中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2014年
8期
1690-1693
,共4页
翟慧%向阳%杨毅宁%马依彤%李晓梅%陈邦党%刘芬%张金宇
翟慧%嚮暘%楊毅寧%馬依彤%李曉梅%陳邦黨%劉芬%張金宇
적혜%향양%양의저%마의동%리효매%진방당%류분%장금우
R65核酶基因%腺相关病毒%高脂血清%增殖%脱噬作用
R65覈酶基因%腺相關病毒%高脂血清%增殖%脫噬作用
R65핵매기인%선상관병독%고지혈청%증식%탈서작용
R65 nuclear enzyme gene%Adeno-associated virus%High lipid serum%Proliferation%Apoptosis
目的 通过重组9型腺相关病毒(rAAV9)载体介导R65核酶基因对体外培养人脐静脉内皮细胞(HUVECs)进行转染,观察R65核酶基因表达对高脂血清刺激后HUVECs增殖和凋亡的影响.方法 体外培养HUVECs,高脂血清诱导建立动脉粥样硬化内皮损伤模型,重组rAAV9介导抗核因子(NF)-κB核酶基因(rAAV9-R65)腺相关病毒载体转染HUVECs.实验分为3组:(1)正常对照组;(2)高脂血清诱导组;(3)高脂血清诱导+病毒转染组.通过Western blot检测HUVECs细胞中p65核蛋白的表达;酶联免疫吸附试验(ELISA)检测NF-κB信号通路下游肿瘤坏死因子(TNF)-α和白细胞介素(IL)-6的变化;Alamar Blue法检测rAAV9-R65转染后HUVECs的体外增殖;rAAV9-R65转染细胞96 h后流式细胞仪检测高脂血清刺激后HUVECs凋亡率的变化.结果 高脂血清诱导HUVECs 24 h后,细胞由卵圆形铺路石样逐渐变成类圆形,Western blot检测显示p65的表达水平分别为:空白组:0.0229 ±0.0024;高脂血清诱导组:0.224 3±0.008 6;rAAV9-R65转染+高脂血清诱导组:0.071 4 ±0.007 0.HUVECs细胞中高脂血清诱导后p65蛋白表达明显增高,而病毒转染后p65水平降低.ELISA检测结果显示HUVEC分泌的TNF-α水平3组分别为:15.98 ±0.43、27.94±0.83、19.73±0.43.而3组IL-6水平分别为:3.94 ±0.12、6.32 ±0.21、5.08 ±0.12.高脂血清诱导后TNF-α和IL-6的分泌显著高于空白组,而rAAV9-R65干预+高脂血清诱导后TNF-α和IL-6的浓度明显降低.Alamar blue法检测显示R65基因表达能明显减轻高脂血清刺激后HUVECs的增殖抑制.流式细胞术检测显示与空白组比较,高脂血清诱导后细胞早期凋亡比率为20.4％,晚期凋亡比率为4.7％;而rAAV9-R65转染+高脂血清诱导组早期凋亡比率降至7.2％,晚期凋亡比率降为2.9％,证明R65表达可明显减轻高脂血清刺激后HUVECs的凋亡.结论 R65基因表达能有效减轻高脂血清刺激后HUVECs的增殖抑制,并能抑制高脂血清诱导的HUVECs凋亡.
目的 通過重組9型腺相關病毒(rAAV9)載體介導R65覈酶基因對體外培養人臍靜脈內皮細胞(HUVECs)進行轉染,觀察R65覈酶基因錶達對高脂血清刺激後HUVECs增殖和凋亡的影響.方法 體外培養HUVECs,高脂血清誘導建立動脈粥樣硬化內皮損傷模型,重組rAAV9介導抗覈因子(NF)-κB覈酶基因(rAAV9-R65)腺相關病毒載體轉染HUVECs.實驗分為3組:(1)正常對照組;(2)高脂血清誘導組;(3)高脂血清誘導+病毒轉染組.通過Western blot檢測HUVECs細胞中p65覈蛋白的錶達;酶聯免疫吸附試驗(ELISA)檢測NF-κB信號通路下遊腫瘤壞死因子(TNF)-α和白細胞介素(IL)-6的變化;Alamar Blue法檢測rAAV9-R65轉染後HUVECs的體外增殖;rAAV9-R65轉染細胞96 h後流式細胞儀檢測高脂血清刺激後HUVECs凋亡率的變化.結果 高脂血清誘導HUVECs 24 h後,細胞由卵圓形鋪路石樣逐漸變成類圓形,Western blot檢測顯示p65的錶達水平分彆為:空白組:0.0229 ±0.0024;高脂血清誘導組:0.224 3±0.008 6;rAAV9-R65轉染+高脂血清誘導組:0.071 4 ±0.007 0.HUVECs細胞中高脂血清誘導後p65蛋白錶達明顯增高,而病毒轉染後p65水平降低.ELISA檢測結果顯示HUVEC分泌的TNF-α水平3組分彆為:15.98 ±0.43、27.94±0.83、19.73±0.43.而3組IL-6水平分彆為:3.94 ±0.12、6.32 ±0.21、5.08 ±0.12.高脂血清誘導後TNF-α和IL-6的分泌顯著高于空白組,而rAAV9-R65榦預+高脂血清誘導後TNF-α和IL-6的濃度明顯降低.Alamar blue法檢測顯示R65基因錶達能明顯減輕高脂血清刺激後HUVECs的增殖抑製.流式細胞術檢測顯示與空白組比較,高脂血清誘導後細胞早期凋亡比率為20.4％,晚期凋亡比率為4.7％;而rAAV9-R65轉染+高脂血清誘導組早期凋亡比率降至7.2％,晚期凋亡比率降為2.9％,證明R65錶達可明顯減輕高脂血清刺激後HUVECs的凋亡.結論 R65基因錶達能有效減輕高脂血清刺激後HUVECs的增殖抑製,併能抑製高脂血清誘導的HUVECs凋亡.
