sEng、TGFβ1与妊娠期高血压疾病发生的相关性研究
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摘要
妊娠期高血压疾病(Hypertensive disorder complicating pregnancy, HDCP)是妊娠期特有疾病,病因和发病机制尚未完全阐明,已有研究表明,转化生成因子TGFβ1及其受体与妊娠期高血压疾病发生的中心环节--血管内皮损伤及胎盘血管重铸过程密切相关,本研究将通过观察TGFβ1及其可溶性受体成份-sEng对体外培养的脐静脉内皮细胞及滋养细胞的影响,探讨其在妊娠期高血压疾病发生、发展中的作用。
第一部分sEng及TGFβ1与内皮细胞损伤的相关性研究目的：观察可溶性Endoglin (soluble endoglin, sEng)及转化生长因子β1(TGFβ1)对体外培养的人脐静脉内皮细胞(human umbilical vein endothelial cell, HUVEC)增殖、凋亡的影响,及其对HUVEC产生一氧化氮(nitric oxide, NO)量,内皮型一氧化氮合酶(endothelial nitric oxide synthase, eNOS)1177位点丝氨酸磷酸化程度的影响,探讨sEng及TGFβ1参与内皮细胞损伤的可能机制。
方法：体外培养HUVEC,将3代以内的HUVEC接种于96孔培养板,分4组：对照组(单纯RPMI-1640完全培养液培养)、sEng组、TGFβ1及(sEng+TGFβ1)组：根据四甲基偶氮唑蓝(MTT)结果,将sEng组、TGFβ1及(sEng+TGFβ1)组按加药浓度分别分为110、100μg/L三个亚组,收集6h、12h,24h时间点的细胞及培养液。ELISA法测定细胞培养液中sEng及TGFβ1含量;MTT比色法检测对照组、不同浓度sEng、TGFβ1及(sEng+TGFβ1)培养6h、12h、24h后HUVEC的细胞的存活率,流式细胞技术观察各组凋亡率和细胞周期分布；硝酸酶还原法测定细胞培养液中NO含量,Western印迹法检测各组细胞eNOS蛋白相对表达量及其1177位点丝氨酸磷酸化情况,实时荧光PCR检测各组细胞eNOS mRNA的相对表达量。
结果：(1)细胞培养液中sEng及TGFβ1的含量：24h、48h、72hHUVEC培养液中未测得sEng;24h、48h、72h细胞培养液中TGFβ1含量分别为(3.12±0.78)、(3.33±0.91)、(3.08±0.87)ng/L,三个时间段TGFβ1含量无明显变化(F=2.10,p>0.05)。(2)细胞存活率：sEng组HUVEC存活率在各个浓度点及时间点均低于对照组和其它各组,且与时间和加药浓度均呈负相关;TGFβ1组,加1μg/L时HUVEC表现为增殖,存活率高于对照组,加10、100μg/L时,HUVEC存活率与时间和加药浓度均呈负相关(sEng+TGFβ1)组HUVEC存活率与对照组比较无显著性差异,无时间及浓度依赖性。(3)细胞凋亡率和周期分布：sEng组,HUVEC凋亡率明显增加,与加药浓度及作用时间均呈正相关,sEng组G1期细胞比例显著高于对照组,亦与浓度及时间呈正相关；TGFβ1各组HUVEC凋亡率与对照组各点比较均无明显差异,1μg/L组G1期细胞比例显著低于对照组并与作用时间呈负相关,10、100μg/L组G1期细胞比例显著高于对照组且与作用浓度及时间呈正相关；(sEng+TGFβ1)组HUVEC凋亡率、G1期细胞比例、存活率与对照组比较无显著性差异,且无浓度及时间依赖性。(4)细胞培养液中NO含量变化：对照组培养液内NO含量24h内无明显变化(F=2.30,p>0.05), sEng组细胞培养液中NO含量低于对照组和其它各组并与作用时间和加药浓度均呈负相关;TGFβ1组,加1gg/L时,细胞培养液中NO含量显著高于对照组及其它各组,与作用时间呈正相关,加10、100gg/L时,细胞培养液中NO含量与时间和加药浓度均呈负相关；(sEng-TGFβ1)组细胞培养液中NO含量与对照组比较无显著性差异,亦无时间及浓度依赖性。(5) eNOS蛋白及mRNA表达水平变化：sEng组,HUVEC eNOS蛋白及mRNA表达在各个时间点及浓度点均低于对照组及其它各组,且与作用时间及加药浓度呈明显的负相关;TGFβ1组,加1μg/L时eNOS蛋白及mRNA表达水平在各个时间点均高于对照组及其它各组,且与时间呈正相关,加10、100μg/L时,eNOS蛋白及mRNA表达水平在各个浓度点及时间点均低于对照组但高于sEng组,且与作用时间及浓度呈负相关(sEng+TGFβ1)组各个时间点及浓度点细胞eNOS蛋白及mRNA表达水平与对照组比较无显著性差异。(6) eNOS蛋白活化情况：sEng组,eNOS-Ser(p)1177/eNOS在各个时间点及浓度点均低于对照组及其它各组,并与作用时间及加药浓度呈负相关；TGFβ1组,加1μg/L时eNOS-Ser(p)1177/eNOS在各个时间点均高于对照组及其它各组并与时间呈正相关,加10、100μg/L时,eNOS-Ser(p)1177在各个浓度点及时间点均低于对照组但高于sEng组,且与时间及浓度呈负相关;(sEng+TGFβ1)组各个时间点及浓度点细胞eNOS-Ser(p)1177/eNOS与对照组比较无显著性差异。
结论：1、加入外源性sEng可与内源性产生的TGFβ1结合阻断TGFβ1的信号传导,通过促进凋亡,并使细胞受阻于G1期,延缓其从Gl期向S期过渡的方式,抑制HUVEC的增殖.同时通过下调eNOS1177位点丝氨酸的磷酸化水平,抑制eNOS的活性,使HUVEC分泌NO功能下降,进而影响内皮细胞的舒缩功能。2、加入外源性的TGFβ1,对HUVEC的影响取决于作用浓度.高浓度TGFβ1主要通过使内皮细胞受阻于G1期.延缓其从G1期向S期过渡的方式,抑制HUVEC的增殖,同时通过下调eNOS1177位点丝氨酸的磷酸化水平,抑制eNOS的活性,使HUVEC分泌NO的能力下降,影响内皮细胞的舒缩功能,低浓度的TGFβ1对HUVEC的作用与之相反。3、(sEng+TGFβ1)组HUVEC的增殖、eNOS活性、NO的产生与对照组比较无显著差异,说明两因子等量加入细胞培养液,sEng作为TGFβ1的受体,二者几乎达到1：1结合,结合后二者均不能发挥前述作用,推断受体与配体成比例结合有助于内皮细胞发挥正常的作用,任一因子过量使比例失调,则对HUVEC产生不同程度的影响,进而参与内皮细胞功能的紊乱。
第二部分sEng与TGFβ1对人早孕绒毛膜滋养细胞增殖及浸润的影响
目的：探讨可溶性Endoglin与TGFβ1对人早孕绒毛膜滋养细胞增殖能力和浸润功能的影响。
方法：采用胰蛋白酶-DNase消化法培养人早孕期(孕6-8周)绒毛膜滋养层细胞,将3代以内的滋养细胞接种于96孔培养板,分4组：对照组(单纯DMEM完全培养液培养)、sEng组、TGFβ1及(sEng+TGFβ1)组ELISA法测定细胞培养液中sEng及TGFβ1含量；MTT法观察滋养细胞的增殖情况并确定后续试验中sEng、TGFβ1及(sEng-TGFβ1)(?)的加药浓度及作用时间：MTT法观察各组细胞存活率,流式细胞技术观察各组细胞的细胞周期变化,Transwell技术检测各组滋养细胞的浸润功能；Western印迹法检测各组滋养细胞MMP-2、MMP-9蛋白的表达；RT-PCR法检测各组滋养细胞MMP-2、MMP-9mRNA的表达。
