【作者】 刘敏；

【导师】 周容；

【作者基本信息】 四川大学 ， 妇产科学， 2022， 博士

【摘要】 目的:重度子痫前期（severe preeclampsia,SPE）是妊娠期高血压疾病的一种类型,严重威胁母儿生命健康。SPE患者发病的原因具有复杂性,兼有多机制致病的综合征发病性质。然而,目前对其发病机制的探究尚不完善。本研究在人胎盘组织上,检测胎盘组织的基底细胞粘附分子（basal cell adhesion molecules,BCAM）表达水平,分析胎盘BCAM表达与SPE重要临床指标之间的相关性。在细胞水平（HTR-8/SVneo和JAR细胞）上,探究BCAM表达变化对滋养细胞生物学行为的影响;通过生物信息学网站预测靶向BCAM的miRNAs,证实miR-199a-5p介导BCAM低表达及滋养细胞生物学行为改变;通过RNA测序筛选及实验证实BCAM调控的下游PIK3R6/p-STAT3信号通路。进一步在动物实验上,建立BCAM稳定低表达的孕鼠模型,检测孕鼠血压、多器官（胎盘、肾脏和肝脏）的形态结构和功能、胎鼠的一般情况（胎重和身长）等,确证胎盘BCAM低表达可致机体SPE样表型,并进一步证实BCAM/PIK3R6/p-STAT3通路参与调控SPE的分子机制。材料和方法:胎盘组织:通过RT-qPCR和western blot检测正常对照组与SPE组胎盘间BCAM的表达差异。收集、整理临床样本资料,分析SPE胎盘BCAM表达水平与重要临床指标（24-h尿蛋白、血压和BMI等）的相关性。结合多个生物信息学网站,预测与BCAM存在直接靶向作用的mi RNAs,利用RT-qPCR初步验证这些miRNAs在胎盘组织上的表达。此外,利用western blot验证在细胞学实验上所探究的PIK3R6/p-STAT3信号通路。细胞水平:在HTR-8/SVneo与JAR细胞上,分别用BCAM siRNA（70 nM）和BCAM pc DNA（3.1+）质粒（3μg）处理细胞24 h后,采用CCK8检测细胞增殖能力,Transwell小室培养（有/无带基质胶）检测细胞迁移及侵袭,ibidi血管生成载玻片培养（基质胶）检测细胞的成管能力,流式细胞术检测细胞凋亡水平的变化。通过双荧光素酶报告基因验证miR-199a-5p与BCAM之间是否存在直接结合。滋养细胞经miR-199a-5p mimic或inhibitor处理后,检测细胞增殖、迁移、侵袭、成管和凋亡的影响。经BCAM siRNA转染的HTR-8/SVneo细胞进行RNA测序,通过生物信息学分析后,利用RT-qPCR和western blot检测与JAK-STAT信号通路和PI3K活性调控相关的差异基因（PIK3IP、PIK3CB、PIK3R6、STAT1/3和SOCS1）的表达;检测可调控细胞生物学行为且表达变化最大的几个基因（UBD、PDE6B、XAF1和SAA1）的表达。为验证BCAM/PIK3R6/p-STAT3信号通路的上下游关系,HTR-8/SVneo与JAR细胞经PIK3R6 siRNA和BCAMpcDNA（3.1+）质粒共转染处理,利用western blot检测BCAM、PIK3R6、STAT3和p-STAT3蛋白水平变化。动物实验:分别对孕鼠和非孕鼠进行相同组别设置,均包括Ad-shBCAM实验组、Ad-sFlt-1阳性对照组、Ad-GFP对照组及生理盐水（normal saline,NS）对照组,共有8组。在孕第9.5天（gestational 9.5 days,GD9.5）建立动物模型,各组大鼠尾静脉注射2×10⁹ PFU腺病毒,NS组尾静脉注射等量生理盐水。在GD6、GD9、GD12、GD15和GD17对孕鼠进行称重、24-h尿蛋白收集与检测,并通过鼠尾袖套系统监测无创血压,包括收缩压（systolic blood pressure,SBP）和舒张压（diastolic blood pressure,DBP）;非孕鼠在尾静脉处理后的相同时间内进行同等检测。GD19孕鼠麻醉后检测有创血压,再用异氟烷安乐死后取材,收集孕鼠血浆、尿液及胎盘、肝肾等组织器官,仔鼠计数、称重和测量身长;除胎盘和胎鼠的相关取材外,非孕鼠进行类似操作。通过苏木精-伊红（hematoxylin-eosin,HE）染色、糖原（periodic acid schiff,PAS）染色和马松三色（masson′s trichrome,MST）染色观察大鼠的各脏器的形态结构变化,酶联免疫吸附法测定血浆胎盘生长因子（placenta growth factor,PlGF）和可溶性fms样酪氨酸激酶-1（solublefms-like tyrosine kinase 1,sFlt-1）水平,生化仪检测血浆天冬氨酸转氨酶（aspartate transaminase,AST）、丙氨酸转氨酶（alanine aminotransferase,ALT）和乳酸脱氢酶（lactic dehydrogenase,LDH）水平。此外,利用western blot检测孕鼠胎盘组织BCAM/PIK3R6/p-STAT3通路的蛋白表达。结果:研究对象临床特征:与正常对照组相比,SPE组的重要临床指标,如SBP、DBP、24-h尿蛋白和体重指数显著升高,血浆中AST、ALT、LDH、总胆红素水平、尿素和肌酐水平也显著升高（P<0.05,P<0.01）,血小板计数、血清白蛋白和新生儿身长和体重均显著减少（P<0.05,P<0.01）;SPE组的孕周显著小于正常对照组,而两组间的年龄无统计学差异。胎盘组织:（1）组织免疫荧光结果提示,BCAM在胎盘组织中有较高的基础表达丰度,并分布在滋养细胞胞膜上;RT-q PCR和western blot检测发现,与对照组相比,SPE患者胎盘组织BCAM表达水平显著降低（P<0.01）。（2）SPE胎盘组织BCAM表达水平与SBP和24-h尿蛋白呈显著负相关（P<0.05,P<0.01）;BCAM表达水平与血清白蛋白和新生儿体重呈显著正相关（P<0.05,P<0.01）;而BCAM表达水平与其他临床指标（如BMI、AST和ALT等）之间不具有显著相关性。（3）RT-qPCR检测结果发现,与对照组相比,SPE胎盘组织中miR-199a-5p表达显著增加（P<0.05）,miR-199a-5p和BCAM表达水平呈显著负相关（P<0.05）;Western blot结果显示,SPE胎盘组织中PIK3R6的蛋白水平及p-STAT3^Y705/STAT3蛋白比值均显著降低（P<0.05,P<0.01）。