凋亡、坏死合体滋养细胞微粒对内皮细胞增殖与凋亡的影响

目的:探讨凋亡、坏死合体滋养细胞微粒对人脐静脉内皮细胞(HUVECs)增殖、凋亡的影响。方法:培养原代HUVECs,物理方法诱导滋养细胞凋亡、坏死。采用三步超速离心法制备凋亡合体滋养细胞微粒(a STBM)、坏死合体滋养细胞微粒(n STBM)(实验组),同时制备RBC膜(对照组),并检测胎盘碱性磷酸酶(PLAP)含量。将不同浓度(20、80、320μg/ml)a STBM、n STBM和RBC膜分别与HUVECs共同孵育4、16、32h。采用MTT法检测细胞增殖抑制率,采用流式细胞仪AV-PI双标记观察细胞凋亡情况。结果:(1)80μg/ml a STBM组作用4、16、32h时的细胞增殖抑制率分别为29.19±0.47、44.73±0.69、54.01±1.61;80μg/ml n STBM组作用4、16、32h的细胞增殖抑制率分别为57.15±0.70、64.35±0.89、71.67±0.63。a STBM、n STBM均能明显抑制HUVEC增殖,抑制作用呈浓度时间依赖性;n STBM对HUVECs的增殖抑制性明显高于a STBM,差异有统计学意义(P0.001)。RBC膜处理组对HUVECs增殖无明显影响,与空白组比较差异无统计学意义(P0.05)。(2)80μg/ml a STBM组的细胞早期凋亡率、晚期凋亡-坏死率分别为29.05%和7.46%。80μg/ml n STBM组分别为15.93%和30.31%。RBC膜处理组对HUVECs的凋亡无明显影响,与空白对照组相比,差异无统计学意义(P0.05)。a STBM、n STBM组均能引起HUVECs凋亡,n STBM作用HUVECs的晚期凋亡率高于a STBM组,两者有统计学意义(P0.001)。a STBM主要诱导HUVECs早期凋亡。结论:a STBM、n STBM对内皮细胞有抑制细胞增殖作用,a STBM主要以促进内皮细胞早期凋亡为主,而n STBM以内皮细胞晚期凋亡为主,n STBM对内皮细胞造成的损害强于a STBM。     

子痫前期患者血浆对人脐静脉内皮细胞生长活性及Edg4表达的影响

目的:探讨子痫前期患者血浆对人脐静脉内皮细胞(HUVECs)生长活性的影响以及与溶血磷脂酸受体蛋白内皮分化基因-4(Edg4)的关系。方法:体外培养HU-VECs,取对数生长的细胞,分组实验:对照组,正常晚孕组,轻度子痫前期组,重度子痫前期组。用MTT比色法和流式细胞仪检测各组细胞增殖及凋亡情况,免疫组化链霉菌抗生物素蛋白-过氧化物酶(SP)法和逆转录聚合酶链反应(RT-PCR)检测各组细胞Edg4蛋白及mRNA表达水平。结果:(1)子痫前期组HUVECs增殖减少,细胞凋亡增加;各组间细胞增殖率及细胞凋亡率差异显著(P<0.05);(2)Edg4蛋白主要表达于HUVECs的细胞浆和细胞膜,子痫前期病情越重,其血浆诱导的HUVECs Edg4蛋白表达越强;(3)子痫前期组Edg4 mRNA表达明显升高,与对照组和正常晚孕组相比显著差异(P<0.05);重度子痫前期组Edg4 mRNA表达较轻度组显著升高(P<0.05)。结论:子痫前期患者血浆可诱导HUVECs Edg4蛋白表达增强,并抑制HUVECs细胞增殖,促进细胞凋亡,提示子痫前期患者溶血磷脂酸升高可能与血管内皮细胞功能异常有关。     

