中华结核和呼吸杂志
中華結覈和呼吸雜誌
중화결핵화호흡잡지
Chinese Journal of Tuberculosis and Respiratory Diseases
2010年
7期
524-529
,共6页
白晶%刘先胜%徐永健%张珍祥%谢敏%倪望
白晶%劉先勝%徐永健%張珍祥%謝敏%倪望
백정%류선성%서영건%장진상%사민%예망
细胞外信号调节激酶类%肌细胞,平滑肌%寡核苷酸类,反义%哮喘
細胞外信號調節激酶類%肌細胞,平滑肌%寡覈苷痠類,反義%哮喘
세포외신호조절격매류%기세포,평활기%과핵감산류,반의%효천
Extracellular signal regulated kinases%Myocytes,smooth muscle%Oligonucleotides,antisense%Asthma
目的 观察细胞外信号调节激酶(ERK)反义寡核苷酸(ODNs)对支气管哮喘(简称哮喘)血清被动致敏的人气道平滑肌细胞(HASMCs)增殖和凋亡的影响.方法 用10%哮喘患者血清被动致敏HASMCs(空白对照组),并用脂质体将反义(AODNs组)、正义(SODNs组)及错配ERKODNs(RODNs组)导入HASMCs,以10%非哮喘患者血清为对照(对照血清组).采用流式细胞仪、四甲基偶氮唑盐(MTT)法、3H-TdR掺入法及增殖细胞核抗原(PCNA)免疫荧光技术检测HASMCs的增殖,原位末端标记法和Annexin-V FITC PI双染色法检测细胞凋亡,RT-PCR和Western blot检测ERKmRNA和ERK1/2、磷酸化ERK1/2蛋白的表达.结果 用ERK ODNs干预经哮喘血清致敏的HASMCs后,AODNs组S+G2/M期细胞所占比例、吸光度(A490)值、细胞DNA合成量和PCNA蛋白表达量[分别为(14.21±1.21)%、(0.271±0.021)、(2811±182)cpm/106和(5.25±0.60)]均低于空白对照组[分别为(22.48±2.04)%、0.507±0.090、(3869±396)cpm/106和11.25±1.21](F值分别为65.594、39.676、61.111、120.321,均P<0.01),AODNs组的凋亡指数和早期凋亡细胞百分率[分别为13.96±1.72和(9.17±0.47)%]均高于空白对照组[分别为5.37±0.05和(3.26±0.04)%],差异有统计学意义(F值分别为98.181和65.444,均P<0.01),AODNs组的ERK mRNA和ERK活化率[分别为0.43±0.06和(63±6)%]均低于空白对照组[分别为0.89±0.09和(87±8)%](F值分别为78.043和87.288,均P<0.01).而正义和错配ERK ODNs没有上述作用.结论 反义ERK可通过抑制ERK mRNA表达和翻译抑制哮喘血清被动致敏的HASMCs的增殖,促进其凋亡,ERK信号通道可能对哮喘患者HASMCs增殖与凋亡具有重要的调控作用.
目的 觀察細胞外信號調節激酶(ERK)反義寡覈苷痠(ODNs)對支氣管哮喘(簡稱哮喘)血清被動緻敏的人氣道平滑肌細胞(HASMCs)增殖和凋亡的影響.方法 用10%哮喘患者血清被動緻敏HASMCs(空白對照組),併用脂質體將反義(AODNs組)、正義(SODNs組)及錯配ERKODNs(RODNs組)導入HASMCs,以10%非哮喘患者血清為對照(對照血清組).採用流式細胞儀、四甲基偶氮唑鹽(MTT)法、3H-TdR摻入法及增殖細胞覈抗原(PCNA)免疫熒光技術檢測HASMCs的增殖,原位末耑標記法和Annexin-V FITC PI雙染色法檢測細胞凋亡,RT-PCR和Western blot檢測ERKmRNA和ERK1/2、燐痠化ERK1/2蛋白的錶達.結果 用ERK ODNs榦預經哮喘血清緻敏的HASMCs後,AODNs組S+G2/M期細胞所佔比例、吸光度(A490)值、細胞DNA閤成量和PCNA蛋白錶達量[分彆為(14.21±1.21)%、(0.271±0.021)、(2811±182)cpm/106和(5.25±0.60)]均低于空白對照組[分彆為(22.48±2.04)%、0.507±0.090、(3869±396)cpm/106和11.25±1.21](F值分彆為65.594、39.676、61.111、120.321,均P<0.01),AODNs組的凋亡指數和早期凋亡細胞百分率[分彆為13.96±1.72和(9.17±0.47)%]均高于空白對照組[分彆為5.37±0.05和(3.26±0.04)%],差異有統計學意義(F值分彆為98.181和65.444,均P<0.01),AODNs組的ERK mRNA和ERK活化率[分彆為0.43±0.06和(63±6)%]均低于空白對照組[分彆為0.89±0.09和(87±8)%](F值分彆為78.043和87.288,均P<0.01).而正義和錯配ERK ODNs沒有上述作用.結論 反義ERK可通過抑製ERK mRNA錶達和翻譯抑製哮喘血清被動緻敏的HASMCs的增殖,促進其凋亡,ERK信號通道可能對哮喘患者HASMCs增殖與凋亡具有重要的調控作用.