结果：24h、48h、72h滋养细胞培养液中未测得sEng:24h、48h、72h细胞培养液中TGFβ1含量分别为(5.03±0.54)、(4.09+0.27)、(5.91+0.68)ng/L,三个时间段TGFβ1含量无明显变化(F=3.09,p>0.05);10μg/LsEng、TGFβ1分别作用6h、12h、24h,各组细胞存活率与对照组比较均无明显差异：作用36h、48h,存活率明显降低,分别为(71.44±6.07)%、(51.44±6.34)%(36h),(62.37±8.12)%、(23.48±4.36)%(48h),无明显的时间及浓度依赖性;(sEng+TGFβ1)组滋养细胞存活率在各个时间点及浓度点与对照组比较无明显差异.确定各组加药浓度为10μg/L;作用48h,各组均未见明显凋亡;sEng组G1期细胞比例(88.24±2.11)%显著高于对照组(55.23±1.22)%%但低于TGFβ1组(95.63±2.98)%,(均p<0.05);TGFβ1组G1期细胞比例显著高于其它各组(均p<0.05);(sEng+TGFβ1)组G1期细胞比例(58.11±1.25)%与对照组比较无明显差异；各组加药10μg/L、作用24h, sEng组滋养细胞穿透Transwell基底膜的细胞数为(87+6.3)个/5HP显著低于对照组(143±41.3)个/5HP, TGFβ1组滋养细胞穿透Transwell基底膜的细胞数为(157-±-55.9)个/5HP显著高于对照组,而(sEng+TGFβ1)组细胞穿透数与对照组比较无显著性差异(p>0.05)。sEng组滋养细胞MMP-2和MMP-9蛋白及mRNA的表达明显低于对照组,TGFβ1组滋养细胞MMP-2和MMP-9蛋白及mRNA的表达明显高于对照组,差异均有统计学意义(均p<0.05),而(sEng+TGFβ1)组MMP-2和MMP-9蛋白及mRNA的表达与对照组无显著差异(p>0.05)。
结论：sEng和TGFβ1可能通过调节滋养细胞的G、期细胞比例,影响其增殖能力,但并不导致细胞的死亡;通过调节滋养细胞MMP-2和MMP-9的表达影响其浸润能力；在体外细胞培养环境下,sEng和TGFβ1可达到1:1的结合,并失云各自对滋养细胞的影响,据此推测,早孕期绒毛局部两种因子比例失调,可能不同程度的影响滋养细胞向子宫肌层的正常浸润,造成“胎盘浅着床”引起妊娠期高血压等相关疾病。
Hypertensive disorder complicating pregnancy is a pregnancy-specific disease which etiology and pathogenesis has not been fully elucidated. Previous studies have been shown that angiogenic factors TGFβ1and its receptor are closely related to the central steps of hypertensive disorders in pregnancy:vascular endothelial injury and placental vascular remodeling process. This study will observe the impact of TGFβ1and its soluble receptor components (sEng) in cultured human umbilical vein endothelial cells and trophoblasts explore the roles of these two factors in the development of hypertensive disorder complicating pregnancy.
Section Ⅰ. Research on the correlation between soluble endoglin. transforming growth factor β1and injury of vein endothelial cell
Objectives:To discuss the correlation between soluble endoglin. transforming growth factor β1and injury of vein endothelial cell. investigating the effects of these two factors on human umbilical vein endothelial cell (HUVEC). included apoptosis. proliferation, nitric oxide (NO) production and nitric oxide synthase (eNOS) phosphoryiation.
Methods:Cells (within3passages) were seeded in culture plates of96wells. There are4groups:controlled group with RPMI-1640culture medium only. sEng group. TGF(31group and (sEng-TGFβ1) group. These groups were divided into3subgroups (1、10、100μg/L) respectively on account of MTT results. Collecting the cells and cell cultures at6h.12h and24h for follow-up experiments. The concentration of soluble endoglin and TGFβ1in cell culture was evaluated by ELISA. Cell apoptosis and cell cycle were detected by flow cytometry. Cell viability was determined by methyl thiazolyl tetrazolium assay (MTT). The concentration of the metabolites of nitric oxide in each group was measured by nitrate reductase method. The expression of eNOS and eNOS-Ser(p)1177protein were detected by Western blot. eNOS mRNA in each group was detected by real-time fluorescence reverse transcription polymerase chain reaction (PCR).
