摘要
1.目的:金刚纂是对癌症具有比较客观疗效的一味中药,本文拟以其提取液,来处理肝癌细胞株HepG-2,以初步探讨金刚纂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3×10~5个/ml的细胞悬液,按0.1ml/孔分种于96孔板,置37℃、饱和湿度、5%CO_2培养箱内培养。
2.2金刚纂提取物对HepG-2细胞的毒性作用实验:将金刚纂水提物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,将金刚纂醇提物原液作系列倍比稀释成以下七个浓度100、25、6.25、1.56、0.39、0.098、0.024g/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3×10~5个/ml的细胞悬液,按0.1ml/孔分种于96孔板,置37℃、饱和湿度、5%CO_2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%CO_2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金刚纂提取物对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1×10~4/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,用完全培养基将金刚纂水提物稀释至浓度为60g/L和30g/L,将金刚纂醇提物稀释至浓度为9g/L和4.5g/L,对照组用2%DMEM取代金刚纂水提物及醇提物。37℃、5%CO_2、饱和湿度条件下于CO_2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40 μl,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金刚纂提取物对HepG-2细胞AFP分泌的影响:将金刚纂水提物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。将金刚纂醇提物原液作系列倍比稀释成以下八个浓度9、4.5、2.25、1.125、0.563、0.281、0.141、0.070g/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3×10~5个/ml的细胞悬液,按0.1ml/孔分种于96孔板,置37℃、饱和湿度、5%CO_2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%CO_2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金刚纂水提物对HepG-2细胞ALT和LDH的影响:用0.06%胰蛋白酶将HepG-2
1. Objective: Euphorbia Antiquorum Linn. is a Chinese 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 3×10~5/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°C, humidity saturated, 5% CO_2 incubator.2.2 Observing the cytotoxic effect of Euphorbia Antiquorum Linn. extract on the HepG-2 cell line: Multiple proportions of diluted Euphorbia Antiquorum Linn. 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); similarly, multiple proportions of diluted Euphorbia antiquorum Linn. alcohol extract stock solution were divided into seven concentrations (100g/L, 25g/L, 6.25g/L, 1.56g/L, 0.39g/L, 0.098g/L, and 0.024g/L). After separating the HepG-2 cells into a single cell suspension using 0.06% parenzyme, its density was prepared into a 3×10~5/ml cell suspension using a 10% fetal calf serum DMEM culture solution. The cells were inoculated in a 96 pore plate at 0.1 ml/pore and cultivated in a 37°C, humidity saturated, 5% CO_2 incubator. Cell adherence occurred after 24 hours. After 2 days the culture medium was changed to a medium containing the Chinese herbal extract at 0.1 ml/pore with 2 pores for each concentration. A control group also was set. The cells were cultivated in a 37℃, humidity saturated, 5% CO_2 incubator. The supernatant was abscised after 12 days. Cytotoxity on the remnant cells were detected using MTT.23 The effect of Euphorbia Antiquorum Linn. extract on the growth curve of the HepG-2 cell line: Logarithmic phase cells were acquired and prepared to 1×10~4/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 lml/pore. Cells adherence occurred after 24 hours at which time the Euphorbia Antiquorum Linn, 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°C, 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 40ul per pore were obtained and lOul 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 Euphorbia Antiquorum Linn, extract on HepG-2 cell AFP secretion: Multiple proportions of diluted Euphorbia Antiquorum Linn, aqueous extract stock solution was divided into eight concentrations (60g/L, 30g/L, 15g/L, 7.5g/L, 3.75lg/L, 1.875g/L, 0.938g/L, and 0.469g/L). Likewise, multiple proportions of diluted Euphorbia antiquorum linn, alcohol extract stock solution were divided into seven concentrations (9g/L, 4.5g/L, 2.25g/L, 1.125g/L, 0.563g/L, 0.281g/L, 0.141g/L, and 0.070g/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 3xl05/ml. The cells then were inoculated in a 96 pore plate at 0.1 ml/pore and cultivated in a 37°C, 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 0. lml/pore with 2 pores per concentration. A control group was established. Cell cultivation continued in a 37°C, 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 Euphorbia Antiquorum Linn, 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 3xl05/ml. The cells were inoculated into a 96 pore plate at 0.2ml/pore and cultivated in a 37°C, humidity saturated, 5% CO2 incubator. Cell adherence occurred after 24 hours. The culture solution was abscised and 60g/L and 30g/L of the Euphorbia Antiquorum Linn, aqueous extract was added with 2 pores per concentration. A control group was established using 2% DMEM to replace the Euphorbia Antiquorum Linn, 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 ELIS A.2.6 The apoptotic effect of Euphorbia Antiquorum Linn, 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°C, with saturated humidity and 5% CO2 for 24 hours. The culture was a DMEM solution containing 10% fetal calf solution and lOOu/ml mycillin. The culture solution was changed to contain 5 ml of Euphorbia Antiquorum Linn, 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. 