中华围产医学杂志
中華圍產醫學雜誌
중화위산의학잡지
CHINESE JOURNAL OF PERINATAL MEDICINE
2011年
11期
682-687
,共6页
董微%许群星%韩玉环%尹利荣%张立军%华绍芳
董微%許群星%韓玉環%尹利榮%張立軍%華紹芳
동미%허군성%한옥배%윤리영%장립군%화소방
受体%细胞表面%抗原%CD%脐静脉%内皮细胞%一氧化氮%一氧化氮合酶
受體%細胞錶麵%抗原%CD%臍靜脈%內皮細胞%一氧化氮%一氧化氮閤酶
수체%세포표면%항원%CD%제정맥%내피세포%일양화담%일양화담합매
Receptors%cell surface%Antigens%CD%Umbilical veins%Endothelial cells%Nitricoxide%Nitric oxide synthase
目的 观察可溶性endoglin(soluble endoglin,sEng)对体外培养的人脐静脉内皮细胞产生一氧化氮(nitric oxide,NO)、内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)及其1177位点丝氨酸磷酸化程度的影响. 方法 将3代以内的人脐静脉内皮细胞接种于96孔培养板,分别加入完全细胞培养液(对照组)和不同浓度的sEng(1、10、100 μg/L),作用6、12和24 h后收取细胞培养液及细胞,硝酸酶还原法测定细胞培养液中NO代谢产物亚硝酸盐浓度反映NO含量,Western印记法检测各组细胞eNOS的相对表达量及其1177位点丝氨酸磷酸化情况,实时荧光聚合酶链反应技术检测各组细胞eNOS mRNA的相对表达量.采用方差分析、LSD法及Pearson相关分析法对各组进行比较. 结果 (1)1、10、100μg/L sEng作用6h,细胞培养液中NO浓度分别为(59.25±1.63)、(41.08±2.71)和(30.38±1.63)μmol/L;作用12 h,细胞培养液中NO浓度分别为(54.98±3.34)、(35.00±8.60)和(19.82±3.75)μmol/L；作用24 h,细胞培养液中NO浓度分别为(46.14±4.93)、(30.24±2.08)和(12.78±5.01)μmol/L,而对照组NO浓度随着时间的推移无明显改变(F=2.30,P=0.14).与sEng共培养后,细胞培养液中NO浓度明显降低,并与sEng作用时间(r=0.98,P＜0.05)及浓度(r=-0.88,P＜0.05)呈明显的负相关.(2)1、10、100 μg/L sEng作用6 h eNOS相对表达量分别为0.71±0.00、0.47±0.00和0.32±0.00；作用12 h,eNOS相对表达量分别为0.58±0.00、0.42±0.00和0.25±0.00；作用24 h,eNOS相对表达量分别为0.49±0.00、0.33±0.00和0.18±0.00.而对照组eNOS相对表达量及1177位点丝氨酸磷酸化eNOS吸光度/总eNOS吸光度随时间推移无明显改变(F分别为3.59和0.37,P分别为0.09和0.80).与sEng共培养后,细胞中eNOS蛋白表达量较对照组明显下降,其1177位点丝氨酸磷酸化程度明显减弱,与sEng作用时间(r分别为-0.98和-0.96,P均＜0.05)及浓度(r分别为-0.76和-0.79,P均＜0.05)呈明显负相关.(3)与sEng共培养后,细胞培养液中eNOS mRNA的表达量明显下降,亦与sEng作用时间(r=-0.51,P＜0.05)及浓度(r=-0.82,P＜0.05)呈明显的负相关. 结论 sEng 可能通过抑制eNOS 1177位点丝氨酸磷酸化,导致eNOS激活被抑制,NO生成减少,引起血管舒缩失调.
