Apelin在正常妊娠及妊娠期高血压疾病胎盘组织中的表达及其与一氧化氮关系的研究
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摘要
研究背景
妊娠期高血压疾病(HDCP)是一种发生在妊娠中晚期的常见并发症,是孕产妇和围生儿发病及死亡的主要原因,由于其病因及发病机理仍不明确,目前尚无一种确切而有效的预防和治疗方法。近两个世纪以来,国内外学者对有关妊娠期高血压疾病的病因及发病机理进行了大量研究,根据流行病学调查和实验研究提出了多种病因学假说,如:免疫学说、胎盘浅着床学说、血管内皮受损学说、遗传学说、营养缺乏学说、胰岛素抵抗学说等。大量研究表明,妊娠期高血压疾病基本发病机制为广泛的血管内皮细胞损伤。维持内皮细胞正常功能的几大因子平衡失调是该病发病的关键,主要有一氧化氮(nitric oxide,NO)和内皮素(ET)、前列环素(PGI2)、血栓素A2 (TXA2)、纤溶酶原激活抑制因子(PAI)和组织纤溶酶原激活因子(tPA)等。平衡失调的直接后果是缩血管因子(ET和TXA2)分泌增加,扩血管因子(NO和PGI2)分泌减少,导致患者出现全身小动脉痉挛,使各脏器的血流灌注减少。目前,多数学者们认为胎盘缺血缺氧是导致内皮细胞激活和损伤的更深层次原因,它在妊娠期高血压疾病的发病机制中应该更接近于起始病因。探讨胎盘缺血缺氧的有关因素,将有可能为该病的防治提供新的思路。
Apelin为新近发现的一种血管活性多肽,是孤儿G蛋白偶联性受体APJ的内源性配体,具有扩张血管、增强心肌收缩力、促进NO合成的特性,在调节水盐代谢以及介导免疫调节方面也起重要作用。已确认Apelin多肽存在多种亚型,其中以Apelin-36及Apelin-13较为常见,目前对Apelin及其受体APJ的研究刚刚开始,在妊娠方面的研究报道极少。
NO是血管内皮源性舒张因子,在调节胎儿胎盘循环中起重要作用。胎盘是妊娠期一氧化氮合成的主要场所,NOS是NO合成的最重要的限速步骤,以往的研究证实妊娠期NO合成减少与HDCP发病密切相关,有关Apelin与胎盘绒毛组织NO生成的关系未见报道。
妊娠期高血压疾病是妊娠期特有疾病,而胎盘是妊娠期特有的器官,因此研究胎盘的生理生化改变,将有可能发现引起子宫胎盘缺血缺氧及血管内皮细胞受损的真正原因。本课题选择胎盘为研究对象,采用免疫组织化学方法和实时荧光定量聚合酶链反应(荧光定量RT-PCR)检测Apelin、APJ在绒毛及胎盘组织中的表达、硝酸还原酶法检测胎盘组织NO的水平,分光光度法测定胎盘组织中NOS的活性。探讨Apelin在妊娠期高血压疾病中的作用及其与NO之间的关系。并采用Apelin-36对体外培养的胎盘绒毛进行干预,观察Apelin对胎盘绒毛NO-NOS的影响,以期为妊娠期高血压疾病的防治提供新的思路。
本研究内容包括三部分,现概述如下:
第一部分Apelin及受体APJ在正常妊娠早孕绒毛和晚孕胎盘及脐血管组织中的表达及定位研究
目的探讨血管活性肽Apelin及其受体APJ在早期妊娠绒毛、晚期妊娠胎盘及脐血管组织中的表达和定位。方法分别取10例妊娠7～9周妇女(早孕组)的绒毛组织和15例妊娠37～41周(晚孕组)妇女的胎盘及脐带组织,采用实时荧光定量逆转录聚合酶链反应法(荧光定量RT-PCR)及免疫组织化学染色分析法(SP)检测两组孕妇胎盘Apelin及其受体APJ mRNA和蛋白的表达情况,并用高清晰度彩色图文分析系统(HPIAS - 1000)对其进行定量分析。同时用免疫组织化学染色分析法观察Apelin-36及其受体APJ在正常妊娠脐带血管组织中的定位表达情况。结果(1)荧光定量RT-PCR结果显示,晚孕组胎盘绒毛组织Apelin mRNA水平显著高于早孕组绒毛组织(P<0.05),而APJ mRNA在不同妊娠期绒毛组织中的差异无统计学意义(P >0.05)。(2)免疫组化研究结果显示,Apelin-36在早孕绒毛组织中的表达部位为绒毛合体滋养细胞及细胞滋养细胞,少数间质细胞中也可见到Apelin-36弱阳性表达;在晚孕胎盘组织中,主要表达于胎盘滋养细胞,部分血管内皮细胞及少数间质细胞也可见到Apelin-36的阳性表达,阳性染色主要位于胞浆。APJ蛋白在早孕绒毛组织及晚孕胎盘组织的表达主要位于合体滋养细胞及细胞滋养细胞,其次表达的部位为血管内皮细胞及间质细胞,少数血管平滑肌细胞中也有APJ的表达,阳性染色主要位于胞核。(3)彩色图文分析系统结果显示,Apelin-36在早孕绒毛组织中表达较弱,而在晚孕胎盘组织中阳性表达明显增强,差异有统计学意义(P < 0.01) ,受体APJ在早孕组与晚孕组间的表达差异无统计学意义(P > 0.05)。(4)免疫组化研究结果显示,Apelin-36及受体APJ蛋白在脐静脉血管内皮均有阳性表达,在脐动脉血管内皮细胞中的表达则明显减弱。在脐血管平滑肌组织中,受体APJ散在表达于内皮细胞周围的平滑肌细胞中,未发现Apelin-36的阳性表达。结论正常早孕绒毛及晚孕胎盘组织中有Apelin及其受体APJ的表达,即胎盘组织内存在Apelin- APJ体系的自分泌调节系统,晚孕期胎盘组织Apelin mRNA转录水平增高,可能与妊娠晚期胎盘血流灌注量的需求增加有关。在晚期妊娠的脐静脉血管内皮细胞中,Apelin-36及受体APJ均呈阳性表达,脐动脉血管内皮细胞则表达减弱,这种表达的差异性,可能是由于妊娠晚期脐静脉较脐动脉内皮含有更丰富的活性物质,以适应胎儿生长发育的需要。
第二部分Apelin与妊娠期高血压疾病发病及NO关系的研究
目的检测血管活性肽Apelin及其受体APJ在正常妊娠和妊娠期高血压疾病(HDCP)患者胎盘组织中的表达,探讨Apelin在妊娠期高血压疾病发生、发展中的作用及其与一氧化氮(NO)的关系。方法选择孕周为36～41周的妊娠期高血压疾病患者36例,其中妊娠期高血压10例,轻度子痫前期11例,重度子痫前期15例;选择同期正常孕妇15例作为对照组。采用免疫组织化学染色分析法检测两组孕妇胎盘组织中Apelin-36及受体APJ的表达,荧光定量RT-PCR检测Apelin及APJmRNA的表达。硝酸还原酶法检测胎盘组织NO的水平,分光光度法测定胎盘组织中NOS的活性,结果(1)妊娠期高血压疾病组胎盘组织Apelin-36及受体APJ在表达部位上与对照组(正常晚孕组)无明显差异,主要表达的部位为绒毛合体滋养细胞及细胞滋养细胞。(2)妊娠期高血压疾病组胎盘Apelin mRNA及Apelin-36的表达量显著低于对照组(P < 0.01),且随着病情程度的加重,各组胎盘中Apelin mRNA及Apelin-36的表达水平呈下降的趋势,轻度、重度子痫前期胎盘Apelin mRNA及Apelin-36的表达水平均显著低于妊娠期高血压组(P < 0.01,P < 0.01),轻、重度子痫前期两组之间Apelin mRNA及Apelin-36的表达水平比较,差异也有统计学意义(P < 0.05)。(3)妊娠期高血压疾病各组胎盘组织APJmRNA及蛋白水平的表达与对照组比较差异无统计学意义(P >0.05)。(4)妊娠期高血压疾病组胎盘组织NO水平与对照组相比显著降低(P < 0.01),且随着病情程度的加重,各组胎盘组织中NO水平呈下降的趋势,轻度、重度子痫前期组胎盘组织NO水平均低于妊娠期高血压组(P < 0.01, P < 0.01),轻、重度子痫前期两组之间比较,差异也有统计学意义(P < 0.05)。妊娠期高血压组胎盘组织NO水平与对照组比较,差异无统计学意义(P > 0.05)。(5)妊娠期高血压疾病组胎盘组织NOS水平与对照组相比显著降低(P < 0.01),且随着病情程度的加重,各组胎盘组织中NOS水平呈下降的趋势,妊娠期高血压疾病组轻度、重度子痫前期胎盘组织NO水平均低于妊娠期高血压组(P < 0.05),妊娠期高血压组胎盘组织NO水平与对照组比较,差异无统计学意义(P > 0.05)。(6)妊娠期高血压疾病组胎盘组织NO水平及NOS活性与Apelin mRNA水平呈正相关,相关系数分别为0.823,0.901(P <0.01, P <0.01)。结论(1)血管活性肽Apelin及其受体APJ在妊娠期高血压疾病患者胎盘组织中均有表达,妊娠期高血压疾病患者胎盘组织中Apelin mRNA及Apelin-36表达水平低下,并与病情程度相关,可能在妊娠期高血压疾病的发病中起重要作用。(2)妊娠期高血压疾病组胎盘组织NO水平及NOS活性显著降低,与胎盘组织Apelin mRNA表达水平呈正相关关系,提示胎盘组织Apelin水平下降,NOS活性低下,使NO合成下降,进一步加重胎盘缺血缺氧,促进妊娠期高血压疾病的发生发展。
