml/孔分种于96孔板,置37℃、饱和湿度、5%CO2培养箱内培养,培养24小时细胞贴壁,2d后换含药培养液0.1ml/孔,每个浓度2孔,并设不加药物的对照。置37℃、饱和湿度、5%C02培养箱内继续培养；12d后收集上清,用人血清甲胎蛋白(AFP)酶联定量诊断试剂盒检测上清中AFP的分泌量。2.5疏肝清毒汤对HepG-2细胞ALT和LDH的影响：用0.06%胰蛋白酶将HepG-2细胞分散成单个细胞悬液,用含10%胎牛血清的DMEM培养液配成浓度为3×105个/ml的细胞悬液,将细胞悬浮液加入96孔培养板中,每孔200μl,置37℃、饱和湿度、5%CO2培养箱内培养,培养24小时细胞贴壁。弃培养液,加入浓度为60g/L和30g/L的疏肝清毒汤,每个浓度2孔,对照组用2%DMEM取代疏肝清毒汤。分别培养24 h和48 h后离心收集上清,用酶联免疫法(ELISA)检测ALT、LDH。2.6疏肝清毒汤对HepG-2肝癌细胞凋亡的影响：取对数生长期的细胞,0.06%胰酶消化后制成单细胞悬液,0.4%台盼蓝染色,计算拒染的活细胞率为97.5%。将细胞悬液接种至50cm2培养瓶中,106个细胞/瓶,培养液为含10%FCS、100U/ml青链霉素的10%胎牛血清的DMEM,在37℃、5%CO2、饱和湿度条件下培养24小时后,更换培养液,药物组：每瓶培养液5 ml中加入疏肝清毒汤终浓度为60g/L、30g/L；对照组加入等量2%DMEM。加药后于24小时、48小时每个时间点各取3瓶细胞,进行检测。凋亡细胞的检测：小心弃掉培养液,0.06%胰酶消化3分钟,轻轻吹打细胞成单细胞悬液,1000r/min离心10分钟,Binding Buffer重悬细胞至1×106/ml,取100μl细胞悬液(HepG-2细胞)至5ml FACS管中,加入5μl Annexin V-FITC,10μlF混匀,室温下避光孵育15分钟后每管加入400μl Binding Buffer,1小时内上机进行流式细胞分析。同时设对照管(主细胞)和补偿设置管,即未染色的细胞,单染Annexin V-FIT的管和单染PI的管。FCM流式细胞术分析：采用FACS Calibur(美国BECKMAN COULTE公司)ALTRA型流式细胞仪,488nm激发光源。先将对照管上样,通过调整参数FSC和SSC,在散射光点图(FSC/SSC)中清获显示出一个清晰、集中的细胞群体,对细胞群体进行设门(Gate),再以门中细胞进行后续荧光信号检测。将对照管(裸细胞)的荧光强度设定为非特异性本底荧光,并在此基础上确定阳性荧光表达比率。调定流式细胞仪,使荧光散入图中裸细胞集中分布在左下象限。保持FL1、FL2、FL3不变分别将单染AV-FITC的管和单染PI的管上样,调整仪器荧光补偿值,进行光谱重叠校正后即可分析样本。每个样本上获取10,000个细胞,采用CellQuest功能软件进行分析。2.7AO/EB荧光染色法测定疏肝清毒汤诱导HepG-2细胞凋亡：将1×106/ml HepG-2细胞接种于含10%胎牛血清的DMEM培养液中,加入疏肝清毒汤终浓度为60g/L、30g/L,置37℃、5%CO2浓度及饱和湿度的恒温培养箱中悬浮培养,孵育后分别于24及48小时观察结果。AO/EB染色及观察结果：取细胞悬液100μl,加入AO/EB染料4μl混匀,置1滴于载玻片,上覆盖玻片,荧光显微镜下观察结果并计数200个细胞。2.8疏肝清毒汤对细胞凋亡调控基因p53的影响：细胞爬片分组以24孔培养板每孔置入一块无菌玻片,取对数生长期HepG-2细胞消化制备成细胞悬液,调整细胞浓度为1×105/ml,接种24孔板内lml/孔。置培养箱24小时后,中药组加入疏肝清毒汤终浓度分别为60g/L和30g/L,对照组加入2%DMEM,以上各组均平行三孔,继续培养48小时后,取出爬满的细胞的玻片,用PBS漂洗二遍,经4%多聚甲醛固定30分钟,PBS洗涤后自然风干,-20℃保存备用。检测方法按试剂盒说明书进行,主要步骤有：滴入过氧化物酶阻断剂50μl；37℃10分钟；PBS反复洗三次,滴入正常非免疫羊血清50μl；37℃10分钟,直接甩干,分别加入p53一抗,37℃2小时；PBS洗3次,滴入生物标记的二抗,37℃10分钟；PBS洗3次,滴入链霉菌抗生素蛋白-过氧化物酶溶液,37℃10分钟；PBS洗3次,滴入新鲜配制的DAB显色溶液,光学显微镜下观察显色效果,至显色明显,用自来水冲洗后,苏木素复染。中性树胶封固,摄像。2.9疏肝清毒汤对HepG-2肝癌细胞的Fas/FasL表达的影响：细胞的准备：取对数生长期细胞用10%胎牛血清的DMEM培养液配成细胞数为1×104/ml的单细胞悬液,在24孔培养板中接种细胞,每孔1ml。培养24小时细胞贴壁后,用完全培养基将疏肝清毒汤稀释至浓度为60g/L和30g/L,对照组用2%DMEM取代疏肝清毒汤。37℃,5%CO2、饱和湿度条件下于CO2培养箱中培养。Fas/FasL mRNA相对表达量的变化：分别抽提对照组HepG-2,疏肝清毒汤60g/L、30g/L组HepG-2三组细胞总RNA,逆转录为cDNA。取逆转录反应产物1μl作为模板,共扩增Fas基因片段和内参β-actin。PCR反应条件：94℃预变性5min,94℃变性45s,64℃退火30 s,72℃延伸1min,经35个循环。扩增产物在1.8%琼脂糖凝胶中电泳,将结果在凝胶呈像系统中扫描其辉度值,以Fas基因与内参基因的辉度比值作为其相对表达量,比较各组之间的差别。同样实验共重复3次。取逆转录反应产物1μl作为模板,共扩增FasL基因片段和内参β-actin。PCR反应条件：94℃预变性5 min,94℃变性45 s,58℃退火30 s,72℃延伸1 min,经38个循环。扩增产物在1.8%琼脂糖凝胶中电泳,将结果在凝胶呈像系统中扫描辉度值,以Fas L基因与内参基因的辉度比值作为其相对表达量,比较三组之间的差别。同样实验共重复3次。3.结果3.1疏肝清毒汤对HepG-2细胞的毒性作用实验：(1)疏肝清毒汤在设定的八个浓度(100、50、25、12.5、6.25、3.13、1.56、0.78g/L)时,对HepG-2细胞的药物半数毒性浓度TC50为62.65 g/L,对HepG-2细胞的破坏率分别为107.66、37.83、5.82、-0.97、-3.88、-15.52、4.85、21.34%；(2)疏肝清毒汤在浓度100g/L,细胞出现空泡、萎缩、脱落,破坏率分别达107.66%和88.90%；疏肝清毒汤浓度为50g/L,镜下可见细胞形态大致正常。3.2疏肝清毒汤对HepG-2细胞生长曲线的影响：疏肝清毒汤浓度为60g/L,在0-7天时,其细胞数分别为1、1.7、1.8、1.2、0.9、0.7、0.4、0.3万/ml,浓度为30g/L,其细胞数分别为1、1.7、1.9、1.6、1.4、0.9、0.6、0.4万/ml,细胞增殖缓慢。可见,疏肝清毒汤(60g/L、30g/L)能抑制’HepG-2细胞的生长。3.3疏肝清毒汤对HepG-2细胞AFP分泌的影响：疏肝清毒汤浓度为60g/L和30g/L时,HepG-2细胞AFP的分泌量为13.22 ng/ml和18.08 ng/ml,而对照组为214.52 ng/ml。根据正常参考值为小于20ng/ml的标准,可以看出当疏肝清毒汤浓度在60、30 g/L时,能抑制HepG-2细胞AFP的分泌,在体外对肝癌细胞HepG-2的AFP分泌有抑制作用。故疏肝清毒汤在体外抑制肝癌细胞HepG-2分泌AFP的作用较好。