PEDF蛋白对子痫前期发病影响的研究
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摘要
研究背景
妊娠期高血压疾病是妊娠期特发的全身性疾病,我国发病率9.4%,国外报道7%-12%,迄今为止仍然是母婴发病率及死亡率较高的主要原因。子痫前期(preeclampsia)是该疾病最常见的类型,流行病学调查结果显示,子痫前期的发病率约为5-10%,疾病的病因及发生发展过程还不是十分明确,目前主要认为免疫失衡机制,胎盘浅着床,血管内皮细胞损伤和胎盘滋养细胞缺血、胰岛素抵抗、钙平衡失调等机制可能具有重要的作用。有研究认为,子痫前期患者存在子宫蜕膜血管重铸障碍,绒毛的数量减少、绒毛内的血管发育不良及间质纤维化等,胎盘缺血缺氧,引起广泛的血管内皮细胞的损伤,最终导致多器官、多系统功能损害。而正常胎盘血管床生成依赖于胎盘内血管生成诱导因子与抑制因子的复杂联系。
色素上皮衍生因子(pigment epithelium-derived factor,PEDF)是目前已知最强的血管生成抑制剂,它是一种分子量大小约为50kDa的分泌型糖蛋白。PEDF不仅可以抑制新生血管的形成,还可逆转已经形成的血管。而且其抑制血管生成的活性具有明显的选择性,它可抑制生成病理性血管,但不影响生理性血管的形成。目前研究发现,PEDF是一种多功能蛋白,广泛分布于成人和胎儿多种组织(如眼、脑、肝、脂肪、肌肉、胎盘、卵巢、子宫内膜组织等等),具有营养神经、抗血管生成、抗血管的通透性、抗氧化、抗肿瘤生成、调节脂肪细胞及胰岛素生成等特性。2008年美国Beth等学者发现PEDF在正常妊娠妇女的脉管系统和胎盘的滋养层高度表达,很可能就是胎盘血管形成过程的静止因子,对维持血管的静止状态和确保正常血管的完整性具有重要作用。
以往的大量研究表明,整个妊娠时期母体的胎盘组织以及血清中都可能有血管内皮生长因子(VEGF)及其受体(VEGF-R)表达。VEGF是一种有效的血管生成诱导物,VEGF与其受体结合后可通过旁分泌途径调控血管内皮细胞分化,促进内皮细胞的增生、迁移与浸润,同时VEGF可增加血管的通透性,造成大量血浆蛋白外渗,提供了促进血管生成所需要的暂时性基质。VEGF还能诱导内皮细胞中具有抗凋亡作用的Bcl-2的表达,抑制内皮细胞的凋亡,可见,VEGF在促进血管形成和维持血管内皮细胞的功能方面具有重要的作用,VEGF在胚胎着床、胎盘血管发育的过程中也非常关键。正常妊娠早期胎盘组织以及血清中VEGF水平都处于相对升高的状态,可能一方面因为妊娠早期滋养细胞处于相对缺氧状态,低氧通过一系列信号转导通路,刺激VEGF水平上调,另一方面,在胎儿及胎盘生长发育过程中,需要充足血液和血氧供应,VEGF的表达增加是为了适应妊娠的生理改变。研究认为孕早期VEGF水平下降与子痫前期发病密切相关,子痫前期患者胎盘组织中和母体外周血清中VEGF水平明显下降,可能是子痫前期的发病因素之一。PEDF能够通过作用于Fas/FasL介导的信号转导通路,诱导血管内皮细胞的凋亡,从而抑制血管新生；此外,PEDF还可抑制血管紧张素Ⅱ对VEGF的上调,可见,PEDF是VEGF天然的负性调节剂。PEDF是否类似于VEGF也参与子痫前期的发病过程,目前尚未见文献报道。
正常妊娠时胚泡着床到子宫内膜及子宫肌层的过程主要由分化完全的滋养细胞来实现的,对滋养细胞的严密调控在形成正常的胎盘形成中非常重要,这一过程受到许多细胞因子、粘附分子、激素、氧张力等精确的调节,滋养细胞的功能异常是一系列病理妊娠的重要原因,尤其是胎儿宫内生长受限及子痫前期,其中子痫前期又被称为滋养细胞功能障碍疾病。人早孕绒毛滋养细胞是一种具有侵袭和破坏能力的细胞,类似于肿瘤细胞,但不具有肿瘤细胞的成瘤特性和转移能力。研究已表明,滋养细胞的迁移、侵袭能力的下降可能是子痫前期形成的一个重要原因。文献报道,PEDF能通过下调基质金属蛋白酶(MMP-9)的表达水平,以降低肿瘤细胞的侵袭性,从而具有抗肿瘤的功效。目前研究也证实了MMP-9基因的表达下降,能够降低滋养细胞侵袭能力,与子痫前期的发病密切相关。但PEDF是否能够通过类似的作用机制降低滋养细胞的侵袭能力,是否参与子痫前期的发病过程,这一切都需进一步验证。
因此,我们推测色素上皮衍生因子(PEDF)很可能在子痫前期发病中发挥重要作用,且其对子痫前期发病影响的研究,目前国内外尚未见报道。故本课题从影响胎盘血管床生成因素为切入点,研究子痫前期患者胎盘中PEDF蛋白的表达情况,及其与胎盘组织中血管内皮生长因子(VEGF)及胎盘微血管密度的关系；并进一步从滋养细胞层面上研究PEDF蛋白在体外培养的缺氧的滋养细胞中的表达特点,及缺氧诱导后滋养细胞功能的变化；通过将构建的载有PEDF基因的质粒转染到滋养细胞中,研究PEDF对滋养细胞功能的影响,从而探讨PEDF对子痫前期发病的影响,为进一步寻找子痫前期的发病原因及机理提供基础。
第一部分PEDF蛋白在子痫前期与正常妊娠胎盘组织中的表达
[目的]探讨色素上皮衍生因子(PEDF)、血管内皮生长因子(VEGF)在子痫前期患者胎盘组织的表达及其与胎盘血管病变的关系。
[方法]随机选取2011年10月至2013年1月南方医院住院分娩的子痫前期患者60例(其中子痫前期轻度mPE组30例,子痫前期重度sPE组30例)为研究对象,同期分娩的健康孕妇40例作为对照组,用western blot、免疫荧光组织化学双重标记方法检测子痫前期患者和正常妊娠妇女(对照组)胎盘组织中PEDF、VEGF的表达,并通过免疫荧光方法计数胎盘微血管密度(MVD),同时进行相关性分析。采用完全随机设计资料的多组样本均数比较的单因素方差分析(Oneway-Anova),两两比较用LSD(least significant difference)检验,指标间采用双变量相关分析中的pearson相关分析。
[结果](1)胎盘的滋养细胞和血管内皮细胞中均表达PEDF、VEGF,主要表达于胞质及胞膜上,且两个因子表达位置大体相同。(2)子痫前期各组患者胎盘组织中PEDF阳性表达高于正常对照组,差异有统计学意义(P<0.05),而且sPE组PEDF阳性表达高于mPE组(P<0.05)；(3)与正常对照组相比,子痫前期轻度组和子痫前期重度组患者胎盘组织中VEGF表达减少,分别为0.0840±0.1006,0.0785±0.0748,0.0767±0.0738,差异有统计学意义(P<0.05),而mPE组与sPE组VEGF表达无差异(P>0.05)；(4)正常对照组,mPE组,sPE组MVD计数依次递减,分别为136.06±9.07,106.40±8.51,92.65±7.89。子痫前期各组与正常对照组比较差异有统计学意义(P<0.05),mPE组与sPE组差异有统计学意义P<0.05)；(5)子痫前期各组和正常对照组胎盘组织中PEDF表达与VEGF表达及MVD计数呈显著负相关(r=-0.365,P<0.05；r=-0.655,P<0.05),VEGF表达与MVD数值的变化则呈显著正相关(r=0.448,P<0.05)。
[结论]PEDF与VEGF在胎盘的滋养细胞和血管内皮细胞中表达位置大体相同。PEDF蛋白在子痫前期患者胎盘组织中表达升高,而VEGF及MVD表达下降,后两者均与PEDF表达呈负相关。这提示PEDF可影响胎盘血管重铸,可能参与子痫前期的发生和发展过程。
第二部分缺氧对滋养细胞中PEDF的表达及细胞功能的影响
[目的]检测低氧浓度体外培养的人早孕绒毛滋养细胞株HTR-8/SVneo细胞中PEDF蛋白及其mRNA表达的差异,并研究缺氧对滋养细胞功能的影响,以探求PEDF蛋白对子痫前期发病的影响。
