中国药理学通报
中國藥理學通報
중국약이학통보
CHINESE PHARMACOLOGICAL BULLETIN
2014年
9期
1287-1292
,共6页
王艳华%张慧萍%田珏%周龙霞%陈久凯%马文斌%孔繁琪%赵丽%刘现梅%韩学波%杨晓玲%姜怡邓
王豔華%張慧萍%田玨%週龍霞%陳久凱%馬文斌%孔繁琪%趙麗%劉現梅%韓學波%楊曉玲%薑怡鄧
왕염화%장혜평%전각%주룡하%진구개%마문빈%공번기%조려%류현매%한학파%양효령%강이산
一氧化氮%N-硝基-L-精氨酸甲酯%人胎盘滋养细胞%凋亡%氧化应激%妊娠期高血压疾病
一氧化氮%N-硝基-L-精氨痠甲酯%人胎盤滋養細胞%凋亡%氧化應激%妊娠期高血壓疾病
일양화담%N-초기-L-정안산갑지%인태반자양세포%조망%양화응격%임신기고혈압질병
nitric oxide%N- nitro-L-arginine methyl ester ( L-NAME)%human cytotrophoblast cells%apopto-sis%oxidative stress%hypertension disorder complica-ting pregnancy
目的：探讨一氧化氮( NO)水平下降诱导人胎盘滋养细胞凋亡的可能机制。方法体外培养人胎盘滋养细胞株(HTR-8),分别加入10、100、500、1000μmol·L-1 L-NAME,并设置对照组(0μmol·L-1 L-NAME),孵育48 h；四甲基偶氮唑蓝法( MTT)检测L-NAME对细胞存活率的影响；采用硝酸还原酶法测定细胞中NO的含量；利用透射电镜、流式细胞术和Annexin-V FITC染色检测L-NAME对HTR-8细胞凋亡的影响；Fe3+还原比色法检测总抗氧化能力( T-AOC)、黄嘌呤氧化酶法测定超氧化物歧化酶( SOD)活性及硫代巴比妥酸比色法测定 MDA 含量。结果与对照组比较,100、500、1000μmol·L-1 L-NAME组细胞存活率及NO水平明显降低(P<0.05,P<0.01)；Annexin-V FITC染色和流式分析均显示L-NAME能诱导细胞凋亡,并且呈剂量依赖性；NO水平与细胞凋亡数呈负相关( r=-0.5210)；透射电镜结果显示：与对照组比较,实验组细胞核形态不规则,核膜固缩呈不规则状,染色质凝集或边集,线粒体肿胀或浓缩、嵴断裂或溶解,甚至消失,形成空泡,尤其在100μmol · L-1 L-NAME组可见凋亡小体；同时,HTR-8细胞内T-AOC、SOD水平明显降低(P<0.05),MDA的含量明显升高(P<0.05)；细胞凋亡数与T-AOC(r=-0.3212)、SOD(r=-0.2779)水平均呈负相关,与MDA(r=0.2807)含量呈正相关。结论氧化应激在NO水平降低引起的人胎盘滋养细胞凋亡中起重要作用。
目的：探討一氧化氮( NO)水平下降誘導人胎盤滋養細胞凋亡的可能機製。方法體外培養人胎盤滋養細胞株(HTR-8),分彆加入10、100、500、1000μmol·L-1 L-NAME,併設置對照組(0μmol·L-1 L-NAME),孵育48 h；四甲基偶氮唑藍法( MTT)檢測L-NAME對細胞存活率的影響；採用硝痠還原酶法測定細胞中NO的含量；利用透射電鏡、流式細胞術和Annexin-V FITC染色檢測L-NAME對HTR-8細胞凋亡的影響；Fe3+還原比色法檢測總抗氧化能力( T-AOC)、黃嘌呤氧化酶法測定超氧化物歧化酶( SOD)活性及硫代巴比妥痠比色法測定 MDA 含量。結果與對照組比較,100、500、1000μmol·L-1 L-NAME組細胞存活率及NO水平明顯降低(P<0.05,P<0.01)；Annexin-V FITC染色和流式分析均顯示L-NAME能誘導細胞凋亡,併且呈劑量依賴性；NO水平與細胞凋亡數呈負相關( r=-0.5210)；透射電鏡結果顯示：與對照組比較,實驗組細胞覈形態不規則,覈膜固縮呈不規則狀,染色質凝集或邊集,線粒體腫脹或濃縮、嵴斷裂或溶解,甚至消失,形成空泡,尤其在100μmol · L-1 L-NAME組可見凋亡小體；同時,HTR-8細胞內T-AOC、SOD水平明顯降低(P<0.05),MDA的含量明顯升高(P<0.05)；細胞凋亡數與T-AOC(r=-0.3212)、SOD(r=-0.2779)水平均呈負相關,與MDA(r=0.2807)含量呈正相關。結論氧化應激在NO水平降低引起的人胎盤滋養細胞凋亡中起重要作用。
목적：탐토일양화담( NO)수평하강유도인태반자양세포조망적가능궤제。방법체외배양인태반자양세포주(HTR-8),분별가입10、100、500、1000μmol·L-1 L-NAME,병설치대조조(0μmol·L-1 L-NAME),부육48 h；사갑기우담서람법( MTT)검측L-NAME대세포존활솔적영향；채용초산환원매법측정세포중NO적함량；이용투사전경、류식세포술화Annexin-V FITC염색검측L-NAME대HTR-8세포조망적영향；Fe3+환원비색법검측총항양화능력( T-AOC)、황표령양화매법측정초양화물기화매( SOD)활성급류대파비타산비색법측정 MDA 함량。결과여대조조비교,100、500、1000μmol·L-1 L-NAME조세포존활솔급NO수평명현강저(P<0.05,P<0.01)；Annexin-V FITC염색화류식분석균현시L-NAME능유도세포조망,병차정제량의뢰성；NO수평여세포조망수정부상관( r=-0.5210)；투사전경결과현시：여대조조비교,실험조세포핵형태불규칙,핵막고축정불규칙상,염색질응집혹변집,선립체종창혹농축、척단렬혹용해,심지소실,형성공포,우기재100μmol · L-1 L-NAME조가견조망소체；동시,HTR-8세포내T-AOC、SOD수평명현강저(P<0.05),MDA적함량명현승고(P<0.05)；세포조망수여T-AOC(r=-0.3212)、SOD(r=-0.2779)수평균정부상관,여MDA(r=0.2807)함량정정상관。결론양화응격재NO수평강저인기적인태반자양세포조망중기중요작용。
