摘要
本实验用标记基因ｌａｃＺ５型重组腺病毒（Ａｄ５ＣＭＶｌａｃＺ）转染培养的ＮＧ细胞系，Ｘ－ｇａｌ染色检测转染效率．在培养的ＮＧ细胞系，当病毒滴度为２×１０８时，转集率达到５０％，当滴度为２×１０９时，转染率达１００％，有较好的量效关系；固定病毒液度为１０１０，培养２～１６ｈ，细胞的转染率随时间延长而提高，有较好的时效关系。将Ａｄ５ＣＭＶｌａｃＺ注射到大鼠黑质部位后，分别于注射后３～１２０ｄ取脑、切片、Ｘ－ｇａｌ染色，发现黑质局部从第７ｄ开始有部分蓝染，第１０ｄ达高峰，注射局部感染率１００％；９０ｄ时开始下降，持续至１２０ｄ；纹状体等其它部位无蓝染．上述结果提示，腺病毒载体介导的标记基因可在培养的神经细胞系和中脑黑质部位高效表达，为进一步开展中枢神经系统退变性疾病尤其是帕金森氏病的基因治疗奠定基础。
The present study was undertaken to investigate the efficiency of transfection of adenovirus vector-mediated foreignmarker gene by observing the expression of adenovirus-lac Z(Ad5CMYlacZ) in cultured NG cell line and in rat substantia nigra. In cultured NG cell line, the transfective rate is about 50% when the virus titer is 2 × 108 pfu/ml .whereas up to 100%when the virus titer is 2 × 109 pfu/ml, indicating a well-established dose-effect relationship. Furthermore, there is a perfecttime-effect relationship when the NG cell is cultured from 2～16 hours after Ad5CMYlacZ transfection with virus titer fixedat 1010 pfu/ml. In vivo experiment of rat, the recombinant virus is stereotaxically injected into substantia nigra with 4 μl of2 × 1010 pfu/ml. Brain slices at 15 μm thickness is collected at 3 days to 120 days after injection respectively. Then the slicesis stained with X-gal. If infected with Ad5CMVlacZ, the transfected cells (including neurons and glia cells) will be stained blue. It is found that the blue staining appears at the day 7, reaches peak at day 10 with the infections rate of nearly 100%,comes down from 90 days and sustains at day 120. No blue staining is found in striatum and other areas for the whole experiment. The above results indicate that the recombinant adenovirus Ad5CMVlacZ can trans feet and express in the cultured NGcell line as well as in the dopaminergic neurons of rat substantia nigra with high efficiency. These results give us a clue to thebasic research on the adenovirus-mediated gene therapy of Parkinson's disease.
出处
《神经解剖学杂志》
CAS
CSCD
北大核心
1996年第4期407-412,共6页
Chinese Journal of Neuroanatomy
基金
国家自然科学基金
国家教委跨世纪优秀人才基金
卫生部特别基金
香港北美医学基金
关键词
腺病毒载体
标记基因
中枢神经系疾病
基因治疗
adenovirus vector, marker gene, gene expression, NG cell line, substantia nigra
