肿瘤坏死因子诱导蛋白8对滋养细胞凋亡和侵袭影响研究

目的探讨肿瘤坏死因子诱导蛋白8(TNFAIP8)对HTR8/SVneo滋养细胞凋亡和侵袭、迁移的影响。方法应用免疫组化检测2016年11月至2017年6月福建省立医院南院门诊行人工流产术的7～10周单胎正常妊娠妇女30例的正常早孕期绒毛中TNFAIP8的表达情况。构建针对TNFAIP8的慢病毒载体,感染HTR8/SVneo细胞后沉默TNFAIP8的表达,运用qRT-PCR、Western blot检验沉默效果。Transwell实验、细胞划痕实验、FCM法检测沉默TNFAIP8表达后的HTR8/SVneo细胞的侵袭、迁移能力及凋亡的影响。结果 TNFAIP8在正常早孕期绒毛外滋养细胞中阳性表达,且在绒毛组织与外周血淋巴细胞中表达呈正相关。运用qRT-PCR和Western blot检测显示,shTNFAIP8转入HTR8/SVneo细胞后,TNFAIP8蛋白和mRNA水平明显下调(P 0.05)。Transwell实验、细胞划痕实验结果显示沉默HTR8/SVneo细胞中TNFAIP8的表达明显抑制细胞的侵袭和迁移能力(P 0.001)。FCM法检测结果显示,TNFAIP8沉默组细胞凋亡显著增加(P0.05)。结论在早孕期绒毛外滋养细胞中,阳性表达的TNFAIP8可促进HTR8/SVneo滋养细胞的侵袭及迁移并抑制细胞的凋亡。     

内皮脂酶与重度子痫前期的相关性及其对滋养层细胞功能影响的研究

目的:探讨内皮脂酶(EL)基因在重度子痫前期(PE)患者胎盘组织中的表达,以及EL对人胎盘滋养层细胞生物学功能的影响及其调控机制。方法:收集12例重度PE患者和12例正常孕妇的胎盘组织。Real time-PCR、Western blot法检测EL mRNA和蛋白水平表达。脂质体转染人正常绒毛膜滋养层细胞系HTR8/SVneo干扰EL表达,Real timePCR、Western blot法检测EL转录及表达水平。用Annexin V-FITC、Transwell、Invasion、Tube formation等检测EL表达降低对HTR8/SVneo细胞凋亡、侵袭、迁移及血管化等能力的影响。Real time-PCR探究HTR8/SVneo功能改变的分子机制。结果:PE胎盘组织中EL表达水平下降;EL表达下降影响滋养细胞凋亡、侵袭、迁移等生物活性,MMP-9表达降低。结论:EL可能通过改变MMP-9表达影响人滋养层细胞的生物学功能,并通过S1PR-SPHK-e NOS通路参与胎盘血管的浅着床过程,从而促进PE的发生发展。     

不明原因早期复发性流产患者蜕膜自然杀伤细胞对滋养层细胞系侵袭功能的影响

目的研究早孕期复发性流产(RSA)患者与正常早孕妇女的蜕膜自然杀伤细胞(d NK)对滋养层细胞系HTR-8/SVneo侵袭能力的影响。方法收集正常早孕人工流产妇女(正常早孕组,n=10)和不明原因RSA患者(RSA组,n=8)的早孕蜕膜组织,用密度梯度离心法分离出蜕膜淋巴细胞,免疫磁珠法进一步分选出CD56+CD16-NK细胞,即蜕膜NK细胞,将蜕膜NK细胞与HTR-8/SVneo共培养24 h后,通过MTT法和Transwell侵袭实验检测蜕膜NK细胞对滋养层细胞系HTR8/SVneo黏附及侵袭能力的影响,用Real-time PCR检测共培养后HTR-8/SVneo细胞的MMP-2和MMP-9 mRNA水平的表达情况。结果与正常早孕妇女相比,不明原因早孕期RSA患者的蜕膜NK细胞对滋养层细胞系HTR8/SVneo侵袭能力有减弱作用,并使HTR-8/SVneo表达促侵袭分子MMP-2和MMP-9表达降低。结论妊娠早期局部母-胎界面d NK细胞的功能异常可能是导致不明原因RSA的一个原因。     

