TH和GDNF基因工程细胞脑内移植治疗PD大鼠的实验研究

AN EXPERIMENTAL STUDY ON GENE THERAPY OF PARKINSON'S DISEASE WITH TWO GRAFTS OF GENETICALLY ENGINEERED CELLS EXPRESSING TYROSINE HYDROXYLASE AND GLIAL CELL LINE-DERIVED NEUROTROPHIC FACTOR IN THE RAT

目的 观察酪氨酸羟化酶（ＴＨ）和胶质细胞系源性神经营养因子（ＧＤＮＦ）基因工程细胞联合移植到纹状体内对帕金森病（ＰＤ）大鼠的治疗作用。方法 重组携带ＴＨ基因的质粒表达载体ｐＣＭＶＴＨ及携带ＧＤＮＦ基因的质粒表达载体ｐＣｌ－ｎｅｏ，分别转染原代培养的成纤维细胞，筛选阳性细胞植入大鼠帕金森病模型纹状体内，动态观察行为学变化，并用高效液相色谱于第４、８、１２、１６、２０周测定多巴胺、３，４－二羟苯乙酸（ＤＯＰＡＣ）及高香草酸（ＨＶＡ）含量的变化。结果 联合移植ＴＨ及ＧＤＮＦ转基因细胞致ＰＤ大鼠纹状体内可明显减少模型单侧旋转行为，可降至移植前旋转圈数的４４．３±５．２％（Ｐ＜０．０１），其效果优于单独移植 ＴＨ（６５．６± ６．１％）或 ＧＤＮＦ（６４．８± ５．８％）转基因细胞的 ＰＤ模型（Ｐ＜０．０５）；显著提高纹状体内多巴胺含量，恢复至移植前水平的２１．４±３．４７％（Ｐ＜０．００１），其程度高于单独移植ＴＨ（１２．９＝１．４％）或ＧＤＮＦ（１．５±２．２％）转基因细胞的 ＰＤ模型（Ｐ＜０．０５）。结论 ＴＨ及 ＧＤＮＦ联合基因治疗具有潜在的临床应用价值。

Objective To observe the improvement of motor ab normalities and striatal levels of dopamine and its metabolites in rat model of Parkinson's disease by combining intracerebral grafts of two kinds of geneticall y modified cells expressing tyrosinehydroxylase and glial cell line-derived n eu rotrophic factor.Methods The cultured primary fibrob lasts were sele cted for genetic modification and intracerebral grafting.The recombinant vectors ,pCMVTH containing TH gene and pCI-neo containing GDNF gene,were transferred in t o the fibroblasts in vitro by using lipofectin technique.The cells expressing TH and the cells expressing GDNF were then grafted ipsilaterally into the striatum in PD models rats.The levels of the striatal dopamine(DA), 3,4-dihydroxyphenyl ac etic acid(DOPAC)and homovanillic acid(HVA)in the rat models were determined by u sing high performance liquid chromatography(HPLC).The tests were done in 4, 8, 1 2,16 and 20 weeks after intracerebral transplantation.Results The g rafted genetically modified cells expressing TH and GDNF could substantially red uce the asymmetric rotational behavior of PD rats with a long-term.The scores o f asymmetric rotation of these rats after apomorphine administration decreased to 44.3±5.2%(P＜0.01).The levels dopamine in the striatum decr ease d to 1.92±0.15% and was up to 21.4±3.47%(P＜0.001)compare d wi th its normal level.Conclusion The combining transp lantation of TH gene-expressed and GDNF gene-expressed cultured fibroblasts is useful with re gard to the treatment of PD and had a potential usage in the clinic.