[方法]低氧浓度体外培养人早孕绒毛滋养细胞株HTR-8/SVneo细胞,倒置显微镜观察缺氧对滋养细胞形态的影响,并采用细胞免疫荧光化学技术、实时荧光定量PCR检测各组滋养细胞中PEDF蛋白及mRNA表达的差异。用Tunnel法测定细胞凋亡情况,CKK-8法检测各组滋养细胞的增殖能力,Transwell板测定各组滋养细胞的侵袭能力。采用析因设计资料的方差分析,两组间比较采用独立样本t检验(Independent-Samples T Test),指标间采用双变量相关分析中的spearman相关分析。
[结果](1)在1%低氧浓度环境下体外培养HTR8/SVneo细胞的生长情况：HTR8-SVneo细胞对缺氧反应敏感,细胞形态发生明显的改变,细胞由多边形或梭形逐渐向圆形变化,突起变小,甚至部分消失。(2)细胞免疫荧光结果显示：PEDF蛋白在各组HTR8-Svneo细胞均有表达,其免疫反应产物表达主要定位于胞浆和胞膜中；与常氧组相比,缺氧24小时滋养细胞中PEDF蛋白的表达量无显著差异(P>0.05)；但缺氧48小时后,与常氧组相比,低氧组PEDF蛋白的表达升高,差异有统计学意义(P<0.05),且缺氧时间越长,PEDF蛋白升高更多(P<0.05)。(3)实时荧光定量PCR结果显示：与常氧组相比,低氧组PEDF mRNA表达水平在缺氧24小时差异不显著(P>0.05),但缺氧48、72小时PEDF mRNA表达均升高,差异有统计学意义(P<0.05)；缺氧时间越长,PEDF mRNA表达越高(P<0.05)。以上结果表明缺氧对滋养细胞PEDF蛋白和其mRNA的表达呈时间依赖性。(4)Tunnel法测定滋养细胞凋亡：与常氧组相比,低氧组细胞凋亡数量在缺氧24、48小时无显著差异(P>0.05),但缺氧72小时细胞凋亡增多,差异有统计学意义(P<0.05)；(5)CKK-8法检测各组滋养细胞的增殖能力：各组滋养细胞的增殖能力无显著差异(P>0.05)。(6)Transwell测定滋养细胞侵袭能力：显微镜下可见各组均有数量不等的滋养细胞浸润至膜下室,与常氧组相比,1%低氧组滋养细胞在培养48、72小时浸润至膜下室的细胞减少,差异有统计学意义(P<0.05)。(7)各组HTR-8/SVneo细胞中PEDF蛋白表达、mRNA表达均与凋亡的滋养细胞呈显著正相关(r=0.772,P<0.05；r=0.839,P<0.05),PEDF蛋白表达与mRNA表达呈显著正相关(r=0.876,P<0.05)。(8)各组HTR-8/SVneo中PEDF蛋白表达与滋养细胞侵袭能力呈显著负相关(r=-0.615,P<0.05)。
[结论]我们结果提示缺氧环境能导致HTR8-SVneo滋养细胞中PEDF蛋白及mRNA表达增加,且随着缺氧时间的延长,PEDF表达呈显著增多趋势。缺氧可诱导滋养细胞的凋亡,而且明显抑制HTR8-SVneo滋养细胞的侵袭力,并随着缺氧时间的延长其侵袭能力进一步下降。但缺氧对滋养细胞的增殖影响不大。
第三部分载有PEDF基因的质粒转染到滋养细胞对滋养细胞增殖侵袭能力的影响
[目的]构建载有PEDF基因的质粒(pcDNA-PEDF),并将其转染到人早孕绒毛滋养细胞中,研究PEDF对滋养细胞凋亡、增殖与侵袭功能的影响。
[方法]将构建好的载有PEDF基因的质粒(pcDNA-PEDF)转染到人早孕绒毛滋养细胞中,通过细胞免疫荧光及实时荧光定量PCR,观察转染后24小时滋养细胞中PEDF蛋白及mRNA表达情况；并用Tunnel法测定细胞凋亡,CKK-8法检测各组滋养细胞的增殖能力,Transwell板测定各组滋养细胞的侵袭能力。多组间比较采用单因素方差分析(One-Way ANOVA),两两比较用LSD(least significant difference)检验,相关分析采用双变量的spearman相关分析.
[结果](1)与正常对照组相比,转染PEDF质粒24小时后,HTR-8/SVneo滋养细胞中PEDF蛋白及PEDFmRNA表达增多,差异有统计学意义(P<0.05),而阴性对照组与正常对照组比较无显著差异(P>0.05)；(2)与正常对照组相比,转染PEDF基因质粒的滋养细胞组的细胞凋亡增多,分别为9.67±1.52、15.67±2.52,差异有统计学意义(P<0.05),而阴性对照组与正常对照组比较无显著差异(P>0.05)；(3)转染PEDF基因质粒后,各组HTR-8/SVneo滋养细胞生长增殖无显著差异(P>0.05)；(4)与正常对照组相比,转染PEDF基因质粒的滋养细胞进入Transwell膜下室的细胞数降低,分别为39.00±4.58、20.33±2.52,差异有统计学意义(P<0.05),而阴性对照组与正常对照组比较无显著差异(P>0.05)；(5)PEDF蛋白表达、mRNA表达均与凋亡的滋养细胞呈显著正相关(r=0.970,P<0.05；r=0.745,P<0.05),PEDF蛋白表达与mRNA表达呈显著正相关(r=0.672,P<0.05)；(6)PEDF蛋白表达与滋养细胞侵袭能力呈显著负相关(产-0.882,P<0.05)。
[结论]PEDF对维持正常的人早孕绒毛滋养细胞功能非常重要,PEDF表达增多,可增加滋养细胞的凋亡,降低滋养细胞的侵袭能力,且PEDF蛋白表达水平与滋养细胞的侵袭能力呈显著负相关。
Research Background
Hypertensive disorder complicating pregnancy is the systemic disease which is idiopathetic in the gestational period, the morbidity in China is9.4%, while it is7%～12%abroad. Up to now, the disease is still the main reason for the high incidence and mortality rate in the pregnant women and fetus. Preeclampsia is the most frequent type of the disease. It is shown by the epidemiologic survey, that morbidity rate of preeclampsia is5-10%. The etiological factors and process is not yet full clear to date, at present the main reasons are considered as the immuno-unbalance, superficial embedded placenta, the lesions of vascular endothelial cell, the poorly developed vascular net of placenta and decidua and so on. The research has shown that there is the defective vascular remoldeling of uterine decidua in preclampsia, the decreased amount of chorionic villus, angiodysplasia in chorionic villus, and interstitial fibrosis, which induce the hypoxia of placenta and wide lesions of vascular endothelial cell. It lead the functional lesion of multiorgan and multisystem finally.Normal placental vascular development depends upon the complex interactions between angiogenic inducers and inhibitors within the placenta.
