GFP,TH双基因在NIH-3T3细胞及帕金森病大鼠脑内的表达

Co expression of both GFP and HTH 1 genes in the NIH 3T3 and in the brains of Parkinson ,s disease rats.

目的以绿色荧光蛋白（ＧＦＰ）作为一个标记，观察ＧＦＰ标记的ＨＴＨ１基因修饰的ＮＩＨ－３Ｔ３细胞在体外及植入脑内后的生长情况，判断ＧＦＰ基因能否作为一种新的筛选标记基因。方法构建同时表达ＧＦＰ和ＨＴＨ１双基因的多基因表达载体ＧＣ－ＧＦＰ－Ｐ－ＨＴＨ１－ＳＮ。并转染ＮＩＨ－３Ｔ３细胞，且移植入Ｐａｒｋｉｎｓｏｎ病模型大鼠纹状体内。结果用荧光显微镜、流式细胞仪、免疫组化、ｗｅｓｔｅｒｎｂｌｏｔｉｎｇ等方法均检测到ＧＦＰ、ＨＴＨ１在体外和脑内的表达。结论ＧＦＰ可作为一个筛选标记基因，可对目的基因或植入细胞的其他功能性产物的表达进行观察和检测。

Objective To observe co expression of GFP gene and human tyrosine hydroxylase type 1(HTH 1)gene in vitro or in brains.Methods The multiple gene expression vector GC GFP P HTH 1 SN which contained both GFP cDNA and HTH 1 cDNA was constructed and transfected into NIH 3T3 cells.NIH 3T3 cells which co expressed GFP and HTH 1 were then implanted into the striatum of PD rats.The expression of GFP and HTH 1 in vitro or in brain was detected by fluoresent microscope,flow cytometry,immunohistochemical staining and western blotting etc.Results The NIH 3T3 cells which transfectd with GFP and HTH 1 genes showed strong green fluorescent while positive with TH immunohistochemical staining.The HTH 1 protein was also detected by western blotting and the results showed there was protein band in 60 KD.The TH activity was detected by HPLC ECD.After been implanted into the striatum of PD rats,a large number of green fluorescent cells and TH positive cells were found in the implanted sites.Conclusion GFP gene can be used as a marker gene in observing growth condition of donor cells which implanted into brain.