CAJ | 학술논문

目的探讨转染组织因子胞内段小片段干扰RNA(siRNA)对血管内皮细胞凋亡的影响.方法体外设计组织因子胞内段siRNA Ⅰ和siRNAⅡ,插入质粒DNA后用脂质体将其转入人脐静脉内皮细胞株(HUVEC)中.3组HUVEC分别转染siRNAⅠ质粒(siRNAⅠ组)、siRNAⅡ质粒(siRNAⅡ组)和pc DNA~(TM)6.2GW/-miR质粒(对照组).取各组HUVEC细胞,分别与CD8~+ T淋巴细胞进行混合淋巴细胞反应.用流式细胞仪检测混合淋巴细胞反应中HUVEC的凋亡率,用磁珠法检测混合淋巴细胞反应上清液中活化部分凝血活酶时间(APTT).结果插入的siRNA经过测序,证实为正确序列.HUVEC转染siRNA后24 h及48 h,siRNAⅠ组和siRNAⅡ组HUVEC的凋亡率均低于对照组(P<0.01).siRNA Ⅰ组HUVEC的凋亡率低于siRNAⅡ组(P<0.05).3组上清液的APTT较对照(RPMI 1640培养基)缩短(4±0.46)s(P<0.05).siRNA Ⅰ组和siRNAⅡ组与对照组相比较,APTT的差异无统计学意义(P>0.05).结论组织因子胞内段siRNA构建成功.转染该siRNA可在不影响凝血功能的情况下对混合淋巴细胞反应中的内皮细胞起到一定保护作用,减少内皮细胞的凋亡.
목적 탐토전염조직인자포내단소편단간우RNA(siRNA)대혈관내피세포조망적영향.방법 체외설계조직인자포내단siRNA Ⅰ화siRNAⅡ,삽입질립DNA후용지질체장기전입인제정맥내피세포주(HUVEC)중.3조HUVEC분별전염siRNAⅠ질립(siRNAⅠ조)、siRNAⅡ질립(siRNAⅡ조)화pc DNA~(TM)6.2GW/-miR질립(대조조).취각조HUVEC세포,분별여CD8~+ T림파세포진행혼합림파세포반응.용류식세포의검측혼합림파세포반응중HUVEC적조망솔,용자주법검측혼합림파세포반응상청액중활화부분응혈활매시간(APTT).결과 삽입적siRNA경과측서,증실위정학서렬.HUVEC전염siRNA후24 h급48 h,siRNAⅠ조화siRNAⅡ조HUVEC적조망솔균저우대조조(P<0.01).siRNA Ⅰ조HUVEC적조망솔저우siRNAⅡ조(P<0.05).3조상청액적APTT교대조(RPMI 1640배양기)축단(4±0.46)s(P<0.05).siRNA Ⅰ조화siRNAⅡ조여대조조상비교,APTT적차이무통계학의의(P>0.05).결론 조직인자포내단siRNA구건성공.전염해siRNA가재불영향응혈공능적정황하대혼합림파세포반응중적내피세포기도일정보호작용,감소내피세포적조망.
Objective To investigate the effect of small interfering RNA (siRNA) targeting cytoplasmic domain of tissue factor on apoptosis of vascular endothelial cells. Methods Specific siRNA targeting cytoplasmic domain of tissue factor were designed, and synthetic oligos were inserted into plasmid DNA. The siRNA constructs were transfected into human umbilical vascular endothelial cells (HUVEC) with liposome. The HUVEC were transfected with the constructs encoding siRNA Ⅰ, siRNA Ⅱ and pcDNA~(TM)6.2 GW/-miR plasmid separately. The transfected HUVEC were mixed with CD8~+ T lymphocytes. The apoptotic rate of tranfected HUVEC mixed with lymphocytes was analyzed by flow cytometry. Magnetic beads were used to measure PT of the supematant in the mixed lymphocytes culture. Results The siRNA constructs were confirmed by DNA sequence analysis. The apoptotic rate of HUVEC transfected with siRNA Ⅰ and Ⅱ plasmids was decreased significantly as compared with the empty control group (P<0.01). The apoptosis rate of HUVEC transfected with siRNA Ⅰ plasmid was lower than that of HUVEC transfected with siRNA Ⅱ plasmid (P<0.05). APTT of the culture supernatants in the three transfection groups was lower in the control groups (P <0.05), but there was significant difference among the three transfection groups. Conclusion The siRNA targeting cytoplasmic domain of tissue factor were successfully constructed, siRNA can protect HUVEC, and reduce the apoptotic rate of endothelial cells in mixed lymphocyte reaction without influencing the coagulation function.