Pigment epithelium-derived factor (PEDF) is one of the most potent angiogenic inhibitors identified to date., which is a multifunctional50kDa secreted glycoprotein. Not only pigment epithelium-derived factor (PEDF) could inhibit the formation of new vessels, but also it can reverse the vessels which have been formative. And it is selective to inhibiting the formation of vessels, it inhibits the production of pathologic new vessels, while not affect the formation of physiologic vessels. The findings that pigment epithelium-derived factor(PEDF) is multifunctional protein have been identified to date, it has the function to give the nutrition to nerve, to resist the tumor, to reject the new vessels, to resist the vasopermeability, and it also has the personality of antioxygen and adjusting the cytopoiesis of adipose cells. In the year of2008, Beth and so on,who are American scholars, demonstrated that pigment epitheliumderived factor (PEDF), a potent inhibitor of angiogenesis, is expressed in both the vasculature and trophoblasts of placentas obtained from women with normal pregnancies. Pigment epitheliumderived factor (PEDF) is the factor responsible for inducing vascular quiescence and it is essential to maintain both vascular quiescence and to ensure normal vascular integrity.
A lot of previous study has indicated that, there are vascular endothelial growth factor(VEGF) and its receptor(VEGF-R) expressed in the placentas and maternal serum in the whole pregnanct periods. VEGF is an effective inducer of the production of vessels. After the combination of VEGF and its receptor, it could regulate the differentiation of vascular endothelial cells by the paracrine secretion, and promote the hyperplasy、migration and invasion of endothelial cells. Meanwhile VEGF could increase the permeability of vessels, which induced a lot of plasma protein extravasation, to afford the temporary base which is necessary to promote the information of vessels. VEGF induces the expression of anti-apoptosis material named Bcl-2on the endothelial cells, and inhibite the apoptosis of endothelial cells.So it is evident that VEGF is essential to promote the information of vessels and matain the function of vascular endothelial cells. And VEGF also plays an important role in the implantation of embryo and the developtment of placental vessels. In the early trimester of pregnancy, the level of VEGF in placentas and serum occupies the relative elevated condition. One reason is that there is relative hypoxia stasis in the early trimester of pregnancy, which induces the up-regulation of the level of VEGF by a series of signal transduction pathways. The other reason is to adapt the physiologyical changes of pregnancy, because it needs adequate sufficient blood and oxygen supply in the growth and development of fetus and placentas.The study has showed that there is intimate correlation between the desecended level of VEGF in the earlier pregnant period and the pathogenesis of preeclampsia.The obvious low level of VEGF in the placenta tissues and maternal peripheral blood-serum, which maybe one of the etiological factors for preeclampsia disease. PEDF could induce the apoptosis of vascular endothelial cells and inhibit the formation of new vessels by the signal transduction pathway, which is mediated by Fas/FasL. Furthermore, PEDF could inhibit the up-regulation of VEGF by the angiotonin Ⅱ. So PEDF is the natural negative regulator to VEGF. There is no report about whether PEDF, similarity like VEGF. involves in the pathogenesis of preeclampsia.
The implantation of blastocyst to the endometrium and myometrium is completed by the well differentiated trophoblast cells in normal pregnancy. It is essential important for the regulation to the trophoblast cells during the formation of normal placenta, the dysfunction of trophoblast cell is main cause to a series of pathogenic pregnancy, such as intrauterine growth retardation (IUGR) and pre-eclampsia. Human first trimester trophoblast cell is the cell,which has the invasive and destructive characteristics, similarity to tumor cell, but not tumorigenicity and metastasis. It has been shown in research,that the decreased ability of migration and invasion for the trophoblast cell plays an important role in preeclampsia. It was reported by literacy that PEDF could down-regulate the level of expression of matrix metalloproteinase (MMP-9),and then decrease the invasion ability of tumor cells, so it has the antineoplastic function.At present,the study has confirmed that the decreased expression level of matrix metalloproteinase induced the invasion ability of trophoblast cells, so to involve in the pathogenesis of preeclampsia. But it is still need to be verificated whether PEDF could also depressed the invasion ability of trophoblast cells and involve in the pathogenesis of preeclampsia by the similar mechanism,
Therefore, we infer that pigment epithelium-derived factor(PEDF) may play an important role in the onset of preeclampsia disease, while it has not yet been reported all over the world. So our research is to begin with the factors which affect the the generations of the placental vascular beds. It will investigate the expression of pigment epithelium-derived factor (PEDF) in the placentas of preeclampsia women, and furthermore, it will study the effect of pigment epithelium-derived factor(PEDF) to the preeclampsia disease on the deck of vascular endothelial cells and trophoblast cells. It will provide the base of searching for the reason and mechanism for the pathogenesis of preeclampsia disease.
[Objective] To investigate the expression of pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor(VEGF) in the placentas of preeclampsia patients, and analyze the relationship between the vasculopathy in placentas with PEDF and VEGF.
[Method] A study was performed in60cases of pregnant women with preeclampsia in the obstetrical department of Nanfang Hospital from Octorber2011to January2013, in which30cases were mild preeclampsia(mPE) and30cases were severe preeclampsia(sPE).40normal pregnant women who also be hospitalized and delivered contemporitely were selected as control group. The expression of PEDF, VEGF and MVD in placentas of the norma pregnant group were assayed by using western blot and immunofluorescence double labeling method, mild preeclampsia group and sever preeclampsia group, the relationship between PEDF,VEGF and MVD was analyzed. One-Way ANOVA is adopted to analyze the datas in the multiple groups, LSD test is applied to analyzed the comparison between two groups, the pearson coefficient is used in the correlation between two variables
[Results](1) PEDF and VEGF are both expressed on the trophoblast cells and vascular endothelial cells in the placentas, mainly expressed in the cytoplasm and cell membrane, and the two factors almost were expressed in the same positon.(2) The expression of PEDF in preeclampsia group obviously increased and was significantly higher than that in normal group (P<0.05), which in severe preeclampsia group was significantly higher than that in mild preeclampsia group (P <0.05).(3) compared to normal group, the expression of VEGF in preeclampsia group was significantly lower (P<0.05),but there was no difference between mild preeclampsia group and severe preeclampsia group (P>0.05).(4) The microvessel density (MVD) reduced in preeclampsia group,which was significantly lower than that in normal group(P<0.05), and in severe preeclampsia group it was lower than that in mild preeclampsia group (P<0.05).(5) The expression of PEDF was negatively correlated with the expression of VEGF and MVD (r=-0.365, P<0.05; r=-0.655, P<0.05)in preeclampsia group, and the expression of VEGF was positively correlated with the expression of MVD(r=0.448,P<0.05).
