CAJ | 학술논문

背景：掌握转染的最佳感染复数和产生较强荧光强度的时间，可为后期观察人骨髓间充质干细胞在动物模型体内的示踪奠定基础。目的：探讨HIV-1来源的慢病毒携带增强型红色荧光蛋白转染人骨髓间充质干细胞的可行性。方法：将第4代人骨髓间充质干细胞分成空白组和感染复数为2，3，4组，以细胞数5.0×105个接种于12孔培养皿，添加含有体积分数为1%胎牛血清的成人骨髓间质干细胞完全培养基1 mL。调整慢病毒携带增强型红色荧光蛋白感染滴度为1.0×1011 TU/L，加入各组培养皿中病毒液体积分别为10，15，20μL，感染复数分别为2，3，4，空白组加入10μL PBS。转染后24，72 h荧光倒置显微镜观察细胞红色荧光表达情况并计算出转染率。结果与结论：增强型红色荧光蛋白在骨髓间充质干细胞中稳定表达，转染后24 h倒置荧光显微镜下可见红色荧光，72 h荧光达到最强，细胞转染率与感染复数值呈线性增长关系。转染后21 d内，各转染实验组的人骨髓间充质干细胞数量与空白组比较差异无显著性意义(P >0.05)，以上结果提示采用 HIV-1来源的慢病毒载体介导增强型红色荧光蛋白转染标记人骨髓间充质干细胞是可行的，当感染复数为4时转染效率高并可持续表达至少21 d，且标记后对其增殖活性无影响。
배경：장악전염적최가감염복수화산생교강형광강도적시간，가위후기관찰인골수간충질간세포재동물모형체내적시종전정기출。목적：탐토HIV-1래원적만병독휴대증강형홍색형광단백전염인골수간충질간세포적가행성。방법：장제4대인골수간충질간세포분성공백조화감염복수위2，3，4조，이세포수5.0×105개접충우12공배양명，첨가함유체적분수위1%태우혈청적성인골수간질간세포완전배양기1 mL。조정만병독휴대증강형홍색형광단백감염적도위1.0×1011 TU/L，가입각조배양명중병독액체적분별위10，15，20μL，감염복수분별위2，3，4，공백조가입10μL PBS。전염후24，72 h형광도치현미경관찰세포홍색형광표체정황병계산출전염솔。결과여결론：증강형홍색형광단백재골수간충질간세포중은정표체，전염후24 h도치형광현미경하가견홍색형광，72 h형광체도최강，세포전염솔여감염복수치정선성증장관계。전염후21 d내，각전염실험조적인골수간충질간세포수량여공백조비교차이무현저성의의(P >0.05)，이상결과제시채용 HIV-1래원적만병독재체개도증강형홍색형광단백전염표기인골수간충질간세포시가행적，당감염복수위4시전염효솔고병가지속표체지소21 d，차표기후대기증식활성무영향。
BACKGROUND:To grasp the optimal multiplicity of infection (MOI) and the time when stronger fluorescence intensities produce can lay the foundation for tracing observation of human bone marrow mesenchymal stem celsin vivo in animal models. OBJECTIVE:To investigate the feasibility of HIV-1 lentivirus carrying enhanced red fluorescent protein to transfect human bone marrow mesenchymal stem cels. METHODS:Human bone marrow mesenchymal stem cels at passage 4 were divided into blank group and MOI 2, 3, 4 groups. After that, the cels were seeded into 12-wel plates at a density of 5.0×105 , and cultured in 1 mL complete medium for adult bone marrow mesenchymal stem cels containing 1% fetal bovine serum. The infectious titer of lentivirus-carried enhanced red fluorescent protein was adjusted to 1.0×10 11 TU/L. Lentivirus solution 10, 15, 20 μL at MOI=2, 3, 4 were respectively added into the MOI 2, 3, 4 groups, and 10μL PBS was added into the blank group. At 24 and 72 hours after transfection, the expression of red fluorescence was observed under an inverted fluorescence microscope and the transfection efficiency was calculated. RESULTS AND CONCLUSION:Enhanced red fluorescent protein expressed stably in bone marrow mesenchymal stem cels. At 24 hours after transfection, red fluorescence could be seen under the inverted fluorescence microscope and achieved the peak at 72 hours after transfection. Within 21 days after transfection, there were no differences in the number of human bone marrow mesenchymal stem cels between the MOI 2, 3, 4 groups and blank group (P > 0.05). These results show that the HIV-1 lentivirus carrying enhanced red fluorescent protein is feasible to transfect human bone marrow mesenchymal stem cels, with the highest transfection efficiency when the MOI=4, which can express at least for 21 days and have no effects on the proliferative activity of labeled cels.