第三部分Apelin-36对胎盘绒毛NOS活性、NO及β-HCG分泌的影响
目的探讨Apelin对胎盘绒毛NOS活性和NO生成的影响。方法体外培养正常足月胎盘绒毛,用血管紧张素(AngⅡ)及不同浓度的Apelin-36作用于胎盘绒毛,采用双抗体夹心酶联免疫吸附法(ELISA)测定胎盘绒毛培养液中β-HCG水平,硝酸还原酶法检测胎盘绒毛NO的水平,分光光度法测定胎盘绒毛NOS的活性。结果Apelin-36各组绒毛培养上清液β-hCG水平与对照组比较,差异无统计学意义(P均<0.05),即Apelin-36对足月妊娠胎盘绒毛分泌β-hCG无影响。加入Apelin培养的各组绒毛NO水平较对照组均明显升高(P <0.01),差异有统计学意义。与10ng/ml Apelin组比较,50ng/ml及250ng/ml Apelin两组的NO水平明显升高,差异有统计学意义(P <0.01)。50ng/ml及250ng/ml Apelin两组之间的NO水平差异无统计学意义(P >0.05)。加入Apelin培养的各组绒毛总NOS ( tNOS)活性较对照组均明显升高(P <0.01),其中主要是内皮型NOS (eNOS)活性的升高,各浓度组均有升高,差异有统计学意义(P <0.01),而诱导型NOS (iNOS) )活性无明显改变(P >0.05)。与对照组比较,AngⅡ可显著减少胎盘绒毛NO水平(P<0.01),差异有统计学意义。在AngⅡ作用基础上,分别加入10ng/ml Apelin,250ng/ml Apelin后,可见Apelin促进NO释放,其中250ng/ml Apelin抑制AngⅡ的作用更明显(P < 0.01)。结论Apelin-36对足月妊娠胎盘绒毛分泌β-hCG无影响。Apelin-36可能通过增强胎盘绒毛内皮型NOS的活性,增加NO的生成。AngⅡ可使胎盘绒毛的NOS活性及NO生成显著下降,而加用Apelin后,可使NOS活性逐渐恢复,NO生成增加,从而发挥其可能的生物学作用。
Background Hypertensive disorder complicating pregnancy(HDCP) is a common complication during the late pregnancy and the leading cause of maternal and fetal morbidity and the mortality. But the exact cause and pathogenesis of HDCP have not been clear out, there is not any sure or effective treatment for HDCP. Recently, a great many studies about HDCP have been made and some hypotheses were drawn out, such as immune, placenta shallow invasion, blood vessel endothelium damaged, theory of heredity, theory of innutrition, theory of insulin resistance. From plenty of research, we know the key-link for HDCP is the vascular endothelial damage and the damage is wide in body. The imbalance of nitric oxide (NO), endothelin(ET), prostacyclin(PGI2), thromboxane A2(TXA2), plasminogen activation inhibition factor(PAI), tissue plasminogen activation et al, will result in the ET and TXA2 secreting increased, on the other hand NO and PGI2 decreased which leads to all small arteries spasm, then every organ blooding perfuse is deficient. Now, ischemic and hypoxia of placenta is the major reason leading to endothelia cells damaged. To search the factors about placenta ischemic and hypoxia would get a new treatment for HDCP.
Apelin is an active cardiovascular peptide which was founded recently. It is a endogenic ligand of G protein couple receptor APJ. In the cardiovascular system, Apelin exerts potent vasodilator and positive inotropic activities. It can promote NO synthesis too. In modulating water-electrolyte metabolism and mediating immuno- regulation. Apelin plays a significant role. There are several isoforms about Apelin and the isoform Apelin-36 and Apelin-13 are common. Now the study about Apelin and APJ is just beginning. There are little reports about Apelin on pregnancy.
NO is a relaxing factor of blood vessel endothelium. It has a significant role in regulating fetoplacental circulation. During gestational period, placenta is the principle site for synthesis of NO. Nitric oxide synthase(NOS) is the capital rate-limiting step for synthesizing NO. Studies have confirmed that the reduction of NO in pregnancy period has close relationship to HDCP. But there is no report about the relationship between Apelin and the synthesis NO by placenta villus during pregnancy.
