中华烧伤杂志
中華燒傷雜誌
중화소상잡지
16
2011年
2期
156-160
,共5页
孙荣距%汪琦瑛%张建波%果应菲%赵晓东
孫榮距%汪琦瑛%張建波%果應菲%趙曉東
손영거%왕기영%장건파%과응비%조효동
烧伤%内皮细胞%细胞增殖%细胞凋亡%La核糖核蛋白6%钙/钙调蛋白依赖性丝氨酸蛋白激酶
燒傷%內皮細胞%細胞增殖%細胞凋亡%La覈糖覈蛋白6%鈣/鈣調蛋白依賴性絲氨痠蛋白激酶
소상%내피세포%세포증식%세포조망%La핵당핵단백6%개/개조단백의뢰성사안산단백격매
Burns%Endothelial cells%Cell proliferation%Apoptosis%Acheron%Calcium/calmodulin-dependent serine protein kinase
目的 研究La核糖核蛋白6(Achn)在人血管内皮细胞增殖和凋亡中的调节作用.方法 (1)DMEM无血清培养基培养人血管内皮细胞株Eahy926细胞,按随机数字表法(下同)分为Achn抑制组(转染Achn抑制表达载体psi-Achn)、psi4.1空载体组(转染psi4.1)、Achn诱导组(转染Achn诱导表达载体pcDNA-Achn)、pcDNA3.1空载体组(转染pcDNA3.1)、Achn与钙/钙调蛋白依赖性丝氨酸蛋白激酶(CASK)共转染组(转染pcDNA-Achn与CASK抑制表达载体psi-CASK)、空白对照组(PBS处理),分别于转染后1、24、48、72 h用噻唑蓝法测定各组细胞570 nm波长下的吸光度值.(2)取Eahy926细胞,裂解细胞总蛋白,二辛丁酸法定量后分为蛋白质印迹组(总蛋白量为20μg)、Achn蛋白沉淀组、CASK蛋白沉淀组、IgG对照组,后3组细胞蛋白总量各为100μg,免疫共沉淀法检测各组Achn、CASK蛋白水平.(3)取Eahy926细胞分为LPS组(5 mol/L LPS处理)、氯化钾组(5 mol/L氯化钾处理)、空白对照组(5 mol/L PBS处理)、Achn诱导转染组(转染pcDNA-Achn)、Achn与CASK共转染组(转染pcDNA-Achn与psi-CASK),转染组转染24 h后加入LPS刺激12 h,免疫组织化学法检测各组半胱氨酸天冬氨酸蛋白酶3(caspase-3)蛋白表达.(4)取Eahy926细胞分为Achn诱导组(转染pcDNA-Achn)、Achn抑制组(转染psi-Achn)、对照组(PBS处理),24 h后加入烧伤患者血清处理12 h,流式细胞仪检测各组细胞凋亡率.对实验数据行t检验和单因素方差分析.结果 (1)Achn抑制组细胞增殖水平从24 h开始低于psi4.1空载体组,48、72 h时差异均有统计学意义(t值分别为10.777、6.112,P值均小于0.05);转染后24、48、72 h Achn诱导组细胞增殖水平均显著高于pcDNA3.1空载体组(t值分别为5.367、6.053、9.831,P值均小于0.05);Achn与CASK共转染组细胞增殖水平48、72 h均显著低于Achn诱导组(t值分别为5.481、9.517,P值均小于0.05).(2)CASK蛋白沉淀组CASK抗体沉淀物中可同时检测到CASK蛋白和Achn蛋白,而Achn蛋白沉淀组Achn抗体沉淀物中可同时检测到Achn蛋白和CASK蛋白.(3)Achn诱导转染组血管内皮细胞caspase-3阳性表达率为(15.6±0.5)%,低于LPS组[(32.8±2.6)%,t=10.083,P＜0.05];Achn与CASK共转染组caspase-3阳性细胞表达率[(7.0±2.0)%]进一步降低,显著低于LPS组(t=9.827,P＜0.01).(4)Achn抑制组细胞凋亡率为(45.6±10.9)%,显著高于对照组的(13.2±4.3)%,t=7.043,P＜0.05;Achn诱导组的细胞凋亡率为(5.3±2.9)%,显著低于对照组(t=6.499,P＜0.05).结论Achn能促进入血管内皮细胞增殖,抑制LPS或烧伤血清诱导细胞凋亡并与CASK的作用相关联.