[Conclusion] PEDF and VEGF were together expressed in the trophocyte and vascular endothelial cell of the placenta. The expression of PEDF in preeclampsia placenta was increased, while VEGF and MVD expression was reduced, both of which were negatively correlated with PEDF. It indicates that PEDF may be involved in pathogenesis of preeclampsia, which possibly through affecting the placental vascular reconstruction.
[Objective] To detect the expression of pigment epithelium-derived factor (PEDF) and its mRNA in the first trimester human trophoblast cells(HTR-8/SVneo) in the deficiency oxygen surroundings. then investigate the effect of hypoxia on the first trimester human trophoblast cells, therefore study the effect of PEDF on the preeclampsia disease.
[Method] the first trimester human trophoblast cells (HTR-8/SVneo) were cultured in the deficiency environment, observe the morphologicalchanges of trophoblast cells by inverted microscope. Then detect the expression of pigment epithelium-derived factor (PEDF) and its mRNA in the first trimester human trophoblast cells by using the technology of cell immunohistochemistry and real-time fluorescent quantitative PCR.To detect the apoptosis and the proliferation activity of all the trophoblast cells by the technology of TUNNEL and CKK-8, respectively. And compare the invasion ability of all the trophoblast cells using transwell plate. One-Way ANOVA for Factorial design datas is adopted to analyze the datas in the multiple groups, Independent-Samples T Test is applied in the two groups comparison, the spearman coefficient is used in the correlation between two variables.
[Result](1)the growth status of HTR8/SVneo cell under the1%oxygen concentration:HTR-8/SVneo cells were very sensitive to hypoxia, the shapes of cells changed from polygon or Fusiform shape to round shape, the prominence shrinked,and some of them disappeared, some cells assembled and fused together.(2) cell immunohistochemistry results showed that, PEDF were expressed in each group, the immunological reaction products were expressed mainly in kytoplasm and cell membrane; compared to the normal oxygen concentration, there was no difference on the expression of PEDF in cytotrophoblast cell within hypoxia24hours (P>0.05), but the expression of PEDF increased after under hypoxia48hours, where there is statistical significance (P<0.05), and with the prolonged hypoxia, the expression of PEDF increased more obviously (P<0.05).(3) the real time fluorescent quantitation PCR results showed that, compared to the normal oxygen concentration, there was no difference on the expression of PEDF mRNA in cytotrophoblast cell within hypoxia24hours (P>0.05); but the expression of PEDF mRNA increased after under hypoxia48hours, where there is statistical significance (P<0.05), and with the prolonged hypoxia, the expression of PEDF mRNA increased more obviously (P<0.05).The above results showed that expression of PEDF protein and mRNA reacted to hypoxia as time dependent.(4) to detect apoptosis by the TUNNEL method:compared to the normal oxygen concentration, there was no difference on the amount of apoptosis in cytotrophoblast cell within hypoxia48hours (P>0.05); but the apoptosis cells increased after under hypoxia72hours, where there is statistical significance (P<0.05)(5) to detect the proliferation activity of trophoblast cells by the technology of TUNNEL:there was no difference on the proliferation activity each group (P>0.05).(6) to detect the invasion ability by Transwell:there were disparate trophoblast cells,which infiltrated to the membrane below chamber. compared to the normal oxygen concentration, the infiltrated cells obviously decreased under hypoxia48hours and72hours, where there is statistical significance (P<0.05).(7) The expression of PEDF protein and PEDF mRNA in HTR-8/Svneo trophoblast cells of different groups were postively correlated with the number of apoptosis cells respectively (r=0.772, P<0.05; r=0.839, P<0.05), while the expression of PEDF protein was positively correlated with the expression PEDF mRNA((r=0.876,P<0.05).(8) The expression of PEDF protein in HTR-8/Svneo trophoblast cells of different groups were negatively correlated with the invasion ability of cells respectively(r=-0.615, P<0.05).
[Conclusions] Our results infer that hypoxia can induce the obvious increased expression of pigment epithelium-derived factor (PEDF) protein, as well as its mRNA. And with the prolonged hypoxia time, the tendency of the expression level also increase significantly. Hypoxia could induce the apoptosis of trophoblast cell, and inhibit the invasion activity obviously on the trophoblast cell. The prolonged hypoxia time is followed by the decreased invasion activity.But PEDF do not affect the proliferation of trophoblast cells.
[Objective] To construct the plasmid with PEDF gene (pcDNA-PEDF), then transfect the plasmid to the the first trimester human trophoblast cells, detect the apoptosis of trophoblast cells, then study the effect of the proliferation and invasion activity on the trophoblast cells by PEDF.
[Methods] The first trimester human trophoblast cells were transfected by the the plasmid with PEDF gene, detect the expression of PEDF protein and its mRAN in the trophoblast cells after transfected24hours, through the the technology of cell-mediated immunochemistry and real time fluorescent quantitative PCR. To detect the apoptosis and the proliferation activity of all the trophoblast cells by the technology of TUNNEL and CKK-8, respectively. And compare the invasion ability of all the trophoblast cells using transwell plate. One-Way ANOVA is adopted to analyze the datas in the multiple groups, LSD test is applied to analyzed the comparison between two groups, the spearman coefficient is used in the correlation between two variables
[Result]](1)Compared to the normal control group, after transfected24hours by the PEDF plasmid, the expression of PEDF protein and its mRAN in the HTR8/SVneo cytotrophoblast were increased obviously, where there is statistic significance (P<0.05),while there was no difference between the level in negative control group and normal control group(P>0.05).(2)After the transfection24hours, the apoptosis of trophoblast cell increased, while there was no difference on the proliferation activity in each group(P>0.05).(3) Compared to the normal control group, the trophoblast cells reduced which filtrated to the below chamber of transwell membrane and were transfected by the plasmid with PEDF gene (P<0.05), while there was no difference on the proliferation activity in each group (P>0.05).The result showed the cell invasion activity reduced obviously.(4) The expression of PEDF protein and PEDF mRNA in HTR-8/Svneo trophoblast cells of different groups were postively correlated with the number of apoptosis cells respectively (r=0.970, P<0.05; r=0.745, P<0.05), while the expression of PEDF protein was positively correlated with the expression PEDF mRNA(r=0.672,P<0.05).(5)The expression of PEDF protein was negatively correlated with the invasion ability of the cytoblast(r=-0.882, P<0.05).
[Conclusions] Pigment epithelium-derived factor (PEDF) is very essential to maintain the normal function of the first trimester human trophoblast cells. The increase of PEDF expression could increase the apoptosis of trophoblast cells and reduce their invasion ability. The expression of PEDF protein is negatively correlated with the invasion ability of the trophoblast cells.