子痫前期患者血管内皮祖细胞数量及生物学功能的变化

目的:观察子痫前期患者血管内皮祖细胞(endothelial progenitor cells,EPCs)数量与功能的变化。方法:选取孕28~40周子痫前期孕妇、健康孕妇各20例,抽取外周静脉血20m l,经密度梯度离心法分离单个核细胞,通过CD133/CD34双荧光标记及Ⅷ因子鉴定细胞;用流式细胞仪检测细胞数量,用MTT比色法、改良Boyden小室法、黏附能力测定实验,分别观察EPCs的增殖、迁移及黏附功能的变化。结果:与健康孕妇相比,子痫前期患者外周血EPCs数量明显减少(4.29%±1.21%vs15.32%±2.00%,P<0.01),其增殖能力(增殖率13.45%±1.68%vs 18.45%±1.67%)、迁移能力[迁移细胞(37.25±7.28)个/视野vs(67.10±9.55)个/视野]、黏附能力[黏附细胞(20.65±5.19)个/视野vs(34.40±6.72)个/视野]明显受损。结论:子痫前期患者外周血EPCs数量减少、生物学功能减退。     

川芎嗪对子痫前期脐血清诱导脐静脉内皮细胞NF-κB活性及VCAM-1表达的影响

目的:探讨川芎嗪对子痫前期患者脐静脉血清诱导脐静脉内皮细胞(HU-VEC)核因子-κB(NF-κB)活性及其靶基因血管细胞黏附分子-1(VCAM-1)表达变化的影响。方法:用胰酶消化法培养正常妊娠HUVEC,传代后待细胞长满至70%~80%,加或不加川芎嗪作用30min后分别加入正常妊娠及子痫前期脐静脉血清,培养48h后,MTT测定细胞活力,流式细胞学测定细胞凋亡率,Western-blot测定HUVEC NF-κB的表达,酶联免疫检测VCAM-1的表达。结果:子痫前期患者脐静脉血清培养HUVEC的增殖活力明显低于正常妊娠组,胞核NF-κB及VCAM-1表达明显高于正常妊娠组(P<0.01),子痫前期患者脐静脉血清培养HUVEC的细胞凋亡率明显高于正常妊娠组(P<0.01)。加川芎嗪预处理后,子痫前期组HUVEC增殖活力明显增加,细胞凋亡率、胞核NF-κB及VCAM-1表达明显下降(P<0.01)。结论:川芎嗪可抑制子痫前期患者脐静脉血清诱导的HU-VEC凋亡,NF-κB的核转位及VCAM-1的表达。     

应用两种不同建模方式构建子痫前期SD大鼠动物模型的实验研究

目的:探讨分析应用两种不同建模方式来构建子痫前期SD大鼠的动物模型的特异性。方法:SD孕鼠随机均分3组,空白对照组同前饲养,合体滋养细胞微绒毛(STBM)组用STBM诱导,STBM+亚硝基左旋精氨酸甲酯(L-NAME)组用STBM+L-NAME诱导。检测蛋白尿(U-TP)、血压(BP)、部分凝血活酶时间(APTT)、凝血酶原时间(PT)和D-二聚体(D-D),HE染色胎盘组织,Real time PCR和Western blot测胎盘组织可溶性血管内皮生长因子受体-1(s Flt-1)、可溶性内皮因子(SEng)、胎盘生长因子(PLGF)mRNA及蛋白表达,滋养细胞分离、培养后行功能学实验(EDU测增殖、FCM测凋亡、Transwell测侵袭)。结果:U-TP、BP、APTT、PT及D-D在孕10天3组比较差异无统计学意义(P0.05),孕15天及20天U-TP、BP STBM+L-NAME组较STBM组显著增加(P0.05),孕20天APTT、PT STBM+L-NAME组较STBM组显著缩短,D-D显著增加,空白对照组无明显变化。STBM+L-NAME组HE染色见胎盘蜕膜带水肿明显,炎性细胞浸润,基带和迷路带见滋养细胞增生。3组比较s Flt-1、SEng、PLGF mRNA及蛋白表达差异有统计学意义(P0.05)。STBM+L-NAME组s Flt-1、SEng mRNA及蛋白比STBM组明显增高(P0.05),PLGF mRNA及蛋白明显减低(P0.05)。功能学实验中3组细胞增殖和凋亡差异均有统计学意义(P0.05)。STBM+L-NAME组滋养细胞增殖和侵袭最少,凋亡最多。与STBM组比较差异有统计学意义(P0.05)。结论:在构建子痫前期动物模型中,STBM合用L-NAME诱导比单独应用STBM特异性更强,为临床子痫前期诊疗提供可靠模式研究依据。     