목적 통과중조9형선상관병독(rAAV9)재체개도R65핵매기인대체외배양인제정맥내피세포(HUVECs)진행전염,관찰R65핵매기인표체대고지혈청자격후HUVECs증식화조망적영향.방법 체외배양HUVECs,고지혈청유도건립동맥죽양경화내피손상모형,중조rAAV9개도항핵인자(NF)-κB핵매기인(rAAV9-R65)선상관병독재체전염HUVECs.실험분위3조:(1)정상대조조;(2)고지혈청유도조;(3)고지혈청유도+병독전염조.통과Western blot검측HUVECs세포중p65핵단백적표체;매련면역흡부시험(ELISA)검측NF-κB신호통로하유종류배사인자(TNF)-α화백세포개소(IL)-6적변화;Alamar Blue법검측rAAV9-R65전염후HUVECs적체외증식;rAAV9-R65전염세포96 h후류식세포의검측고지혈청자격후HUVECs조망솔적변화.결과 고지혈청유도HUVECs 24 h후,세포유란원형포로석양축점변성류원형,Western blot검측현시p65적표체수평분별위:공백조:0.0229 ±0.0024;고지혈청유도조:0.224 3±0.008 6;rAAV9-R65전염+고지혈청유도조:0.071 4 ±0.007 0.HUVECs세포중고지혈청유도후p65단백표체명현증고,이병독전염후p65수평강저.ELISA검측결과현시HUVEC분비적TNF-α수평3조분별위:15.98 ±0.43、27.94±0.83、19.73±0.43.이3조IL-6수평분별위:3.94 ±0.12、6.32 ±0.21、5.08 ±0.12.고지혈청유도후TNF-α화IL-6적분비현저고우공백조,이rAAV9-R65간예+고지혈청유도후TNF-α화IL-6적농도명현강저.Alamar blue법검측현시R65기인표체능명현감경고지혈청자격후HUVECs적증식억제.류식세포술검측현시여공백조비교,고지혈청유도후세포조기조망비솔위20.4％,만기조망비솔위4.7％;이rAAV9-R65전염+고지혈청유도조조기조망비솔강지7.2％,만기조망비솔강위2.9％,증명R65표체가명현감경고지혈청자격후HUVECs적조망.결론 R65기인표체능유효감경고지혈청자격후HUVECs적증식억제,병능억제고지혈청유도적HUVECs조망.
Objective To investigate the effects of type 9 recombinant adeno-associated virus (rAAV9) mediated anti-nuclear factor-κB (NF-κB) ribozyme on the proliferation and apoptosis of high lipid serum-induced human umbilical vein endothelial cells (HUVECs) in vitro,and to provide an important support that R65 nuclear enzyme gene would be an optimal gene for gene therapy in AS.Methods HUVECs were cultured in vitro,endothelial damage model of AS was established by using high lipid serum,and the HUVECs were transfected by the rAAV9-R65 recombinant adenovirus vector.Cells were divided into three experimental groups:control group,high lipid serum induced group and high lipid serum induced + rAAV9-R65 transfected group.The expression of p65 protein was detected by using Western blotting.The cell supernatant fluid was collected for determination of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) by using enzyme linked immunosorbent assay (ELISA).Proliferation of HUVECs transfected by rAAV9-R65 was detected by using Alamar blue.The apoptosis rate was detected by using flow cytometry 96 h after transfection by rAAV9-R65.Results The cells were changed from long spindle into class circle after 24 h induced by high lipid serum.Western blotting showed that the expression of p65 in control group was 0.022 9 ± 0.002 4 ; In high lipid serum induced group was 0.224 3 ± 0.008 6 ; In rAAV-R65 transfected and high lipid serum induced group was 0.071 4 ±0.007 0.The expression of p65 in control group was increased after high lipid serum stimulating HUVECs,and decreased statistically significant in rAAV-R65 transfected and high lipid serum induced group.ELISA showed that the concentrations of TNF-α in the supernatant among three groups were:15.98 ±0.43,27.94 ± 0.83 and 19.73 ± 0.43,respectively.And the concentrations of IL-6 among three groups were:3.94 ±0.12,6.32 ± 0.21 and 5.08 ± 0.12,respectively.The levels of TNF-α and IL-6 were significantly higher than control group after 24 h induced by high lipid serum,while they were decreased significantly in rAAV-R65 transfected and high lipid serum induced group.Alamar blue showed that the expression of R65 gene could significantly reduce the proliferation inhibition of HUVECs after high lipid serum stimulation.Flow cytometry analysis showed that the early apoptosis rate in high lipid serum induced group was 20.4％,and the late apoptosis ratio was 4.7％ compared with the control group.While in rAAV-R65 transfected and high lipid serum induced group it was 7.2％ of early apoptosis rate and late apoptosis ratio was 2.9％.It showed that the apoptosis rate of HUVECs was decreased by R65 gene.Conclusion The expression of R65 gene could decrease the proliferation inhibition of HUVECs after the high lipid serum induction,and could inhibit the apoptosis.These findings carry significant implications for adenovirus mediated R65 for gene therapy of AS.