妊娠期高血压疾病是妊娠期特发的全身性疾病,我国发病率9.4%,国外报道7%-12%,迄今为止仍然是母婴发病率及死亡率较高的主要原因。子痫前期(preeclampsia)是该疾病最常见的类型,流行病学调查结果显示,子痫前期的发病率约为5-10%,疾病的病因及发生发展过程还不是十分明确,目前主要认为免疫失衡机制,胎盘浅着床,血管内皮细胞损伤和胎盘滋养细胞缺血、胰岛素抵抗、钙平衡失调等机制可能具有重要的作用。有研究认为,子痫前期患者存在子宫蜕膜血管重铸障碍,绒毛的数量减少、绒毛内的血管发育不良及间质纤维化等,胎盘缺血缺氧,引起广泛的血管内皮细胞的损伤,最终导致多器官、多系统功能损害。而正常胎盘血管床生成依赖于胎盘内血管生成诱导因子与抑制因子的复杂联系。
色素上皮衍生因子(pigment epithelium-derived factor,PEDF)是目前已知最强的血管生成抑制剂,它是一种分子量大小约为50kDa的分泌型糖蛋白。PEDF不仅可以抑制新生血管的形成,还可逆转已经形成的血管。而且其抑制血管生成的活性具有明显的选择性,它可抑制生成病理性血管,但不影响生理性血管的形成。目前研究发现,PEDF是一种多功能蛋白,广泛分布于成人和胎儿多种组织(如眼、脑、肝、脂肪、肌肉、胎盘、卵巢、子宫内膜组织等等),具有营养神经、抗血管生成、抗血管的通透性、抗氧化、抗肿瘤生成、调节脂肪细胞及胰岛素生成等特性。2008年美国Beth等学者发现PEDF在正常妊娠妇女的脉管系统和胎盘的滋养层高度表达,很可能就是胎盘血管形成过程的静止因子,对维持血管的静止状态和确保正常血管的完整性具有重要作用。
以往的大量研究表明,整个妊娠时期母体的胎盘组织以及血清中都可能有血管内皮生长因子(VEGF)及其受体(VEGF-R)表达。VEGF是一种有效的血管生成诱导物,VEGF与其受体结合后可通过旁分泌途径调控血管内皮细胞分化,促进内皮细胞的增生、迁移与浸润,同时VEGF可增加血管的通透性,造成大量血浆蛋白外渗,提供了促进血管生成所需要的暂时性基质。VEGF还能诱导内皮细胞中具有抗凋亡作用的Bcl-2的表达,抑制内皮细胞的凋亡,可见,VEGF在促进血管形成和维持血管内皮细胞的功能方面具有重要的作用,VEGF在胚胎着床、胎盘血管发育的过程中也非常关键。正常妊娠早期胎盘组织以及血清中VEGF水平都处于相对升高的状态,可能一方面因为妊娠早期滋养细胞处于相对缺氧状态,低氧通过一系列信号转导通路,刺激VEGF水平上调,另一方面,在胎儿及胎盘生长发育过程中,需要充足血液和血氧供应,VEGF的表达增加是为了适应妊娠的生理改变。研究认为孕早期VEGF水平下降与子痫前期发病密切相关,子痫前期患者胎盘组织中和母体外周血清中VEGF水平明显下降,可能是子痫前期的发病因素之一。PEDF能够通过作用于Fas/FasL介导的信号转导通路,诱导血管内皮细胞的凋亡,从而抑制血管新生；此外,PEDF还可抑制血管紧张素Ⅱ对VEGF的上调,可见,PEDF是VEGF天然的负性调节剂。PEDF是否类似于VEGF也参与子痫前期的发病过程,目前尚未见文献报道。
正常妊娠时胚泡着床到子宫内膜及子宫肌层的过程主要由分化完全的滋养细胞来实现的,对滋养细胞的严密调控在形成正常的胎盘形成中非常重要,这一过程受到许多细胞因子、粘附分子、激素、氧张力等精确的调节,滋养细胞的功能异常是一系列病理妊娠的重要原因,尤其是胎儿宫内生长受限及子痫前期,其中子痫前期又被称为滋养细胞功能障碍疾病。人早孕绒毛滋养细胞是一种具有侵袭和破坏能力的细胞,类似于肿瘤细胞,但不具有肿瘤细胞的成瘤特性和转移能力。研究已表明,滋养细胞的迁移、侵袭能力的下降可能是子痫前期形成的一个重要原因。文献报道,PEDF能通过下调基质金属蛋白酶(MMP-9)的表达水平,以降低肿瘤细胞的侵袭性,从而具有抗肿瘤的功效。目前研究也证实了MMP-9基因的表达下降,能够降低滋养细胞侵袭能力,与子痫前期的发病密切相关。但PEDF是否能够通过类似的作用机制降低滋养细胞的侵袭能力,是否参与子痫前期的发病过程,这一切都需进一步验证。
因此,我们推测色素上皮衍生因子(PEDF)很可能在子痫前期发病中发挥重要作用,且其对子痫前期发病影响的研究,目前国内外尚未见报道。故本课题从影响胎盘血管床生成因素为切入点,研究子痫前期患者胎盘中PEDF蛋白的表达情况,及其与胎盘组织中血管内皮生长因子(VEGF)及胎盘微血管密度的关系；并进一步从滋养细胞层面上研究PEDF蛋白在体外培养的缺氧的滋养细胞中的表达特点,及缺氧诱导后滋养细胞功能的变化；通过将构建的载有PEDF基因的质粒转染到滋养细胞中,研究PEDF对滋养细胞功能的影响,从而探讨PEDF对子痫前期发病的影响,为进一步寻找子痫前期的发病原因及机理提供基础。
第一部分PEDF蛋白在子痫前期与正常妊娠胎盘组织中的表达
[目的]探讨色素上皮衍生因子(PEDF)、血管内皮生长因子(VEGF)在子痫前期患者胎盘组织的表达及其与胎盘血管病变的关系。
[方法]随机选取2011年10月至2013年1月南方医院住院分娩的子痫前期患者60例(其中子痫前期轻度mPE组30例,子痫前期重度sPE组30例)为研究对象,同期分娩的健康孕妇40例作为对照组,用western blot、免疫荧光组织化学双重标记方法检测子痫前期患者和正常妊娠妇女(对照组)胎盘组织中PEDF、VEGF的表达,并通过免疫荧光方法计数胎盘微血管密度(MVD),同时进行相关性分析。采用完全随机设计资料的多组样本均数比较的单因素方差分析(Oneway-Anova),两两比较用LSD(least significant difference)检验,指标间采用双变量相关分析中的pearson相关分析。
[结果](1)胎盘的滋养细胞和血管内皮细胞中均表达PEDF、VEGF,主要表达于胞质及胞膜上,且两个因子表达位置大体相同。(2)子痫前期各组患者胎盘组织中PEDF阳性表达高于正常对照组,差异有统计学意义(P<0.05),而且sPE组PEDF阳性表达高于mPE组(P<0.05)；(3)与正常对照组相比,子痫前期轻度组和子痫前期重度组患者胎盘组织中VEGF表达减少,分别为0.0840±0.1006,0.0785±0.0748,0.0767±0.0738,差异有统计学意义(P<0.05),而mPE组与sPE组VEGF表达无差异(P>0.05)；(4)正常对照组,mPE组,sPE组MVD计数依次递减,分别为136.06±9.07,106.40±8.51,92.65±7.89。子痫前期各组与正常对照组比较差异有统计学意义(P<0.05),mPE组与sPE组差异有统计学意义P<0.05)；(5)子痫前期各组和正常对照组胎盘组织中PEDF表达与VEGF表达及MVD计数呈显著负相关(r=-0.365,P<0.05；r=-0.655,P<0.05),VEGF表达与MVD数值的变化则呈显著正相关(r=0.448,P<0.05)。
[结论]PEDF与VEGF在胎盘的滋养细胞和血管内皮细胞中表达位置大体相同。PEDF蛋白在子痫前期患者胎盘组织中表达升高,而VEGF及MVD表达下降,后两者均与PEDF表达呈负相关。这提示PEDF可影响胎盘血管重铸,可能参与子痫前期的发生和发展过程。
第二部分缺氧对滋养细胞中PEDF的表达及细胞功能的影响
[目的]检测低氧浓度体外培养的人早孕绒毛滋养细胞株HTR-8/SVneo细胞中PEDF蛋白及其mRNA表达的差异,并研究缺氧对滋养细胞功能的影响,以探求PEDF蛋白对子痫前期发病的影响。
[方法]低氧浓度体外培养人早孕绒毛滋养细胞株HTR-8/SVneo细胞,倒置显微镜观察缺氧对滋养细胞形态的影响,并采用细胞免疫荧光化学技术、实时荧光定量PCR检测各组滋养细胞中PEDF蛋白及mRNA表达的差异。用Tunnel法测定细胞凋亡情况,CKK-8法检测各组滋养细胞的增殖能力,Transwell板测定各组滋养细胞的侵袭能力。采用析因设计资料的方差分析,两组间比较采用独立样本t检验(Independent-Samples T Test),指标间采用双变量相关分析中的spearman相关分析。