HDCP is a special disorder of pregnancy and placenta is a special organ of pregnancy. So to study the physiological and biochemical change of placenta will find the real cause which resulting in uteroplacental ischemia and hypoxia and to blood vessel endothelial cell damage. In our study, placenta was selected as the research object. For the goal of understanding the possible role of Apelin in the pathogenesis of HDCP and the relation with NO. Real-time fluorescence quantitative RT-PCR method (FQ-RT-PCR) was used to detect the expression of Apelin and APJ mRNA, and the expression of Apelin-36 and APJ protein were examined by immunohistochemistry SP and image analysis method in villus and placenta tissue. The stable metabolic end products of NO was measured with nitrate reductase and The NOS activity was assayed by spectrophotometry in placenta tissue. Apelin-36 was used to interfere in placenta villus tissue in vitro, and to observe whether Apelin can exert NO-NOS from placenta villus tissue. So to expect to offer a new find thinking for prevention and treatment measures about HDCP.
This study is divided into three parts:
PartⅠ
Expression and localization of Apelin and APJin human placenta and umbilical vessels
Objective To explore the expression and localization of endogenous vasoactive Apelin and its receptor APJ in first trimester placenta, term placenta and umbilical vessels. Methods Ten villus specimens were taken from the pregnant women conceived about 7～9 weeks, and fifteen placenta specimens and umbilical vessels were taken from pregnant women conceived about 37～41 weeks. Real-time fluorescence quantitative RT-PCR method (FQ-RT-PCR) was used to detect the expression of Apelin and APJ mRNA, the expression and localization of Apelin-36 and APJ protein were examined by immunohistochemistry SP and image analysis methods (HPIAS-1000). Results (1) The expression of Apelin mRNA was significantly increased in placentas tissue from term pregnancy compared to first trimester placenta (P <0.05), but the expression of APJ mRNA in two groups had not statistical significant (P>0. 05). (2) The result of immunohistochemistry demonstrated that Apelin-36 was predominantly localized in villous syncytiotrophoblasts and cytotrophoblasts, with weak staining of villous stroma at first trimester. In late trimester, Apelin-36 was was predominantly localized in villous syncytiotrophoblasts, it can be detected in capillaries and a few cells of villous stroma too. APJ predominantly localized in nuclei of villous syncytiotrophoblasts and cytotrophoblasts, secondly, localized in villous endothelium and stroma of placental tissue. Moreover there are stainings of APJ in some vessel muscle cells. (3)There is weak expression of Apelin-36 in villus in first trimester , but its expression increased significantly in placental tissue in the late trimester, and the difference had statistical significant (P <0. 01). The level of APJ expression has no difference between two groups (P >0.05). (4)The results showed that Apelin-36 and APJ appeared in the endothelial cells of human umbilical vessels, APJ staining was detected in some vascular smooth muscle cells of human umbilical vessels. But staining of Apelin-36 and APJ were weaker in umbilical vein than that in umbilical artery. Conclusion Both Apelin and APJ receptor are expressed in placental villus from the first to the third trimester of gestation, suggesting that the autocrine regulation system about Apelin-APJ exists in placenta, and the level of mRNA transcripts for Apelin increased may be associated with the need to increase blood priming volume in placenta in late trimester of gestation. Apelin and and its recptor APJ are positively expressed in the endothelial cells of human umbilical veins. However this expression decreased in umbilical artery. More active compounds may exist in umbilical artery blood to meet the need of fetal growth, and this causes that difference.