细胞水平:在HTR-8/SVneo细胞和JAR细胞上,（1）BCAMsiRNA和BCAMpcDNA（3.1+）过表达质粒可分别有效沉默和过表达细胞的BCAM表达水平（P<0.01）。（2）CCK8检测结果显示,BCAM沉默可显著减弱细胞在各时间点（24、48和72 h）上的增殖能力,而BCAM过表达可增加细胞增殖能力（P<0.05,P<0.01）。（3）Transwell小室培养实验表明,BCAM沉默可抑制细胞的迁移、侵袭能力,而BCAM过表达可促进细胞迁移、侵袭能力（P<0.05,P<0.01）。（4）小管形成实验提示,BCAM沉默可显著减弱细胞的血管生成能力（P<0.05,P<0.01）,而BCAM过表达有增加细胞血管生成的趋势（P=0.06）。（5）流式细胞术结果表明,BCAM沉默及过表达均不会对细胞凋亡产生显著影响。（6）MiR-199a-5p mimic转染细胞后可有效增加细胞miR-199a-5p的表达,并降低BCAM的mRNA与蛋白表达水平;MiR-199a-5p inhibitor可有效降低细胞mi R-199a-5p的表达,并增加细胞BCAM的m RNA与蛋白表达水平（P<0.05,P<0.01）。（7）双荧光素酶报告基因结果显示,miR-199a-5p mimic与野生型质粒组共转染的荧光活性显著低于miR-199a-5p mimic与突变型质粒组共转染组的荧光活性（P<0.05,P<0.01）,表明miR-199a-5p与BCAM存在直接结合。（8）MiR-199a-5p表达增加可显著抑制细胞增殖、迁移和侵袭能力（P<0.05,P<0.01）,而对血管生成和细胞凋亡无显著影响。（9）RNA测序与生物信息学分析提示,BCAM表达降低可能影响JAK-STAT信号通路和PI3K活性调控。（10）Western blot结果显示,BCAM沉默可抑制PIK3R6蛋白表达和p-STAT3^Y705/STAT3蛋白比值,BCAM过表达可增加PIK3R6蛋白表达和p-STAT3^Y705/STAT3蛋白比值（P<0.05,P<0.01）。然而,沉默PIK3R6不影响BCAM表达水平,BCAM过表达质粒与PIK3R6 si RNA共转染后,PIK3R6 si RNA可显著逆转BCAM对PIK3R6蛋白表达和p-STAT3^Y705/STAT3蛋白比值的上调作用（P<0.05,P<0.01）。动物实验:与Ad-GFP对照组及NS对照组相比,（1）Ad-shBCAM实验组胎盘组织BCAM蛋白表达显著降低（P<0.01）。（2）Ad-shBCAM实验组和Ad-sFlt-1阳性对照组在处理后的不同时间点（GD12、GD15和GD17）上,孕鼠的收缩压、舒张压、24-h尿蛋白水平均显著升高（P<0.05,P<0.01）。（3）HE染色结果显示,Ad-sh BCAM与Ad-s Flt-1组胎盘迷路区血管分支减少;MST染色结果显示,Ad-shBCAM与Ad-sFlt-1组胎盘绒毛细胞中存在胶原沉积;ELISA检测结果提示,Ad-shBCAM与Ad-sFlt-1组的孕鼠血浆Pl GF水平显著降低,而sFlt-1水平和sFlt-1/PlGF比值显著升高（P<0.05,P<0.01）。（4）PAS染色结果显示,Ad-shBCAM与Ad-sFlt-1组的孕鼠肾脏组织紊乱,肾小管萎缩,间质水肿,内皮细胞肿胀;免疫荧光结果表明肾脏CD31荧光表达显著减弱。（5）Ad-shBCAM实验组与Ad-sFlt-1阳性对照组的孕鼠肝脏小叶间门静脉坏死,周围肝细胞排列紊乱,孕鼠血浆ALT、AST和LDH水平均显著升高（P<0.05,P<0.01）。（6）Ad-shBCAM与Ad-sFlt-1组的胎盘重量、胎鼠重量和身长均显著减少（P<0.05,P<0.01）;而各组间每窝的胎仔数无显著差异。（7）在非孕鼠上进行类似的检测,结果表明,各组大鼠的无创血压和有创血压均无显著差异;肾脏基底膜突出,内皮细胞排列规整;肝脏细胞排列规整,细胞单列呈索状。结论:（1）SPE患者胎盘组织中BCAM表达水平显著降低,并与重要临床指标之间呈显著相关性,表明胎盘BCAM表达降低与SPE发生密切相关。（2）BCAM表达降低可抑制HTR-8/SVneo和JAR细胞增殖、迁移、侵袭能力和HTR-8/SVneo细胞成管能力。（3）MiR-199a-5p与BCAM之间存在直接结合位点,miR-199a-5p表达增加可抑制BCAM表达。（4）MiR-199a-5p表达升高可抑制滋养细胞（HTR-8/SVneo和JAR）增殖、迁移和侵袭能力。（5）BCAM表达降低可抑制PIK3R6/p-STAT3通路。（6）BCAM表达降低可通过PIK3R6/p-STAT3通路诱导孕鼠SPE样表型。更多还原

【Abstract】 Objective:Severe preeclampsia（SPE）is a type of hypertensive disease in pregnancy,which seriously threatens the health of mothers and children.The pathogenesis of SPE in pregnant women is complicated with the multi-mechanism syndrome.However,the study of its pathogenesis is still incomplete.In this study,the expression level of basal cell adhesion molecules（BCAM）in human placental tissues was detected,and the correlation between BCAM expression and important clinical indicators of SPE was analyzed.In vitro（HTR-8/SVneo and JAR cells）,the effects of changes in BCAM expression on the biological behavior of trophoblast cells were investigated,and