目的 研究La覈糖覈蛋白6(Achn)在人血管內皮細胞增殖和凋亡中的調節作用.方法 (1)DMEM無血清培養基培養人血管內皮細胞株Eahy926細胞,按隨機數字錶法(下同)分為Achn抑製組(轉染Achn抑製錶達載體psi-Achn)、psi4.1空載體組(轉染psi4.1)、Achn誘導組(轉染Achn誘導錶達載體pcDNA-Achn)、pcDNA3.1空載體組(轉染pcDNA3.1)、Achn與鈣/鈣調蛋白依賴性絲氨痠蛋白激酶(CASK)共轉染組(轉染pcDNA-Achn與CASK抑製錶達載體psi-CASK)、空白對照組(PBS處理),分彆于轉染後1、24、48、72 h用噻唑藍法測定各組細胞570 nm波長下的吸光度值.(2)取Eahy926細胞,裂解細胞總蛋白,二辛丁痠法定量後分為蛋白質印跡組(總蛋白量為20μg)、Achn蛋白沉澱組、CASK蛋白沉澱組、IgG對照組,後3組細胞蛋白總量各為100μg,免疫共沉澱法檢測各組Achn、CASK蛋白水平.(3)取Eahy926細胞分為LPS組(5 mol/L LPS處理)、氯化鉀組(5 mol/L氯化鉀處理)、空白對照組(5 mol/L PBS處理)、Achn誘導轉染組(轉染pcDNA-Achn)、Achn與CASK共轉染組(轉染pcDNA-Achn與psi-CASK),轉染組轉染24 h後加入LPS刺激12 h,免疫組織化學法檢測各組半胱氨痠天鼕氨痠蛋白酶3(caspase-3)蛋白錶達.(4)取Eahy926細胞分為Achn誘導組(轉染pcDNA-Achn)、Achn抑製組(轉染psi-Achn)、對照組(PBS處理),24 h後加入燒傷患者血清處理12 h,流式細胞儀檢測各組細胞凋亡率.對實驗數據行t檢驗和單因素方差分析.結果 (1)Achn抑製組細胞增殖水平從24 h開始低于psi4.1空載體組,48、72 h時差異均有統計學意義(t值分彆為10.777、6.112,P值均小于0.05);轉染後24、48、72 h Achn誘導組細胞增殖水平均顯著高于pcDNA3.1空載體組(t值分彆為5.367、6.053、9.831,P值均小于0.05);Achn與CASK共轉染組細胞增殖水平48、72 h均顯著低于Achn誘導組(t值分彆為5.481、9.517,P值均小于0.05).(2)CASK蛋白沉澱組CASK抗體沉澱物中可同時檢測到CASK蛋白和Achn蛋白,而Achn蛋白沉澱組Achn抗體沉澱物中可同時檢測到Achn蛋白和CASK蛋白.(3)Achn誘導轉染組血管內皮細胞caspase-3暘性錶達率為(15.6±0.5)%,低于LPS組[(32.8±2.6)%,t=10.083,P＜0.05];Achn與CASK共轉染組caspase-3暘性細胞錶達率[(7.0±2.0)%]進一步降低,顯著低于LPS組(t=9.827,P＜0.01).(4)Achn抑製組細胞凋亡率為(45.6±10.9)%,顯著高于對照組的(13.2±4.3)%,t=7.043,P＜0.05;Achn誘導組的細胞凋亡率為(5.3±2.9)%,顯著低于對照組(t=6.499,P＜0.05).結論Achn能促進入血管內皮細胞增殖,抑製LPS或燒傷血清誘導細胞凋亡併與CASK的作用相關聯.
목적 연구La핵당핵단백6(Achn)재인혈관내피세포증식화조망중적조절작용.방법 (1)DMEM무혈청배양기배양인혈관내피세포주Eahy926세포,안수궤수자표법(하동)분위Achn억제조(전염Achn억제표체재체psi-Achn)、psi4.1공재체조(전염psi4.1)、Achn유도조(전염Achn유도표체재체pcDNA-Achn)、pcDNA3.1공재체조(전염pcDNA3.1)、Achn여개/개조단백의뢰성사안산단백격매(CASK)공전염조(전염pcDNA-Achn여CASK억제표체재체psi-CASK)、공백대조조(PBS처리),분별우전염후1、24、48、72 h용새서람법측정각조세포570 nm파장하적흡광도치.(2)취Eahy926세포,렬해세포총단백,이신정산법정량후분위단백질인적조(총단백량위20μg)、Achn단백침정조、CASK단백침정조、IgG대조조,후3조세포단백총량각위100μg,면역공침정법검측각조Achn、CASK단백수평.(3)취Eahy926세포분위LPS조(5 mol/L LPS처리)、록화갑조(5 mol/L록화갑처리)、공백대조조(5 mol/L PBS처리)、Achn유도전염조(전염pcDNA-Achn)、Achn여CASK공전염조(전염pcDNA-Achn여psi-CASK),전염조전염24 h후가입LPS자격12 h,면역조직화학법검측각조반광안산천동안산단백매3(caspase-3)단백표체.(4)취Eahy926세포분위Achn유도조(전염pcDNA-Achn)、Achn억제조(전염psi-Achn)、대조조(PBS처리),24 h후가입소상환자혈청처리12 h,류식세포의검측각조세포조망솔.대실험수거행t검험화단인소방차분석.결과 (1)Achn억제조세포증식수평종24 h개시저우psi4.1공재체조,48、72 h시차이균유통계학의의(t치분별위10.777、6.112,P치균소우0.05);전염후24、48、72 h Achn유도조세포증식수평균현저고우pcDNA3.1공재체조(t치분별위5.367、6.053、9.831,P치균소우0.05);Achn여CASK공전염조세포증식수평48、72 h균현저저우Achn유도조(t치분별위5.481、9.517,P치균소우0.05).(2)CASK단백침정조CASK항체침정물중가동시검측도CASK단백화Achn단백,이Achn단백침정조Achn항체침정물중가동시검측도Achn단백화CASK단백.(3)Achn유도전염조혈관내피세포caspase-3양성표체솔위(15.6±0.5)%,저우LPS조[(32.8±2.6)%,t=10.083,P＜0.05];Achn여CASK공전염조caspase-3양성세포표체솔[(7.0±2.0)%]진일보강저,현저저우LPS조(t=9.827,P＜0.01).(4)Achn억제조세포조망솔위(45.6±10.9)%,현저고우대조조적(13.2±4.3)%,t=7.043,P＜0.05;Achn유도조적세포조망솔위(5.3±2.9)%,현저저우대조조(t=6.499,P＜0.05).결론Achn능촉진입혈관내피세포증식,억제LPS혹소상혈청유도세포조망병여CASK적작용상관련.