PartⅡ
Study on Apelin and nitric oxide in the pathogenesis of Hypertensive disorder complicating pregnancy
Objective To understand the possible role of Apelin and the relationship with nitric oxide (NO) in the pathogenesis and progression of HDCP. The expression of Apelin peptide and its acceptor in the placental tissue of normal pregnancy and HDCP were detected. methods Select 36 cases from the pregnant-woman of HDCP during 36～41 gestational weeks,including 10 cases of Gestational hypertension(GH) and 11 cases of Mild-Preeclampsia(MP) and 15 cases of Severe-preeclampsia(SP). Meanwhile, selected 15 normal pregnant women as the control group. The expression of Apelin-36 and its acceptor APJ-protein was detected with immunohistochemistry technique. The expression of Apelin and its acceptor APJ mRNA was detected with fluorescent quantitation RT-PCR. The level of NO was detected with nitrate reductase technique and the activity of NOS was detected with spectrophotometry. Results (1)The expression site of Apelin-36 and its acceptor APJ was not obviously different between the placental tissue of HDCP and normal pregnancy. The main expression site of Apelin-36 and its acceptor APJ located in the villous syncytiotrophoblasts. (2)The expression of Apelin-36 mRNA and Apelin-36 in the placental tissue of HDCP was the significantly lower than that in the control group (P <0.01), and it decreased gradually according to GH, MP, SP(P <0.01, P <0.05). The levels of expression of Apelin-36 mRNA and Apelin-36 were declined in each group. The levels of Apelin-36 mRNA and Apelin-36 expression in the group of MP and SP were the notable lower than the group of GH(P <0.01, P <0.01). The levels of Apelin-36 mRNA and Apelin-36 expression has statistical significance between the group of MP and SP (P <0.05). (3)The level of APJ mRNA and its protein expression in HDCP showed no statistical significance compared with the control group(P <0. 05). (4) The level of NO from the placenta HDCP was significant lower than that of the control group (P <0.01). and they were decreased gradually according to GH, MP ,SP (P<0.01, P <0.05). The levels of NO in the placenta tissue of MP and SP were lower than the that in GH(P <0.01, P<0.01). There was significant difference between the MP and SP (P <0.05). There was no significant difference between GH and the control group (P >0.05). (5) The level of NOS in HDCP group was notable lower than that in the control(P <0.01). and they were decreased gradually according to GH, MP,SP (P < 0.01, P < 0.05). The levels of NOS in placenta tissue of MP and SP were lower than the that in GH (P <0.05), There was no significant difference between GH and the control group (P >0.05). (6)In HDCP group, the level of NO and the activity of NOS were positive correlation with Apelin mRNA. The coefficient correlation were 0. 823, 0.901 respectively (P <0.01, P <0.01). Conclusoin (1) Both Apelin and APJ receptor are expressed in placental villus of HDCP. In the placenta tissue of HDCP, the expression of Apelin mRNA and Apelin-36 is lower, and they are correlated with the condition of the HDCP. Apelin may play a important role in the pathogenesis of HDCP. (2) The level of NO and the activity of NOS were significant lower in HDCP, which were positive correlated to Apelin mRNA. These hints proved that the level of Apelin in placenta tissue from HDCP was lower, which results in lower activity of NOS, contributed to the synthesis of NO. The lower NO aggravates the placenta ischemic and hypoxia, indicating that this may be involved in the progression of HDCP.
PartⅢEffect of Apelin on production of NO, HCG and activity of NOS in human placental villus
Objective To investigate the effects of Apelin on nitric oxide synthase (NOS) activity and nitric oxide (NO) production in placental villus. Methods The experiments were performed in human term placental villus in vitro. The cultured villus were treated with Apelin-36 and angiotensin II(Ang II).ELISA was used to detectβ-HCG levels in the medium of the cultures. The amount of NO in placental villus was detected by the nitrate reductase technique. The NOS activity in placental villi was measured with spectrophotometric assay. Resultsβ-HCG levels in the medium of placental villus in each groups were not different with that in control group.(P>0.05). It suggested that the production ofβ-hCG from human term placental villus was not affected by Apelin-36. NO levels in the villus with Apelin-36 were significantly higher than that in control group(P <0.01). In comparison with 10ng/ml Apelin group, the NO levels were significantly higher in 50ng/ml and 250ng/ml Apelin-36 group(P <0.01). There was no significant difference in NO level between 50ng/ml and 250ng/ml Apelin group(P>0.05). Total nitric oxide synthase (tNOS) activities in placental villus in each groups cultured with Apelin were significantly higher than that in control group(P<0.01), and endothelial NOS (eNOS) activities were mainly higher in all concentration groups(P<0. 01), however, the inducible NOS(iNOS) activities were not altered (P>0. 05). the level of NO and the activity of NOS were significantly decreased in the villus induced by AngII(P < 0.01)and Apelin could prevent this decrease(P < 0.05). Conclusion The production ofβ-hCG from human term placental villus was not affected by Apelin-36. Apelin-36 may increase the level of NO by the enhancement of the activity of eNOS in placental villus. AngII can Reduced activity of NOS and production of NO, which could be prevented with Apelin. Apelin may play its possible biological role by inducing production of NO in placenta.