SIRT3在早发型子痫前期胎盘组织中的表达及作用

目的:检测早发型子痫前期(eoPE)患者胎盘组织中SIRT3表达,探讨SIRT3对滋养细胞功能的调控及其可能机制。方法:免疫组化法和免疫印迹法检测eoPE患者胎盘中SIRT3表达。试剂盒检测eoPE患者胎盘组织氧化应激因子水平。应用瞬时转染质粒使HTR-8/SVneo细胞过表达SIRT3,检测滋养细胞的侵袭和迁移能力。通过CoCl₂构建滋养细胞氧化应激模型,研究SIRT3对HTR-8/SVneo细胞抗氧化应激能力的调控。免疫印迹法检测Akt信号通路关键蛋白表达。结果:eoPE患者胎盘组织中SIRT3表达较对照组显著降低,SOD和GSH-Px水平明显降低,MDA水平明显升高。过表达SIRT3可促进HTR-8/SVneo细胞侵袭和迁移,显著缓解CoCl₂诱导的滋养细胞氧化应激。过表达SIRT3可显著上调Akt信号通路上的关键蛋白表达。结论:胎盘组织中SIRT3下调参与eoPE病理过程。SIRT3可能通过Akt信号通路促进滋养层细胞侵袭、迁移以及维持细胞氧化应激处于平衡状态。     

LncRNA H19通过调节miR-let7/MMP-9分子轴促进滋养细胞侵袭及迁移

目的:通过检测妊娠期高血压疾病(HDP)患者胎盘组织中lncRNA H19、miR-let7、MMP-9表达，探索HDP发病机制。方法:收集2019年12月至2021年3月在昆明医科大学第一附属医院分娩的妊娠期高血压疾病(HDP)患者86例(PIH 26例，PE 29例，sPE 31例)和正常妊娠孕妇20例。分别对HTR-8/Svneo细胞进行敲减或过表达H19,过表达H19与miR-let7e-5p mimics共转染。RT-qPCR法检测胎盘组织及HTR-8/Svneo细胞中lncRNA H19、miR-let7、MMP-9 mRNA表达水平，免疫组化法检测胎盘组织中MMP-9蛋白表达水平。Transwell试验检测细胞侵袭力，细胞划痕实验检测细胞迁移力，CCK-8试验检测细胞活力。结果:与正常妊娠组相比，HDP患者胎盘组织中lncRNA H19表达明显上调，miR-let7b-5p、miR-let7e-5p及MMP-9蛋白表达均下调；PIH及sPE患者胎盘组织中MMP-9 mRNA表达下调。HTR/SVneo细胞中敲减H19后，lncRNA H19及MMP-9 mRNA表达减少，...     

PTEN在子痫前期胎盘滋养细胞浸润不足中的作用及机制研究26

目的:检测子痫前期(PE)患者胎盘组织中PTEN表达,探讨PTEN对滋养细胞迁移功能的调控及其可能机制.方法:收集重度PE患者和正常孕妇的胎盘组织各20例,实时定量PCR和Western blot法检测胎盘组织中PTEN表达;将PTEN过表达质粒(pcDNA3.1-HA-PTEN)或特异性siRNA(siPTEN)瞬时转染至HTR-8/SVneo细胞,同时转染pcDNA3.1或siCTL作为对照.采用划痕实验评估细胞迁移能力,Western blot法检测p-Akt(Thr308)、p-Akt(Ser473)、Akt、MMP-2和MMP-9表达.结果:重度PE患者胎盘组织中PTEN mRNA和蛋白表达明显高于正常孕妇,差异均有统计学意义(P<0.01).与转染pcDNA3.1组比较,pcDNA3.1-HA-PTEN瞬时转染HTR-8/SVneo细胞48 h后,细胞迁移率明显降低[(26.42±6.68)%vs(52.92±6.71)%,P<0.01],p-Akt(Thr308)和p-Akt(Ser473)表达显著下调,Akt表达无明显改变,MMP-2和MMP-9蛋白表达下降;与转染siCTL组比较,siPTEN瞬时转染HTR-8/SVneo细胞48 h后,细胞迁移率则明显升高[(50.39±6.84)%vs(30.04±5.40)%,P<0.01],p-Akt(Thr308)和p-Akt(Ser473)的表达明显上调,Akt表达无明显改变,MMP-2和MMP-9蛋白表达升高.结论:重度PE患者胎盘组织中PTEN表达明显升高,PTEN可通过下调Akt活性降低MMP-2和MMP-9表达,抑制滋养细胞的迁移.提示PTEN在PE的发病中发挥了一定的作用.     

