CAJ | 학술논문

背景：体外实验提示LMP-1基因可提高骨质疏松性骨髓间充质干细胞LMP-1蛋白的表达。目的：用含有LIM矿化蛋白1基因的反转录病毒载体(RV-LMP-1-GFP)转染骨质疏松性SD大鼠骨髓间充质干细胞，检测其对骨质疏松性骨髓间充质干细胞LIM矿化蛋白表达的影响。方法：随机取雌性SD大鼠12只，采用双侧卵巢切除法建立骨质疏松性大鼠模型，另取6只大鼠仅切除卵巢周围等量脂肪组织，保留卵巢为假手术组。喂养2个月后取双侧股骨、胫骨、肱骨分离培养其骨髓间充质干细胞。均取3代培养细胞随机分为去势组、LMP-1转染组(转染LMP-1基因)和假手术组。分别行RT-PCR及Western-Blot检测各组细胞LIM矿化蛋白1 mRNA和蛋白的表达。结果与结论：培养出骨质疏松性SD大鼠骨髓间充质干细胞，转染后倒置荧光显微镜下可见绿色荧光表达；3组细胞均可在基因及蛋白水平表达 LIM 矿化蛋白1。与假手术组及去势组比较，LMP-1基因转染组的LIM-1mRNA和蛋白的表达均升高显著(P<0.05)，假手术组与去势组之间差异无显著性意义(P>0.05)。成功实现重组 RV-LMP-1-GFP 基因在骨质疏松性 SD 大鼠骨髓间充质干细胞中 mRNA 和蛋白水平的表达，且LMP-1的表达水平显著提高。结果表明重组LMP-1基因成功导入骨质疏松性大鼠骨髓间充质干细胞基因组中，并使得LMP-1 mRNA和蛋白的表达明显提高。
배경：체외실험제시LMP-1기인가제고골질소송성골수간충질간세포LMP-1단백적표체。목적：용함유LIM광화단백1기인적반전록병독재체(RV-LMP-1-GFP)전염골질소송성SD대서골수간충질간세포，검측기대골질소송성골수간충질간세포LIM광화단백표체적영향。방법：수궤취자성SD대서12지，채용쌍측란소절제법건립골질소송성대서모형，령취6지대서부절제란소주위등량지방조직，보류란소위가수술조。위양2개월후취쌍측고골、경골、굉골분리배양기골수간충질간세포。균취3대배양세포수궤분위거세조、LMP-1전염조(전염LMP-1기인)화가수술조。분별행RT-PCR급Western-Blot검측각조세포LIM광화단백1 mRNA화단백적표체。결과여결론：배양출골질소송성SD대서골수간충질간세포，전염후도치형광현미경하가견록색형광표체；3조세포균가재기인급단백수평표체 LIM 광화단백1。여가수술조급거세조비교，LMP-1기인전염조적LIM-1mRNA화단백적표체균승고현저(P<0.05)，가수술조여거세조지간차이무현저성의의(P>0.05)。성공실현중조 RV-LMP-1-GFP 기인재골질소송성 SD 대서골수간충질간세포중 mRNA 화단백수평적표체，차LMP-1적표체수평현저제고。결과표명중조LMP-1기인성공도입골질소송성대서골수간충질간세포기인조중，병사득LMP-1 mRNA화단백적표체명현제고。
BACKGROUND:In vitro experiments suggest that LIM mineralization protein-1 (LMP-1) gene can increase the expression of LMP-1 protein in osteoporotic bone marrow mesenchymal stem cells. OBJECTIVE:To investigate the LMP-1 expression in osteoporotic bone marrow mesenchymal stem cells transfected with RV-LMP-1-GFP in vitro. METHODS:Twelve SD female rats were selected and subjected to bilateral ovariectomy for establishment of osteoporosis models. After 2 months of feeding, bilateral femurs, tibiae, and humeri of rats were taken to isolate and culture bone marrow mesenchymal stem cells. Passage 3 cells were taken and randomly divided into ovariectomized group and LMP-1 transfection group. Another six rats only underwent removal of the same amount of fat tissue around the ovary, and passage 3 cells which were harvested as those in the former two groups served as sham group. RT-PCR and western blot assay were performed to determine the expression of LMP-1 protein and mRNA. RESULTS AND CONCLUSION:Under an inverted fluorescence microscope, transfected bone marrow mesenchymal stem cells from osteoporotic rats showed green fluorescent expression. The three groups al could express LMP-1 at the protein and mRNA levels. The expression levels of LMP-1 protein and mRNA in the LMP-1 transfection group were significantly higher than those in the ovariectomized group and sham group (P<0.05), but there was no statistical difference between the latter two groups (P>0.05). The successful expression of the RV-LMP-1-GFP gene in osteoporotic SD rat bone marrow mesenchymal stem cells was realized at the protein and mRNA levels;moreover, the expression of LMP-1 was increased dramatical y. These findings indicate that LMP-1 gene is successful y transferred into osteoporotic SD rat bone marrow mesenchymal stem cells, and significantly elevates the expression of LMP-1 mRNA and protein.