[结果](1)在1%低氧浓度环境下体外培养HTR8/SVneo细胞的生长情况：HTR8-SVneo细胞对缺氧反应敏感,细胞形态发生明显的改变,细胞由多边形或梭形逐渐向圆形变化,突起变小,甚至部分消失。(2)细胞免疫荧光结果显示：PEDF蛋白在各组HTR8-Svneo细胞均有表达,其免疫反应产物表达主要定位于胞浆和胞膜中；与常氧组相比,缺氧24小时滋养细胞中PEDF蛋白的表达量无显著差异(P>0.05)；但缺氧48小时后,与常氧组相比,低氧组PEDF蛋白的表达升高,差异有统计学意义(P<0.05),且缺氧时间越长,PEDF蛋白升高更多(P<0.05)。(3)实时荧光定量PCR结果显示：与常氧组相比,低氧组PEDF mRNA表达水平在缺氧24小时差异不显著(P>0.05),但缺氧48、72小时PEDF mRNA表达均升高,差异有统计学意义(P<0.05)；缺氧时间越长,PEDF mRNA表达越高(P<0.05)。以上结果表明缺氧对滋养细胞PEDF蛋白和其mRNA的表达呈时间依赖性。(4)Tunnel法测定滋养细胞凋亡：与常氧组相比,低氧组细胞凋亡数量在缺氧24、48小时无显著差异(P>0.05),但缺氧72小时细胞凋亡增多,差异有统计学意义(P<0.05)；(5)CKK-8法检测各组滋养细胞的增殖能力：各组滋养细胞的增殖能力无显著差异(P>0.05)。(6)Transwell测定滋养细胞侵袭能力：显微镜下可见各组均有数量不等的滋养细胞浸润至膜下室,与常氧组相比,1%低氧组滋养细胞在培养48、72小时浸润至膜下室的细胞减少,差异有统计学意义(P<0.05)。(7)各组HTR-8/SVneo细胞中PEDF蛋白表达、mRNA表达均与凋亡的滋养细胞呈显著正相关(r=0.772,P<0.05；r=0.839,P<0.05),PEDF蛋白表达与mRNA表达呈显著正相关(r=0.876,P<0.05)。(8)各组HTR-8/SVneo中PEDF蛋白表达与滋养细胞侵袭能力呈显著负相关(r=-0.615,P<0.05)。
[结论]我们结果提示缺氧环境能导致HTR8-SVneo滋养细胞中PEDF蛋白及mRNA表达增加,且随着缺氧时间的延长,PEDF表达呈显著增多趋势。缺氧可诱导滋养细胞的凋亡,而且明显抑制HTR8-SVneo滋养细胞的侵袭力,并随着缺氧时间的延长其侵袭能力进一步下降。但缺氧对滋养细胞的增殖影响不大。
第三部分载有PEDF基因的质粒转染到滋养细胞对滋养细胞增殖侵袭能力的影响
[目的]构建载有PEDF基因的质粒(pcDNA-PEDF),并将其转染到人早孕绒毛滋养细胞中,研究PEDF对滋养细胞凋亡、增殖与侵袭功能的影响。
[方法]将构建好的载有PEDF基因的质粒(pcDNA-PEDF)转染到人早孕绒毛滋养细胞中,通过细胞免疫荧光及实时荧光定量PCR,观察转染后24小时滋养细胞中PEDF蛋白及mRNA表达情况；并用Tunnel法测定细胞凋亡,CKK-8法检测各组滋养细胞的增殖能力,Transwell板测定各组滋养细胞的侵袭能力。多组间比较采用单因素方差分析(One-Way ANOVA),两两比较用LSD(least significant difference)检验,相关分析采用双变量的spearman相关分析.
[结果](1)与正常对照组相比,转染PEDF质粒24小时后,HTR-8/SVneo滋养细胞中PEDF蛋白及PEDFmRNA表达增多,差异有统计学意义(P<0.05),而阴性对照组与正常对照组比较无显著差异(P>0.05)；(2)与正常对照组相比,转染PEDF基因质粒的滋养细胞组的细胞凋亡增多,分别为9.67±1.52、15.67±2.52,差异有统计学意义(P<0.05),而阴性对照组与正常对照组比较无显著差异(P>0.05)；(3)转染PEDF基因质粒后,各组HTR-8/SVneo滋养细胞生长增殖无显著差异(P>0.05)；(4)与正常对照组相比,转染PEDF基因质粒的滋养细胞进入Transwell膜下室的细胞数降低,分别为39.00±4.58、20.33±2.52,差异有统计学意义(P<0.05),而阴性对照组与正常对照组比较无显著差异(P>0.05)；(5)PEDF蛋白表达、mRNA表达均与凋亡的滋养细胞呈显著正相关(r=0.970,P<0.05；r=0.745,P<0.05),PEDF蛋白表达与mRNA表达呈显著正相关(r=0.672,P<0.05)；(6)PEDF蛋白表达与滋养细胞侵袭能力呈显著负相关(产-0.882,P<0.05)。
[结论]PEDF对维持正常的人早孕绒毛滋养细胞功能非常重要,PEDF表达增多,可增加滋养细胞的凋亡,降低滋养细胞的侵袭能力,且PEDF蛋白表达水平与滋养细胞的侵袭能力呈显著负相关。
Research Background
Hypertensive disorder complicating pregnancy is the systemic disease which is idiopathetic in the gestational period, the morbidity in China is9.4%, while it is7%～12%abroad. Up to now, the disease is still the main reason for the high incidence and mortality rate in the pregnant women and fetus. Preeclampsia is the most frequent type of the disease. It is shown by the epidemiologic survey, that morbidity rate of preeclampsia is5-10%. The etiological factors and process is not yet full clear to date, at present the main reasons are considered as the immuno-unbalance, superficial embedded placenta, the lesions of vascular endothelial cell, the poorly developed vascular net of placenta and decidua and so on. The research has shown that there is the defective vascular remoldeling of uterine decidua in preclampsia, the decreased amount of chorionic villus, angiodysplasia in chorionic villus, and interstitial fibrosis, which induce the hypoxia of placenta and wide lesions of vascular endothelial cell. It lead the functional lesion of multiorgan and multisystem finally.Normal placental vascular development depends upon the complex interactions between angiogenic inducers and inhibitors within the placenta.