PKC抑制剂对子痫前期血清诱导人脐静脉内皮细胞NF-κB核转位及VCAM-1表达的影响

目的:探讨子痫前期血清诱导人脐静脉内皮细胞(HUVEC)蛋白激酶(PKC)活性改变、核因子-κB(NF-κB)核转位及内皮细胞黏附分子-1(VCAM-1)的表达以及PKC抑制剂对它们的影响。方法:用胰酶消化培养法培养正常妊娠HUVEC,传代后待细胞长满至70%~80%后,加或不加PKC抑制剂多黏菌素B(PMB)作用30m in后分别加入正常妊娠及子痫前期血清,培养2h,W estern B lot测定细胞胞浆PKC、胞膜PKC含量、胞浆核因子κB抑制因子(I-κB)、胞核NF-κBp65含量;培养48h,用MTT测定细胞活力,流式细胞学测定细胞凋亡,酶联免疫法测定HUVEC VCAM-1的表达。结果:子痫前期血清培养的HUVEC胞浆PKC及I-κB含量明显低于对照组(P<0.05),胞膜PKC含量、胞核NF-κBp65含量、VCAM-1表达、细胞凋亡率明显高于对照组(P<0.05),细胞活力明显低于对照组(P<0.05)。加PKC抑制剂预处理后,子痫前期组HUVEC胞浆PKC含量及I-κB含量明显增加(P<0.05);胞膜PKC含量、胞核NF-κBp65含量、VCAM-1表达、细胞凋亡率明显下降(P<0.05),细胞活力明显增加(P<0.05)。结论:子痫前期血清可促进HUVEC NF-κB活性及VCAM-1表达,PKC抑制剂可抑制子痫前期患者血清诱导HUVEC的NF-κB活性及VCAM-1表达,PKC、NF-κB在子痫前期内皮细胞损伤过程中可能起重要的桥梁作用。     

子痫前期血清对滋养-内皮细胞共培养体系滋养细胞侵袭力和内皮细胞凋亡的影响

目的:探讨子痫前期血清对滋养-内皮细胞共培养体系滋养细胞侵袭力改变及对内皮细胞凋亡的影响.方法:选择正常足月妊娠剖宫产孕妇10例(正常对照组),子痫前期患者20例(子痫前期组).采用组织块贴壁法培养人孕早期细胞滋养细胞(CTB),胰酶消化法培养正常妊娠人脐静脉内皮细胞(HUVEC),传代后建立滋养细胞与脐静脉内皮细胞共培养模型,并分别加入正常对照组和子痫前期组患者静脉血清培养24小时;Transwell小室检测细胞滋养细胞的侵袭能力;流式细胞学检测人脐静脉内皮细胞凋亡率;逆转录-聚合酶链反应(RT-PCR)检测细胞滋养细胞MMP-2、MMP-9mRNA及人脐静脉内皮细胞自杀相关因子(Fas)mRNA的表达.结果:子痫前期患者血清共培养体系的细胞滋养细胞的侵袭能力低于正常对照组(P<0.01);人脐静脉内皮细胞凋亡率明显低于正常对照组(P<0.05);MMP-2、MMP-9和FasmRNA的表达明显下降(P<0.05).结论:子痫前期血清可能通过降调细胞滋养细胞MMP-2、MMP-9表达而影响滋养-内皮细胞共培养体系滋养细胞的侵袭能力,并且其滋养-内皮细胞共培养体的内皮细胞Fas的表达异常可能与内皮细胞凋亡下降有关.提示滋养细胞MMP-2、MMP-9、Fas/Fasl的表达异常可能参与了子痫前期子宫螺旋动脉重铸障碍的病理过程.     