the possible causes of changes in BCAM expression and its downstream molecular mechanisms were elucidated.In an animal experiment,we established a stable lower expression of BCAM pregnant rat model,detect the blood pressure of pregnant rats,the structure and function of multiple organs（placenta,kidney,and liver）,the general situation of fetal rats（fetal weight and length）,confirmed the low expression of placenta BCAM can cause the SPE-like phenotype in pregnant rats,and further confirmed BCAM molecular mechanism involved in the regulation of SPE.Materials and Methods:In placental tissues,the expression of BCAM was detected by RT-qPCR and western blot,clinical sample data were collected to analyze the correlation between the expression level of placental BCAM and important clinical indicators（such as 24-h urinary protein,SBP,DBP,etc.）.Combined with several bioinformatics websites,miRNAs directly targeting BCAM were predicted,and the expression of these miRNAs in placental tissues was preliminarily verified by RT-qPCR.In addition,western blot was used to verify the PIK3R6/p-STAT3 signaling pathway explored in vitro.In vitro,HTR-8/SVneo and JAR cells were treated with BCAM siRNA（70 n M）and BCAM pcDNA（3.1+）plasmids（3μg）,respectively.CCK8 was used to detect cell proliferation,cell culture in transwell（with or without matrigel）was used to detect cell migration and invasion,ibidi angiogenesis slide was used to detect angiogenesis,and flow cytometry was used to detect cell apoptosis.Combined with several bioinformatics websites,miRNAs directly targeting BCAM were predicted,and the expression of these miRNAs in HTR-8/SVneo,JAR cells,and placentas was verified by RT-qPCR.Dual-luciferase reporter gene confirmed whether there was direct binding between miR-199a-5p and BCAM.The effects of proliferation,migration,invasion,angiogenesis,and apoptosis of HTR-8/SVneo and JAR cells were detected after miR-199a-5p mimic or inhibitor treatment.HTR-8/SVneo cells transfected by BCAM siRNA were used for RNA sequencing and bioinformatics analysis.The differentially expressed genes（PIK3IP,PIK3CB,PIK3R6,STAT1/3,and SOCS1）were detected by RT-qPCR and western blot.The differentially expressed several genes（UBD,PDE6B,XAF1,and SAA1）closely related to the tumor also were detected.To verify the BCAM/PIK3R6/p-STAT3 signaling pathway,HTR-8/SVneo and JAR cells were co-transfected with PIK3R6 siRNA and BCAM pcDNA（3.1+）plasmid,western blot was used to detect the protein levels of BCAM,PIK3R6,STAT3,and p-STAT3.In animal experiments,pregnant rats and non-pregnant rats were respectively set up in the same group,including the Ad-sh BCAM experimental group,Ad-sFlt-1positive control group,Ad-GFP control