3.4疏肝清毒汤对HepG-2细胞ALT和LDH的影响：加入疏肝清毒汤24h后,疏肝清毒汤60g/L组ALT为12.34 U/L,LDH为182.42 U/L,疏肝清毒汤30g/L组ALT为10.89 U/L,LDH为165.33 U/L,与空白组比较有极显著性差异；加入疏肝清毒汤48 h后,疏肝清毒汤60g/L组ALT为18.60 U/L,LDH为284.40 U/L,疏肝清毒汤30g/L组ALT为15.18 U/L,LDH为218.30 U/L,与空白组比较有显著性差异。3.5疏肝清毒汤对HepG-2肝癌细胞凋亡的影响：疏肝清毒汤随浓度增加,HepG-2细胞的凋亡率升高,出现典型的凋亡峰,与对照组相比差异有显著性(P<0.05)。但本实验凋亡率较小,可能与细胞凋亡发生的整个过程短暂,凋亡时间不同步,部分凋亡细胞碎裂未能检测到有关。但实验结果还是较明确地证明了疏肝清毒汤能直接诱导HepG-2细胞凋亡,这可能是疏肝清毒汤抗肝癌的重要机制之一。3.6AO/EB荧光染色法测定疏肝清毒汤诱导HepG-2细胞凋亡：对照组细胞在24和48小时其凋亡率分别为2.10%和3.21%；疏肝清毒汤60g/L诱导HepG-2细胞凋亡,在24和48小时其凋亡率分别为8.90%和18.32%；疏肝清毒汤30g/L诱导HepG-2细胞凋亡,在24和48小时其凋亡率分别为6.40%和12.54%。3.7疏肝清毒汤对细胞凋亡调控基因p53的影响：疏肝清毒汤组p53表达显色较对照组明显加深,呈强染色,且阳性细胞数明显增多。疏肝清毒汤浓度为60g/L时,p53阳性细胞率为33.56%,疏肝清毒汤浓度为30g/L时,p53阳性细胞率为29.45%,与空白对照组比较,有显著性差异(P<0.05),且呈剂量依赖关系。3.8疏肝清毒汤对HepG-2肝癌细胞的Fas/FasL表达的影响：(1)Fas基因mRNA相对表达量的变化：Fas基因与β-actin RT-PCR共扩增,表明以疏肝清毒汤60g/L剂量处理后的HepG-2细胞其Fas基因的相对表达量较对照组明显增高。(2)FasL基因mRNA相对表达量的变化：FasL基因与β-actin RT-PCR共扩增,表明疏肝清毒汤60g/L剂量组HepG-2细胞Fas L基因的相对表达量较空白对照组和阴性对照组明显增高。4.结论4.1疏肝清毒汤能抑制HepG-2细胞的生长,对HepG-2细胞有明显的细胞毒作用,并且这种改变随着药物作用时间的延长而更加明显。4.2疏肝清毒汤在体外抑制肝癌细胞HepG-2分泌AFP的作用较好。4.3疏肝清毒汤抗癌的作用机制是诱导细胞凋亡,且诱导作用随诱导时间延长,其细胞凋亡率逐渐增高。4.4疏肝清毒汤能上调人肝癌细胞HepG-2凋亡相关基因p53的表达,且呈剂量依赖关系。4.5疏肝清毒汤能上调Fas/FasL的表达,从而诱导肝癌细胞的凋亡。更多还原

【Abstract】 1. Objective:ShuGanQingDuTang is a self-mastery medicinal herb which has a relatively objective therapeutic effect on cancer. This research project used its extract to treat the HepG-2 hepatic carcinoma cell line and investigated in vitro its effective mechanism in fighting hepatic cancer.2. Methodology:2.1 Cell culture:The HepG-2 cell line was separated into a single cell suspension with 0.06% parenzyme. It was concentrated to 3×105/ml using a DMEM culture containing 10% fetal calf serum to attain a cell suspension. The cells were inoculated in a 96 pore plate at 0.1 ml/pore after which the cells were cultivated in a 37℃, humidity saturated,5%CO2 incubator.2.2 Observing the effect of ShuGanQingDuTang to the HepG-2 cell line from the toxic experiment:Multiple proportions of diluted ShuGanQingDuTang. aqueous extract stock solution were divided into eight concentrations (100g/L,50g/L,25g/L,12.5g/L,6.25g/L, 3.13g/L,1.56g/L, and 0.78g/L). After separating the HepG-2 cells into a single cell suspension using 0.06% parenzyme, its density was prepared into a 3×105/ml cell suspension using a 10%fetal calf serum DMEM culture solution. The cells were inoculated in a 96 pore plate at 0.1ml/pore and cultivated in a 37℃, humidity saturated,5%CO2 incubator. Cell adherence occurred after 24 hours. After 2 days the culture medium was changed to a medium containing the Chinese herbal extract at 0.1ml/pore with 2 pores for each concentration. A control group also was set. The cells were cultivated in a 37℃, humidity saturated,5% CO2 incubator. The supernatant was abscised after 12 days. Cytotoxity on the remnant cells were detected using MTT.2.3 The effect of ShuGanQingDuTang extract on the growth curve of the HepG-2 cell line:Logarithmic phase cells were acquired and prepared to 1×104/ml density single cell suspension using a DMEM culture solution containing 10% fetal calf serum. The cells were inoculated in a 24 pore plate at 1ml/pore. Cells adherence occurred after 24 hours at which time