携带肝细胞生长因子基因重组质粒在大鼠体内的组织分布研究

Distribution of recombinant plasmid carrying hepatocyte growth factor gene in rat

目的研究携带肝细胞生长因子基因的重组质粒(pUDKH)经肌肉注射给药后在大鼠体内的组织分布及其与大鼠基因组DNA的整合情况。方法二级Wistar大鼠雌雄各20只,按体重和取样时间的不同随机分为12h、24h、3d、7d、14d、21d的各实验组(给予pUDKH,2.0mg/kg)和注射后3d取样的对照组(给予PBS,200μl/只),依次将各组大鼠摘眼球取血后脱臼处死,按脑、心、肺、胸腺、肝、肠系膜淋巴结(MLN)、脾、肾、生殖腺、左腿肌肉、右腿肌肉的顺序取样。经典酚/氯仿抽提法提取各组织总DNA,应用紫外分光光度计测定其浓度和纯度,PCR方法检测其中的β肌动蛋白基因;巢式PCR检测各组织总DNA中pUDKH的分布情况;ApaLI酶切分离pUDKH分布阳性各组织中的pUDKH DNA和基因组DNA,巢式PCR检测pUDKH DNA与基因组DNA的整合情况。结果各组织总DNA浓度与纯度均符合实验需要,并适于采用PCR或巢式PCR方法进行体内组织分布及基因组DNA整合情况检测。巢式PCR检测显示,在各时间点的右腿肌肉与外周血、注射后3和7d的肝脏、3和14d的生殖腺、7d的胸腺、7和14d的MLN与左腿肌肉等组织中pUDKH分布均呈阳性,而在各时间点的脑、心、肺、脾、肾等组织中pUDKH分布均呈阴性。pUDKH分布阳性的各组织中均未检测到pUDKH DNA与基因组DNA的整合。结论pUDKH经肌肉注射给药后,在各时间点大鼠体内各组织中呈现不同的分布谱。在pUDKH分布阳性的各组织中,pUDKH DNA与基因组DNA发生随机整合的概率较低。

Objective To study the tissue distribution of recombinant plasmid carrying hepatocyte growth factor gene （pUDKH） in rats by intramuscular injection and its genome integration with rats DNA. Methods A total of 40 Wistar rats （20 male and 20 female） were enrolled in this study, and randomly divided into experiment （injected with pUDKH, 2.0 mg/kg） and control （injected with 200 pd of PBS） groups. The rats in experiment group were killed in 12 h, 24 h, 3 d, 7 d, 14 d, and 21 d respectively after the injection, and the control group was sacrificed in 3 days. The tissues of brain, heart, lungs, thymus, liver, MLN, spleen, kidney, genital glands, and muscles of the injected and opposite legs were obtained from the rats. The total DNA extracted from various tissues was determined by classic ＂phenol-chloroform＂ technique, its concentration and purity were measured using ultraviolet spectrophotometer, and the content of β-actin was detected with PCR. Nest-PCR was applied to detect the distribution of pUDKH, pUDKH DNA was isolated from tissue Genomic DNA that was digested by restriction incision enzyme in all the pUDKH distributed tissues, And then Nest-PCR was employed to assay the integrated plasmid. Results The concentration and purity of isolated total DNA from varied tissues satisfied demand of this experiments and can be used to examine gene integrated into genome and distribution in tissues by PCR or Nest-PCR. It was detected in the liver at days 3 and 7, in the genital glands at days 3 and 14, the thymus at day 7, MLN and opposite leg at days 7 and 14, at the injected leg and the peripheral blood at all the time points. Whereas, in the brain, heart, lungs, spleen, and kidney, no pUDKH was found at any of the time points. Plasmid integration was not detected in all the pUDKH distributed tissues. Conclusions By intramuscular injection, the distribution of plasmid pUDKH in rat are different among various tissues at different time points. The plasmid integration is below a detectable level by Nest-PCR assay.