Objective To investigate regulatory effect of Acheron (Achn) on proliferation and apoptosis of human vascular endothelial cell. Methods ( 1 ) Eahy926 cells were cultured in serum-free DMEM medium (96-well plates) and were divided into Achn inhibition group (transfected with plasmid psi-Achn), psi4.1 group (transfected with psi4. 1 empty vector), Achn induction group (transfected with pcDNA-Achn), pcDNA3.1 group (transfected with pcDNA3.1 empty vector), cotransfection group [cotransfected with pcDNA-Achn + psi-calcium/calmodulin-dependent serine protein kinase (CASK)] , blank control group (treated with PBS) according to the random number table (the same method below). The cell proliferation was determined by MTT assay at post transfection hour (PTH) 1, 24, 48, 72, with expression of absorbance value. (2) Total protein of Eahy926 cells were extracted and quantitated by BCA assay, and then they were divided into Achn antibody precipitation group (100 μg protein) , CASK antibody precipitation group ( 100 μg protein), IgG antibody group ( 100 μg protein), Western blot group (20 μg protein).Achn and CASK protein levels were determined by immunoprecipitation and Western blot. (3) Synchronously cultured Eahy926 cells were divided into LPS induction group (treated with 5 mol/L LPS), Achn transfection group (transfected with pcDNA-Achn), cotransfection group (cotransfected with psi-CASK and pcDNA-Achn) , KCl group (treated with 5 mol/L KCl), and blank control group (treated with 5 mol/LPBS). Cells in transfection groups were stimulated by LPS for 12 hours after PTH 24. Caspase-3 protein level was detected by immunohistochemistry. (4) Synchronously cultured Eahy926 cells were divided into Achn inhibition group (transfected with psi-Achn vector), Achn induction group ( transfected with pcDNA-Achn vector), and blank control group ( treated with PBS). Apoptosis rate was determined by FITC/PI with flow cytometry. Data were processed with one-way analysis of variance and t test. Results ( 1 ) The cell proliferation in Achn inhibition group was lower than that in psi4.1 group from PTH 24, and the differences were statistically significant at PTH 48, 72 (with t value respectively 10. 777, 6.112, P values all below 0. 05 ).The cell proliferation in Achn induction group during PTH 24-72 were higher that in pcDNA3. 1 group (with t value respectively 5. 367, 6. 053, 9. 831, P values all below 0.05 ). The cell proliferation in cotransfection group at PTH 48, 72 were significantly lower than that in Achn induction group ( with t value respectively 5.481, 9. 517, P values all below 0. 05). (2) Achn protein was detected in CASK antibody precipitation group while CASK protein was also detected in Achn antibody precipitation group. (3) Caspase-3 level in Achn transfection group was lower [( 15.6 ± 0. 5 ) %] as compared with that in LPS induction group [(32. 8 ±2.6)%, t = 10. 083, P ＜ 0. 05], and that in cotransfection group showed further inhibition [(7.0 ±2.0)%,t =9.827, P ＜0.01]. (4) Apoptosis rate in Achn inhibition group[(45.6 ± 10.9)%] was higher than that in blank control group [(13.2±4.3) %, t =7.043, P ＜0.05]; while that in Achn inductiongroup [(5.3 ±2.9)%] was lower than that in blank control group ( t =6.499, P ＜0.05).Conclusions Achn can promote human vascular endothelial cell proliferation, and inhibit its apoptosis induced by LPS or burn serum, and the effect is related to CASK.