PTEN在子痫前期胎盘滋养细胞浸润不足中的作用及机制研究

目的:检测子痫前期(PE)患者胎盘组织中PTEN表达,探讨PTEN对滋养细胞迁移功能的调控及其可能机制。方法:收集重度PE患者和正常孕妇的胎盘组织各20例,实时定量PCR和Western blot法检测胎盘组织中PTEN表达;将PTEN过表达质粒(pcDNA3.1-HA-PTEN)或特异性siRNA(siPTEN)瞬时转染至HTR-8/SVneo细胞,同时转染pcDNA3.1或siCTL作为对照。采用划痕实验评估细胞迁移能力,Western blot法检测p-Akt(Thr308)、p-Akt(Ser473)、Akt、MMP-2和MMP-9表达。结果:重度PE患者胎盘组织中PTEN mRNA和蛋白表达明显高于正常孕妇,差异均有统计学意义(P0.01)。与转染pcDNA3.1组比较,pcDNA3.1-HA-PTEN瞬时转染HTR-8/SVneo细胞48h后,细胞迁移率明显降低[(26.42±6.68)%vs(52.92±6.71)%,P0.01],p-Akt(Thr308)和p-Akt(Ser473)表达显著下调,Akt表达无明显改变,MMP-2和MMP-9蛋白表达下降;与转染siCTL组比较,siPTEN瞬时转染HTR-8/SVneo细胞48h后,细胞迁移率则明显升高[(50.39±6.84)%vs(30.04±5.40)%,P0.01],p-Akt(Thr308)和p-Akt(Ser473)的表达明显上调,Akt表达无明显改变,MMP-2和MMP-9蛋白表达升高。结论:重度PE患者胎盘组织中PTEN表达明显升高,PTEN可通过下调Akt活性降低MMP-2和MMP-9表达,抑制滋养细胞的迁移。提示PTEN在PE的发病中发挥了一定的作用。     

长链非编码RNA中OIP5-AS1在重度子痫前期孕妇胎盘组织中的表达及意义

目的 探讨长链非编码RNA Opa相互作用蛋白5-反义转录物1(Opa-interactingprotein 5 antisense RNA 1, OIP5-AS1)在重度子痫前期孕妇胎盘组织中的表达及其可能作用。方法 采用实时定量荧光（RT-qPCR）法测定30例重度子痫前期孕妇和30例正常胎盘中OIP5-AS1和miR-29b表达情况;采用过划痕实验和Transwell侵袭实验检测缺氧条件下向人绒毛膜滋养层细胞系（HTR-8/SVneo细胞）内转染OIP5-AS1对HTR-8/SVneo细胞迁移和侵袭能力的影响;采用RT-qPCR法测定转染OIP5-AS1和OIP5-AS1基因的小干扰RNA(OIP5-AS1siRNA)后HTR-8/SVneo细胞微小RNA(microRNA,miRNA)miR-29b的表达情况。结果 在mRNA水平上,重度子痫前期组胎盘组织中OIP5-AS1较对照组表达降低（P <0.01）;过表达OIP5-AS1后,HTR-8/SVneo细胞迁移和侵袭能力增加,差异有统计学意义（P <0.01）;过表达OIP5-AS1后HTR-8/SVneo细胞中...     

胎盘生长因子在妊娠期糖尿病患者胎盘中抑制滋养细胞凋亡的作用研究

目的:探讨胎盘生长因子(PLGF)对妊娠期糖尿病(GDM)患者胎盘滋养细胞凋亡的影响。方法:收集20例GDM和20例正常孕妇胎盘组织,采用HE染色法观察GDM患者胎盘组织形态学变化。TUNEL染色以及Caspase-3活性检测胎盘中细胞凋亡情况。Western blot检测胎盘中PLGF蛋白表达情况,免疫组化观察PLGF的原位表达。离体培养人绒毛滋养细胞(HTR-8/SVneo),分为对照组、高糖组(HG)、PLGF+高糖组(PLGF+HG)和PLGF组,应用流式细胞术检测各组HTR-8/SVneo滋养细胞的凋亡率。结果:与对照组相比,GDM患者胎盘中滋养细胞凋亡明显减少,Caspase-3活性明显减弱。PLGF在GDM胎盘中的表达明显高于对照组;高糖诱导HTR-8/SVneo滋养细胞凋亡增加,PLGF预处理明显逆转高糖诱导的HTR-8/SVneo滋养细胞凋亡增加。结论:GDM患者胎盘中滋养细胞凋亡明显减少,而胎盘中PLGF表达升高。离体实验进一步证明PLGF能够抑制高糖所致HTR-8/SVneo滋养细胞凋亡增加,因此PLGF表达升高可能是GDM患者胎盘中滋养细胞凋亡减少的原因。     