the ShuGanQingDuTang. aqueous extract was diluted to 60g/L and 30g/L and the alcohol extract was diluted to 9g/L and 4.5g/L by complete medium. A control group used 2% DMEM to replace the aqueous and alcohol herbal extracts. The cells then were cultivated in a 37℃, humidity saturated,5%CO2 incubator. After incubation, samples of four pores each were acquired instantly and after the 1st,2nd,3rd,4th,5th,6th, and 7th day. After cellula digestivum samples of 40μl per pore were obtained and 10μl of 0.4% trypanblue staining solution was added. They were placed in a hemocytometer and viable cells which resisted staining were counted. This then was plotted graphically against culture time.2.4 The effect of ShuGanQingDuTang extract on HepG-2 cell AFP secretion:Multiple proportions of diluted ShuGanQingDuTang aqueous extract stock solution was divided into eight concentrations (60g/L,30g/L,15g/L,7.5g/L,3.751g/L,1.875g/L,0.938g/L, and 0.469g/L). After separating the HepG-2 cells into a single cell suspension using 0.06% parenzyme, a DMEM culture solution containing 10% fetal calf serum was used to prepare a cell suspension with a density of 3×105/ml. The cells then were inoculated in a 96 pore plate at 0.1ml/pore and cultivated in a 37℃, humidity saturated,5% CO2 incubator. Cells adherence occurred after 24 hours. After 2 days, the common culture medium was changed to a culture medium containing the herbal extract at O.lml/pore with 2 pores per concentration. A control group was established. Cell cultivation continued in a 37℃, humidity saturated,5%CO2 incubator. Supernatant was collected after 12 days and AFP secretion was detected using an AFP diagnostic kit.2.5 The effect of ShuGanQingDuTang extract on HepG-2 cell ALT and LDH secretions:The HepG-2 cells were separated into a single cell suspension using 0.06% parenzyme. DMEM culture solution containing 10% fetal calf serum was used to prepare the cell suspension to a density of 3×105/ml. The cells were inoculated into a 96 pore plate at 0.2ml/pore and cultivated in a 37℃, humidity saturated,5%CO2 incubator. Cell adherence occurred after 24 hours. The culture solution was abscised and 60g/L and 30g/L of the ShuGanQingDuTang aqueous extract was added with 2 pores per concentration. A control group was established using 2% DMEM to replace the ShuGanQingDuTang. aqueous extract. The cells were cultivated for 24 hours and 48 hours after which supernatant was collected by centrifuge and detected for ALT and LDH by ELISA.2.6 The apoptotic effect of ShuGanQingDuTang aqueous extract on HepG-2 cells: Logarithmic phase cells were acquired and prepared into a single cell suspension by 0.06% parenzyme and stained with 0.4% trypanblue. The viable cell count which resisted staining was 97.5%. The cells were inoculated in 50cm2 culture flasks with 106 cells per bottle and incubated at 37℃, with saturated humidity and 5% CO2 for 24 hours. The culture was a DMEM solution containing 10% fetal calf solution and 100μ/ml mycillin. The culture solution was changed to contain 5 ml of ShuGanQingDuTang aqueous extract per bottle at concentrations of 60g/L and 30g/L. A control group was established which used 2% DMEM to replace the herbal aqueous extract. Add the herbal extracts 24 and 48 hours later,each time point from each of 3 bottles of cell were detected for apoptosis. Apoptosis detection methodology:The culture solution was abscised carefully, trypsinized for 3 minutes by 0.06% parenzyme and lightly tapped to create a single cell suspension from the HepG-2 divided cell line and centrifuged for 10 minutes at 1000rpm. The cells were resuspended to 1×106/ml with Binding Buffer. 