Results:(1) The concentration of sEng in cell culture was0at24h.48h and72h. The concentration of TGFβ1in cell culture was (3.12±0.78).(3.33±0.91). (3.08±0.87)ng/L respectively. There were no significant difference at these three time points (F=2.10, p>0.05).(2) Cell viability:The cell viability was significantly decreased in sEng group. It had negative correlation with time and medicine concentration. The cell viability were significantly increased in1μg/L TGFP1group, but decreased in10and100μg/L groups. It had negative correlation with time and medicine concentration in the latter groups. There was no significant difference between (sEng+TGFβ1) group and controlled group at any concentration and time points.(3) Cell apoptosis and cell cycle:the cell apoptosis rate and the G1phase proportion in sEng group were higher than those in other groups. It had positive correction with time and medicine concentration. The cell G1proportion were significantly decreased in1μg/L TGFβ1group, but increased in10and100μg/L groups. It had positive correlation with time and medicine concentration in the latter groups. But TGFβ1had no business of apoptosis. There was no significant difference between (sEng-TGFβ1) group and controlled group on cell apoptosis and cell cycle at any concentration and time points.(4) Nitric oxide levels in cell culture:sEng decreased the concentration of nitric oxide levels through negative correlation with stimulate time and medicine concentration.1μg/L TGFβ1increased the concentration of nitric oxide levels through positive affection with stimulate time. But10and100μg/L TGFβ1decreased the concentration of nitric oxide levels through negative affection with stimulate time and medicine concentration. There was no significant difference between (sEng+TGFβ1) group and controlled group on nitric oxide levels at any concentration and time points.(5) The expression of eNOS protein and mRNA: The expression of eNOS protein and mRNA decreased significantly in sEng groups. It also had negative correlation with stimulate time and medicine concentration.1μg/L TGFβ1increased the expression of eNOS protein and mRNA through positive affection with stimulate time. But10and100μg/L TGFβ1decreased the expression of eNOS protein and mRNA through negative affection with stimulate time and medicine concentration. There was no significant difference between (sEng+TGFβ1) group and controlled group on the expression of eNOS protein and mRNA at any concentration and time points.(6) Nitric oxide synthase phosphorylation (eNOS-Ser(p)1177/ENOS ratio):eNOS-Ser(p)1177/eNOS ratio decreased significantly in sEng groups. It also had negative correlation with stimulate time and medicine concentration.1μg/L TGFβ1increased the eNOS-Ser(p)1177/eNOS ratio through positive affection with stimulate time. But10and100μg/L TGFβ1decreased the eNOS-Ser(p)1177/eNOS ratio through negative affection with stimulate time and medicine concentration. There was no significant difference between (sEng+TGFβ1) group and controlled group on the eNOS-Ser(p)1177/eNOS ratio at any concentration and time points.
