摘要
研究目的：
本课题根据“癌毒学说”,旨在研究杉蟾藤复方成分对肺癌细胞增殖和凋亡的影响,对凋亡相关蛋白的调控作用以及对肺癌细胞形态学的影响,以初步探讨杉蟾藤复方抗肺癌的有效作用机制。
研究方法：
1、采用MTT法分别检测红豆杉单体、蟾皮单体、雷公藤单体对A549细胞及NCI-H1688细胞的抑制增殖作用,比较其抗癌活性。
2、流式细胞仪检测凋亡,观察红豆杉单体、蟾皮单体、雷公藤单体作用于A549细胞及NCI-H1688细胞24h后对细胞周期的影响。
3、免疫组化法检测杉蟾藤复方成分对肺癌细胞相关蛋白Bad、Bax、Bcl-2蛋白调控的影响。
4、透射电镜观察体外对A549细胞及NCI-H1688细胞形态学的影响以及观察是否出现凋亡小体、核染色质固缩、边集等现象。
5、激光共聚焦显微镜观察杉蟾藤复方成分作用于A549细胞及NCI-H1688细胞的变化。
研究结果：
1、药物作用细胞24、48小时后,与空白对照组相比,不同浓度和作用时间的三种单体对A549细胞及NCI-H1688细胞均具有明显生长抑制作用(P<0.05),尤其高剂量组的抑制作用显著(P<0.01),且在同一浓度水平,随着作用时间的延长,三种单体对A549细胞及NCI-H1688细胞的生长抑制作用呈递增态势。
2、A549细胞和NCI-H1688细胞经过不同剂量的三种单体处理48h后,各剂量组均出现不同程度的凋亡,且细胞的凋亡比例在早、中晚期均呈现出随剂量增高而增长的态势。各剂量组细胞的总凋亡率与正常对照组比较,差异均有统计学意义(P<0.05)。
3、免疫组化显示抑凋亡Bcl-2蛋白表达减弱,总Bax蛋白／Bcl-2蛋白上升,促凋亡Bad蛋白表达增强；抑凋亡基因Bcl-2mRNA表达下调,总Bax mRNA/Bcl-2mRNA上升,促凋亡Bad mRNA表达上调。
4、透射电镜观察显示：三种单体作用A549细胞及NCI-H1688细胞48h后发生体积皱缩,细胞膜不完整,核膜消失,核质萎缩、碎裂,个别形成凋亡小体；部分染色质固缩,沿核膜形成数个团块状边集；细胞器肿胀,空泡状明显增多。正常对照组细胞膜表面光滑、完整,可见微绒毛,胞质密度高,无空泡,核内染色质分布均匀、核仁大较完整,居中或边移；线粒体脊断裂或消失。
5、、激光共聚焦显微镜观察到杉蟾藤复方成分作用与A549细胞和NCI-H1688细胞后,均出现不同程度的磷酯酰丝氨酸外翻,细胞发生早期凋亡现象。
研究结论：
1、杉蟾藤复方成分(红豆杉单体、蟾皮单体、雷公藤单体)体外对肺腺癌A549细胞及小细胞肺癌NCI-H1688细胞均具有抑制增殖作用,且抑制率水平具有浓度和时间的依赖性。
2、杉蟾藤复方成分能够诱导A549细胞和NCI-H1688细胞发生凋亡,并引起细胞发生相应的形态学改变。
3、杉蟾藤复方成分调控蛋白诱导A549细胞和NCI-H1688细胞发生凋亡的机制也可能与调控Bcl-2家族的Bax、Bcl-2、Bad蛋白以及Bax mRNA、Bcl-2mRNA、Bad mRNA的综合表达有关。
Research purpose:
To research the effects of compound components of Shanchanteng on the proliferation and apoptosis of Lung cancer cells, the regulation of apoptosis related protein and effects on Lung cancer cell morphology, to investigate the effective molecular mechanism of compound components of Shanchanteng against lung cancer.
Research methods:
1.To detected in inhibiting the proliferation of Taxus monomer, toad skin monomers, monomers of Tripterygium wilfordii on A549cells and NCI-H1688cells by using MTT method, to compare their anti-cancer activity.
2.Apoptosis was detected by flow cytometry, observed the influence of Taxus monomer, toad skin monomers, monomers of Tripterygium wilfordii on A549cells and NCI-H1688cells in cell cycle.
3.To detect the effect of immunohistochemistry fir toad rattan compound components of lung cancer associated protein Bad, Bax, Bcl-2protein regulation.
4.Transmission electron microscopy observation on A549cells in vitro and NCI-H1688cell morphology and observe whether apoptotic bodies, nuclear chromatin pyknosis, margination phenomenon.
5.Laser confocal microscopy was used to observe the changes of compound components of Shanchanteng composition effects on A549cells and NCI-H1688cells.
Results:
1.Cell drug24, after48hours, compared with the blank control group, different concentration and duration of the three monomers on A549cells and NCI-H1688cells were inhibited (P<0.05), especially the inhibitory effect of high dose group significantly (P<0.01),and in the same concentration, with the extension of time, three inhibited the growth of A549cells was monomer and NCI-H1688cells increase.
2.A549cells and NCI-H1688cells after different doses of three monomer48h after treatment, each dose group showed varying degrees of apoptosis, and the percentage of apoptotic cells in the early, middle and late show with the dose increased and growth. The total cell apoptosis rate in each dose group compared with the control group, the difference was statistically significant (P<0.05).
3.Immunohistochemistry showed that the anti-apoptotic Bcl-2protein expression decreased, increased total Bax protein/Bcl-2protein, apoptosis Bad protein; apoptosis suppressing gene Bcl-2downregulation of mRNA expression, increase in total Bax mRNA/Bcl-2mRNA Bad mRNA expression, apoptosis.
4.Transmission electron microscope observation showed:three monomers of A549cells and NCI-H1688cells after48h in size Shrinkage, the membrane is not complete, the nuclear membrane disappeared, nuclear atrophy, fragmentation, individual and formation of apoptotic bodies; part of chromatin pyknosis, along the nuclear membrane forms a plurality of crumb edge set; cell swelling, vacuole increased significantly. The cell membrane in the normal rats, complete with smooth surface, microvilli, cytoplasmic density high, no bubble, nuclear chromatin distribution, large nucleolus more complete, center or edge shift; mitochondrial ridge broken or disappeared.
5.Laser confocal microscopy to compound components of Shanchanteng with A549cells and NCI-H1688cells, there are varying degrees of phosphatidylserine eversion, apoptosis phenomenon.
Conclusions:
1. Compound components of Shanchanteng (Taxus monomer, toad skin monomers, monomers of Tripterygium wilfordii) in vitro on lung adenocarcinoma A549cells and small cell lung cancer NCI-H1688cells can inhibit proliferation and suppress the dependence rate, concentration and time of.
2.Compound components of Shanchanteng can induce A549cell and NCI-H1688cell apoptosis, and induce cell has the corresponding morphological changes.
3.The mechanism of A549cell and NCI-H1688cell apoptosis induced by Sugi To compound components of regulatory proteins may also be associated with regulation of Bcl-2family Bax, Bcl-2, Bad protein and Bax of mRNA, Bcl-2mRNA, Bad mRNA expression.
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