100μl of cell suspension solution was obtained and placed into 5ml FACS pipes to which was added 5μl of Annexin V-FITC, 10μl F and misce bene. After 15 minutes of incubation away from light.400μl of Binding Buffer was added to each pipe, whereupon flow cytometry was performed within one hour. Simultaneously, a control pipe with adelomorphous cells and a compensation installation pipe with unstained cells, simple staining Annexin V-FIT pipe and a simple staining PI pipe were set. FCM flow cytometry analysis:A FACS Caliber (Beckman Coulte, USA) ALTRA-type flow cytometer was used with a 488nm excitation light source. The control pipe was first used as a sample to obtain a distinct and localized cell colony in the FSC/SSC by calibrating the FSC and SSC parameters. A gate was set for the cell colony after which the continued fluorescence signal detection commenced for the cells in the gate. Fluorescence intensity was set for the control pipe (null cells) for non-specificity background fluorescence. This established the basis for determining the expression ratio of positive fluorescence. The upflow cytometer was calibrated to make the.fluorescence. scatter of the null cell in the graph to be concentrated in the left inferior quadrant. Without altering FL1, FL2, and FL3, the simple staining AV-FITC pipe and the simple staining PI pipe were placed as.samples. Fluorescence compensation was adjusted and analysis commenced after calibrating the optical spectrum overlay.10,000 cells from each sample were obtained and analyzed with CellQuest software.2.7 Determining the ability of ShuGanQingDuTang aqueous extract to induce HepG-2 cell apoptosis using a AO/EB fluorescence staining method:1×106/ml HepG-2 cells were inoculated in 10% fetal calf serum DMEM after which ShuGanQingDuTang aqueous extract was added at densities of 60g/L and 30g/L. The suspension was cultured in a 37℃, humidity saturated,5%CO2 incubator. Results were observed 24 and 48 hours after incubation. Results of AO/EB staining:a 100μl cell suspension was obtained to which was added 4μl of AO/EB colorant and misce bene. One drop was placed on a glass slide and covered with a coverslip.200 cells were counted under a fluorescent microscope.2.8 The effect of ShuGanQingDuTang aqueous extract on the apoptosis control gene p53:Cells slices were divided into groups. Sterile glass slides were inserted in each pore of the 24 pore cultivation plate. HepG-2 logarithmic phase cells were acquired and then digested and prepared into a cell suspension. Cell density was established at 1×105/ml and then inoculated in the 24 pore cultivation plates with 1ml/pore and incubated for 24 hours: ShuGanQingDuTang aqueous extract was added to the herbal group at