目的 觀察可溶性endoglin(soluble endoglin,sEng)對體外培養的人臍靜脈內皮細胞產生一氧化氮(nitric oxide,NO)、內皮型一氧化氮閤酶(endothelial nitric oxide synthase,eNOS)及其1177位點絲氨痠燐痠化程度的影響. 方法 將3代以內的人臍靜脈內皮細胞接種于96孔培養闆,分彆加入完全細胞培養液(對照組)和不同濃度的sEng(1、10、100 μg/L),作用6、12和24 h後收取細胞培養液及細胞,硝痠酶還原法測定細胞培養液中NO代謝產物亞硝痠鹽濃度反映NO含量,Western印記法檢測各組細胞eNOS的相對錶達量及其1177位點絲氨痠燐痠化情況,實時熒光聚閤酶鏈反應技術檢測各組細胞eNOS mRNA的相對錶達量.採用方差分析、LSD法及Pearson相關分析法對各組進行比較. 結果 (1)1、10、100μg/L sEng作用6h,細胞培養液中NO濃度分彆為(59.25±1.63)、(41.08±2.71)和(30.38±1.63)μmol/L;作用12 h,細胞培養液中NO濃度分彆為(54.98±3.34)、(35.00±8.60)和(19.82±3.75)μmol/L；作用24 h,細胞培養液中NO濃度分彆為(46.14±4.93)、(30.24±2.08)和(12.78±5.01)μmol/L,而對照組NO濃度隨著時間的推移無明顯改變(F=2.30,P=0.14).與sEng共培養後,細胞培養液中NO濃度明顯降低,併與sEng作用時間(r=0.98,P＜0.05)及濃度(r=-0.88,P＜0.05)呈明顯的負相關.(2)1、10、100 μg/L sEng作用6 h eNOS相對錶達量分彆為0.71±0.00、0.47±0.00和0.32±0.00；作用12 h,eNOS相對錶達量分彆為0.58±0.00、0.42±0.00和0.25±0.00；作用24 h,eNOS相對錶達量分彆為0.49±0.00、0.33±0.00和0.18±0.00.而對照組eNOS相對錶達量及1177位點絲氨痠燐痠化eNOS吸光度/總eNOS吸光度隨時間推移無明顯改變(F分彆為3.59和0.37,P分彆為0.09和0.80).與sEng共培養後,細胞中eNOS蛋白錶達量較對照組明顯下降,其1177位點絲氨痠燐痠化程度明顯減弱,與sEng作用時間(r分彆為-0.98和-0.96,P均＜0.05)及濃度(r分彆為-0.76和-0.79,P均＜0.05)呈明顯負相關.(3)與sEng共培養後,細胞培養液中eNOS mRNA的錶達量明顯下降,亦與sEng作用時間(r=-0.51,P＜0.05)及濃度(r=-0.82,P＜0.05)呈明顯的負相關. 結論 sEng 可能通過抑製eNOS 1177位點絲氨痠燐痠化,導緻eNOS激活被抑製,NO生成減少,引起血管舒縮失調.
목적 관찰가용성endoglin(soluble endoglin,sEng)대체외배양적인제정맥내피세포산생일양화담(nitric oxide,NO)、내피형일양화담합매(endothelial nitric oxide synthase,eNOS)급기1177위점사안산린산화정도적영향. 방법 장3대이내적인제정맥내피세포접충우96공배양판,분별가입완전세포배양액(대조조)화불동농도적sEng(1、10、100 μg/L),작용6、12화24 h후수취세포배양액급세포,초산매환원법측정세포배양액중NO대사산물아초산염농도반영NO함량,Western인기법검측각조세포eNOS적상대표체량급기1177위점사안산린산화정황,실시형광취합매련반응기술검측각조세포eNOS mRNA적상대표체량.채용방차분석、LSD법급Pearson상관분석법대각조진행비교. 결과 (1)1、10、100μg/L sEng작용6h,세포배양액중NO농도분별위(59.25±1.63)、(41.08±2.71)화(30.38±1.63)μmol/L;작용12 h,세포배양액중NO농도분별위(54.98±3.34)、(35.00±8.60)화(19.82±3.75)μmol/L；작용24 h,세포배양액중NO농도분별위(46.14±4.93)、(30.24±2.08)화(12.78±5.01)μmol/L,이대조조NO농도수착시간적추이무명현개변(F=2.30,P=0.14).여sEng공배양후,세포배양액중NO농도명현강저,병여sEng작용시간(r=0.98,P＜0.05)급농도(r=-0.88,P＜0.05)정명현적부상관.(2)1、10、100 μg/L sEng작용6 h eNOS상대표체량분별위0.71±0.00、0.47±0.00화0.32±0.00；작용12 h,eNOS상대표체량분별위0.58±0.00、0.42±0.00화0.25±0.00；작용24 h,eNOS상대표체량분별위0.49±0.00、0.33±0.00화0.18±0.00.이대조조eNOS상대표체량급1177위점사안산린산화eNOS흡광도/총eNOS흡광도수시간추이무명현개변(F분별위3.59화0.37,P분별위0.09화0.80).여sEng공배양후,세포중eNOS단백표체량교대조조명현하강,기1177위점사안산린산화정도명현감약,여sEng작용시간(r분별위-0.98화-0.96,P균＜0.05)급농도(r분별위-0.76화-0.79,P균＜0.05)정명현부상관.(3)여sEng공배양후,세포배양액중eNOS mRNA적표체량명현하강,역여sEng작용시간(r=-0.51,P＜0.05)급농도(r=-0.82,P＜0.05)정명현적부상관. 결론 sEng 가능통과억제eNOS 1177위점사안산린산화,도치eNOS격활피억제,NO생성감소,인기혈관서축실조.
