Pim-3质粒构建体在大鼠活体肝组织的表达和活性的研究

Construction of a recombinant Pim3-expressing plasmid and expression and activity thereof:experiment with rats

目的构建大鼠 Pim-3绿色荧光蛋白(GFP)表达质粒并观察其在活体肝组织的表达和活性。方法采用逆转录 PCR 的方法获取目的 cDNA,重组质粒经酶切鉴定和测序;大鼠活体基因肝靶向性转染通过尾静脉流体力学注射法完成,肝细胞凋亡的诱导采用腹腔内注射内毒素和 D-半乳糖胺(D-GalN)来实现。动物分为 A、B、C 和 D 组(即正常、对照、空质粒和重组质粒组,每组8只);肝组织 GFP 表达通过荧光显微镜、Pim-3表达通过逆转录(RT)-PCR 方法检测;肝细胞凋亡采用缺口末端标记技术(TUNEL)分析和半胱天冬酶-3活性检测。结果成功构建 GFP 表达质粒 pEGFP-N2/Pim-3;重组质粒和空质粒 DNA 通过流体力学注射法被成功转染入大鼠活体肝组织内;4组 Pim-3的相对表达水平分别为0.06±0.02、0、0、0.49±0.15。D 组与其他3组差异均有统计学意义(均 P<0.01);4组肝细胞的凋亡指数(AI)分别为:(3.1±0.7)%,(72.5±6.1)%、(69.8±5.7)%和(4.9±1.2)%;肝组织半胱天冬酶-3活性分别为(60±15)、(147±55)、(142±50)和(76±27)pmol·min^(-1)·mg^(-1),D 组与 B、C 两组间差异有统计学意义(均 P<0.01)。结论构建的重组体质粒 pEGFP-N2/Pim-3能在大鼠活体肝组织内有效表达,并发挥其对肝细胞凋亡的抑制效应。

Objective To construct a plasmid expressing green fluorescent protein （GFP） and gene of Pim-3, a member of the serine/threonine kinase family, and to investigate the in vivo expression of the construct and its effect on cell apoptosis. Methods Pim-3 gene was cloned from myocardium tissues of Wistar rat by RT-PCR and subcloned into GFP-expressing plasmid vector pEGFP-N2 by restriction enzyme. The recombinant plasmid pEGFP-N2/Pim-3 was constructed by T4-1igase and then identified through enzyme digestion and gene sequencing. Thirty-two Wistar rats were randomly divided into 4 equal groups ： Group A, as control group; Group B, injected intravenously with Ringer＇s solution; Group C, injected with blank vector, and Group D, injected with the recombinant plasmid pEGFP-N2/Pim-3. One day later, endotoxin/ D-galactoamine （D-GaIN） was intraperitoneally injected. 24 hours later the rats were killed. Fluorescence microscopy was used to observe the expression of the reporter gene GFP in the liver tissues. RT-PCR was used to detect the Pim-3 mRNA expression. The hepatic apoptosis was detected by TUNEL assay. The activity of caspase-3 was detected. Results A 998 bp target cDNA fragment with restriction enzyme sites was amplified and inserted into the multiple clone site of pEGFP-N2 successfully. High expression levels of the target gene Pim-3 and reporter gene GFP were achieved in the rat liver after transfer of the recombinant plasmid. The relative Pim-3 expression level of Group D was 0.49 ±0.15, significantly higher than those of Groups A, B, and C （0.06 ±0.02, 0, and 0 respectively, all P 〈0.01 ）. The apoptotic index of Group D was （4.9±1.2）%, significantly lower than those of Groups B and C [（72.5±6. 1）% and （69.8 ± 5.7）% respectively, both P 〈0.01 ] ; however, not significantly different from that of Group A [ （3.1 ± 0. 7 ） % ]. The activity of caspace-3 of Group D was （76 ± 27 ） pmol · min ^- 1 . mg^- 1 , significantly lower than those of Groups B and C [ （ 147 ± 55 ） and （ 142± 50） pmol· min ^- l . mg^- 1, respectively, both P 〈 0.01 ] ; however, not significantly different from that of Group A（60±15）. Condus｜on The recombinant plasmid pEGFP-N2/Pim-3 can achieve high expression in living cells and have an inhibitory effect on hepatic apoptosis.