妊娠期高血压疾病(HDCP)是一种发生在妊娠中晚期的常见并发症,是孕产妇和围生儿发病及死亡的主要原因,由于其病因及发病机理仍不明确,目前尚无一种确切而有效的预防和治疗方法。近两个世纪以来,国内外学者对有关妊娠期高血压疾病的病因及发病机理进行了大量研究,根据流行病学调查和实验研究提出了多种病因学假说,如:免疫学说、胎盘浅着床学说、血管内皮受损学说、遗传学说、营养缺乏学说、胰岛素抵抗学说等。大量研究表明,妊娠期高血压疾病基本发病机制为广泛的血管内皮细胞损伤。维持内皮细胞正常功能的几大因子平衡失调是该病发病的关键,主要有一氧化氮(nitric oxide,NO)和内皮素(ET)、前列环素(PGI2)、血栓素A2 (TXA2)、纤溶酶原激活抑制因子(PAI)和组织纤溶酶原激活因子(tPA)等。平衡失调的直接后果是缩血管因子(ET和TXA2)分泌增加,扩血管因子(NO和PGI2)分泌减少,导致患者出现全身小动脉痉挛,使各脏器的血流灌注减少。目前,多数学者们认为胎盘缺血缺氧是导致内皮细胞激活和损伤的更深层次原因,它在妊娠期高血压疾病的发病机制中应该更接近于起始病因。探讨胎盘缺血缺氧的有关因素,将有可能为该病的防治提供新的思路。
Apelin为新近发现的一种血管活性多肽,是孤儿G蛋白偶联性受体APJ的内源性配体,具有扩张血管、增强心肌收缩力、促进NO合成的特性,在调节水盐代谢以及介导免疫调节方面也起重要作用。已确认Apelin多肽存在多种亚型,其中以Apelin-36及Apelin-13较为常见,目前对Apelin及其受体APJ的研究刚刚开始,在妊娠方面的研究报道极少。
NO是血管内皮源性舒张因子,在调节胎儿胎盘循环中起重要作用。胎盘是妊娠期一氧化氮合成的主要场所,NOS是NO合成的最重要的限速步骤,以往的研究证实妊娠期NO合成减少与HDCP发病密切相关,有关Apelin与胎盘绒毛组织NO生成的关系未见报道。
妊娠期高血压疾病是妊娠期特有疾病,而胎盘是妊娠期特有的器官,因此研究胎盘的生理生化改变,将有可能发现引起子宫胎盘缺血缺氧及血管内皮细胞受损的真正原因。本课题选择胎盘为研究对象,采用免疫组织化学方法和实时荧光定量聚合酶链反应(荧光定量RT-PCR)检测Apelin、APJ在绒毛及胎盘组织中的表达、硝酸还原酶法检测胎盘组织NO的水平,分光光度法测定胎盘组织中NOS的活性。探讨Apelin在妊娠期高血压疾病中的作用及其与NO之间的关系。并采用Apelin-36对体外培养的胎盘绒毛进行干预,观察Apelin对胎盘绒毛NO-NOS的影响,以期为妊娠期高血压疾病的防治提供新的思路。
本研究内容包括三部分,现概述如下:
第一部分Apelin及受体APJ在正常妊娠早孕绒毛和晚孕胎盘及脐血管组织中的表达及定位研究
目的探讨血管活性肽Apelin及其受体APJ在早期妊娠绒毛、晚期妊娠胎盘及脐血管组织中的表达和定位。方法分别取10例妊娠7～9周妇女(早孕组)的绒毛组织和15例妊娠37～41周(晚孕组)妇女的胎盘及脐带组织,采用实时荧光定量逆转录聚合酶链反应法(荧光定量RT-PCR)及免疫组织化学染色分析法(SP)检测两组孕妇胎盘Apelin及其受体APJ mRNA和蛋白的表达情况,并用高清晰度彩色图文分析系统(HPIAS - 1000)对其进行定量分析。同时用免疫组织化学染色分析法观察Apelin-36及其受体APJ在正常妊娠脐带血管组织中的定位表达情况。结果(1)荧光定量RT-PCR结果显示,晚孕组胎盘绒毛组织Apelin mRNA水平显著高于早孕组绒毛组织(P<0.05),而APJ mRNA在不同妊娠期绒毛组织中的差异无统计学意义(P >0.05)。(2)免疫组化研究结果显示,Apelin-36在早孕绒毛组织中的表达部位为绒毛合体滋养细胞及细胞滋养细胞,少数间质细胞中也可见到Apelin-36弱阳性表达;在晚孕胎盘组织中,主要表达于胎盘滋养细胞,部分血管内皮细胞及少数间质细胞也可见到Apelin-36的阳性表达,阳性染色主要位于胞浆。APJ蛋白在早孕绒毛组织及晚孕胎盘组织的表达主要位于合体滋养细胞及细胞滋养细胞,其次表达的部位为血管内皮细胞及间质细胞,少数血管平滑肌细胞中也有APJ的表达,阳性染色主要位于胞核。(3)彩色图文分析系统结果显示,Apelin-36在早孕绒毛组织中表达较弱,而在晚孕胎盘组织中阳性表达明显增强,差异有统计学意义(P < 0.01) ,受体APJ在早孕组与晚孕组间的表达差异无统计学意义(P > 0.05)。(4)免疫组化研究结果显示,Apelin-36及受体APJ蛋白在脐静脉血管内皮均有阳性表达,在脐动脉血管内皮细胞中的表达则明显减弱。在脐血管平滑肌组织中,受体APJ散在表达于内皮细胞周围的平滑肌细胞中,未发现Apelin-36的阳性表达。结论正常早孕绒毛及晚孕胎盘组织中有Apelin及其受体APJ的表达,即胎盘组织内存在Apelin- APJ体系的自分泌调节系统,晚孕期胎盘组织Apelin mRNA转录水平增高,可能与妊娠晚期胎盘血流灌注量的需求增加有关。在晚期妊娠的脐静脉血管内皮细胞中,Apelin-36及受体APJ均呈阳性表达,脐动脉血管内皮细胞则表达减弱,这种表达的差异性,可能是由于妊娠晚期脐静脉较脐动脉内皮含有更丰富的活性物质,以适应胎儿生长发育的需要。
第二部分Apelin与妊娠期高血压疾病发病及NO关系的研究
目的检测血管活性肽Apelin及其受体APJ在正常妊娠和妊娠期高血压疾病(HDCP)患者胎盘组织中的表达,探讨Apelin在妊娠期高血压疾病发生、发展中的作用及其与一氧化氮(NO)的关系。方法选择孕周为36～41周的妊娠期高血压疾病患者36例,其中妊娠期高血压10例,轻度子痫前期11例,重度子痫前期15例;选择同期正常孕妇15例作为对照组。采用免疫组织化学染色分析法检测两组孕妇胎盘组织中Apelin-36及受体APJ的表达,荧光定量RT-PCR检测Apelin及APJmRNA的表达。硝酸还原酶法检测胎盘组织NO的水平,分光光度法测定胎盘组织中NOS的活性,结果(1)妊娠期高血压疾病组胎盘组织Apelin-36及受体APJ在表达部位上与对照组(正常晚孕组)无明显差异,主要表达的部位为绒毛合体滋养细胞及细胞滋养细胞。(2)妊娠期高血压疾病组胎盘Apelin mRNA及Apelin-36的表达量显著低于对照组(P < 0.01),且随着病情程度的加重,各组胎盘中Apelin mRNA及Apelin-36的表达水平呈下降的趋势,轻度、重度子痫前期胎盘Apelin mRNA及Apelin-36的表达水平均显著低于妊娠期高血压组(P < 0.01,P < 0.01),轻、重度子痫前期两组之间Apelin mRNA及Apelin-36的表达水平比较,差异也有统计学意义(P < 0.05)。(3)妊娠期高血压疾病各组胎盘组织APJmRNA及蛋白水平的表达与对照组比较差异无统计学意义(P >0.05)。(4)妊娠期高血压疾病组胎盘组织NO水平与对照组相比显著降低(P < 0.01),且随着病情程度的加重,各组胎盘组织中NO水平呈下降的趋势,轻度、重度子痫前期组胎盘组织NO水平均低于妊娠期高血压组(P < 0.01, P < 0.01),轻、重度子痫前期两组之间比较,差异也有统计学意义(P < 0.05)。妊娠期高血压组胎盘组织NO水平与对照组比较,差异无统计学意义(P > 0.05)。(5)妊娠期高血压疾病组胎盘组织NOS水平与对照组相比显著降低(P < 0.01),且随着病情程度的加重,各组胎盘组织中NOS水平呈下降的趋势,妊娠期高血压疾病组轻度、重度子痫前期胎盘组织NO水平均低于妊娠期高血压组(P < 0.05),妊娠期高血压组胎盘组织NO水平与对照组比较,差异无统计学意义(P > 0.05)。(6)妊娠期高血压疾病组胎盘组织NO水平及NOS活性与Apelin mRNA水平呈正相关,相关系数分别为0.823,0.901(P <0.01, P <0.01)。结论(1)血管活性肽Apelin及其受体APJ在妊娠期高血压疾病患者胎盘组织中均有表达,妊娠期高血压疾病患者胎盘组织中Apelin mRNA及Apelin-36表达水平低下,并与病情程度相关,可能在妊娠期高血压疾病的发病中起重要作用。(2)妊娠期高血压疾病组胎盘组织NO水平及NOS活性显著降低,与胎盘组织Apelin mRNA表达水平呈正相关关系,提示胎盘组织Apelin水平下降,NOS活性低下,使NO合成下降,进一步加重胎盘缺血缺氧,促进妊娠期高血压疾病的发生发展。