Conclusions:1.sEng inhibited the proliferation of HUVEC in vitro through blocking the HUVEC in G1phase and increasing the apoptosis rate. It reduced the production of NO through inhibiting the proliferation of HUVEC and activation of eNOS. sEng participated the injury of endothelial cells.2. TGFβ1inhibited the proliferation of HUVEC in vitro through blocking the HUVEC in G1phase occasionally. The affection on HUVEC was up to medicine concentration. Low dose had promoted affection on HUVEC. But high dose had converse affections.3. sEng together with TGF(31were added in the cell culture seemed like having no affection on HUVEC. but it reflected that appropriate proportion of sEng and TGFβ1was very important to keep normal form and function of endothelial cells. All of above. sEng and TGFβ1participated the injury of endothelial cells by many ways.
Section Ⅱ. Effects of soluble endoglin and TGFβ1on proliferation and invasive ability of cultured cytotrophoblasts from human early pregnancy chorionic villus
Objective:To investigate the effects of soluble endoglin and TGFβ1on proliferation and invasive ability of cultured cytotrophoblasts from human early pregnancy chorionic villus.
Methods:Cytotrophoblasts of normal6to8week pregnancy were cultured by trypsin digestion method. Cells (within3passages) were seeded in culture plates of96wells. There are4groups:controlled group with DMEM culture medium only. sEng group. TGFβ1group and (sEng-TGFβ1) group. The concentration of soluble endoglin and TGFβ1in cell culture was evaluated by ELISA. The medicine concentration at follow-up experiments of these two factors was determined by MTT. The proliferation ability was determined by MTT. Cell cycle was detected by flowcvtometry. The invasive ability was determined by Transwell invasion assay. The expression of MMP-2and MMP-9protein was detected by Western blot. The expression of MMP-2and MMP-9mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR).
Results:The concentration of sEng in cell culture was0at24h,48h and72h. The concentration of TGFfβ1in cell culture was (5.03±0.54),(4.09±0.27). and (5.91±0.68)ng/L respectively. There were no significant difference at these three time points (F=3.09, p>0.05). Cell survival probability were degraded in sEng and TGFβ1groups at36h (71.44±6.07)%.(51.44±6.34)%and48h (62.37±8.12)%.(23.48±4.36)%versus controlled group. There was no significant difference between (sEng±TGFβ1) group and controlled group at any concentration and time points. The medicine concentration was10μg/L in every group on account of MTT. There was no significant cell apoptosis in every group. G1phase proportion in sEng group (88.24±2.11)%was higher than that of (sEng+TGFβ1)(58.11±1.25)%and control groups (55.23±1.22)%. G1phase proportion in TGFβ1group (95.63±2.98)%was higher than any other groups. There had no significant difference between (sEng+TGFβ1) group and control group. Cell numbers penetrating basement membrane was decreased significantly in sEng group (87±6.3) versus controlled group (143±41.3) but increased significantly in TGFβ1group (157±55.9). There was no significant difference between (sEng+TGF±1) group and controlled group. Compared with controlled group, the expression of MMP-2and MMP-9mRNA and protein of cytotrophoblasts was significantly lower in sEng group but higher in TGF(31group. There also was no significant difference between (sEng+TGFβ1) group and controlled group.