concentrations of. 60g/L and 30g/L.2% DMEM was added instead to the control group. Each group had three pores set in parallel and cultivation resumed for 48 hours after which the slides which were full of cells were dislodged and rinsed twice with PBS, and fixed for 30 minutes with 4% paraformaldehyde. They were then air dried after being rinsed with PBS and preserved for later use in -20℃refrigerator. Detection was preformed according to the instructions provided with the kit. The main steps were as follows:50μl peroxydase blocking agent was dropped in and left at 37℃for 10 minutes. They were then rinsed with PBS 3 times after which 50μl of normal non-immune goat blood serum was dropped in and left at 37℃for 10 minutes. They were directly dried and the first p53 antibody as separately added and left at 37℃for 2 hours. They were rinsed with PBS 3 times after which the second biological tag antibody was added and left at 37℃for 10 minutes. After rinsing with PBS 3 times, streptomycete antibiotic albumen-peroxydase solution was dropped in and left at 37℃for 10 minutes. They were rinsed with PBS 3 times and freshly prepared DAB colorate coloration solution was dropped in and the coloration effect was observed under light microscope. When the coloration manifested they were rinsed with tap water and counterstained with hematoxylin. They were then fixed with a neutral resin photographed.2.9The effect of ShuGanQingDuTang on the Fas/FasL expression on HepG-2:Cells preparation:Logarithmic phase cells were acquired and prepared as a 1×104/ml concentration of single cell suspension with 10% fetal calf serum DMEM culture solution. The cells were inoculated in 24-pore plates at 1ml/pore. Cell adherence occurred after 24 hours. ShuGanQingDuTang aqueous extract was diluted to concentrations of 60g/L and 30g/L using complete culture medium. The control group used 2% DMEM to replace the ShuGanQingDuTang aqueous extract. Cells were cultured in a 37℃, humidity saturated, 5% CO2 incubator.Relative expression variation of Fas/FasL mRNA:The cell’s total RNA of the 3 groups (the control group and the 60g/L and 30g/L groups with ShuGanQingDuTang aqueous extract) were separately extracted and reverse transcripted into cDNA. 1μl of the reverse transcription reaction product was taken as a mold and the Fas gene fragment and intra-referenceβ-actin were coamplified. PCR reactive condition:35 cycles were performed of 94℃force-denature for 5 minutes,94℃denature for 45 seconds,64℃anealing for 30 seconds, and 72℃extension for 1 minute. The amplified product was ionophoresised in 1.8% sepharose gel, and its brightness scanned in a gel imaging system. The relative value of the brightness of the Fas gene to the intra-referenceβ-actin gene acted as a comparative contrast among the groups. The same experiment was repeated 3 times. 1μl of reverse transcription reaction product was obtained as a mold and the FasL gene fragment and the intra-referenceβ-actin were coamplified. The PCR reactive condition:38 cycles were performed of 94℃denature for 5minutes,94℃denature for 45 seconds,58℃of anealing for 30 seconds,and 72℃extension for 1 minute. The amplified production was ionophoresised in 1.8% sepharose gel, and its brightness scanned in a gel imaging system. The relative value of the brightness of the FasL gene to the intra-referenceβ-actin gene acted as a comparative contrast among the three groups. The same experiment was repeated 3 times. 