第三部分Apelin-36对胎盘绒毛NOS活性、NO及β-HCG分泌的影响
目的探讨Apelin对胎盘绒毛NOS活性和NO生成的影响。方法体外培养正常足月胎盘绒毛,用血管紧张素(AngⅡ)及不同浓度的Apelin-36作用于胎盘绒毛,采用双抗体夹心酶联免疫吸附法(ELISA)测定胎盘绒毛培养液中β-HCG水平,硝酸还原酶法检测胎盘绒毛NO的水平,分光光度法测定胎盘绒毛NOS的活性。结果Apelin-36各组绒毛培养上清液β-hCG水平与对照组比较,差异无统计学意义(P均<0.05),即Apelin-36对足月妊娠胎盘绒毛分泌β-hCG无影响。加入Apelin培养的各组绒毛NO水平较对照组均明显升高(P <0.01),差异有统计学意义。与10ng/ml Apelin组比较,50ng/ml及250ng/ml Apelin两组的NO水平明显升高,差异有统计学意义(P <0.01)。50ng/ml及250ng/ml Apelin两组之间的NO水平差异无统计学意义(P >0.05)。加入Apelin培养的各组绒毛总NOS ( tNOS)活性较对照组均明显升高(P <0.01),其中主要是内皮型NOS (eNOS)活性的升高,各浓度组均有升高,差异有统计学意义(P <0.01),而诱导型NOS (iNOS) )活性无明显改变(P >0.05)。与对照组比较,AngⅡ可显著减少胎盘绒毛NO水平(P<0.01),差异有统计学意义。在AngⅡ作用基础上,分别加入10ng/ml Apelin,250ng/ml Apelin后,可见Apelin促进NO释放,其中250ng/ml Apelin抑制AngⅡ的作用更明显(P < 0.01)。结论Apelin-36对足月妊娠胎盘绒毛分泌β-hCG无影响。Apelin-36可能通过增强胎盘绒毛内皮型NOS的活性,增加NO的生成。AngⅡ可使胎盘绒毛的NOS活性及NO生成显著下降,而加用Apelin后,可使NOS活性逐渐恢复,NO生成增加,从而发挥其可能的生物学作用。
Background Hypertensive disorder complicating pregnancy(HDCP) is a common complication during the late pregnancy and the leading cause of maternal and fetal morbidity and the mortality. But the exact cause and pathogenesis of HDCP have not been clear out, there is not any sure or effective treatment for HDCP. Recently, a great many studies about HDCP have been made and some hypotheses were drawn out, such as immune, placenta shallow invasion, blood vessel endothelium damaged, theory of heredity, theory of innutrition, theory of insulin resistance. From plenty of research, we know the key-link for HDCP is the vascular endothelial damage and the damage is wide in body. The imbalance of nitric oxide (NO), endothelin(ET), prostacyclin(PGI2), thromboxane A2(TXA2), plasminogen activation inhibition factor(PAI), tissue plasminogen activation et al, will result in the ET and TXA2 secreting increased, on the other hand NO and PGI2 decreased which leads to all small arteries spasm, then every organ blooding perfuse is deficient. Now, ischemic and hypoxia of placenta is the major reason leading to endothelia cells damaged. To search the factors about placenta ischemic and hypoxia would get a new treatment for HDCP.
Apelin is an active cardiovascular peptide which was founded recently. It is a endogenic ligand of G protein couple receptor APJ. In the cardiovascular system, Apelin exerts potent vasodilator and positive inotropic activities. It can promote NO synthesis too. In modulating water-electrolyte metabolism and mediating immuno- regulation. Apelin plays a significant role. There are several isoforms about Apelin and the isoform Apelin-36 and Apelin-13 are common. Now the study about Apelin and APJ is just beginning. There are little reports about Apelin on pregnancy.
NO is a relaxing factor of blood vessel endothelium. It has a significant role in regulating fetoplacental circulation. During gestational period, placenta is the principle site for synthesis of NO. Nitric oxide synthase(NOS) is the capital rate-limiting step for synthesizing NO. Studies have confirmed that the reduction of NO in pregnancy period has close relationship to HDCP. But there is no report about the relationship between Apelin and the synthesis NO by placenta villus during pregnancy.