Pigment epithelium-derived factor (PEDF) is one of the most potent angiogenic inhibitors identified to date., which is a multifunctional50kDa secreted glycoprotein. Not only pigment epithelium-derived factor (PEDF) could inhibit the formation of new vessels, but also it can reverse the vessels which have been formative. And it is selective to inhibiting the formation of vessels, it inhibits the production of pathologic new vessels, while not affect the formation of physiologic vessels. The findings that pigment epithelium-derived factor(PEDF) is multifunctional protein have been identified to date, it has the function to give the nutrition to nerve, to resist the tumor, to reject the new vessels, to resist the vasopermeability, and it also has the personality of antioxygen and adjusting the cytopoiesis of adipose cells. In the year of2008, Beth and so on,who are American scholars, demonstrated that pigment epitheliumderived factor (PEDF), a potent inhibitor of angiogenesis, is expressed in both the vasculature and trophoblasts of placentas obtained from women with normal pregnancies. Pigment epitheliumderived factor (PEDF) is the factor responsible for inducing vascular quiescence and it is essential to maintain both vascular quiescence and to ensure normal vascular integrity.
A lot of previous study has indicated that, there are vascular endothelial growth factor(VEGF) and its receptor(VEGF-R) expressed in the placentas and maternal serum in the whole pregnanct periods. VEGF is an effective inducer of the production of vessels. After the combination of VEGF and its receptor, it could regulate the differentiation of vascular endothelial cells by the paracrine secretion, and promote the hyperplasy、migration and invasion of endothelial cells. Meanwhile VEGF could increase the permeability of vessels, which induced a lot of plasma protein extravasation, to afford the temporary base which is necessary to promote the information of vessels. VEGF induces the expression of anti-apoptosis material named Bcl-2on the endothelial cells, and inhibite the apoptosis of endothelial cells.So it is evident that VEGF is essential to promote the information of vessels and matain the function of vascular endothelial cells. And VEGF also plays an important role in the implantation of embryo and the developtment of placental vessels. In the early trimester of pregnancy, the level of VEGF in placentas and serum occupies the relative elevated condition. One reason is that there is relative hypoxia stasis in the early trimester of pregnancy, which induces the up-regulation of the level of VEGF by a series of signal transduction pathways. The other reason is to adapt the physiologyical changes of pregnancy, because it needs adequate sufficient blood and oxygen supply in the growth and development of fetus and placentas.The study has showed that there is intimate correlation between the desecended level of VEGF in the earlier pregnant period and the pathogenesis of preeclampsia.The obvious low level of VEGF in the placenta tissues and maternal peripheral blood-serum, which maybe one of the etiological factors for preeclampsia disease. PEDF could induce the apoptosis of vascular endothelial cells and inhibit the formation of new vessels by the signal transduction pathway, which is mediated by Fas/FasL. Furthermore, PEDF could inhibit the up-regulation of VEGF by the angiotonin Ⅱ. So PEDF is the natural negative regulator to VEGF. There is no report about whether PEDF, similarity like VEGF. involves in the pathogenesis of preeclampsia.
The implantation of blastocyst to the endometrium and myometrium is completed by the well differentiated trophoblast cells in normal pregnancy. It is essential important for the regulation to the trophoblast cells during the formation of normal placenta, the dysfunction of trophoblast cell is main cause to a series of pathogenic pregnancy, such as intrauterine growth retardation (IUGR) and pre-eclampsia. Human first trimester trophoblast cell is the cell,which has the invasive and destructive characteristics, similarity to tumor cell, but not tumorigenicity and metastasis. It has been shown in research,that the decreased ability of migration and invasion for the trophoblast cell plays an important role in preeclampsia. It was reported by literacy that PEDF could down-regulate the level of expression of matrix metalloproteinase (MMP-9),and then decrease the invasion ability of tumor cells, so it has the antineoplastic function.At present,the study has confirmed that the decreased expression level of matrix metalloproteinase induced the invasion ability of trophoblast cells, so to involve in the pathogenesis of preeclampsia. But it is still need to be verificated whether PEDF could also depressed the invasion ability of trophoblast cells and involve in the pathogenesis of preeclampsia by the similar mechanism,
Therefore, we infer that pigment epithelium-derived factor(PEDF) may play an important role in the onset of preeclampsia disease, while it has not yet been reported all over the world. So our research is to begin with the factors which affect the the generations of the placental vascular beds. It will investigate the expression of pigment epithelium-derived factor (PEDF) in the placentas of preeclampsia women, and furthermore, it will study the effect of pigment epithelium-derived factor(PEDF) to the preeclampsia disease on the deck of vascular endothelial cells and trophoblast cells. It will provide the base of searching for the reason and mechanism for the pathogenesis of preeclampsia disease.
[Objective] To investigate the expression of pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor(VEGF) in the placentas of preeclampsia patients, and analyze the relationship between the vasculopathy in placentas with PEDF and VEGF.
[Method] A study was performed in60cases of pregnant women with preeclampsia in the obstetrical department of Nanfang Hospital from Octorber2011to January2013, in which30cases were mild preeclampsia(mPE) and30cases were severe preeclampsia(sPE).40normal pregnant women who also be hospitalized and delivered contemporitely were selected as control group. The expression of PEDF, VEGF and MVD in placentas of the norma pregnant group were assayed by using western blot and immunofluorescence double labeling method, mild preeclampsia group and sever preeclampsia group, the relationship between PEDF,VEGF and MVD was analyzed. One-Way ANOVA is adopted to analyze the datas in the multiple groups, LSD test is applied to analyzed the comparison between two groups, the pearson coefficient is used in the correlation between two variables
[Results](1) PEDF and VEGF are both expressed on the trophoblast cells and vascular endothelial cells in the placentas, mainly expressed in the cytoplasm and cell membrane, and the two factors almost were expressed in the same positon.(2) The expression of PEDF in preeclampsia group obviously increased and was significantly higher than that in normal group (P<0.05), which in severe preeclampsia group was significantly higher than that in mild preeclampsia group (P <0.05).(3) compared to normal group, the expression of VEGF in preeclampsia group was significantly lower (P<0.05),but there was no difference between mild preeclampsia group and severe preeclampsia group (P>0.05).(4) The microvessel density (MVD) reduced in preeclampsia group,which was significantly lower than that in normal group(P<0.05), and in severe preeclampsia group it was lower than that in mild preeclampsia group (P<0.05).(5) The expression of PEDF was negatively correlated with the expression of VEGF and MVD (r=-0.365, P<0.05; r=-0.655, P<0.05)in preeclampsia group, and the expression of VEGF was positively correlated with the expression of MVD(r=0.448,P<0.05).