목적 관찰세포외신호조절격매(ERK)반의과핵감산(ODNs)대지기관효천(간칭효천)혈청피동치민적인기도평활기세포(HASMCs)증식화조망적영향.방법 용10%효천환자혈청피동치민HASMCs(공백대조조),병용지질체장반의(AODNs조)、정의(SODNs조)급착배ERKODNs(RODNs조)도입HASMCs,이10%비효천환자혈청위대조(대조혈청조).채용류식세포의、사갑기우담서염(MTT)법、3H-TdR참입법급증식세포핵항원(PCNA)면역형광기술검측HASMCs적증식,원위말단표기법화Annexin-V FITC PI쌍염색법검측세포조망,RT-PCR화Western blot검측ERKmRNA화ERK1/2、린산화ERK1/2단백적표체.결과 용ERK ODNs간예경효천혈청치민적HASMCs후,AODNs조S+G2/M기세포소점비례、흡광도(A490)치、세포DNA합성량화PCNA단백표체량[분별위(14.21±1.21)%、(0.271±0.021)、(2811±182)cpm/106화(5.25±0.60)]균저우공백대조조[분별위(22.48±2.04)%、0.507±0.090、(3869±396)cpm/106화11.25±1.21](F치분별위65.594、39.676、61.111、120.321,균P<0.01),AODNs조적조망지수화조기조망세포백분솔[분별위13.96±1.72화(9.17±0.47)%]균고우공백대조조[분별위5.37±0.05화(3.26±0.04)%],차이유통계학의의(F치분별위98.181화65.444,균P<0.01),AODNs조적ERK mRNA화ERK활화솔[분별위0.43±0.06화(63±6)%]균저우공백대조조[분별위0.89±0.09화(87±8)%](F치분별위78.043화87.288,균P<0.01).이정의화착배ERK ODNs몰유상술작용.결론 반의ERK가통과억제ERK mRNA표체화번역억제효천혈청피동치민적HASMCs적증식,촉진기조망,ERK신호통도가능대효천환자HASMCs증식여조망구유중요적조공작용.
Objective Airway smooth muscle (ASM) cell proliferation is a key feature of airway remodeling in asthma. The extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway is one of the most important transduction pathways involved in the proliferation of ASM of asthmatic rats. However, its role in the human ASMCs proliferation remains unclear. Methods HASMCs were cultured in vitro and passively sensitized with 10% serum from asthmatic patients, and non-asthmatic human serum treated HASMCs served as the control. Then, HASMCs were transfected with ERK sense, antisense and mismatched oligodeoxynucleotides(ODN). The proliferations of HASMCs were detected by flow cytometry analysis, MTT colorimetric assay, [3H] thymidine incorporation and proliferating cell nuclear antigen ( PCNA) in immunofluorescence staining respectively. The apoptosis of HASMCs were detected by in situ end labeling and Annexin-V FITC PI double staining. The expressions of ERK mRNA, ERK protein and phosphorylation of ERK1/2(p-ERK1/2) protein were measured by RT-PCR and Western blotting, respectively. Results The percentage of S + G2/M phase, absorbance ( A490) value, DNA synthesis value and the expression of PCNA protein in HASMCs passively sensitized with 10% serum from asthmatic patients were (22. 48 ± 2. 04)% ,(0.507 ±0.090) ,(3869 ±396) cpm/106cells,(11. 25 ±1. 21), respectively. After treated with ERK oligodeoxynucleotides, these measurements were decreased to(14.21 ± 1. 21)% , (0. 271 ±0.021), (2811 ±182) cpm/106 cells and (5.25 ±0.60), respectively ( F = 65. 594, 39. 676,61. 111, 120. 321, respectively, P < 0. 05 ). The apoptotic index (13. 96 ± 1. 72) and the percentage of the early apoptotic cells (9. 17 ±0. 47)% in HASMCs from group AODNs were significantly increased compared to those of chronic asthma group, which were (5. 37 ± 0. 05 ), (3. 26 ± 0. 04 )% , respectively ( F = 98. 181, 65.444, respectively, P<0. 05). The expression of ERK mRNA (0.43 ±0.06) and the activation ratio of ERK (63 ±6) % in HASMCs from group AODNs were significantly decreased compared to those of chronic asthma group, which were (0. 89 ± 0. 09), (87 ± 8) % , respectively ( F = 78.043,87. 288, respectively, P < 0. 05 ). ERK antisense ODN inhibited the proliferation of HASMCs and induced the apoptosis of HASMCs, but the sense and the mismatched ones did not have these effects. Conclusions ERK antisense ODN inhibited the proliferation and increased the apoptosis in cultured HASMCs passively sensitized with 10% serum from asthmatic patients. The result suggests that ERK signaling pathway may contribute to the proliferation and apoptosis of HASMCs in asthmatic patients.