Conclusions:sEng and TGFβ1correlated with the procedure of cytotrophoblasts proliferation through moderating G1phase proportion in vitro. They also regulated cytotrophoblasts invasive ability by affecting the expression of MMP-2and MMP-9. Keeping suitable proportion was very important for cytotrophoblasts to invasive in mvometrium with normal depth at the first trimester.
第一部分sEng及TGFβ1与内皮细胞损伤的相关性研究目的：观察可溶性Endoglin (soluble endoglin, sEng)及转化生长因子β1(TGFβ1)对体外培养的人脐静脉内皮细胞(human umbilical vein endothelial cell, HUVEC)增殖、凋亡的影响,及其对HUVEC产生一氧化氮(nitric oxide, NO)量,内皮型一氧化氮合酶(endothelial nitric oxide synthase, eNOS)1177位点丝氨酸磷酸化程度的影响,探讨sEng及TGFβ1参与内皮细胞损伤的可能机制。
方法：体外培养HUVEC,将3代以内的HUVEC接种于96孔培养板,分4组：对照组(单纯RPMI-1640完全培养液培养)、sEng组、TGFβ1及(sEng+TGFβ1)组：根据四甲基偶氮唑蓝(MTT)结果,将sEng组、TGFβ1及(sEng+TGFβ1)组按加药浓度分别分为110、100μg/L三个亚组,收集6h、12h,24h时间点的细胞及培养液。ELISA法测定细胞培养液中sEng及TGFβ1含量;MTT比色法检测对照组、不同浓度sEng、TGFβ1及(sEng+TGFβ1)培养6h、12h、24h后HUVEC的细胞的存活率,流式细胞技术观察各组凋亡率和细胞周期分布；硝酸酶还原法测定细胞培养液中NO含量,Western印迹法检测各组细胞eNOS蛋白相对表达量及其1177位点丝氨酸磷酸化情况,实时荧光PCR检测各组细胞eNOS mRNA的相对表达量。
结果：(1)细胞培养液中sEng及TGFβ1的含量：24h、48h、72hHUVEC培养液中未测得sEng;24h、48h、72h细胞培养液中TGFβ1含量分别为(3.12±0.78)、(3.33±0.91)、(3.08±0.87)ng/L,三个时间段TGFβ1含量无明显变化(F=2.10,p>0.05)。(2)细胞存活率：sEng组HUVEC存活率在各个浓度点及时间点均低于对照组和其它各组,且与时间和加药浓度均呈负相关;TGFβ1组,加1μg/L时HUVEC表现为增殖,存活率高于对照组,加10、100μg/L时,HUVEC存活率与时间和加药浓度均呈负相关(sEng+TGFβ1)组HUVEC存活率与对照组比较无显著性差异,无时间及浓度依赖性。(3)细胞凋亡率和周期分布：sEng组,HUVEC凋亡率明显增加,与加药浓度及作用时间均呈正相关,sEng组G1期细胞比例显著高于对照组,亦与浓度及时间呈正相关；TGFβ1各组HUVEC凋亡率与对照组各点比较均无明显差异,1μg/L组G1期细胞比例显著低于对照组并与作用时间呈负相关,10、100μg/L组G1期细胞比例显著高于对照组且与作用浓度及时间呈正相关；(sEng+TGFβ1)组HUVEC凋亡率、G1期细胞比例、存活率与对照组比较无显著性差异,且无浓度及时间依赖性。(4)细胞培养液中NO含量变化：对照组培养液内NO含量24h内无明显变化(F=2.30,p>0.05), sEng组细胞培养液中NO含量低于对照组和其它各组并与作用时间和加药浓度均呈负相关;TGFβ1组,加1gg/L时,细胞培养液中NO含量显著高于对照组及其它各组,与作用时间呈正相关,加10、100gg/L时,细胞培养液中NO含量与时间和加药浓度均呈负相关；(sEng-TGFβ1)组细胞培养液中NO含量与对照组比较无显著性差异,亦无时间及浓度依赖性。(5) eNOS蛋白及mRNA表达水平变化：sEng组,HUVEC eNOS蛋白及mRNA表达在各个时间点及浓度点均低于对照组及其它各组,且与作用时间及加药浓度呈明显的负相关;TGFβ1组,加1μg/L时eNOS蛋白及mRNA表达水平在各个时间点均高于对照组及其它各组,且与时间呈正相关,加10、100μg/L时,eNOS蛋白及mRNA表达水平在各个浓度点及时间点均低于对照组但高于sEng组,且与作用时间及浓度呈负相关(sEng+TGFβ1)组各个时间点及浓度点细胞eNOS蛋白及mRNA表达水平与对照组比较无显著性差异。(6) eNOS蛋白活化情况：sEng组,eNOS-Ser(p)1177/eNOS在各个时间点及浓度点均低于对照组及其它各组,并与作用时间及加药浓度呈负相关；TGFβ1组,加1μg/L时eNOS-Ser(p)1177/eNOS在各个时间点均高于对照组及其它各组并与时间呈正相关,加10、100μg/L时,eNOS-Ser(p)1177在各个浓度点及时间点均低于对照组但高于sEng组,且与时间及浓度呈负相关;(sEng+TGFβ1)组各个时间点及浓度点细胞eNOS-Ser(p)1177/eNOS与对照组比较无显著性差异。
结论：1、加入外源性sEng可与内源性产生的TGFβ1结合阻断TGFβ1的信号传导,通过促进凋亡,并使细胞受阻于G1期,延缓其从Gl期向S期过渡的方式,抑制HUVEC的增殖.同时通过下调eNOS1177位点丝氨酸的磷酸化水平,抑制eNOS的活性,使HUVEC分泌NO功能下降,进而影响内皮细胞的舒缩功能。2、加入外源性的TGFβ1,对HUVEC的影响取决于作用浓度.高浓度TGFβ1主要通过使内皮细胞受阻于G1期.延缓其从G1期向S期过渡的方式,抑制HUVEC的增殖,同时通过下调eNOS1177位点丝氨酸的磷酸化水平,抑制eNOS的活性,使HUVEC分泌NO的能力下降,影响内皮细胞的舒缩功能,低浓度的TGFβ1对HUVEC的作用与之相反。3、(sEng+TGFβ1)组HUVEC的增殖、eNOS活性、NO的产生与对照组比较无显著差异,说明两因子等量加入细胞培养液,sEng作为TGFβ1的受体,二者几乎达到1：1结合,结合后二者均不能发挥前述作用,推断受体与配体成比例结合有助于内皮细胞发挥正常的作用,任一因子过量使比例失调,则对HUVEC产生不同程度的影响,进而参与内皮细胞功能的紊乱。
第二部分sEng与TGFβ1对人早孕绒毛膜滋养细胞增殖及浸润的影响
目的：探讨可溶性Endoglin与TGFβ1对人早孕绒毛膜滋养细胞增殖能力和浸润功能的影响。
方法：采用胰蛋白酶-DNase消化法培养人早孕期(孕6-8周)绒毛膜滋养层细胞,将3代以内的滋养细胞接种于96孔培养板,分4组：对照组(单纯DMEM完全培养液培养)、sEng组、TGFβ1及(sEng+TGFβ1)组ELISA法测定细胞培养液中sEng及TGFβ1含量；MTT法观察滋养细胞的增殖情况并确定后续试验中sEng、TGFβ1及(sEng-TGFβ1)(?)的加药浓度及作用时间：MTT法观察各组细胞存活率,流式细胞技术观察各组细胞的细胞周期变化,Transwell技术检测各组滋养细胞的浸润功能；Western印迹法检测各组滋养细胞MMP-2、MMP-9蛋白的表达；RT-PCR法检测各组滋养细胞MMP-2、MMP-9mRNA的表达。
