摘要
阿尔茨海默病(Alzheimer’s disease,AD)是一种中枢神经系统的退行性疾病,其原因和发病机制至今未明。β淀粉样蛋白(amyloid proteinβ, Aβ)的神经毒作用是导致AD的共同通路,大量研究表明Aβ诱导的细胞凋亡是AD发病的重要病理生理学机制。
细胞膜氯通道的激活可能是介导细胞凋亡的重要离子机制,本实验利用氯通道的阻断剂DIDS和Phloretin研究Aβ25-35诱导PC12细胞凋亡这一AD模型中氯通道的作用。
JNK信号通路是丝裂原活化蛋白激酶(mitogen-activated protein kinases, MAPKs)中重要的通路之一,是参与应激诱导的细胞凋亡的重要信号转导机制,本实验初探Aβ25-35诱导PC12细胞凋亡过程中是否有JNK蛋白的磷酸化激活,以及氯通道阻断剂DIDS和Phloretin与JNK的磷酸化激活的关系。
由于Aβ诱导神经元凋亡的机制十分复杂,至今尚未找到可以抑制细胞凋亡的关键性靶点。因此,阐明机制找到靶点,将为AD的防治带来希望。
目的:
1.观察氯通道阻断剂DIDS能否抑制Aβ25-35诱导的PC12细胞凋亡,了解氯通道在Aβ诱导PC12细胞凋亡过程中的作用。
2.观察对VSOR Cl-通道有相对特异性阻断作用的Phloretin能否抑制Aβ25-35诱导的PC12细胞凋亡,从而了解VSOR Cl-通道是否参与下调Aβ毒性作用。
3.探讨JNK在Aβ25-35诱导的PC12细胞凋亡中的作用,以及氯通道阻断剂与JNK的关系。
方法:
1.以传代培养的PC12细胞为研究对象,实验设立阴性组(正常对照组),阳性对照组(仅给Aβ25-35诱导细胞凋亡处理),及实验组(Aβ25-35诱导细胞凋亡+氯通道阻断剂DIDS或Phloretin)。
2.噻唑兰(3-( 4, 5-dimethylthiazol-2-yl) -2, 5-diphenyl tetrazolium bromide,MTT)比色法观察PC12细胞存活率;用荧光显微镜观察Hoechst33258荧光染色后的细胞核的形态学变化;采用荧光素检测乳酸脱氢酶(lactate dehydrogenase, LDH)水平,反映PC12细胞膜完整性的改变;DNA琼脂糖凝胶电泳,检测PC12细胞凋亡的发生。
3.采用Western Blot印迹法检测Aβ25-35诱导PC12细胞凋亡时不同时间点P-JNK的表达和不同处理组P-JNK的表达。
4.所有数据应用SPSS11.0统计软件进行数据分析处理。
结果:
1.实验结果验证了Aβ25-35作用于PC12细胞时细胞发生凋亡。并且发现DIDS和Phloretin能够有效地抑制Aβ25-35诱导的PC12细胞凋亡。
2. 40μmol/L Aβ25-35作用于PC12细胞(作用24 h)后,MTT实验显示细胞存活率(58.4 %±9.3 %)降低,与阳性对照组相比细胞存活率在Aβ25-35+DIDS组(86.0%±7.2%)或Aβ25-35+ Phloretin组(94.1%±9.3%),均有增加,且差异有统计学意义(P < 0.01)。
3. Hoechst33258荧光染色显示Aβ25-35作用于PC12细胞时,细胞出现明显的皱缩,胞核染色质断裂、聚集、固缩等典型的凋亡形态学变化,而实验组凋亡细胞明显减少。
4. LDH释放水平:阴性组与Aβ25-35阳性对照组(433.1±41.5 U/L)比较差异有统计学意义(P < 0.01);Aβ25-35阳性对照组与Aβ25-35+Phloretin实验组(354.4±34.3 U/L)比较差异有统计学意义(P < 0.01);Aβ25-35阳性对照组与Aβ25-35 +DIDS实验组(366.7±28.3 U/L),比较差异有统计学意义(P < 0.01)。
5. DNA琼脂糖凝胶电泳结果:Aβ25-35阳性组可以见到明显的DNA“梯带”,阴性组和实验组没有DNA“梯带”。
6. Western Blot印迹结果:6 h时Aβ25-35诱导PC12 P-JNK蛋白表达量增高,与0 h比较差异有统计学意义(P < 0.01)。两个实验组与阳性对照组比较,P-JNK蛋白表达量的差异均有统计学意义(P < 0.01)。
结论:
本研究表明:氯通道阻断剂DIDS和Phloretin可以抑制Aβ25-35诱导的PC12细胞凋亡,而且,应用氯通道阻断剂可使P-JNK表达降低。因相对特异性的VSOR Cl-通道阻断剂Phloretin可以对Aβ诱导的PC12细胞凋亡起到保护作用,从而推测VSOR Cl-通道在Aβ诱导PC12细胞凋亡时可能被激活。且推测氯通道阻断剂对Aβ25-35诱导PC12细胞凋亡的下调作用是通过抑制JNK磷酸化实现的。
AD is a common degenerative disease of the central nervous system .Its causes and mechanism are still unclear. A number of experiments indicate that Aβinduced apoptosis is one of the pathophysiology mechanisms by which Aβinduces AD.