RNA干扰技术抑制HLX1基因表达对滋养细胞生物学性能的影响

目的:研究小分子干扰RNA(siRNA)技术抑制人类同源异型盒基因HLX1的表达对胎盘滋养细胞株HTR-8/SVneo细胞生物学性能的影响。方法:常规体外培养HTR-8/SVneo细胞,设实验组(转染HLX1基因的特异性siRNA)、阴性对照组(转染阴性对照siRNA)及空白对照组(除不含siRNA片段,余试剂与另两组相同)3组分别进行瞬时转染。用流式细胞术测定siRNA转染效率,应用实时定量PCR技术和蛋白印迹法测定转染后各组HTR-8/SVneo细胞中HLX1 mRNA和蛋白的表达;应用MTT比色实验、Matrigel侵袭实验分别检测转染后各组细胞的增殖能力与侵袭能力的变化。结果:(1)转染24h后测得siRNA转染效率达(86.3±2.6)%。(2)转染48h后HLX1 mRNA的表达水平下调(77.0±1.2)%(P<0.01),转染72h后HLX1蛋白的表达水平下调(82.6±1.2)%(P<0.01),与HLX1 mRNA的表达水平下调相一致。(3)转染HLX1 siRNA 24h后,HTR-8/SVneo细胞的增殖活性已受到抑制,转染72h后细胞增殖抑制最显著,抑制率达到(58.1±4.4)%(P<0.01)。(4)转染HLX1 siRNA后,HTR-8/SVneo细胞侵袭Matrigel基质胶的能力受到显著抑制,其穿膜细胞数为(29±3)个,与空白对照组(穿膜细胞数为[53±8]个)相比,差异有统计学意义(P<0.01)。结论:HLX1基因表达水平的下降可以显著抑制滋养细胞的增殖活性与侵袭能力;HLX1基因表达异常可能通过影响滋养细胞增殖、侵袭等过程参与IFGR、子痫前期等疾病的发生。     

脂氧素对子痫前期患者巨噬细胞增殖和分泌功能的影响

目的 探讨脂氧素对子痫前期患者巨噬细胞增殖和分泌功能的影响.方法 对温州医学院附属第一医院产科2007年3-7月收治的24例子痫前期孕妇(子痫前期组)和24例正常孕妇(正常妊娠组)的腹腔巨噬细胞进行体外培养.采用酶联免疫吸附试验(ELISA)测定不同浓度脂氧素(0、10、100 nmol/L)作用两组孕妇腹腔巨噬细胞48 h后,上清液中肿瘤坏死因子α(TNF-α)的水平.用细胞生长抑制实验法检测两组不同浓度脂氧素(0、10、100 nmol/L)作用24 h后的巨噬细胞增殖抑制率.结果 (1)TNF-α水平:子痫前期组0、10、100 nmol/L浓度的脂氧素作用下,TNF-α水平分别为(1867.5±47.3)、(1836.9±4.5)、(1800.5±2.7)ng/L.正常妊娠组0、10、100 nmol/L浓度的脂氧素作用下,TNF-α水平分别为(791.3±62.2)、(789.4±2.3)、(781.5±1.9)ng/L.子痫前期组TNF-α水平显著高于正常妊娠组,两组比较,差异有统计学意义(P＜0.05).脂氧素对子痫前期组TNF-α水平的影响呈剂量依赖性抑制(P＜0.05),而其对正常妊娠组TNF-α水平变化无明显影响(P＞0.05).(2)增殖抑制率:子痫前期组0、10、100 nmol/L浓度的脂氧素作用下,增殖抑制率分别为(14.8±6.3)%、(32.9±3.6)%、(36.7±3.8)%.正常妊娠组0、10、100 nmol/L浓度的脂氧素作用下,增殖抑制率分别为(16.8±6.9)%、(16.7±5.4)%、(15.9±2.1)%.子痫前期组增殖抑制率比正常妊娠组明显升高,两组比较,差异有统计学意义(P＜0.05),脂氧素呈剂量依赖性抑制子痫前期组孕妇腹腔巨噬细胞的增殖抑制率(P＜0.05),而对正常妊娠组的增殖抑制率则无明显影响(P＞0.05).结论 脂氧素呈剂量依赖性地抑制子痫前期患者的巨噬细胞增殖和TNF-α分泌.     

