【摘要】：背景:掌握轉(zhuǎn)染的最佳感染復(fù)數(shù)和產(chǎn)生較強(qiáng)熒光強(qiáng)度的時(shí)間,可為后期觀察人骨髓間充質(zhì)干細(xì)胞在動(dòng)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(qiáng)型紅色熒光蛋白轉(zhuǎn)染人骨髓間充質(zhì)干細(xì)胞的可行性。方法:將第4代人骨髓間充質(zhì)干細(xì)胞分成空白組和感染復(fù)數(shù)為2,3,4組,以細(xì)胞數(shù)5.0×105個(gè)接種于12孔培養(yǎng)皿,添加含有體積分?jǐn)?shù)為1%胎牛血清的成人骨髓間質(zhì)干細(xì)胞完全培養(yǎng)基1 m L。調(diào)整慢病毒攜帶增強(qiáng)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(xì)胞紅色熒光表達(dá)情況并計(jì)算出轉(zhuǎn)染率。結(jié)果與結(jié)論:增強(qiáng)型紅色熒光蛋白在骨髓間充質(zhì)干細(xì)胞中穩(wěn)定表達(dá),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(dá)到最強(qiáng),細(xì)胞轉(zhuǎn)染率與感染復(fù)數(shù)值呈線性增長(zhǎng)關(guān)系。轉(zhuǎn)染后21 d內(nèi),各轉(zhuǎn)染實(shí)驗(yàn)組的人骨髓間充質(zhì)干細(xì)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(qiáng)型紅色熒光蛋白轉(zhuǎn)染標(biāo)記人骨髓間充質(zhì)干細(xì)胞是可行的,當(dāng)感染復(fù)數(shù)為4時(shí)轉(zhuǎn)染效率高并可持續(xù)表達(dá)至少21 d,且標(biāo)記后對(duì)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(guó)家青年科學(xué)基金項(xiàng)目(31200726) 廣州市應(yīng)用基礎(chǔ)研究項(xiàng)目(2013J4100101)~~
【分類號(hào)】：R329.2;R373

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 Song Pan;Jingzhi Wan;Shaosheng Liu;Song Zhang;Hao Xiong;Jun Zhou;Wu Xiong;Kunfei Yu;Yong Fu;;Lentivirus carrying the Atoh1 gene infects normal rat cochlea[J];Neural Regeneration Research;2013年17期

2 Yuxin Wu;Jinghan Zhang;Xiaoming Ben;;Neuronal-like cell differentiation of non-adherent bone marrow cell-derived mesenchymal stem cells[J];Neural Regeneration Research;2013年22期

3 張穎;辛毅;汪勁松;許秀芳;羅毅;黃益民;;綠色熒光蛋白和紅色熒光蛋白共轉(zhuǎn)染骨髓間充質(zhì)干細(xì)胞、乳鼠心肌細(xì)胞、Eahy926細(xì)胞表達(dá)特征的共聚焦分析[J];新鄉(xiāng)醫(yī)學(xué)院學(xué)報(bào);2011年02期

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

1 崔岳毅;宋純理;譚杰;付鑫;陳仲強(qiáng);劉忠軍;黨耕町;;局部植入辛伐他汀誘導(dǎo)自體骨髓間充質(zhì)干細(xì)胞歸巢修復(fù)大鼠顱骨缺損[J];國(guó)際骨科學(xué)雜志;2013年06期

2 Hu-Yan Li;He Li;Bing-jie Wang;Qun Gu;Zhi-Qiang Jiang;Xue-Dong Wu;;Synthesis and properties of poly(3-hydroxybutyrate-co-3-hydroxyvalerate)/chitin nanocrystals composite scaffolds for tissue engineering[J];Chinese Chemical Letters;2014年12期

3 李翰翔;丁曉玲;徐茂義;沈忠飛;;人骨髓間充質(zhì)干細(xì)胞成骨誘導(dǎo)的研究進(jìn)展[J];吉林醫(yī)學(xué);2013年31期

4 劉文忠;王栓科;陳永剛;任小軍;董平;;三磷酸腺苷與骨髓間充質(zhì)干細(xì)胞聯(lián)合移植對(duì)損傷脊髓中神經(jīng)元特異性烯醇化酶表達(dá)的影響[J];蘭州大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2014年01期

5 高賀云;魏明發(fā);王燕;吳曉娟;朱天琦;;膠質(zhì)細(xì)胞源性神經(jīng)營(yíng)養(yǎng)因子和神經(jīng)營(yíng)養(yǎng)素-3雙基因轉(zhuǎn)染大鼠骨髓間充質(zhì)干細(xì)胞體外誘導(dǎo)分化為神經(jīng)樣細(xì)胞的研究[J];實(shí)用兒科臨床雜志;2011年11期

6 王淑輝;尚延昌;于美娟;張雅妮;張成;;Micro-dystrophin基因修飾的骨髓間充質(zhì)干細(xì)胞移植治療后mdx鼠MyoD和devMHC的變化[J];臨床和實(shí)驗(yàn)醫(yī)學(xué)雜志;2014年12期

7 謝尚U，

本文編號(hào)：2179960