3 bottles of cells were obtained 24 and 48 hours after adding the herbal extracts whereupon they underwent detection 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 lOOOrpm. The cells were resuspended to lxlO6/ml with Binding Buffer. lOOul of cell suspension solution was obtained and placed into 5ml FACS pipes to which was added 5ul of Annexin V-FTTC, lOul F and misce bene. After 15 minutes of incubation away from light. 400ul 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-FTT 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 Euphorbia Antiquorum Linn, aqueous extract to induce HepG-2 cell apoptosis using a AO/EB fluorescence staining method: lxl06/ml HepG-2 cells were inoculated in 10% fetal calf serum DMEM after which Euphorbia Antiquorum Linn, aqueous extract was added at densities of 60g/L and 30g/L. The suspension was cultured in a 37°C, humidity saturated, 5% CO2 incubator. Results were observed 24 and 48 hours after incubation. Results of AO/EB staining: a lOOul cell suspension was obtained to which was added 4ul 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 Euphorbia Antiquorum Linn, 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 lxlO5/ml and then inoculated in the 24 pore cultivation plates with 1 ml/pore and incubated for 24 hours. Euphorbia Antiquorum linn, 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°C refrigerator. Detection was preformed according to the instructions provided with the kit. The main steps were as follows: 50ul peroxydase blocking agent was dropped in and left at 37°C for 10 minutes. They were then rinsed with PBS 3 times after which 50ul of normal non-immune goat blood serum was dropped in and left at 37°C for 10 minutes. They were directly dried and the first p53 antibody as separately added and left at 37°C for 2 hours. They were rinsed with PBS 3 times after which the second biological tag antibody was added and left at 37°C for 10 minutes. After rinsing with PBS 3 times, streptomycete antibiotic albumen-peroxydase solution was dropped in and left at 37°C 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.3. Results:3.1 Experiment on the cytotoxic effect of Euphorbia Antiquorum Linn, extract on HepG-2:(l) The TC50 of HepG-2 cells was 62.65g/L with concentrations of Euphorbia Antiquorum Linn, aqueous extract at lOOg/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%. At concentrations of 100, 25, 6.25, 1.56, 0.39, 0.098, and 0.024g/L of Euphorbia Antiquorum linn, alcohol extract, TC50 was 9.10g/L with a HepG-2 cell destruction rate of 88.90%, 85.40%, 36.90%, 7.25%, 6.00%, 4.75%, and 1.50 %. (2) Cells manifested vacuoles, atrophy, and abscission, with destruction rates of 107.66% and 88.90%, respectively, with Euphorbia antiquorum Iinn.aqueous extract and alcohol extracts at lOOg/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 Euphorbia Antiquorum Linn, extract on the growth curve of HepG-2 cells: With an Euphorbia Antiquorum Linn, 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. At an alcohol extract density of 9g/L, cell populations from 0-7 days were 1, 1.7, 3.1, 6.7, 9.5, 13.4, 21.6, and 39.8 105/ml. At a density of 4.5g/L, cell populations from days 0-7 were 1, 1.7, 3.4, 6.9, 10.4, 17.6, 23.5, and 40.6 105/ml. Cell proliferation was fast. This shows that Euphorbia Antiquorum Linn, aqueous extract (60g/L and 30g/L) could inhibit the growth of HepG-2 cells while its alcohol extract (9g/L and 4.5g/L) had no transparent inhibiting effect on cell multiplication.3.3 The effect of Euphorbia Antiquorum Linn, extract HepG-2 cell AFP secretion: With densities of Euphorbia Antiquorum linn, aqueous extract at 60g/L and 30g/L, the volume of AFP secretion of HepG-2 cells were 13.22ng/ml and 18.08rig/ml. Comparatively the volume of AFP secretions of the control group was 214.52t|g/ml. The standard reference values is less than 20r|g/ml showing that Euphorbia Antiquorum Linn, aqueous extract at concentrations of 60g/L and 30g/L could inhibit AFP secretions of the HepG-2 cells in vitro. But its alcohol extract had no effect on AFP secretion on HepG-2 cells in vitro. Thus, the effect of the aqueous extract in inhibiting AFP secretion in vitro was superior to that of the alcohol extract which had no inhibitory effect.3.4 The effect of Euphorbia Antiquorum Linn, extracts on ALT and LDH secretions of HepG-2 cells: Different concentrations of Euphorbia Antiquorum Linn, 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 Euphorbia Antiquorum Linn, aqueous extract on HepG-2 apoptosis: HepG-2 cell apoptosis increased proportionately with the increase in the concentrations of Euphorbia Antiquorum Linn, 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 Euphorbia Antiquorum linn, inhibits hepatic carcinoma.3.6 Determining whether Euphorbia Antiquorum Linn, aqueous extract induces HepG-2 apoptosis using the AO/EB fluorescence staining method: The apoptotic
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