3. Results:3.1 Experiment on the cytotoxic effect of ShuGanQingDuTang extract on HepG-2:(1) The TC50 of HepG-2 cells was 62.65g/L with concentrations of ShuGanQingDuTang aqueous extract at 100g/L,50g/L,25g/L,12.5g/L,6.25g/L,3.13g/L,1.56g/L, and 0.78g/L. The rate of HepG-2 cell line destruction was 107.66,37.83%,5.82%,-0.97%,-3.88%,-15.52%,4.85%, and 21.34%. (2) Cells manifested vacuoles, atrophy, and abscission, with destruction rates of 107.66% and 88.90%, respectively, with ShuGanQingDuTang aqueous extract and alcohol extracts at 100g/L. With an aqueous extract density of 50g/L and an alcohol extract density of 6.25 g/L the cell structure appeared roughly normal under the microscope.3.2 The effect of ShuGanQingDuTang extract on the growth curve of HepG-2 cells: With an ShuGanQingDuTang aqueous extract density of 60g/L, cell populations from days 0-7 were 1,1.7,1.8,1.2,0.9,0.7,0.4, and 0.3 105/ml. At a density of 30g/L cell populations from days 0-7 were 1,1.7,1.9,1.6,1.4,0.9,0.6, and 0.4 105/ml. Cell proliferation was. slow. Cell proliferation was fast. This shows that ShuGanQingDuTang aqueous extract (60g/L and 30g/L) could inhibit the growth of HepG-2 cells while transparent inhibiting effect on cell multiplication.3.3 The effect of ShuGanQingDuTang extract HepG-2 cell AFP secretion:With densities of ShuGanQingDuTang. aqueous extract at 60g/L and 30g/L, the volume of AFP secretion of HepG-2 cells were 13.22ηg/ml and 18.08ηg/ml. Comparatively the volume of AFP secretions of the control group was 214.52ηg/ml. The standard reference values is less than 20ηg/ml showing that ShuGanQingDuTang aqueous extract at concentrations of 60g/L and 30g/L could inhibit AFP secretions of the HepG-2 cells in vitro.3.4 The effect of ShuGanQingDuTang extracts on ALT and LDH secretions of HepG-2 cells:Different concentrations of ShuGanQingDuTang aqueous extract were added and measured after 24 hours. At a concentration of 60g/L there was 12.34 U/L of ALT and 182.42 U/L of LDH. At a concentration of 30g/L, the ALT was 10.89 U/L and LDH was 165.33 U/L which was significantly difference than the control group.48 hours after adding a 60g/L concentration of the aqueous extract the ALT was 18.60 U/L and LDH was 284.40 U/L whereas the 30g/L group measured ALT at 15.18 U/L and LDH at 18.30 U/L which also was significantly difference than the control group.3.5 The effect of ShuGanQingDuTang aqueous extract on HepG-2 apoptosis:HepG-2 cell apoptosis increased proportionately with the increase in the concentrations of ShuGanQingDuTang aqueous extract showing a typical apoptotic peak. This was significantly different than the control group (P<0.05). However, the apoptotic rate was small which might be because the entire apoptotic process was temporary, the apoptotic times were uneven, or portions of apoptotic cell fragments failed to be detected. Nevertheless the experiment results still demonstrated that Euphorbia Antiquorum Linn. could directly induce HepG-2 cell apoptosis which likely could be one of the important mechanisms by which ShuGanQingDuTang inhibits hepatic carcinoma. 