HDCP is a special disorder of pregnancy and placenta is a special organ of pregnancy. So to study the physiological and biochemical change of placenta will find the real cause which resulting in uteroplacental ischemia and hypoxia and to blood vessel endothelial cell damage. In our study, placenta was selected as the research object. For the goal of understanding the possible role of Apelin in the pathogenesis of HDCP and the relation with NO. Real-time fluorescence quantitative RT-PCR method (FQ-RT-PCR) was used to detect the expression of Apelin and APJ mRNA, and the expression of Apelin-36 and APJ protein were examined by immunohistochemistry SP and image analysis method in villus and placenta tissue. The stable metabolic end products of NO was measured with nitrate reductase and The NOS activity was assayed by spectrophotometry in placenta tissue. Apelin-36 was used to interfere in placenta villus tissue in vitro, and to observe whether Apelin can exert NO-NOS from placenta villus tissue. So to expect to offer a new find thinking for prevention and treatment measures about HDCP.
This study is divided into three parts:
PartⅠ
Expression and localization of Apelin and APJin human placenta and umbilical vessels
Objective To explore the expression and localization of endogenous vasoactive Apelin and its receptor APJ in first trimester placenta, term placenta and umbilical vessels. Methods Ten villus specimens were taken from the pregnant women conceived about 7～9 weeks, and fifteen placenta specimens and umbilical vessels were taken from pregnant women conceived about 37～41 weeks. Real-time fluorescence quantitative RT-PCR method (FQ-RT-PCR) was used to detect the expression of Apelin and APJ mRNA, the expression and localization of Apelin-36 and APJ protein were examined by immunohistochemistry SP and image analysis methods (HPIAS-1000). Results (1) The expression of Apelin mRNA was significantly increased in placentas tissue from term pregnancy compared to first trimester placenta (P <0.05), but the expression of APJ mRNA in two groups had not statistical significant (P>0. 05). (2) The result of immunohistochemistry demonstrated that Apelin-36 was predominantly localized in villous syncytiotrophoblasts and cytotrophoblasts, with weak staining of villous stroma at first trimester. In late trimester, Apelin-36 was was predominantly localized in villous syncytiotrophoblasts, it can be detected in capillaries and a few cells of villous stroma too. APJ predominantly localized in nuclei of villous syncytiotrophoblasts and cytotrophoblasts, secondly, localized in villous endothelium and stroma of placental tissue. Moreover there are stainings of APJ in some vessel muscle cells. (3)There is weak expression of Apelin-36 in villus in first trimester , but its expression increased significantly in placental tissue in the late trimester, and the difference had statistical significant (P <0. 01). The level of APJ expression has no difference between two groups (P >0.05). (4)The results showed that Apelin-36 and APJ appeared in the endothelial cells of human umbilical vessels, APJ staining was detected in some vascular smooth muscle cells of human umbilical vessels. But staining of Apelin-36 and APJ were weaker in umbilical vein than that in umbilical artery. Conclusion Both Apelin and APJ receptor are expressed in placental villus from the first to the third trimester of gestation, suggesting that the autocrine regulation system about Apelin-APJ exists in placenta, and the level of mRNA transcripts for Apelin increased may be associated with the need to increase blood priming volume in placenta in late trimester of gestation. Apelin and and its recptor APJ are positively expressed in the endothelial cells of human umbilical veins. However this expression decreased in umbilical artery. More active compounds may exist in umbilical artery blood to meet the need of fetal growth, and this causes that difference.
