大鼠JNK3超表达细胞模型的建立及特性研究

In vitro establishment of the overexpressing rat c-Jun N-terminal Kinase model in SHSY5Y

目的构建大鼠c-JunN末端激酶3（JNK3）在SH-SY5Y细胞的超表达模型，从而研究帕金森病（PD）和阿尔茨海默病（AD）等神经退行性疾病的发生机制与JNK3的相互关系以及建立新的PD和AD治疗药物筛选平台。方法RT-PCR获得大鼠JNK3（rJNK3）基因片段．连人真核表达载体pcDNA3．1／hisC构建真核表达质粒pcDNA3．1／hisC-rJNK3。脂染法把质粒转染到SHSY5Y细胞内，经过G418筛选出稳定表达rJNK3的单克隆细胞系。Western blot检测其蛋白表达量。通过观察细胞形态、测定细胞生长率、MPP诱导的细胞凋亡以及细胞免疫荧光定位等实验对细胞系SHSY5Y-rJNK3进行细胞株特性研究。结果重组质粒pcDNA3．1／hisC-rJNK3转染SHSY5Y细胞构建成稳定表达rJNK3蛋白的细胞系SHSY5Y-rJNK3。SHSY5Y-rJNK3和SHSY5Y细胞形态上差异显著，MTT结果显示生长率也不相同，SHSY5Y-rJNK3比SHSY5Y细胞对MPP的刺激更敏感。结论JNK3蛋白在细胞内的过量表达会导致细胞形态发生一定程度的改变，同时导致细胞对外界因素诱导的凋亡更加敏感，因此在细胞内过表达rJNK3的SHSY5Y-rJNK3更能客观准确的反映JNK3作用下的神经细胞凋亡现象和本质。

Objective To establish a PD model ofoverexpressing rat c-Jun N-terminal kinase 3 （JNK3） in SHSY5Y（SHSY5Y-rJNK3）, and study the relationship between JNK3 and occuring of PD and AD. Methods Extract purificat RNA and get Rat JNK3 through RT-PCR. The pcDNA3. 1/hisC-rJNK3 vector was established and stably transfected into SHSY5Y overexpressing rat JNK3. SHSYSY-rJNK3 was selected by Western blotting analysis. The growth rate and the sensitivity to MPP＋ of SHSY5Y-rJNK3 were further evaluated by morphological observation, immunofluorescence and MTT assay. Results Successfully established SHSY5Y-rJNK3 series by transfected pcDNA3.1/hisC-rJNK3 vector in SHSY5Y over expressing rJNK3. There were morphological differences between SHSYSY and SHSYSY-rJNK3. The result of MTT showed that there were little differences between growth rate of both. Stimulated by MPP＋ of different concentrations, the SHSY5Y-rJNK3 made more morphological changes in 100 μm MPP＋ than SHSY5Y, and the results of MTT also demonstrated that SHSY5Y-rJNK3 was more sensitive to MPP＋ compared to SHSY5Y with lower cell viability. Conclusions Overexpressing JNK3 in SHSY5Y makes morphological changes on cells, also makes cells more sensitive to the poisons. Therefore, studying on SHSY5Y-rJNK3 which is overexpressed JNK3 can reflect phenomenons and substance ofapotosis which related to YNK3 more objective and accurately.