3.6 Determining whether ShuGanQingDuTang aqueous extract induces HepG-2 apoptosis using the AO/EB fluorescence staining method:The apoptotic rate of the cells in the control group at 24 hours was 2.10% and at 48 hours was 3.21%. Comparatively, the rate at which the ShuGanQingDuTang aqueous extract (60g/L concentration) induced apoptosis at 24 hours was 8.90% and at 48 hours was 18.32%. The apoptotic rate of the aqueous extract at 30g/L concentration at 24 hours was 6.40% and at 48 hours was 12.54%.3.7 The effect of ShuGanQingDuTang aqueous extract on the apoptosis control gene-p53:The coloration of p53 expression of the ShuGanQingDuTang group was obviously deeper than that of the control group. It had stronger staining and increased proliferation in the positive cell population. At an herbal extract density of 60g/L, the p53 positive cell rate was 33.56%. At a 30g/L concentration, the p53 positive cell rate was 29.45%. This was significantly different than the control group (P<0.05) and showed dose dependency.3.8 The effect of ShuGanQingDuTang on Fas/FasL expression in HepG-2: (1)Relative expression variation of Fas mRNA:The Fas gene andβ-actin coamplified by RT-PCR showed a significantly higher relative value of Fas gene expression in the HepG-2 cells treated by ShuGanQingDuTang at a 60g/L concentration in comparison to the control group.The amplified product was electrophoresis in 1.8% agarose. Its brightness was scanned using a gel imaging system. The relative value of the brightness of Fas gene compared to the intra-referenceβ-actin was used to differentiate the groups. The same experiment was repeated 3 times.(2)Relative expression variation of FasL mRNA:The FasL gene and B-actin coamplified by RT-PCR showed a significantly higher relative value of the Fas gene expression in the HepG-2 cells treated by ShuGanQingDuTang at a concentration of 60g/L compared to the control group and negative control group.4. Conclusion:4.1 ShuGanQingDuTang aqueous extract can inhibit the growth of the HepG-2 cell line while its alcohol extract has no apparent inhibition on cell multiplication. The aqueous extract has an obvious cytotoxic effect on HepG-2 cells. Further, the effect increases obviously with the prolongation over time of the drug action.4.2 The inhibitory effect in vitro of the ShuGanQingDuTang. aqueous extract on HepG-2 AFP secretions is much better than that of its alcohol extract which has no inhibition on AFP secretion.The anticarcinogenic mechanism of ShuGanQingDuTang is apoptosis. With induction time extended,the effect of induction was increasing, the cell apoptosis rate is higher. 4.3 The anticarcinogenic mechanism of ShuGanQingDuTang is apoptosis. The rate at which it induces apoptosis increases gradually over time.4.4 ShuGanQingDuTang aqueous extract can up-regulate the expression of the apoptosis related gene, p53, in HepG-2 cells in a dose dependent manner.4.5 ShuGanQingDuTang aqueous extract can up-regulate Fas/FasL expression thereby inducing apoptosis of hepatic carcinoma cells.更多还原