PartⅡ
Study on Apelin and nitric oxide in the pathogenesis of Hypertensive disorder complicating pregnancy
Objective To understand the possible role of Apelin and the relationship with nitric oxide (NO) in the pathogenesis and progression of HDCP. The expression of Apelin peptide and its acceptor in the placental tissue of normal pregnancy and HDCP were detected. methods Select 36 cases from the pregnant-woman of HDCP during 36～41 gestational weeks,including 10 cases of Gestational hypertension(GH) and 11 cases of Mild-Preeclampsia(MP) and 15 cases of Severe-preeclampsia(SP). Meanwhile, selected 15 normal pregnant women as the control group. The expression of Apelin-36 and its acceptor APJ-protein was detected with immunohistochemistry technique. The expression of Apelin and its acceptor APJ mRNA was detected with fluorescent quantitation RT-PCR. The level of NO was detected with nitrate reductase technique and the activity of NOS was detected with spectrophotometry. Results (1)The expression site of Apelin-36 and its acceptor APJ was not obviously different between the placental tissue of HDCP and normal pregnancy. The main expression site of Apelin-36 and its acceptor APJ located in the villous syncytiotrophoblasts. (2)The expression of Apelin-36 mRNA and Apelin-36 in the placental tissue of HDCP was the significantly lower than that in the control group (P <0.01), and it decreased gradually according to GH, MP, SP(P <0.01, P <0.05). The levels of expression of Apelin-36 mRNA and Apelin-36 were declined in each group. The levels of Apelin-36 mRNA and Apelin-36 expression in the group of MP and SP were the notable lower than the group of GH(P <0.01, P <0.01). The levels of Apelin-36 mRNA and Apelin-36 expression has statistical significance between the group of MP and SP (P <0.05). (3)The level of APJ mRNA and its protein expression in HDCP showed no statistical significance compared with the control group(P <0. 05). (4) The level of NO from the placenta HDCP was significant lower than that of the control group (P <0.01). and they were decreased gradually according to GH, MP ,SP (P<0.01, P <0.05). The levels of NO in the placenta tissue of MP and SP were lower than the that in GH(P <0.01, P<0.01). There was significant difference between the MP and SP (P <0.05). There was no significant difference between GH and the control group (P >0.05). (5) The level of NOS in HDCP group was notable lower than that in the control(P <0.01). and they were decreased gradually according to GH, MP,SP (P < 0.01, P < 0.05). The levels of NOS in placenta tissue of MP and SP were lower than the that in GH (P <0.05), There was no significant difference between GH and the control group (P >0.05). (6)In HDCP group, the level of NO and the activity of NOS were positive correlation with Apelin mRNA. The coefficient correlation were 0. 823, 0.901 respectively (P <0.01, P <0.01). Conclusoin (1) Both Apelin and APJ receptor are expressed in placental villus of HDCP. In the placenta tissue of HDCP, the expression of Apelin mRNA and Apelin-36 is lower, and they are correlated with the condition of the HDCP. Apelin may play a important role in the pathogenesis of HDCP. (2) The level of NO and the activity of NOS were significant lower in HDCP, which were positive correlated to Apelin mRNA. These hints proved that the level of Apelin in placenta tissue from HDCP was lower, which results in lower activity of NOS, contributed to the synthesis of NO. The lower NO aggravates the placenta ischemic and hypoxia, indicating that this may be involved in the progression of HDCP.
PartⅢEffect of Apelin on production of NO, HCG and activity of NOS in human placental villus
Objective To investigate the effects of Apelin on nitric oxide synthase (NOS) activity and nitric oxide (NO) production in placental villus. Methods The experiments were performed in human term placental villus in vitro. The cultured villus were treated with Apelin-36 and angiotensin II(Ang II).ELISA was used to detectβ-HCG levels in the medium of the cultures. The amount of NO in placental villus was detected by the nitrate reductase technique. The NOS activity in placental villi was measured with spectrophotometric assay. Resultsβ-HCG levels in the medium of placental villus in each groups were not different with that in control group.(P>0.05). It suggested that the production ofβ-hCG from human term placental villus was not affected by Apelin-36. NO levels in the villus with Apelin-36 were significantly higher than that in control group(P <0.01). In comparison with 10ng/ml Apelin group, the NO levels were significantly higher in 50ng/ml and 250ng/ml Apelin-36 group(P <0.01). There was no significant difference in NO level between 50ng/ml and 250ng/ml Apelin group(P>0.05). Total nitric oxide synthase (tNOS) activities in placental villus in each groups cultured with Apelin were significantly higher than that in control group(P<0.01), and endothelial NOS (eNOS) activities were mainly higher in all concentration groups(P<0. 01), however, the inducible NOS(iNOS) activities were not altered (P>0. 05). the level of NO and the activity of NOS were significantly decreased in the villus induced by AngII(P < 0.01)and Apelin could prevent this decrease(P < 0.05). Conclusion The production ofβ-hCG from human term placental villus was not affected by Apelin-36. Apelin-36 may increase the level of NO by the enhancement of the activity of eNOS in placental villus. AngII can Reduced activity of NOS and production of NO, which could be prevented with Apelin. Apelin may play its possible biological role by inducing production of NO in placenta.
引文
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