神经节苷脂GD1a在无血清条件下诱导FBJ细胞凋亡

摘要

神经节苷脂(Gangliosides)是一类含唾液酸的鞘糖脂,存在于细胞膜的外表面。在胚胎形成、细胞分化、细胞增殖、转化和细胞转移等生理现象中发挥着重要作用。本文阐述了神经节苷脂GD1a的一种新的功能即诱导FBJ病毒感染的小鼠骨肉瘤细胞产生凋亡。在无血清培养条件下,富含GD1a的FBJ-S1细胞趋向死亡,而缺乏GD1a的FBJ-LL细胞却却未见明显影响。通过流式细胞仪对细胞进行膜联蛋白V异硫氰酸荧光素(FITC-AnnexinV)和碘化丙啶(PI)双染检测和细胞周期分析发现FBJ-S1细胞在无血清条件下趋向死亡是因凋亡而引起的。因此,我们推测GD1a可诱导FBJ细胞凋亡。为了验证这一推论,外源性GD1a在无血清培养条件下处理FBJ-LL细胞,发现FBJ-LL细胞出现死亡。FBJ-S1细胞通过加入葡萄糖苷神经酰胺合成酶抑制剂(D-PDMP)处理和转染入唾液酸转移酶(st3ga15)的siRNA的来抑制GD1a的合成,发现在无血清条件下经D-PDMP处理后FBJ-S1细胞抵抗死亡,且转染siRNA到FBJ-S1细胞后得到的单克隆细胞也同样抵抗死亡。另外,很多文章报道神经酰胺和神经节苷脂GD3可诱导凋亡,但在发生凋亡的FBJ细胞中神经酰胺的表达含量并没有变化,而GD3并未见在FBJ细胞中表达,这说明神经酰胺和GD3不是引起FBJ细胞凋亡的因素。
　　 窖蛋白1(caveolin-1,亦译为小窝蛋白1)是一种肿瘤抑制蛋白。本文研究发现,窖蛋白1参与了GD1a诱导的FBJ细胞凋亡并起着关键作用。FBJ-S1细胞在无血清条件下趋向死亡,而FBJ-LL细胞并未出现此现象,逆转录PCR法检测发现FBJ-S1细胞中窖蛋白1的表达量是FBJ-LL细胞的5倍。FBJ-LL细胞在外源性GD1a处理后促进其趋向死亡,免疫印迹法(Western blot)发现窖蛋白1的表达量也明显增加。另外,利用siRNA技术沉降FBJ-S1细胞中窖蛋白1的表达量,无血清培养条件下,结果使其存活。数据表明窖蛋白1可促进FBJ细胞的凋亡。存活蛋白(survivin,亦译为生存蛋白或生存素)是一种凋亡抑制蛋白,研究发现窖蛋白1可以调节存活蛋白,且存活蛋白也参与了GD1a诱导的FBJ细胞凋亡。
　　 FBJ-LL细胞可在无血清培养条件下存活,我们推测FBJ-LL细胞可能分泌一种或多种生长因子。因此我们随机检测了FBJ-LL和FBJ-S1细胞中一些生长因子的mRNA水平表达,发现FBJ-LL细胞中生长因子如PDGFα-TNFα和VEGFB的表达量是FBJ-S1细胞中的数倍,且FBJ-S1细胞在无血清培养基中加入PDGFα或TNFα培养后使其存活。数据表明内源性GD1a可调节生长因子的表达同时也可诱导凋亡。另外,通过信号传导抑制剂和细胞内吞抑制剂来研究GD1a诱导FBJ细胞凋亡的机制,发现p38 MAPK途径和细胞内吞途径对GD1a诱导的细胞凋亡发挥着重要作用。

目录

文摘

英文文摘

Chapter 1. Introduction

1.1. Gangliosides and ganglioside GD 1 a

1.2. Caveolae and caveolin-1

1.3. Cell death and apoptosis

1.4. MAPKs and PI3K pathway

1.5. Cellular endocytosis

1.6. Summary

Chapter 2. Metefials and methods

2.1. Cell lines and cell culture

2.2. Chemicals

2.3. Glycolipid extraction and high performace thin layer chromatography (HPTLC)

2.4. Small interference RNA (siRNA) and siRNA transfection

2.4.1. SiRNA sequence

2.4.2. The protocols of siRNA tranfection

2.5. RNA extraction and reverse transcriptase PCR (RT-PCR)

2.5.1. The protocol of total RNA extraction

2.5.2. RT-PCR

2.5.3. Agarose eletrophoresis

2.6. SDS-PAGE electrophoresis and western blot

2.7. Analysis of apoptosis by FACS (Fluorescence-Activated Cell Sorter)

2.7.1. Double staining

2.7.2. Propidium iodide staining

2.8. Cell viability assay (or WST assay)

2.9. Statistical analysis

Chapter 3. GDla induces FBJ-cell apoptosis under serum-free conditions

3.1. GDla is responsible for cell death in FBJ cells

3.2. FBJ-S1 cells undergo apoptosis under serum starvation

3.3. FBJ cells undergo apoptosis due to neither ceramide or GD3

Chapter 4. Caveolin-1 is responsible for FBJ-cell apoptosis under serum-free conditions

4.1. Caveolin-1 expression is responsible for FBJ-cell apoptosis

4.2. Survivin is related to FBJ-Cell apoptosis induced by GDla

Chapter 5. The mechanisms of GD1a-induced FBJ-cell apoptosis

5.1. FBJ-LL cell-conditioned medium helps FBJ-S1 cells to survive in serum-free medium

5.2. Inhibition of p38 MAPK reverses the sensitivity of FBJ-S1 cells to apoptosis

5.3. Inhibitors of endocytosis promote FBJ-S1 cells survival

5.4. Cyclosporin A makes FBJ-S1 cells to survive under serum starvation

Chapter 6. Exogenous gangliosides induce FBJ-LL cell death under serum-free conditions

Chapter 7. Conclusions and discussion

References

Published paper

Acknowledgements