group,and saline control group,a total of eight groups.The animal model was established on gestational 9.5 days（GD9.5）.Rats in each group with 2×10⁹ PFU adenovirus by tail vein injection and pregnant rats of the saline group were injected with saline of equal volume.The pregnant rats were weighed on GD6,GD9,GD12,GD15,and GD17,and the 24-h urine protein was collected and detected.Non-invasive blood pressure was monitored by the tail-cuff system.At the same time,non-pregnant mice were tested after tail vein treatment.Pregnant rats of GD19 were euthanized by isoflurane,and the plasma,urine,tissues,and organs（such as placenta,liver,and kidney）of pregnant rats were collected.The fetal rats were counted,weighed,and measured.The morphological and structural changes of multiple organs were observed by HE,PAS,and MST staining.The levels of plasma Pl GF and sFlt-1 were determined by ELISA assay,and levels of plasma AST,ALT,and LDH were determined by biochemistry analyzer.In addition,a western blot was used to detect the protein expression of the BCAM/PIK3R6/p-STAT3pathway in the placentas of pregnant rats.Results:Clinical characteristics of subjects:Compared with the control group,the important clinical indicators of SPE,such as systolic blood pressure,diastolic blood pressure,24-h urine protein,body mass index,serum AST,ALT,LDH,total bilirubin levels,urea,and creatinine levels were also significantly increased（P<0.05,P<0.01）.However,platelet count,serum albumin,fetal length,and fetal weight were significantly decreased（P<0.05,P<0.01）.The pregnancy weeks of SPE group were lower than those of in control group.Then,there was no significant difference in age of pregnant women between the control and SPE groups.Placental tissue:（1）Immunofluorescence showed that BCAM expression was increased in placental tissue and distributed in trophoblast cells.RT-qPCR and western blot results showed that the expression level of BCAM in placental tissue of patients with SPE was significantly reduced than that of the control group（P<0.01）.（2）There were significant negative correlations between the expression level of BCAM and serum albumin,SBP,24-h urine protein in patients with SPE（P<0.05,P<0.01）.The expression level of BCAM was significantly positively correlated with serum albumin and fetal weight（P<0.05,P<0.01）.There was no significant correlation between BCAM expression level and other clinical indicators,such as BMI,AST,and ALT.（3）Compared with the control group,the expression of miR-199a-5p was significantly increased in the placentas of SPE（P<0.05）,and there was a significant negative correlation between miR-199a-5p and BCAM expression（P<0.05）.Western blot showed that PIK3R6 protein level and p-STAT3/STAT3 ratio in SPE placentas were significantly decreased（P<0.05,P<0.01）.In vitro（HTR-8/SVneo and JAR cells）:（1）BCAM siRNA and BCAM pcDNA（3.1+）overexpressed plasmid could effectively silence the expression level of BCAM（P<0.01）.