结果：24h、48h、72h滋养细胞培养液中未测得sEng:24h、48h、72h细胞培养液中TGFβ1含量分别为(5.03±0.54)、(4.09+0.27)、(5.91+0.68)ng/L,三个时间段TGFβ1含量无明显变化(F=3.09,p>0.05);10μg/LsEng、TGFβ1分别作用6h、12h、24h,各组细胞存活率与对照组比较均无明显差异：作用36h、48h,存活率明显降低,分别为(71.44±6.07)%、(51.44±6.34)%(36h),(62.37±8.12)%、(23.48±4.36)%(48h),无明显的时间及浓度依赖性;(sEng+TGFβ1)组滋养细胞存活率在各个时间点及浓度点与对照组比较无明显差异.确定各组加药浓度为10μg/L;作用48h,各组均未见明显凋亡;sEng组G1期细胞比例(88.24±2.11)%显著高于对照组(55.23±1.22)%%但低于TGFβ1组(95.63±2.98)%,(均p<0.05);TGFβ1组G1期细胞比例显著高于其它各组(均p<0.05);(sEng+TGFβ1)组G1期细胞比例(58.11±1.25)%与对照组比较无明显差异；各组加药10μg/L、作用24h, sEng组滋养细胞穿透Transwell基底膜的细胞数为(87+6.3)个/5HP显著低于对照组(143±41.3)个/5HP, TGFβ1组滋养细胞穿透Transwell基底膜的细胞数为(157-±-55.9)个/5HP显著高于对照组,而(sEng+TGFβ1)组细胞穿透数与对照组比较无显著性差异(p>0.05)。sEng组滋养细胞MMP-2和MMP-9蛋白及mRNA的表达明显低于对照组,TGFβ1组滋养细胞MMP-2和MMP-9蛋白及mRNA的表达明显高于对照组,差异均有统计学意义(均p<0.05),而(sEng+TGFβ1)组MMP-2和MMP-9蛋白及mRNA的表达与对照组无显著差异(p>0.05)。
结论：sEng和TGFβ1可能通过调节滋养细胞的G、期细胞比例,影响其增殖能力,但并不导致细胞的死亡;通过调节滋养细胞MMP-2和MMP-9的表达影响其浸润能力；在体外细胞培养环境下,sEng和TGFβ1可达到1:1的结合,并失云各自对滋养细胞的影响,据此推测,早孕期绒毛局部两种因子比例失调,可能不同程度的影响滋养细胞向子宫肌层的正常浸润,造成“胎盘浅着床”引起妊娠期高血压等相关疾病。
Hypertensive disorder complicating pregnancy is a pregnancy-specific disease which etiology and pathogenesis has not been fully elucidated. Previous studies have been shown that angiogenic factors TGFβ1and its receptor are closely related to the central steps of hypertensive disorders in pregnancy:vascular endothelial injury and placental vascular remodeling process. This study will observe the impact of TGFβ1and its soluble receptor components (sEng) in cultured human umbilical vein endothelial cells and trophoblasts explore the roles of these two factors in the development of hypertensive disorder complicating pregnancy.
Section Ⅰ. Research on the correlation between soluble endoglin. transforming growth factor β1and injury of vein endothelial cell
Objectives:To discuss the correlation between soluble endoglin. transforming growth factor β1and injury of vein endothelial cell. investigating the effects of these two factors on human umbilical vein endothelial cell (HUVEC). included apoptosis. proliferation, nitric oxide (NO) production and nitric oxide synthase (eNOS) phosphoryiation.
Methods:Cells (within3passages) were seeded in culture plates of96wells. There are4groups:controlled group with RPMI-1640culture medium only. sEng group. TGF(31group and (sEng-TGFβ1) group. These groups were divided into3subgroups (1、10、100μg/L) respectively on account of MTT results. Collecting the cells and cell cultures at6h.12h and24h for follow-up experiments. The concentration of soluble endoglin and TGFβ1in cell culture was evaluated by ELISA. Cell apoptosis and cell cycle were detected by flow cytometry. Cell viability was determined by methyl thiazolyl tetrazolium assay (MTT). The concentration of the metabolites of nitric oxide in each group was measured by nitrate reductase method. The expression of eNOS and eNOS-Ser(p)1177protein were detected by Western blot. eNOS mRNA in each group was detected by real-time fluorescence reverse transcription polymerase chain reaction (PCR).
