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目的:构建基质细胞衍生因子1α-绿色荧光蛋白(SDF-1α-GFP)重组腺病毒载体并摸索其转染大鼠心肌细胞系的最佳条件。方法:人工合成SDF-1α基因并插入线性化表达载体,转化感受态E.coliDH5a细胞。筛选阳性菌落,采用DNA测序技术测定目的基因序列。用获得的重组腺病毒(adenovirus,Ad)载体转染293T细胞,采用qPCR技术测定SDF-1α的mRNA表达水平。用不同滴度感染复数Ad-SDF-1α-GFP转染H9C2心肌细胞系,采用倒置相差显微镜及荧光显微镜分别在24和48h检测细胞形态及GFP蛋白阳性的细胞比率。结果:DNA测序证实SDF-1α基因构建成功。qPCR结果显示,与阴性对照组相比,MOI 2000组的SDF-1α的表达水平增加了约10 000倍,MOI 8000组的SDF-1α表达水平增加了约210 000倍。转染H9C2心肌细胞系的结果显示,MOI值为2 000、3 000、4 000、5 000、6 000、7 000及8 000的SDF-1α-GFP转染H9C2细胞48h后,其转染率分别为(45±5.04)%、(75±5.70)%、(80±5.67)%、(85±6.45)%、(90%±6.90)%、(90±5.22)%和(95±5.36)%,但是当MOI值区间为5 000~8 000时,细胞形态上出现明显损伤。结论:MOI值为4 000的Ad-SDF-1α-GFP转染H9C2细胞系48h能达到最佳的转染效率,并最小程度损伤细胞。
OBJECTIVE: To construct the recombinant adenovirus vector containing stromal cell-derived factor 1α-green fluorescent protein (SDF-1α-GFP) and explore the optimal conditions for its transfection into rat cardiomyocyte cell line. Methods: SDF-1α gene was synthesized and inserted into a linearized expression vector to transform competent E. coli DH5a cells. Positive colonies were screened and DNA sequencing was used to determine the target gene sequence. 293T cells were transfected with the obtained recombinant adenovirus (Ad) vector and the expression of SDF-1α mRNA was detected by qPCR. H9C2 cells were transfected with Ad-SDF-1α-GFP transfected with different titers of Ad-SDF-1α-GFP. The cell morphology and GFP-positive cell ratio were detected by inverted phase contrast microscope and fluorescence microscope at 24 and 48 hours respectively. Results: DNA sequencing confirmed the successful construction of SDF-1α gene. The qPCR results showed that the expression level of SDF-1α in MOI 2000 group increased by about 10 000 times compared with the negative control group, and the expression level of SDF-1α in MOI 8000 group increased by about 210 000 folds. The results of transfection of H9C2 cardiomyocytes showed that the transfection efficiency of H9C2 cells transfected with SDF-1α-GFP with MOI values of 2,000, 3,000, 4,000, 5,000,000, 6,000, 7,000 and 8,000 Were (45 ± 5.04)%, (75 ± 5.70)%, (80 ± 5.67)%, (85 ± 6.45)%, (90 ± 6.90)%, (90 ± 5.22)% and (95 ± 5.36) %, But when the MOI value ranged from 5 000 to 8 000, significant morphological damage was observed. CONCLUSION: H9C2 cells transfected with Ad-SDF-1α-GFP at an MOI of 4 000 for 48 h achieve the best transfection efficiency with minimal damage to cells.