天然产物诱导肝星状细胞凋亡的研究

论文摘要

目的:研究芦荟大黄素和隐丹参酮的诱导大鼠肝星状细胞（t-HSC/C1-6）细胞凋亡信号通路以及观察丹参标准组分对COl4诱导的肝纤维化大鼠的恢复作用,期望为肝纤维化的中药治疗研究提供新的思路。 方法:以MTT法检测芦荟大黄素和隐丹参酮引起的t-HSC/C1-6细胞生存率下降,利用DNA片断化,caspase活性分析,以标记了的膜联蛋白-v和碘化丙啶作为荧光探针的流式细胞技术和免疫印迹法进一步探讨细胞凋亡所经的通路。采用CCl4诱导大鼠急性和亚急性肝受损模型,造模结束后给予丹参标准组分进行治疗,测定血清学指标和肝组织丙二醛（MDA）含量,进行H&E染色和α-SMA免疫染色。 结果:（1）不同浓度的芦荟大黄素（12.5-50μM）和隐丹参酮（2.5-10μM）作用于t-HSC/C1-6细胞后明显抑制t-HSC/C1-6细胞生长率,呈剂量和时间依赖性。（2）经两药物处理后琼脂糖凝胶电泳可见到明显的DNA梯带形成,透射电镜观察到细胞皱缩、核染色质浓缩沿核膜排列和凋亡小体形成等。流式细胞测试中,早期细胞凋亡阶段的细胞百分比随浓度和时间增加而增多。（3）两者均激活caspase-3和caspase-9,而不激活caspase-8;蛋白印迹法实验进一步证明caspase-3和caspase-9的激活形式的出现和poly（ADP-ribose）polymerase的分裂;细胞色素c在线粒体中表达减少,而在细胞质中增多。芦荟大黄素处理后增加的Bax,不变化的Bcl-2导致Bax/Bcl-2比率增高。隐丹参酮处理后全长Bax只位于线粒体中,续而分裂成p18/Bax,并不是从细胞质转移到线粒体。（4）丹参标准组分给药后,明显降低CCl4诱导的肝纤维化大鼠血清GOT和GPT的活性,有效抑制α-SMA。

论文目录

• Abstract
• Chapter 1. Aloe Emodin-Induced Apoptosis in t-HSC/CI-6 Cells Involves a Mitochondria-Mediated Pathway
• 1. Introduction
• 2. Materials and Methods
• 2.1 Chemical reagents
• 2.2 Cell culture
• 2.3 Cell viability assay
• 2.4 Observation of morphology changes
• 2.5 DNA fragmentation analysis
• 2.6 Flow cytometry analysis
• 2.7 Measurement of mitochondrial membrane potential
• 2.8 Assay of caspase activity
• 2.9 Preparation of whole cell lysates
• 2.10 Isolation of cytosol and mitochondrial extract
• 2.11 Western blot analysis
• 2.12 Statistical analysis
• 3. Results
• 3.1 Growth inhibition
• 3.2 Effect of AE apoptosis in t-HSC/CI-6 cells
• 3.3 Caspase activation in AE-induced apoptosis
• 3.4 Expression of Bax and Bcl-2 proteins induced by AE treatment
• 3.5 Involvement of a mitochondrial pathway in AE-induced apoptosis
• 4. Discussion
• Chapter 2. Cryptotanshinone Induces Apoptosis in t-HSC7CI-6 Cells and salvia miltionhiza Inhibits Hepatic Fibrosis Induced by Carbon Tetrachloride
• 1. Introduction
• 2. Materials and Methods
• 2.1 Chemical reagents
• 2.2 Preparation of salvia miltiorrhiza standard fraction
• 2.3 Cell culture
• 2.4 Cell growth assay
• 2.5 DNA fragmentation analysis
• 2.6 Now cytometry analysis of annexin v-FTTC binding
• 2.7 Caspase activity assay
• 2.8 Cytosol and mitochondrial fraction isolation
• 2.9 Western blot analysis
• 2.10 Animals
• 4-induced acute liver injury in rats'>2.11 salvia miltiorrhiza standard fraction pre-treatment in CCl₄-induced acute liver injury in rats
• 4-induced subacute liver injury in rats'>2.12 salvia milliorrhiza standard fraction pre-treatment in CCl₄-induced subacute liver injury in rats
• 2.13 Determination of serum biochemical parameters
• 2.14 Determination of lipid peroxidation levels in liver
• 2.15 Hislological and immunohistochemistry examination
• 2.16 Statistical analysis
• 3. Results
• 3.1 Anti-proliferative effects of salvia miltiorrhiza standard fraction tanshinone I. tanshinone HA and cryptotanshinone
• 3.2 Occurrence of apoptosis induced by cryptotanshinone
• 3.3 Cryptotanshinone-induced caspase-3. -9 activation. PARP cleavage and cytochromee release
• 3.4 Cryptotanshinone induced the cleavage of Bax residing exclusively in the mitochondria
• 3.5 Cryptotanshinone-induced apoptosis is independent of the antiapoptotie protein Bcl-2
• 4-induced acute liver injury in vivo'>3.6 Protective effective of salvia milliorrhiza standard fraction pre-treatment in CCl₄-induced acute liver injury in vivo
• 4-induced subacute liver injury in vivo'>3.7 Protective effective of salvia miltiorrhiza standard fraction pre-treatmenti CCl₄-induced subacute liver injury in vivo
• 4.Discussion
• Conclusions
• References
• Figure legends
• Table
• Figure
• Review
• Chinese Appendix
• Abstract
• Review
• Acknowledgement