Results:(1) The concentration of sEng in cell culture was0at24h.48h and72h. The concentration of TGFβ1in cell culture was (3.12±0.78).(3.33±0.91). (3.08±0.87)ng/L respectively. There were no significant difference at these three time points (F=2.10, p>0.05).(2) Cell viability:The cell viability was significantly decreased in sEng group. It had negative correlation with time and medicine concentration. The cell viability were significantly increased in1μg/L TGFP1group, but decreased in10and100μg/L groups. It had negative correlation with time and medicine concentration in the latter groups. There was no significant difference between (sEng+TGFβ1) group and controlled group at any concentration and time points.(3) Cell apoptosis and cell cycle:the cell apoptosis rate and the G1phase proportion in sEng group were higher than those in other groups. It had positive correction with time and medicine concentration. The cell G1proportion were significantly decreased in1μg/L TGFβ1group, but increased in10and100μg/L groups. It had positive correlation with time and medicine concentration in the latter groups. But TGFβ1had no business of apoptosis. There was no significant difference between (sEng-TGFβ1) group and controlled group on cell apoptosis and cell cycle at any concentration and time points.(4) Nitric oxide levels in cell culture:sEng decreased the concentration of nitric oxide levels through negative correlation with stimulate time and medicine concentration.1μg/L TGFβ1increased the concentration of nitric oxide levels through positive affection with stimulate time. But10and100μg/L TGFβ1decreased the concentration of nitric oxide levels through negative affection with stimulate time and medicine concentration. There was no significant difference between (sEng+TGFβ1) group and controlled group on nitric oxide levels at any concentration and time points.(5) The expression of eNOS protein and mRNA: The expression of eNOS protein and mRNA decreased significantly in sEng groups. It also had negative correlation with stimulate time and medicine concentration.1μg/L TGFβ1increased the expression of eNOS protein and mRNA through positive affection with stimulate time. But10and100μg/L TGFβ1decreased the expression of eNOS protein and mRNA through negative affection with stimulate time and medicine concentration. There was no significant difference between (sEng+TGFβ1) group and controlled group on the expression of eNOS protein and mRNA at any concentration and time points.(6) Nitric oxide synthase phosphorylation (eNOS-Ser(p)1177/ENOS ratio):eNOS-Ser(p)1177/eNOS ratio decreased significantly in sEng groups. It also had negative correlation with stimulate time and medicine concentration.1μg/L TGFβ1increased the eNOS-Ser(p)1177/eNOS ratio through positive affection with stimulate time. But10and100μg/L TGFβ1decreased the eNOS-Ser(p)1177/eNOS ratio through negative affection with stimulate time and medicine concentration. There was no significant difference between (sEng+TGFβ1) group and controlled group on the eNOS-Ser(p)1177/eNOS ratio at any concentration and time points.