Objective To investigate the effects of soluble endoglin(sEng)on nitric oxide (NO)production and endothelial nitric oxide synthase(eNOS)phosphorylation in cultured human umbilical vein endothelial cells.Methods Human umbilical vein endothelial cells within 3 passages seeded in culture plates of 96 wells,were stimulated by total culture medium(control group)or sEng (1,10 and 100 μg/L)respectively.Cells and medium were collected after cells were cultured for 6,12 and 24 hours respectively.The concentration of the metabolites of NO in each group was measured by nitrate reductase method.The expression of eNOS and eNOS-Ser(p)1177 were detected by Western blot.The expression of eNOS mRNA in each group was detected by real-time fluorescence reverse transcription-polymerase chain reaction.Analysis of variance,LSD method and pearson correlation were used to compare the difference between groups.Results(1)The concentration of the metabolites of NO in 1,10 and 100μg/L sEng groups was(59.25±1.63),(41.08±2.71)and (30.38±1.63)μmol/L respectively after cultured for 6 hours;(54.98±3.34),(35.00±8.60)and (19.82±3.75)μmol/L for 12 hours; and(46.14±4.93),(30.24±2.08)and(12.78±5.01)μmol/L for 24 hours.There was no significant changes in control group with time going by(F=2.30,P=0.14).The concentration of the metabolites of NO was significantly lower in sEng group,and which had negative correlation with culture time(r=-0.98,P＜0.05)and dose(r=-0.88,P＜0.05).(2)The expression of eNOS in 1,10,100 μg/L sEng groups was 0.71 ± 0.00,0.47 ± 0.00 and 0.32±0.00 after cultured for 6 hours; 0.58±0.00,0.42±0.00 and 0.25±0.00 for 12 hours; and 0.49±0.00,0.33±0.00 and 0.18±0.00 for 24 hours.While the expression of eNOS and eNOS-Ser (p)1177/eNOS had no significant changes in control group with time going by(F=3.59 and 0.37,P=0.09 and 0.80).The expression of eNOS protein and eNOS-Ser(p)1177 decreased significantly in sEng groups,which had negative correlation with culture time(r=0.98 and-0.96,P＜0.05)and dose(r=-0.76 and-0.79,P＜0.05).(3)The expression of eNOS mRNA decreased significantly in sEng groups.Which also had negative correlation with culture time(r=-0.51,P＜0.05)and dose(r=-0.82,P＜0.05).Conclusions sEng might inhibit eNOS activity by blocking 1177 Ser phosphorylation to decrease NO production.
