【摘要】：背景:掌握轉(zhuǎn)染的最佳感染復(fù)數(shù)和產(chǎn)生較強熒光強度的時間,可為后期觀察人骨髓間充質(zhì)干細胞在動物模型體內(nèi)的示蹤奠定基礎(chǔ)。目的:探討HIV-1來源的慢病毒攜帶增強型紅色熒光蛋白轉(zhuǎn)染人骨髓間充質(zhì)干細胞的可行性。方法:將第4代人骨髓間充質(zhì)干細胞分成空白組和感染復(fù)數(shù)為2,3,4組,以細胞數(shù)5.0×105個接種于12孔培養(yǎng)皿,添加含有體積分數(shù)為1%胎牛血清的成人骨髓間質(zhì)干細胞完全培養(yǎng)基1 m L。調(diào)整慢病毒攜帶增強型紅色熒光蛋白感染滴度為1.0×1011TU/L,加入各組培養(yǎng)皿中病毒液體積分別為10,15,20μL,感染復(fù)數(shù)分別為2,3,4,空白組加入10μL PBS。轉(zhuǎn)染后24,72 h熒光倒置顯微鏡觀察細胞紅色熒光表達情況并計算出轉(zhuǎn)染率。結(jié)果與結(jié)論:增強型紅色熒光蛋白在骨髓間充質(zhì)干細胞中穩(wěn)定表達,轉(zhuǎn)染后24 h倒置熒光顯微鏡下可見紅色熒光,72 h熒光達到最強,細胞轉(zhuǎn)染率與感染復(fù)數(shù)值呈線性增長關(guān)系。轉(zhuǎn)染后21 d內(nèi),各轉(zhuǎn)染實驗組的人骨髓間充質(zhì)干細胞數(shù)量與空白組比較差異無顯著性意義(P0.05),以上結(jié)果提示采用HIV-1來源的慢病毒載體介導(dǎo)增強型紅色熒光蛋白轉(zhuǎn)染標記人骨髓間充質(zhì)干細胞是可行的,當感染復(fù)數(shù)為4時轉(zhuǎn)染效率高并可持續(xù)表達至少21 d,且標記后對其增殖活性無影響。
[Abstract]:Background: mastering the optimal number of infections and the time of producing strong fluorescence intensity can lay a foundation for the later observation of human bone marrow mesenchymal stem cells (BMSCs) tracer in animal models. Aim: to investigate the feasibility of transfection of human bone marrow mesenchymal stem cells (BMSCs) with lentivirus derived from HIV-1 and enhanced red fluorescent protein. Methods: the fourth generation of human bone marrow mesenchymal stem cells (BMSCs) were divided into blank group and infected plural group (2). The cells were inoculated in 12 well culture dish with 5. 0 脳 10 ~ 5 cells. The adult bone marrow mesenchymal stem cells containing 1% fetal bovine serum were added to the complete culture medium of 1 mL of adult bone marrow mesenchymal stem cells. The titer of lentivirus carrying enhanced red fluorescent protein infection was adjusted to 1.0 脳 1011TU / L, the volume of virus solution in the culture dish was 10 ~ 1520 渭 L, the complex number of infection was 2 ~ 3 ~ 3 ~ (4), and the blank group was added 10 渭 L PBS. The expression of red fluorescence was observed by fluorescence inverted microscope at 24 ~ 72 h after transfection and the transfection rate was calculated. Results and conclusion: the enhanced red fluorescent protein was stably expressed in bone marrow mesenchymal stem cells. The fluorescence intensity of 72 h was the strongest under the inverted fluorescence microscope 24 h after transfection, and the cell transfection rate was linearly increased with the infection complex value. Within 21 days after transfection, There was no significant difference in the number of human bone marrow mesenchymal stem cells between each transfection group and the blank group (P0.05). The above results suggested that the lentivirus vector derived from HIV-1 was used to mediate the transfection of enhanced red fluorescent protein to label human bone marrow mesenchymal cells (BMSCs). Stem cells are feasible. When the complex number of infection is 4, the transfection efficiency is high and the expression can be sustained for at least 21 days, and the labeling has no effect on its proliferation activity.
【作者單位】： 廣州醫(yī)科大學(xué)附屬第三醫(yī)院骨科;
【基金】：國家青年科學(xué)基金項目(31200726) 廣州市應(yīng)用基礎(chǔ)研究項目(2013J4100101)~~
【分類號】：R329.2;R373

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