Conclusions:1.sEng inhibited the proliferation of HUVEC in vitro through blocking the HUVEC in G1phase and increasing the apoptosis rate. It reduced the production of NO through inhibiting the proliferation of HUVEC and activation of eNOS. sEng participated the injury of endothelial cells.2. TGFβ1inhibited the proliferation of HUVEC in vitro through blocking the HUVEC in G1phase occasionally. The affection on HUVEC was up to medicine concentration. Low dose had promoted affection on HUVEC. But high dose had converse affections.3. sEng together with TGF(31were added in the cell culture seemed like having no affection on HUVEC. but it reflected that appropriate proportion of sEng and TGFβ1was very important to keep normal form and function of endothelial cells. All of above. sEng and TGFβ1participated the injury of endothelial cells by many ways.
Section Ⅱ. Effects of soluble endoglin and TGFβ1on proliferation and invasive ability of cultured cytotrophoblasts from human early pregnancy chorionic villus
Objective:To investigate the effects of soluble endoglin and TGFβ1on proliferation and invasive ability of cultured cytotrophoblasts from human early pregnancy chorionic villus.
Methods:Cytotrophoblasts of normal6to8week pregnancy were cultured by trypsin digestion method. Cells (within3passages) were seeded in culture plates of96wells. There are4groups:controlled group with DMEM culture medium only. sEng group. TGFβ1group and (sEng-TGFβ1) group. The concentration of soluble endoglin and TGFβ1in cell culture was evaluated by ELISA. The medicine concentration at follow-up experiments of these two factors was determined by MTT. The proliferation ability was determined by MTT. Cell cycle was detected by flowcvtometry. The invasive ability was determined by Transwell invasion assay. The expression of MMP-2and MMP-9protein was detected by Western blot. The expression of MMP-2and MMP-9mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR).
Results:The concentration of sEng in cell culture was0at24h,48h and72h. The concentration of TGFfβ1in cell culture was (5.03±0.54),(4.09±0.27). and (5.91±0.68)ng/L respectively. There were no significant difference at these three time points (F=3.09, p>0.05). Cell survival probability were degraded in sEng and TGFβ1groups at36h (71.44±6.07)%.(51.44±6.34)%and48h (62.37±8.12)%.(23.48±4.36)%versus controlled group. There was no significant difference between (sEng±TGFβ1) group and controlled group at any concentration and time points. The medicine concentration was10μg/L in every group on account of MTT. There was no significant cell apoptosis in every group. G1phase proportion in sEng group (88.24±2.11)%was higher than that of (sEng+TGFβ1)(58.11±1.25)%and control groups (55.23±1.22)%. G1phase proportion in TGFβ1group (95.63±2.98)%was higher than any other groups. There had no significant difference between (sEng+TGFβ1) group and control group. Cell numbers penetrating basement membrane was decreased significantly in sEng group (87±6.3) versus controlled group (143±41.3) but increased significantly in TGFβ1group (157±55.9). There was no significant difference between (sEng+TGF±1) group and controlled group. Compared with controlled group, the expression of MMP-2and MMP-9mRNA and protein of cytotrophoblasts was significantly lower in sEng group but higher in TGF(31group. There also was no significant difference between (sEng+TGFβ1) group and controlled group.
Conclusions:sEng and TGFβ1correlated with the procedure of cytotrophoblasts proliferation through moderating G1phase proportion in vitro. They also regulated cytotrophoblasts invasive ability by affecting the expression of MMP-2and MMP-9. Keeping suitable proportion was very important for cytotrophoblasts to invasive in mvometrium with normal depth at the first trimester.
引文
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