正常妊娠中细胞增殖细胞凋亡情况的研究

目的　研究细胞增殖和细胞凋亡在正常妊娠不同孕期胎盘绒毛、蜕膜生长发育中的作用。方法　对 40例正常早孕吸宫流产病例和 40例正常晚孕分娩病例的胎盘绒毛和蜕膜进行分析 :免疫组织化学链霉菌抗生物素蛋白 -过氧化物酶法 (S -P法 )检测细胞增殖 ;用DNA缺口原位末端标记技术 (TUNEL法 )检测细胞凋亡。结果　正常早孕绒毛细胞滋养细胞、合体滋养细胞、蜕膜中的增殖指数 (PI)分别为 (70 74± 1 34 ) %、0、(89 32± 1 75 ) % ,凋亡指数 (AI)分别为 (10 2 7± 0 35 ) %、(15 2 8± 2 43) %、(5 2 3± 1 35 ) %。正常晚孕胎盘细胞滋养细胞、合体滋养细胞和蜕膜中增殖指数 (PI)分别为 (2 10± 1 2 8) %、0、(2 0 4± 1 31) % ;凋亡指数 (AI)分别是 (1 0 6± 0 94) %、(41 79±1 48) %、(6 0 81± 1 41) %。即 :正常早、晚孕绒毛和蜕膜组织中都有一定量的细胞增殖和细胞凋亡发生 ,但早孕时以细胞增殖为主 (P <0 0 1) ,晚孕时以细胞凋亡为主 (P <0 0 1)。结论　细胞增殖和细胞凋亡都是细胞生长代谢的形式 ,随着妊娠进展 ,胎盘细胞增殖下降、细胞凋亡增加。提示 :细胞增殖和凋亡对胎盘绒毛的正常发育和老化起重要作用     

Decrease in lipid levels of syncytiotrophoblast micro-particles reduced their potential to inhibit endothelial cell proliferation

Background Preeclampsia is characterized by damage to the maternal endothelium that has been suggested to be mediated in part by elevated shedding of inflammatory placental syncytiotrophoblast micro-particles (STBM) into the maternal circulation. Previously, we have shown that STBM, prepared by three different methods: mechanical dissection, in vitro placental explants culture and perfusion of placenta, can inhibit endothelial cell proliferation. Only mechanically prepared STBM induced apoptosis in the endothelial cells. Now, we have examined lipid levels in the three STBM preparations and their differential responses on endothelial cells. Methods We examined the lipid levels in the three STBM preparations using thin layer chromatography. Furthermore, the effects of reduced lipid levels in the three STBM preparations using the pharmacological agent methyl-β-cyclodextrin were examined on endothelial cell proliferation and apoptosis. Results Among the three STBM preparations, mechanical STBM contained highest levels of lipids. The reduction in lipid levels in mechanical STBM reduced their potential to inhibit human umbilical vein endothelial cells (HUVEC) proliferation and blocked their potential to induce apoptosis. No similar effect was observed following lipid reduction in the two other STBM preparations. Conclusions As it has been suggested that mechanically derived STBM may more closely resemble placental micro-particles generated in preeclampsia, our data suggest that lipid content may play a role in the anti-endothelial defects present in this disease.     

A comparative study of the effect of three different syncytiotrophoblast micro-particles preparations on endothelial cells

Pre-eclampsia is a pregnancy-associated multi-system disorder of unknown etiology, characterized by damage to the maternal endothelium. The latter facet has been suggested to be mediated in part by elevated shedding of inflammatory placental syncytiotrophoblast micro-particles (STBM) into the maternal circulation. In this study, we have examined STBM prepared by three different methods: mechanical dissection, in vitro placental explant culture and perfusion of placental cotyledons. All three preparations yielded morphologically similar STBM, as confirmed by scanning electron microscopy, and all contained syncytiotrophoblast-specific proteins as determined by the presence of placental alkaline phosphatase. The functional properties of the three STBM preparations were examined on human umbilical vein endothelial cells (HUVEC), where the mechanically prepared particles were found to inhibit proliferation to the greatest extent. Furthermore, only mechanically prepared STBM lead to the detachment and apoptosis of HUVEC cells. Our study, therefore, suggests that STBM prepared from placental perfusion or in vitro explant culture are biologically different from mechanically prepared ones, and may provide a better approximation of physiologically produced placental micro-particles.     