Chloride channel is a kind of important channel in the cellular membrane by which apoptosis can be mediated. We will investigate what role the chloride channel plays when PC12 apoptosis is induced by Aβ25-35.
MAPK signal transduction pathway can perform significant functions in cell apoptosis. JNK signal transduction pathway is an important branch of MAPK signal transduction pathway. We will try to find out what role JNK plays when PC12 cells apoptosis is induced by Aβ25-35 ,also ,we will try to find out the relationship between chloride channel blockers and JNK.
The mechanisms by which Aβinduces cell apoptosis are very complex, People are still unclear about the key point that can be made use of to prevent cell apoptosis. So if the mechanism and the key point are known, it is possible that AD can be cured.
Aims:
1. To investigate whether DIDS can inhibite PC12 apoptosis induced by Aβ25-35.
2. To investigate whether Phloretin , a relatively specific VSOR Cl- channel blocker , can inhibite PC12 apoptosis induced by Aβ25-35.
3. To find out what role JNK plays when PC12 apoptosis is induced by Aβ25-35 and to find out the relationship between chloride channel blockers and JNK.
Methods:
1. Serial subcultivation PC12 cells were the objects for research , cells were divided into different groups: the negative control group(normal),positive group(treated with Aβonly) and experimental groups(treated with Aβ+chloride channel blockers DIDS or Phloretin).
2. Cell survival rate was estimated by MTT , Fluorescence microscope was used to observe the morphological change of the nuclear after stained by Hoechst33258 , the integrity of cell member was surveyed by measuring released LDH level, apoptosis was further proved by agarose gel electrophoresis of DNA.
3. The expressions of P-JNK of PC12 cells induced by Aβon different time points were detected by Western Blot.The expressions of P-JNK in different groups were detected by Western Blot.
4. All data were analyzed by SPSS11.0.
Results:
1. The experiment results indicated that apoptosis occurred when PC12 cells were treated by Aβ. The experiment results also indicated that DIDS and Phloretin could protect cells from apoptosis induced by Aβ.
2. MTT manifested low cell survival rate(58.4%±9.3%) when cells were exposed to 40μmol/L Aβ25-35, cell survival rates in experimental groups were : Aβ25-35+DIDS(86.0%±7.2%) and Aβ25-35+ Phloretin (94.1±9.3%), and the difference possessed statistical significance(P<0.01).
3. Hoechst33258 displayed obvious cell shrinkage , and the breakage , aggregation , pyknosis of nuclear chromatin when cells were exposed to 40μmol/L Aβ25-35 and the number of cells with typical apoptosis morphological changes decreased in the experimental groups.
4. The level of released LDH : the difference between the negative group(328.7±18.9 U/L) and positive group(433.1±41.5 U/L)possessed statistical significance (P < 0.01);the differences between the positive group and experimental groups (Aβ25-35+Phloretin:354.4±34.3 U/L and Aβ25-35 + DIDS:366.7±28.3 U/L) both possessed statistical significance(P < 0.01).
5. Agarose gel electrophoresis of DNA manifested obvious DNA Ladder in the positive group , while none could be seen in the negative group or experimental groups .
6. P-JNK expression after PC12 cells had been exposed to Aβ25-35 for 6h was increased compared with that of 0h and the difference possessed statistical significance(P < 0.01).Also ,the differences of P-JNK expressions between two experimental groups and the positive group meant statistical significance(P < 0.01).
Conclusion:
Both DIDS and Phloretin could prohibit Aβ25-35 induced PC12 apoptosis and the protective role may correlate with down regulating the phosphorylation of JNK. As Phloretin , a relatively specific VSOR Cl- channel blocker , could protect cells from apoptosis , it was supposed that VSOR Cl- channel was activated when PC12 cells were exposed to Aβ.Also, it was supposed that chloride channel blockers exerted protective role by inhibiting the phosphorylation of JNK.
引文
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