论文标题:蜂房水提液对体外培养的肝癌细胞SMMC-7721增殖和凋亡的实验研究
A study of the honeycomb water extracts on the proliferation and apoptosis of the in vitro cultured livertumor cell SMMC-7721
论文作者 刘志伟
论文导师 顾万清,论文学位 硕士,论文专业 肝胆外科
论文单位 军医进修学院,点击次数 101,论文页数 54页File Size2066k
2001-06-01论文网 http://www.lw23.com/lunwen_509279422/ 肝癌,中药,蜂房,细胞培养,细胞增殖,细胞周 期,凋亡
liver neoplasms,tradition Chinese herbs,honeycomb, cell culture,cell proliferation,cell cycle,apoptosis.
传统中医药在恶性肿瘤的治疗方面具有重要的地位。采 用来自天然的抗肿瘤中草药治疗肿瘤，具有疗效确切，毒副 作用小的特点，是晚期肿瘤的主要治疗手段，也是其他抗肿 瘤治疗措施的有益补充。本实验的目的是探讨中药蜂房的体 外抗肿瘤作用。材料和方法：1、人肝癌细胞SMMC-7721的 培养：于含10%胎牛血清(FCS)、100U/ml青霉素、链霉素 的RPMI640培养液中，在37℃、5%CO2、饱和湿度条件下， 于CO2培养箱中进行培养。2、MTT比色试验：选择在对数 生长期生长良好的细胞，制成单细胞悬液，接种细胞于96 孔培养板中。培养24小时后，药物组：以4个稀释度1：40， 1：80，1：160，1：320将蜂房水提液加入培养液，对照组 加入等体积的无菌蒸馏水，于加药后第48小时、72小时加 入MTT检测；3、生长曲线法：取1×104/ml的单细胞悬液， 每孔lml接种在24孔培养板中。药物组按1：80稀释度加 入蜂房水提液，对照组加等量灭菌蒸馏水，绘制细胞生长曲 线；4、集落形成法：单细胞悬液用RPMI 1640完全培养基 稀释成每毫升500个细胞，每孔2ml接种至6孔培养板，药 物组每孔各加蜂房水提液20μ1，对照组加灭菌蒸馏水20μ1， 静置培养8天，计数集落；5、细胞周期分析：将细胞悬液 接种于50cm2培养瓶中，每瓶5ml，药物组，培养液中加入 蜂房水提液60ul；对照组，培养液中加入等体积灭菌蒸馏水。 作用24小时、48小时，DNA染色后流式细胞仪检测，用ModFit 软件分析；6、凋亡检测：将细胞悬液接种至50cm2培养瓶 中，106个细胞/瓶，培养24小时后，更换培养液，药物组： 每瓶培养液5ml中加入露蜂房水提液60ul；对照组加入等量 3 灭菌蒸馏水。加药后于24．J’时、48．J’时按Annexin V仔I试 剂盒操作说明流式细胞仪进行检测。采用CellQuest功能软 件进行分析。结果：l、MTT法测定蜂房水提液明显抑制肝 癌细胞的生长，作用48小时，l：40的药物稀释浓度，细胞 生长抑制率为85．4％o＜o．01人 作用72 小时，1：4O 的药 物浓度，抑制率达 97．3％…＜0刀 1）；并存在效应－剂量、效应 －时间依赖关系；2、生长曲线显示药物组细胞生长缓慢，表 现为低平型曲线；3、集落形成实验结果显示药物组肝癌细 胞集落形成明显受抑，给药组集落形成率为 18石％士 2．42％， 对照组集落形成率为58．6％士15．0儿HO．0102，差异显著； 4、细胞周期分析结果：药物作用 48小时后，GO七 l期细胞 占细胞总数的 85．71％，高于对照组的 70．96％，P＜0．of；S期 的细胞占总数的 门．61％，低于对照组17．92％，P＜0．01；GZ－ M 期 的细胞 占细胞总数的2．68％，低于对照组 门．09％，P＜0．of；5、凋亡检测结果：对照组48 J时后，凋 亡率为2．09％士0．79趴 药物处理后48小时，凋亡率为10．51 士0．53（P＜0．05），中期凋亡率为 4．96士口．15，P＜0．01。结 论：蜂房水提液作用于肝癌细胞S删C｛721的S期，阻止细 胞进入GZ仇期，并诱导细胞凋亡，抑制体外培养的人肝癌 细胞的增殖。
The traditional Chinese medicine plays an important role inthe treatment of malignant tumors. The experiment is to studythe anti-tumor effects exerted by honeycomb in vitro.Material and Methods: The liver tumor cell SMMC-7721were cultured in RPMI-1640 media with 10% FCS, 100u/mlpenicillin and streptomycin on the condition of 37℃ 5% CO2.There were two group in the experiment. One is experimentgroup which the different concentration of honeycomb waterextracts was dissolved in the culture media; the other is controlgroup which the same volume of aseptic distilled water as thatof experiment group was dissolved in culture media. 1. MTTassay: The suspended cells obtained from the log phase culturedcells were incubated into 96-well plates. 24 hours later, the drugwas added into media at the concentration of1:40,1:80,1:160,1:320. At the end of 48 hours and 72 hours,MTT was added, then to test. 2. Growth curves: Single cell.suspension 1×104/ml was incubated into 24 well plates, thewater extracts of honeycomb were added at the concentration of1:80, then to draw the cell growth curves. 3. Colony-formingtest: Single cell suspension 500 cells/ml was incubated into 6well plates, added 20ul drug in the experiment group and 20ulwater in the control group, cultured for 8 days, then count thenumber of colony. 4. Cell cycle analysis: The cell suspensionswere cultured in 50 cm2 glass flasks, 5 ml each flask, 20ul drugand 20ul water were added in experiment and control grouprespectively After 24 hours and 48 hours medication, performedDNA staining then examined them with flow cytometer, andanalyzed the result by Modifit software. 5. Apoptosis detection:l X l0e cells were planted into the flask. After adding 20ul drugand 20ul water, the cells were cultured fOr 24 and 48 hours, thendetection was carried with the flow cytometer according to theAnnexin V/Pi apoptosis kit direction, and the resuIts wereanalyzed by CellQuest software.Result8: l. MTT showed the honeycomb water extractssignificantly suppressed the growth of liver tumor cells, and thedose-effect dependence, time-effect dependence relationshipwere existed. At 48 hours after medication, the cell growthinhibition rate was 85.4% in the l:40 diluted medicine group,P<0.01; At 72 hours the rate was 97.3%, P<0.0l; 2.Growthcurve showed the cells of experiment group grew slowly; 3.CoIony forming test indicated that the cell colony fOrming wassignificantly inhibited by the honeycomb. The forming rate wasl8.6% f2.42% in the experiment group, 58.6%t l5.0% in thecontrol group, P=0.0l2; 4.CelI cyc1e anaIysis showed: In theexperiment group, at 48 hours, G0-Gl phase celI was 85.7%which higher than that in the control group, P<0.0l, S phasecells were l l .6l% and G2-M phase cells were 2.68%, lower thanthese in the control group, P<0.0l; 5. Apoptosis detectionresults suggested the honeycomb induced the apoptosis of Iivertumor cells. 48 hours after treatment, the cell apoptosis rate wasl0.5l% i 0.53% in the experiment group, and 2.09%f0.79% inthe control group, P<0.05.Conclusion:The honeycomb water extracts influenced the S-phase ofliver tumor cell SMMC-7721, blocked the cells going into G2-M phase, induced the apoptosis of the tumor cells and inhibitedthe cuItured tumor cells proliferation in vitro.
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