（2）The results of CCK8 showed that BCAM silencing significantly reduced cell proliferation at 24,48,and 72 h,respectively,while BCAM overexpression significantly increased cell proliferation（P<0.05,P<0.01）.（3）Transwell experiment showed that BCAM silencing decreased the migration and invasion ability of trophoblast cells,while BCAM overexpression increased their ability of migration and invasion（P<0.05,P<0.01）.（4）Tubule formation experiment suggested that BCAM silencing significantly reduced the angiogenesis of trophoblast cells（P<0.05,P<0.01）,while BCAM overexpression tended to increase the angiogenesis of cells（P=0.06）.（5）Flow cytometry results showed that both BCAM silencing and overexpression had no significant effect on apoptosis.（6）miR-199a-5p mimic could effectively increase the expression levels of miR-199a-5p but decrease the expression levels of BCAM,and miR-199a-5p inhibitor could significantly decrease the levels of miR-199a-5p but increase the expression levels of BCAM,both in mRNA and protein levels（P<0.05,P<0.01）.（7）The results of the double luciferase reporter gene revealed that the fluorescence activity of miR-199a-5p mimic and wild-type plasmid co-transfected group was significantly lower than that of miR-199a-5p mimic and mutant plasmid co-transfected group（P<0.05,P<0.01）.It suggested that there was at least one direct binding between miR-199a-5p and BCAM.（8）Increased miR-199a-5p expression significantly depressed cell proliferation,migration,and invasion（P<0.05,P<0.01）,but had no significant effect on angiogenesis and apoptosis.（9）RNA sequencing and bioinformatics analysis suggested that decreased BCAM expression might affect the Jak-STAT signaling pathway and PI3K activity regulation.The mRNA and protein of PIK3R6 and STAT3 were inhibited by reduction of BCAM,while the mRNA and protein of PIK3R6 and STAT3 were increased by BCAM overexpression（P<0.05,P<0.01）.（10）Western blot showed that BCAM silencing significantly inhibited the protein expression of PIK3R6 and p-STAT3^S705,while BCAM overexpression significantly increased the protein expression of PIK3R6 and p-STAT3^S705,but had no significant effect on the protein expression of STAT3（P<0.05,P<0.01）;Silencing PIK3R6 did not affect the expression level of BCAM,while PIK3R6 siRNA could significantly reverse the up-regulation of PIK3R6 and p-STAT3 protein expressions regulated by BCAM overexpression after co-transfection of BCAM overexpressed plasmid and PIK3R6 siRNA（P<0.05,P<0.01）.Animal experiments:Compared with the Ad-GFP control group and saline control groups,（1）Ad-sh BCAM group placental tissue BCAM protein significantly decreased（P<0.01）.