中华生物医学工程杂志
中華生物醫學工程雜誌
중화생물의학공정잡지
CHINESE JOURNAL OF BIOMEDICAL ENGINEERING
2011年
1期
25-28
,共4页
林晓圳%董颀%张弋%陈敏生
林曉圳%董頎%張弋%陳敏生
림효수%동기%장익%진민생
神经肽Y%Ca2+-钙调素依赖性蛋白激酶Ⅱ%肌细胞,心脏%心肌病,肥厚性%大鼠
神經肽Y%Ca2+-鈣調素依賴性蛋白激酶Ⅱ%肌細胞,心髒%心肌病,肥厚性%大鼠
신경태Y%Ca2+-개조소의뢰성단백격매Ⅱ%기세포,심장%심기병,비후성%대서
Neuropeptide Y%Calcium/calmodulin-dependent protein kinase Ⅱ%Myocytes,cardiac%Cardiomyopathy,hypertrophic%Rats
目的 探讨神经肽Y对大鼠心肌细胞ca2+-钙调素依赖性蛋白激酶Ⅱ(CaMK Ⅱ)的影响.方法 原代培养SD乳鼠心肌细胞,分为对照组(n=6)和神经肽Y组(n=6),神经肽Y组加入终浓度为100 nmol/L的神经肽Y,对照组不进行处理.处理15 min后采用同位素32P掺人法检测心肌细胞CaMK Ⅱ活性,免疫印迹法(Western blotting)检测磷酸化CaMK Ⅱ(p-CaMK Ⅱ)蛋白表达;处理24 h后激光共聚焦显微镜下记录心肌细胞静息状态下细胞核Ca2+-浓度,实时定量PCR检测心肌细胞β-肌球蛋白重链(β-MHC)、心钠素mRNA表达.结果 神经肽Y刺激心肌细胞15 min后,神经肽Y组CaMK Ⅱ活性较对照组明显升高(336 094.80±10 744.61比273 301.50±16 737.99,P＜0.05),P-CaMK Ⅱ蛋白表达明显增加.神经肽Y刺激心肌细胞24 h后,神经肽Y组心肌细胞胞核Ca2+-浓度较对照组明显增加(226.87±17.37比84.04±6.50,P＜0.01);实时定量PCR显示β-MHC、心钠素mRNA表达亦明显增加.结论 神经肽Y通过增加心肌细胞胞核Ca2+浓度活化CaMK Ⅱ.CaMK Ⅱ可能在神经肽Y诱导心肌细胞肥大效应中起着重要作用.
目的 探討神經肽Y對大鼠心肌細胞ca2+-鈣調素依賴性蛋白激酶Ⅱ(CaMK Ⅱ)的影響.方法 原代培養SD乳鼠心肌細胞,分為對照組(n=6)和神經肽Y組(n=6),神經肽Y組加入終濃度為100 nmol/L的神經肽Y,對照組不進行處理.處理15 min後採用同位素32P摻人法檢測心肌細胞CaMK Ⅱ活性,免疫印跡法(Western blotting)檢測燐痠化CaMK Ⅱ(p-CaMK Ⅱ)蛋白錶達;處理24 h後激光共聚焦顯微鏡下記錄心肌細胞靜息狀態下細胞覈Ca2+-濃度,實時定量PCR檢測心肌細胞β-肌毬蛋白重鏈(β-MHC)、心鈉素mRNA錶達.結果 神經肽Y刺激心肌細胞15 min後,神經肽Y組CaMK Ⅱ活性較對照組明顯升高(336 094.80±10 744.61比273 301.50±16 737.99,P＜0.05),P-CaMK Ⅱ蛋白錶達明顯增加.神經肽Y刺激心肌細胞24 h後,神經肽Y組心肌細胞胞覈Ca2+-濃度較對照組明顯增加(226.87±17.37比84.04±6.50,P＜0.01);實時定量PCR顯示β-MHC、心鈉素mRNA錶達亦明顯增加.結論 神經肽Y通過增加心肌細胞胞覈Ca2+濃度活化CaMK Ⅱ.CaMK Ⅱ可能在神經肽Y誘導心肌細胞肥大效應中起著重要作用.
목적 탐토신경태Y대대서심기세포ca2+-개조소의뢰성단백격매Ⅱ(CaMK Ⅱ)적영향.방법 원대배양SD유서심기세포,분위대조조(n=6)화신경태Y조(n=6),신경태Y조가입종농도위100 nmol/L적신경태Y,대조조불진행처리.처리15 min후채용동위소32P참인법검측심기세포CaMK Ⅱ활성,면역인적법(Western blotting)검측린산화CaMK Ⅱ(p-CaMK Ⅱ)단백표체;처리24 h후격광공취초현미경하기록심기세포정식상태하세포핵Ca2+-농도,실시정량PCR검측심기세포β-기구단백중련(β-MHC)、심납소mRNA표체.결과 신경태Y자격심기세포15 min후,신경태Y조CaMK Ⅱ활성교대조조명현승고(336 094.80±10 744.61비273 301.50±16 737.99,P＜0.05),P-CaMK Ⅱ단백표체명현증가.신경태Y자격심기세포24 h후,신경태Y조심기세포포핵Ca2+-농도교대조조명현증가(226.87±17.37비84.04±6.50,P＜0.01);실시정량PCR현시β-MHC、심납소mRNA표체역명현증가.결론 신경태Y통과증가심기세포포핵Ca2+농도활화CaMK Ⅱ.CaMK Ⅱ가능재신경태Y유도심기세포비대효응중기착중요작용.
Objective To explore the effect of neuropeptide Y (NPY) on calcium/calmodulindependent protein kinase Ⅱ (CaMK Ⅱ ) in rat cardiomyocytes.Methods The cardiomyocytes from neonatal Sprague-Dawley (SD) rats were primarily cultured and divided into the control and NPY groups (n=6 each).The NPY group was added with 100 nmol/L NPY (final concentration), while the control group was left untreated.After 15 min, the activity of CaMK Ⅱ was detected by isotope 32P incorporation assay, and the expression of phospho - CaMK Ⅱ (p - CaMK Ⅱ ) protein by Western blotting.After 24 h, intranuclear concentration of Ca2+ in resting cardiomyocytes was recorded under laser scanning confocal microscope (LSCM).In addition, real-time PCR was used to detect the expressions of P-myosin heavy chain (β-MHC)mRNA and atrial natriuretic factor (ANF) mRNA in cardiomyocytes.Results After NPY stimulation for 15 min, the NPY group showed significantly elevated activity of CaMK Ⅱ (336 094.80±0 744.61 vs 273 301.50±±16 737..99, P＜0.05) and increased expression of p-CaMK Ⅱ protein in cardiomyocytes, compared with the control group.After 24 h, the NYP group showed a significant increase in intranuclear Ca2+ concentration compared wiith the control group (226.87±17.37 vs 84.04±6.50,P＜0.01).Moreover, real-time PCR also showed signficant increase in expressions of β- MHC and ANF mRNA.Conclusion The CaMK Ⅱ is activated with the increase of intranuclear Ca2+ concentration by NPY, which may play a major role in NYP-induced cardiomyocyte hypertrophy.