Effect of syncytiotrophoblast microvillous membrane treatment on gene expression in human umbilical vein endothelial cells

OBJECTIVE: Syncytiotrophoblast membrane fragments (STBM) exist in the peripheral circulation in pregnant women and it has been shown that the level of circulating STBM is significantly increased with pre-eclampsia compared with uncomplicated pregnancies. STBM could be one of the factors which directly causes the endothelial cell dysfunction of pre-eclampsia. This study investigates the effect of STBM on endothelial cell gene expression. DESIGN: Human umbilical vein endothelial cells were cultured in the presence and absence of STBM. At specified time points, total RNA was purified from the cultures and analysed on microarrays. SETTING: A laboratory investigation using placentas obtained from a hospital delivery ward. SAMPLE: Placentas from nine healthy women were obtained. STBM vesicles were isolated from the placentas and umbilical vein endothelial cell cultures were established from the umbilical cords. METHODS: Gene expression was screened by Affymetrix GeneChips and confirmed with real-time polymerase chain reaction or enzyme-linked immunosorbent assay. MAIN OUTCOME MEASURES: Fold changes in gene expression levels between treated and control cultures were calculated from the microarray results. RESULTS: Overall, the results do not show any great changes in gene expression in endothelial cells after STBM treatment (28 genes changed two-fold or more out of approximately 10,000 genes examined by microarray). In general, the changes observed are consistent with inhibition of proliferation of endothelial cells by exposure to STBM. The unfolded protein response in particular may be involved. CONCLUSIONS: STBM may influence endothelial cell function during pregnancy but STBM alone cannot account for the entire range of endothelial dysfunctions observed in pre-eclampsia.     

Endothelial cell apoptosis is induced by fetal plasma from pregnancy with umbilical placental vascular disease

OBJECTIVE: Vascular disease in the umbilical placental circulation that is detected by umbilical artery Doppler study is associated with adverse fetal outcome. Endothelial cell activation and platelet consumption are features of this pathologic condition. We postulated that this was due to the local release of factors that cause endothelial cell injury and that these would spill into the fetal circulation. To test this hypothesis, we examined for the presence in fetal plasma of factors that induced endothelial cell apoptosis in pregnancies that were complicated by umbilical placental vascular disease. STUDY DESIGN: Isolated and cultured human umbilical vein endothelial cells were exposed to fetal plasma from the 3 fetal groups: normal pregnancy (n = 32 patients), pregnancy with umbilical placental vascular disease that was identified by an abnormal umbilical artery Doppler study (n = 38 patients), and pregnancy with maternal preeclampsia and normal umbilical artery Doppler study (n = 16 patients). Early apoptosis can be recognized by a loss of plasma membrane asymmetry with membrane uptake of annexin V. This was measured with annexin V and propidium iodide staining by fluorescent-activated cell scanning. Cells that underwent early apoptosis stained positive for annexin V and negative for propidium iodide (in contrast with cells that underwent necrosis). Cytosolic proteolytic activity was also measured. The lysates from endothelial cells that were stimulated by fetal plasma from umbilical placental vascular disease were tested for caspase-3 and caspase-8 activities by a fluorescent assay with spectrofluorophotometry. RESULTS: The percentage of endothelial cells that underwent apoptosis was significantly higher (P <.05) when stimulated with fetal plasma from pregnancies with umbilical placental vascular disease (17.71% +/- 1.31%) than with fetal plasma from normal pregnancies (9.76% +/- 0.87%). In the presence of maternal preeclampsia with normal umbilical artery Doppler study, the percent of apoptotic cells (11.31% +/- 1.59%) was similar to that of the normal group. In the group with abnormal umbilical artery Doppler study, there was no difference between pregnancies with preeclampsia (n = 17 pregnancies) and without preeclampsia (n = 21 pregnancies). The protease activity of caspase-3 was significantly enhanced in the group with umbilical placental vascular disease compared with normal pregnancy (0.79 +/- 0.06 vs 0.45 +/- 0.08 microMol/L). However, no difference in caspase-8 activity was detected (0.66 +/- 0.05 vs 0.56 +/- 0.05 microMol/L). CONCLUSION: Endothelial cell apoptosis is a feature of umbilical placental vascular disease. Our study demonstrates the presence of factors in the fetal plasma that caused endothelial cells to undergo early apoptosis. This increased apoptosis was only seen in the presence of placental vascular disease and was independent of the presence or absence of maternal preeclampsia. Our results indicate that programmed endothelial cell death occurs in the fetal circulation as a part of the injury that is associated with the development of umbilical placental vascular disease. The caspase-3, rather than caspase-8, signal transduction pathway appears to be involved in the mediation of endothelial cell apoptosis that was detected in our study.     