（2）On GD12,GD15,and GD17,non-invasive blood pressure（SBP and DBP）and 24-h urine protein level of pregnant rats in the Ad-sh BCAM group and Ad-sFlt-1 positive control groups were increased significantly（P<0.05,P<0.01）.（3）HE staining showed that vascular branches in the placental labyrinth were decreased in Ad-sh BCAM and Ad-sFlt-1 groups.MST staining showed collagen deposition in placental trophoblast cells in Ad-sh BCAM and Ad-sFlt-1 groups.Plasma PlGF level in Ad-sh BCAM and Ad-sFlt-1 groups was significantly decreased,while plasma sFlt-1 level and sFlt-1/Pl GF ratio were increased significantly（P<0.05,P<0.01）.（4）The periodic acid-schiff staining showed that renal tissue disorder,renal tubule atrophy,interstitial edema,and endothelial cell swelling were observed in Ad-shBCAM and Ad-sFlt-1 groups.Immunofluorescence found that the expression of CD31 in the kidney was significantly decreased in Ad-sh BCAM and Ad-sFlt-1 groups.（5）In the Ad-sh BCAM and Ad-sFlt-1 positive control groups,liver interlobular portal vein necrosis,surrounding stem cells disorder,the levels of plasma ALT,AST and LDH were significantly increased（P<0.05,P<0.01）.（6）The placental weight,fetal weight,and fetal body length in Ad-shBCAM and Ad-sFlt-1 groups were decreased significantly（P<0.05,P<0.01）;There was no significant difference in litter size between all groups.（7）There was no significant difference between invasive blood pressure and noninvasive blood pressure in non-pregnant rats.The basement membrane of the kidney was prominent and the endothelial cells appeared neatly arranged.The liver cells are arranged in a regular,single row of cells in a cable shape.Conclusion:（1）The expression levels of BCAM in placentas of patients with SPE were significantly decreased and were significantly correlated with important clinical indicators,suggesting that the expression levels of BCAM were closely related to the occurrence of SPE.（2）InHTR-8/SVneo and JAR cells,a lower expression level of BCAM could inhibit proliferation,migration,invasion,and angiogenesis of trophoblast cells.（3）There was at least one direct binding site between miR-199a-5p and BCAM,and increased miR-199a-5p expression inhibited BCAM expression.（4）Increased miR-199a-5P expression inhibited proliferation,migration,and invasion of trophoblast cells（HTR-8/SVneo and JAR）.（5）Decreased BCAM expression can inhibit the PIK3R6/p-STAT3 signaling pathway.（6）Decreased BCAM expression could induce SPE-like phenotype in pregnant rats through the PIK3R6/p-STAT3pathway.更多还原