小檗碱抗人高转移性肺癌细胞诱导的人脐静脉血管内皮细胞凋亡的作用研究

【作者】 何萍；

【导师】 黄启福； 王毅；

【作者基本信息】 北京中医药大学 ， 中西医结合基础， 2008， 硕士

【摘要】 肿瘤转移是临床抗肿瘤治疗失败和患者死亡的主要原因,因此,控制转移是决定癌症患者预后的关键因素。肿瘤转移是一个复杂的、多步骤的过程。血行转移是肿瘤转移最重要的途径。肿瘤细胞与血管内皮细胞问的相互作用是血行转移最复杂、最重要的环节。已有研究表明,肿瘤细胞转移过程中伴随着血管内皮细胞的皱缩和凋亡,且血管内皮细胞内第二信使Ca²⁺浓度及pH值的改变与细胞凋亡密切相关。小檗碱（Berberine）是消化系统感染常用药。近来研究发现,小檗碱还具有抑制肿瘤细胞生长的作用。我们也对其抗肿瘤作用进行了初步研究,发现小檗碱具有抑制肺癌细胞增殖,抑制实体瘤生长及减少肺内转移灶的作用。但其抗肿瘤转移的机制还有待进一步深入研究。激光扫描共聚焦显微镜（Laser Scanning Confocal Microscope,LSCM）是现代生物学中用于无损伤观察活细胞和组织的一种重要方法。与特异性荧光探针结合,广泛应用于在活细胞层次监测一些重要生化成分的空间分布和随时间的变化,从而获得生命活动过程的信息。本实验将利用共聚焦显微镜研究小檗碱抗人高转移性肺癌细胞（PG细胞）诱导的人脐静脉血管内皮细胞（HUVEC）凋亡的作用,阐明其抗肿瘤转移的可能机制。1目的研究小檗碱抗PG细胞上清液诱导的HUVEC凋亡的作用,阐明小檗碱抑制肿瘤细胞转移的机理。利用共聚焦荧光成像技术观察小檗碱对肿瘤转移的影响,从细胞、亚细胞水平上阐明药物与细胞的作用机制,为中药研究开辟一条新的研究途径。2方法首先用MTT法筛选出有效影响HUVEC活性的PG细胞培养上清液浓度与作用时间,建立PG细胞上清液诱导HUVEC凋亡模型。用共聚焦荧光成像技术观察小檗碱抗HUVEC凋亡效应,具体方法:药物防治组用不同浓度小檗碱处理HUVEC后,再与PG细胞培养上清液作用。经MTT法检测不同药物浓度、不同药物作用时间下HUVEC的活性,以确定小檗碱防治的最佳药物剂量和最佳作用时间。应用激光扫描共聚焦显微镜技术测定HUVEC凋亡百分率、细胞核形态、细胞内Ca²⁺浓度及pH值变化。3结果（1）小檗碱能够明显降低PG细胞上清液诱导的HUVEC凋亡百分率。（2）小檗碱可有效减轻HUVEC细胞核染色质浓缩等凋亡形态变化。（3）小檗碱可缓解PG细胞培养上清液引起的HUVEC内Ca²⁺浓度升高。（4）小檗碱可改善PG细胞培养上清液引起的HUVEC内pH值下降。4结论小檗碱能够抗PG细胞诱导的HUVEC凋亡,使细胞结构和功能趋于恢复正常,并呈现剂量依赖关系。其作用机制可能是通过调控细胞内Ca²⁺浓度,改善pH值,从而抑制内皮细胞凋亡,防止肿瘤转移。更多还原

【Abstract】 Tumor metastasis is the primary cause of failures in antineoplaston and death of patients;therefore,how to control tumor metastasis is the key to decide the prognosis of cancer patients.The tumor metastasis is a complex and multi-step process.Hematogenous metastasis is the most important way.The interaction of tumor cell and the vascular endothelial cell is the most complex and the most important link in hematogenous metastasis.Some research indicate that tumor metastasis is followed with vascular endothelial cell’s shrinkage and apoptosis, the second messenger Ca²⁺density and the pH change in the vascular endothelial cell have a close correlation with apoptosis.The berberine is a common medication in the digestive system’s infection.Some researches indicate that berberine also has the function to restrain the cancer cell’s growth in resent years.We have also conducted preliminary study in this function,discovering that the berberine has the function to restrain the lung cancer cell’s multiplication,the growth of solid tumor and to reduce lung-metastasis. But its mechanism in anti-tumor metastasis also needs further research.The Laser Scanning Confocal Microscope（LSCM）is a important method in the modern biology used in the non-damage observation of living cell and organization.Combined with specificity fluorescent probe,it widely applies in monitoring some important biochemical ingredient in spatial distribution and change along with the time in the living cell level,thus to obtain the information of the vital movement process.This experiment will use LSCM to research the protective function that berberine on the PG cell to cause the apoptosis of human umbilical vein endothelial cell（HUVEC）,expounds the mechanism to resist tumor metastasis.1 GoalTo research the protective function which berberine to PG cell causes HUVEC apoptosis,expounded the mechanism that berberine resist tumor metastasis.Using LSCM to observe influence that berberine on the tumor metastasis,to expound the mechanism of action between drug and cell,and open a new research way for the traditional Chinese medicine research. 2 MethodFirst we use the MTT method to screen the most appropriate density and the time which function between the HUVEC and PG cell to culture supernatant,with the PG cell to culture supernatant and function with the HUVEC,establishing the tumor attacking model.The medicine protection group is using the berberine to process HUVEC before the PG cell culture supernatant function with the HUVEC.We use the MTT method to detect the cell’s survival percentage in different medicine density and different medicine response time,to determine that the best medicine dosage and the best response time with the berberine.Then we carries on the observation and analysis aiming directly at the HUVEC percentage of apoptosis,the shape of nucleolus,the Ca2+ density and the pH value change and so.3 Result（1）Berberine can obviously reduce the apoptosis percentage of HUVEC attacked by tumor.（2）Berberine can relieve the deformation of nucleolus,the concentration of chromatin and so on.（3）Berberine can relieve the calcium overload of HUVEC.（4）Berberine can restrain the depression of pH value.4 ConclusionThe berberine can obviously reduce the HUVEC damage caused by tumor,making the cell structure and the function restore normally.One of these kinds of possibly protective mechanisms is to restrain apoptosis through reducing the excessive load of the Ca²⁺in the cell and the pH reducing.更多还原