Loss of Binding and Entry of Liposome-DNA Complexes Decreases Transfection Efficiency in Differentiated Airway Epithelial Cells*
Loss of Binding and Entry of Liposome-DNA Complexes Decreases Transfection Efficiency in Differentiated Airway Epithelial Cells*
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脂质体-DNA 复合物结合和进入的丧失会降低分化气道上皮细胞的转染效率*
DOI：
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发表时间：
1997
影响因子：
4.8
通讯作者：
R. Boucher
中科院分区：
文献类型：
--
作者：
H. Matsui;L. Johnson;S. Randell;R. Boucher
囊性纤维性肺病基因治疗的靶细胞是位于气道腔内的分化良好的细胞。通过培养像“岛”一样生长的呼吸道上皮细胞，并表现出连续的细胞分化，我们研究了使高分化细胞比低分化细胞更难转染阳离子脂质体的机制。岛边缘的低分化细胞可被脂质体-β复合体(pCMVDNA：Lipofec-ACE=1：5(w/w))所感染，而岛中心分化较高的细胞则不能。对脂质介导的转导步骤的评估表明，边缘细胞结合更多的脂质体-DNA复合体，部分原因是表面电荷更负(通过阳离子铁蛋白结合来衡量)，边缘细胞内化比中央细胞内化更多的脂质体-DNA复合体。边缘细胞具有受体介导的低密度脂蛋白内吞作用、10 nm微球的吞噬作用和2-μm微球的吞噬作用，而中央细胞仅具有受体介导的内吞作用。细胞松弛素B能抑制65%的吞噬作用和93%的吞噬作用，使边缘细胞脂质体-DNA复合体的进入减少50%。钾耗竭可使吞噬功能减少90%，但对吞噬作用无影响，可抑制边缘细胞脂质体-DNA复合体进入71%。这些结果表明，脂质体-DNA复合体通过吞噬作用进入边缘细胞，这一途径在中央细胞中检测不到。总之，相对分化的细胞表面负电荷的减少和吞噬作用内化途径的缺失可能解释了阳离子脂质体介导的呼吸道上皮细胞基因转移的分化依赖性减少。
The target cells for gene therapy of cystic fibrosis lung disease are the well differentiated cells that line airway lumens. Employing cultures of airway epithelial cells that grow like “islands” and exhibit a continuum of cellular differentiation, we studied the mechanisms that render well differentiated cells more difficult to transfect with cationic liposomes than poorly differentiated cells. The poorly differentiated cells at the edge of the islands were transfectable with liposome-DNA complexes (pCMVβ:LipofectACE = 1:5 (w/w)), whereas the more differentiated cells in the center of the islands were not. Evaluation of the steps leading to lipid-mediated transfection revealed that edge cells bound more liposome-DNA complexes, in part due to a more negative surface charge (as measured by cationized ferritin binding), and that edge cells internalized more liposome-DNA complexes than central cells. Edge cells exhibited receptor-mediated endocytosis of LDL, pinocytosis of 10-nm microspheres, and phagocytosis of 2-μm microspheres, whereas central cells were only capable of receptor-mediated endocytosis. Cytochalasin B, which inhibited pinocytosis by 65% and phagocytosis by 93%, decreased edge cell liposome-DNA complex entry by 50%. Potassium depletion, which decreased phagocytosis by >90% but had no effect on pinocytosis, inhibited edge cell liposome-DNA complex entry by 71%. These results indicate that liposome-DNA complexes enter edge cells via phagocytosis and that this pathway is not detectable in central cells. In conclusion, both reduced negative surface charge and absence of phagocytosis internalization pathways in relatively differentiated cells may explain differentiation-dependent decrements in cationic liposome-mediated gene transfer in airway epithelia.
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影响因子：
158.5
作者：
KNOWLES, MR;HOHNEKER, KW;BOUCHER, RC
通讯作者：
BOUCHER, RC
脂质体介导的动脉基因转移后基因表达增加，与内膜平滑肌细胞增殖相关。
DOI：
10.1172/jci117017
发表时间：
1994
期刊：
The Journal of clinical investigation
影响因子：
--
作者：
Takeshita,S;Gal,D;Leclerc,G;Pickering,JG;Riessen,R;Weir,L;Isner,JM
通讯作者：
Isner,JM
影响因子：
3.4
作者：
ZHOU, XH;HUANG, L
通讯作者：
HUANG, L
DOI：
10.1073/pnas.81.19.6054
发表时间：
1984-10
影响因子：
11.1
作者：
M. Williams
通讯作者：
M. Williams
体外臭氧暴露调节支气管上皮细胞屏障功能。
影响因子：
10.4
作者：
Yu,XY;Takahashi,N;Croxton,TL;Spannhake,EW
通讯作者：
Spannhake,EW