[Conclusion] PEDF and VEGF were together expressed in the trophocyte and vascular endothelial cell of the placenta. The expression of PEDF in preeclampsia placenta was increased, while VEGF and MVD expression was reduced, both of which were negatively correlated with PEDF. It indicates that PEDF may be involved in pathogenesis of preeclampsia, which possibly through affecting the placental vascular reconstruction.
[Objective] To detect the expression of pigment epithelium-derived factor (PEDF) and its mRNA in the first trimester human trophoblast cells(HTR-8/SVneo) in the deficiency oxygen surroundings. then investigate the effect of hypoxia on the first trimester human trophoblast cells, therefore study the effect of PEDF on the preeclampsia disease.
[Method] the first trimester human trophoblast cells (HTR-8/SVneo) were cultured in the deficiency environment, observe the morphologicalchanges of trophoblast cells by inverted microscope. Then detect the expression of pigment epithelium-derived factor (PEDF) and its mRNA in the first trimester human trophoblast cells by using the technology of cell immunohistochemistry and real-time fluorescent quantitative PCR.To detect the apoptosis and the proliferation activity of all the trophoblast cells by the technology of TUNNEL and CKK-8, respectively. And compare the invasion ability of all the trophoblast cells using transwell plate. One-Way ANOVA for Factorial design datas is adopted to analyze the datas in the multiple groups, Independent-Samples T Test is applied in the two groups comparison, the spearman coefficient is used in the correlation between two variables.
[Result](1)the growth status of HTR8/SVneo cell under the1%oxygen concentration:HTR-8/SVneo cells were very sensitive to hypoxia, the shapes of cells changed from polygon or Fusiform shape to round shape, the prominence shrinked,and some of them disappeared, some cells assembled and fused together.(2) cell immunohistochemistry results showed that, PEDF were expressed in each group, the immunological reaction products were expressed mainly in kytoplasm and cell membrane; compared to the normal oxygen concentration, there was no difference on the expression of PEDF in cytotrophoblast cell within hypoxia24hours (P>0.05), but the expression of PEDF increased after under hypoxia48hours, where there is statistical significance (P<0.05), and with the prolonged hypoxia, the expression of PEDF increased more obviously (P<0.05).(3) the real time fluorescent quantitation PCR results showed that, compared to the normal oxygen concentration, there was no difference on the expression of PEDF mRNA in cytotrophoblast cell within hypoxia24hours (P>0.05); but the expression of PEDF mRNA increased after under hypoxia48hours, where there is statistical significance (P<0.05), and with the prolonged hypoxia, the expression of PEDF mRNA increased more obviously (P<0.05).The above results showed that expression of PEDF protein and mRNA reacted to hypoxia as time dependent.(4) to detect apoptosis by the TUNNEL method:compared to the normal oxygen concentration, there was no difference on the amount of apoptosis in cytotrophoblast cell within hypoxia48hours (P>0.05); but the apoptosis cells increased after under hypoxia72hours, where there is statistical significance (P<0.05)(5) to detect the proliferation activity of trophoblast cells by the technology of TUNNEL:there was no difference on the proliferation activity each group (P>0.05).(6) to detect the invasion ability by Transwell:there were disparate trophoblast cells,which infiltrated to the membrane below chamber. compared to the normal oxygen concentration, the infiltrated cells obviously decreased under hypoxia48hours and72hours, where there is statistical significance (P<0.05).(7) The expression of PEDF protein and PEDF mRNA in HTR-8/Svneo trophoblast cells of different groups were postively correlated with the number of apoptosis cells respectively (r=0.772, P<0.05; r=0.839, P<0.05), while the expression of PEDF protein was positively correlated with the expression PEDF mRNA((r=0.876,P<0.05).(8) The expression of PEDF protein in HTR-8/Svneo trophoblast cells of different groups were negatively correlated with the invasion ability of cells respectively(r=-0.615, P<0.05).
[Conclusions] Our results infer that hypoxia can induce the obvious increased expression of pigment epithelium-derived factor (PEDF) protein, as well as its mRNA. And with the prolonged hypoxia time, the tendency of the expression level also increase significantly. Hypoxia could induce the apoptosis of trophoblast cell, and inhibit the invasion activity obviously on the trophoblast cell. The prolonged hypoxia time is followed by the decreased invasion activity.But PEDF do not affect the proliferation of trophoblast cells.
[Objective] To construct the plasmid with PEDF gene (pcDNA-PEDF), then transfect the plasmid to the the first trimester human trophoblast cells, detect the apoptosis of trophoblast cells, then study the effect of the proliferation and invasion activity on the trophoblast cells by PEDF.
[Methods] The first trimester human trophoblast cells were transfected by the the plasmid with PEDF gene, detect the expression of PEDF protein and its mRAN in the trophoblast cells after transfected24hours, through the the technology of cell-mediated immunochemistry and real time fluorescent quantitative PCR. To detect the apoptosis and the proliferation activity of all the trophoblast cells by the technology of TUNNEL and CKK-8, respectively. And compare the invasion ability of all the trophoblast cells using transwell plate. One-Way ANOVA is adopted to analyze the datas in the multiple groups, LSD test is applied to analyzed the comparison between two groups, the spearman coefficient is used in the correlation between two variables
[Result]](1)Compared to the normal control group, after transfected24hours by the PEDF plasmid, the expression of PEDF protein and its mRAN in the HTR8/SVneo cytotrophoblast were increased obviously, where there is statistic significance (P<0.05),while there was no difference between the level in negative control group and normal control group(P>0.05).(2)After the transfection24hours, the apoptosis of trophoblast cell increased, while there was no difference on the proliferation activity in each group(P>0.05).(3) Compared to the normal control group, the trophoblast cells reduced which filtrated to the below chamber of transwell membrane and were transfected by the plasmid with PEDF gene (P<0.05), while there was no difference on the proliferation activity in each group (P>0.05).The result showed the cell invasion activity reduced obviously.(4) The expression of PEDF protein and PEDF mRNA in HTR-8/Svneo trophoblast cells of different groups were postively correlated with the number of apoptosis cells respectively (r=0.970, P<0.05; r=0.745, P<0.05), while the expression of PEDF protein was positively correlated with the expression PEDF mRNA(r=0.672,P<0.05).(5)The expression of PEDF protein was negatively correlated with the invasion ability of the cytoblast(r=-0.882, P<0.05).
[Conclusions] Pigment epithelium-derived factor (PEDF) is very essential to maintain the normal function of the first trimester human trophoblast cells. The increase of PEDF expression could increase the apoptosis of trophoblast cells and reduce their invasion ability. The expression of PEDF protein is negatively correlated with the invasion ability of the trophoblast cells.
引文
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[85]Elayappan B, Ravinarayannan H, Pasha SP, et al.PEDF inhibits VEGF-and EPO- induced angiogenesis in retinal endothelial cells through interruption of PI3K/Akt phosphorylation.Angiogenesis.2009;12(4):313-24.
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