Aim To investigate the possible mecha-nisms of the levels of NO decrease induced apoptosis in human placental trophoblast cells. Methods Human placental trophoblast cells ( HTR-8 ) were cultured in 5 ml DMEM-F12 culture medium with 37℃ 5% CO2 . Then, the old culture medium was discarded and re-placed with 10,100,500,1 000 μmol·L-1 L-NAME, and the group without L-NAME was set as the control group, cultured for 48h. The effects of L-NAME on the survival of cells were detected by methylthiazolyldiphe-nyl tetrazolium bromide ( MTT); the content of NO in cells was tested by nitrate reductive enzymatic;trans-mission electron microscopy, flow cytometry analysis and Annexin-V FITC dyeing were used to test the effects of L-NAME on apoptosis in HTR-8 cells;restore Fe3+ colorimetric assay was applied for detection of to-tal antioxidant capacity ( T-AOC ) , xanthine oxidase for detection of superoxide dismutase ( SOD) activity, and thiobarbituric acid colorimetry for determination of content of MDA. Results Compared with the control group, the survival rate of HTR-8 cells and the levels of NO in 100,500,1 000 μmol·L-1 L-NAME group were significantly reduced(P<0.05,P<0.01). Flow analysis and Annexin-V FITC staining showed that L-NAME could induce cell apoptosis in a dose-dependent manner. The number of cell apoptosis was negatively correlated with the content of NO ( r = -0.5210 ) in HTR-8 cells. Transmission electron microscopy results showed that compared with the control group, the ex-perimental group's cell nucleus shape was irregular, nuclear pyknosis in irregular shape, the chromatin ag-glutination or side the collection, mitochondrial swell-ing or enrichment, crest fracture or dissolved, even vanished, forming the vacuole, especially in 100 μmol ·L-1 L-NAME group, the apoptotic bodies obviously appeared. At the same time, T-AOC, SOD levels in HTR-8 cells decreased ( P <0.05 ) , and the MDA content increased ( P<0.05 ) . The number of cell ap-optosis was negatively correlated with the level of T-AOC ( r= -0.3212 ) , SOD ( r= -0.2779 ) in HTR-8 cells , while positively correlated with the content of MDA(r=0.2807). Conclusion Oxidative stress may play an important role in the levels of NO decrease in-duced apoptosis in human placental trophoblast cells.