Enhanced angiogenic capacity of human umbilical vein endothelial cells from women with preeclampsia

Maternal and placental angiogenic abnormalities are a common feature of preeclampsia. The aim of this study was to determine if endothelial cells from women with preeclampsia exhibit different angiogenic responses compared to healthy cells. Using the endothelial tube formation assay, we have shown that primary human umbilical vein endothelial cells (HUVECs) isolated from women with preeclampsia display greater levels of in vitro angiogenic branching compared to cells from healthy women. A comparable increase in tube formation was observed in healthy cells cultured at 0.5% O(2). Vascular endothelial growth factor (VEGF) receptor inhibition resulted in a decrease in angiogenesis in both healthy hypoxic cells and cells from women with preeclampsia. These findings demonstrate that HUVECs from women with preeclampsia exhibit inherent differences in their angiogenic capacity which are apparent in the absence of placental or maternal factors.     

子痫前期重度患者血清损伤后血管内皮细胞上清对脐带间充质干细胞的趋化作用

目的探讨子痫前期重度患者血清培育后的血管内皮细胞上清是否对人脐带间充质干细胞(UC-MSCs)有趋化作用。方法 2009年10月至11月在中国医科大学附属盛京医院将内皮细胞以1×105个/mL的浓度接种于6孔培养板,进行分组干预:每组用含15%子痫前期重度患者血清(P组)、健康孕妇血清(H组)、不含血清的DMEM培养液(N组)培养24h,取各组上清液于无菌EP管中,离心20min后加入Transwell小室下室,取第5代UC-MSCs用含5%胎牛血清的DF-12调整细胞浓度为1×105个/mL,加入预处理后的Transwell小室上室,将Tran-swell小室置于37℃、5%CO2孵箱孵育24h。取出后4%多聚甲醛固定、伊红染色后于20倍镜下随机记数5个无重叠细胞视野的透膜细胞数,取其平均数代表该组细胞的侵袭力。结果 N组越膜细胞数:40.87±6.33;H组越膜细胞数:45.47±6.89;P组越膜细胞数:56.07±6.76。P组越膜细胞最多,差异有统计学意义(P<0.01,F=20.53)。结论 UC-MSCs具有向子痫前期重度患者血清培育后血管内皮细胞趋化的生理特性。     

Plasma from preeclamptic women increases human endotheial cell prostacyclin production without changes in cellular enzyme activity or mass

OBJECTIVE: We investigated differencesin prostacyclin production by endothelial cells exposed to plasma from either preeclamptic women or normal pregnant women.STUDY DESIGN: A case-control study of matched preeclamptic and normal pregnancies was used to compare prostacyclin synthesis by human umbilical vein endothelial cells incubated with pregnancy plasma for 24 hours. Prostacyclin concentrations in conditioned media were measured by radioimmunoassay of its stable metabolite (6-keto-prostaglandin F_1α). Human umbilical vein endothelial cell lysates were used to determine concentrations of the enzymes cyclooxygenase and prostacyclin synthase.RESULTS: Prostacyclin production by human umbilical vein endothelial cells incubated with plasma from preeclamptic women was significantly greater than that by cells exposed to normal pregnancy plasma. Differences in prostacyclin production under the two experimental conditions could be explained neither by differences in enzyme mass nor activities of cyclooxygenase and prostacycline synthase.CONCLUSION: The stimulatory effect of preeclampsia plasma on prostacycline biosynthesis in human umbilical vein endothelial cells appears to be manifested at a step(s) proximal to the activation of cyclooxygenase. Possible mechanisms are increased phospholipase A₂, lipoprotein, or lipid peroxide activities in preeclampsia.