Matsumura J S, Kim R, Shively V P, MacDonald R C, Pearce W H
Department of Surgery and the Feinberg Cardiovascular Research Institute, Northwestern University Medical School, Chicago, Illinois, 60611, USA.
J Surg Res. 1999 Aug;85(2):339-45. doi: 10.1006/jsre.1999.5678.
Cationic liposomes are an alternative non-viral vector for gene therapy, but several factors affect transfection efficiency. A novel cationic lipid, o-ethyldioleoylphosphatidylcholinium (EDOPC), was studied for characterization of the time course and effects of lipid composition, concentration, charge ratio, mixing techniques, passage number, and stimulated state on transfection of human vascular cells, represented by human umbilical vein endothelial cells (HUVEC).
HUVEC cultures were seeded at a density of 45,000 cells/well in 24-well plates and incubated overnight. Triplicate wells were transfected with samples of EDOPC/reporter plasmid for 2 h, followed by a 24-h expression time, which was the peak expression time point in an initial time-course experiment. Measuring luciferase in cell lysates quantitated gene expression.
Transfection of HUVEC with EDOPC was optimal with a concentration of 100 microgram lipid/well, ratio of 3:1 EDOPC:plasmid, fractional mixing of lipid and plasmid, centrifugation, and incubation in serum-free media. Transfections in sequential passages showed striking decreases in gene expression and regression analysis revealed the relationship: RLU = 120,000 - (10, 400 x passage number), r(2) = 0.947. HUVEC activated by cytokine stimulation remain susceptible to gene transfer specifically with EDOPC.
During transfection of HUVEC with cationic lipid species, an increase in passage number is associated with linear reduction in luciferase expression, and hence passage number must be controlled in comparative experiments. Characteristics of EDOPC may permit site-specific efficient transfection of activated human vascular cells that can be isolated from serum by mechanical methods.
阳离子脂质体是基因治疗中一种替代性的非病毒载体，但有几个因素会影响转染效率。我们研究了一种新型阳离子脂质，即邻乙基二油酰磷脂酰胆碱（EDOPC），以表征脂质组成、浓度、电荷比、混合技术、传代次数和刺激状态对以人脐静脉内皮细胞（HUVEC）为代表的人血管细胞转染的时间进程和影响。
将HUVEC培养物以45,000个细胞/孔的密度接种于24孔板中并孵育过夜。用EDOPC/报告质粒样品转染一式三份的孔2小时，随后进行24小时的表达时间，这是初始时间进程实验中的峰值表达时间点。通过测量细胞裂解物中的荧光素酶来定量基因表达。
用EDOPC转染HUVEC时，脂质浓度为100微克/孔、EDOPC与质粒的比例为3:1、脂质与质粒分步混合、离心以及在无血清培养基中孵育时效果最佳。连续传代转染显示基因表达显著下降，回归分析揭示了这种关系：相对光单位（RLU）= 120,000 -（10,400×传代次数），r² = 0.947。细胞因子刺激激活的HUVEC仍然对基因转移敏感，特别是对EDOPC。
在用阳离子脂质转染HUVEC的过程中，传代次数的增加与荧光素酶表达的线性降低相关，因此在比较实验中必须控制传代次数。EDOPC的特性可能允许对可通过机械方法从血清中分离的活化人血管细胞进行位点特异性高效转染。
