阿托伐他汀促进体外培养大鼠乳鼠皮层神经元树突生长及突触相关蛋白表达

Effect of atorvastatin on the development of dendrite of cultured cortical neuronsin vitro and the expression of the synapse-associated proteins

目的探讨阿托伐他汀(atorvastatin,Ato)对体外培养大鼠乳鼠大脑皮层神经元树突生长及突触相关蛋白是否有影响。方法选用出生0～24 h Sprague-Dawley(SD)大鼠乳鼠,取大脑皮层,进行皮层神经元体外培养,神经元培养4 d后用于实验。实验分为对照组、不同浓度Ato处理组、Ato处理不同时间组;对照组:加入等量含0.1%DMSO培养基;Ato处理组:培养4 d神经元,加入不同浓度Ato(0.1、1、5、10μmol.L-1)作用48 h;Ato处理时间组:将Ato 5μmol.L-1加入培养4 d神经元,分别作用12、24、48 h。应用倒置相差显微镜和微管相关蛋白-2(MAP2)免疫荧光染色观察树突的生长,应用Tsview软件测量神经元树突分支总长度(totaldendritic branch length,TDBL)和一级树突数目(primary-or-der dendrite number,PDN);应用免疫荧光染色法和Westernblot检测皮层神经元突触素(synaptophysin,SYP)、突触后致密蛋白95(postsynaptic density protein-95,PSD-95)蛋白表达的改变。结果给予不同浓度Ato(0.1、1、5、10μmol.L-1)作用48 h后,神经元TDBL较对照组明显增加(P<0.01),神经元PDN较对照组呈浓度依赖性增加,Ato(0.1、1μmol.L-1)时可增加神经元PDN(P<0.05),Ato(5、10μmol.L-1)时明显增加神经元PDN(P<0.01);Ato 5μmol.L-1作用24 h后可增加神经元TDBL、PDN(P<0.05),Ato5μmol.L-1作用48 h后可明显增加神经元TDBL、PDN(P<0.01);应用倒置相差显微镜和微管相关蛋白-2(MAP2)免疫荧光染色观察树突TDBL和PDN明显增加;免疫荧光和Western blot结果显示Ato可增加SYP、PSD-95表达水平。结论 Ato促进体外培养大鼠乳鼠皮层神经元树突生长及突触相关蛋白表达。

Aim To investigate whether atorvastatin can promote the grow of neurons dendrite and the quantities of synapse-associated proteins cultured by the milk rat cortical neurons in vitro. Method The cerebral cor- tex of 0 -24 hour-old Sprague-Dawley （SD） rats were collected under sterile conditions to cultured cortical neuron in vitro. After being cultured for 4 days, corti- cal neurons were divided into control group, treatment of atorvastatin different concentrations, treament group of atorvastatin at different time points. The equivalent cultured medium containing 0. 1% DMSO was added to control group; after the neurons were cultured for 4 days, atorvastatin of different concentrations （0. 1,1,5, 10 μmol · L-1） were added to the atorvastatin treat- ment group and were then observed 48 hours later; The atorvastin 5 μmol · L-1 was put into the medium cul- tured neuron from the last group after it was cultured for 4 days, the reactions were observed respectively after 12 h,24 h,48 h. Inverted phase contrast microscope and tiny tubes related proteins-2 （MAP2） immunofluo- rescence stain were employed to observe the grow of dendrite, and the software was applied to measure the total dendrite branch length （TDBL） and number of primary-order dendrite （PDN）. Immunofluoreseeneeand Western blot were used to measure the change in the expression of synaptophysin （ SYP）, postsynaptic density protein 95 （PSD95）. Results After 48 h, the TDBL increased singnificantly compared with control group（P 〈0. 01 ）. The PDN increased in a concentra- tion-dependent manner compared with control groups. Atorvastatin（ 0. 1,1 μmol · L-l）increased the PDN of neuron（P 〈 0.05 ）. Atorvastatin （5,10 μmol · L-1 ） remarkably increased the PDN of neuron （P 〈 0. 01 ）. 24 h after atorvastatin （5 μmol · L- 1） was added, the TDBL and the PDN increased （ P 〈 0.05 ）. 48 h after atorvastatin （5 μmol · L-l） was added,the TDBL and the PDN remarkably increased （P 〈 0. O1 ） . The TDBL and the PDN were found to have increased singnificant- ly under the observation of inverted phase contrast mi- croscope and MAP2 immunofluorescence staining. Im- munofluorescence and Western blot showd that atorvas- tatin improved the levels of SYP and PSD-95 expres- sions. Conclusion Atorvastatin can promote the devel- opment of dendrite of cultured cortical neurons in vitro and the expression of the synapse-associated proteins.