基质金属蛋白酶3及其抑制剂3在子痫前期患者胎盘中的表达

目的:通过检测子痫前期患者胎盘组织中基质金属蛋白酶3(MMP-3)及其抑制剂3(TIMP-3)的表达,以探讨两者的关系及其在子痫前期发病中的作用。方法:收集34例子痫前期患者(子痫前期组)及32例正常晚期妊娠妇女(正常对照组)的胎盘组织,用免疫组化法检测MMP-3及TIMP-3在胎盘组织中的表达。同时进行HE染色,选取5个高倍视野,计数绒毛血管数。结果:①MMP-3和TIMP-3在胎盘组织的蜕膜细胞和绒毛滋养细胞均有表达。②子痫前期组胎盘蜕膜细胞和绒毛滋养细胞中MMP-3的表达均显著低于对照组(P<0.01)。TIMP-3在子痫前期组胎盘蜕膜细胞和绒毛滋养细胞中的表达量均显著高于对照组(P<0.01)。③子痫前期组胎盘绒毛血管密度明显低于对照组(P<0.01)。结论:子痫前期患者胎盘中MMP-3与TIMP-3的表达情况与正常晚期妊娠妇女存在差异,提示妊娠妇女胎盘组织中MMP-3和TIMP-3的表达水平变化可能与子痫前期的发生、发展相关。     

Immunocytochemistry of human chorionic gonadotropin in human chorionic villi

The immunocytochemical localization of human chorionic gonadotropin was investigated in chorionic villi from the seventh to twelfth week of gestation. By the light microscopic peroxidase-antiperoxidase technique, positive reactions of human chorionic gonadotropin were found exclusively in the syncytiotrophoblast. Immunoelectron microscopy by means of the protein A-gold technique reveals localization of the immunoreactive gold particles in two kinds of membrane-bound granular inclusions in this cell; one type is granules of 200 to 300 nm in diameter with moderate electron density and the other is large electron-dense bodies of 500 to 1000 nm. The former seems to be Golgi-derived secretory granules that play a role in the release of human chorionic gonadotropin from the syncytiotrophoblast. Although the origin of the latter is still uncertain, a certain amount of this hormone might be stored or treated by lysosomal digestion in the large bodies during these stages.     

Education about human papillomavirus and human papillomavirus vaccines in adolescents

PURPOSE OF REVIEW: The purpose of this article is to review recent literature that may help guide the development of effective, evidence-based strategies to educate adolescents about human papillomavirus (HPV) and HPV vaccines. Educational strategies are essential, given several new and highly effective technologies to prevent HPV and related diseases such as cervical cancer. RECENT FINDINGS: Although little has been published regarding adolescent knowledge about HPV and HPV vaccines, studies conducted primarily in adult women demonstrate that knowledge generally is poor. Studies of adolescent attitudes about HPV vaccines have identified several modifiable factors associated with intention and confidence in one's ability to receive the vaccine, including higher perceived severity of cervical cancer and fewer barriers to vaccination. Studies of clinician attitudes about HPV vaccines have demonstrated that although clinicians generally support vaccination, some report concerns; for example, adolescents may practice riskier sexual behaviors after vaccination. Studies also show that clinicians believe that educational materials developed specifically for adolescents are essential. SUMMARY: The recent literature on adolescent knowledge about HPV and attitudes about HPV vaccines supports the importance of designing developmentally appropriate educational materials for adolescents about HPV and HPV vaccines, and provides guidance for the development of key educational messages.     

Presence of a human chorionic gonadotropin--like substance in human sperm

An hCG-like material has been extracted from human sperm. These experiments were designed to characterize this material. Sperms of 10 volunteers were separated from seminal fluid, washed in PBS three times, and resuspended in 0.5 ml of the same buffer. Samples were pooled; cells were disrupted by sonication and extracted in alkaline buffer by constant agitation at 4 degrees C. The extract was ultracentrifuged at 4 degrees C. Supernate was lyophilized and reconstituted in 2 cc of distilled water. This material presented a dose-response curve parallel to those of IS2-hCG and CR119 in beta hCG RIA. When chromatographed in a Sephadex G-150 column the extract eluted within the hCG range and immunoreacted in the specific beta hCG RIA. When absorbed onto a concanavalin A--Sepharose column, all recovered immunoreactive material eluted after exposure to alpha-D-methylglucoside, indicating that it is a glycoprotein. The extract stimulated progesterone and testosterone secretion in porcine granulosa cells and decapsulated rat testis, respectively, indicating its biologic potency.     

Cytogenetic investigations on human oocytes and early human embryonic stages

The chromosome analysis of undivided oocytes and polyploid embryos within a human in vitro fertilization and embryo replacement program offers a unique opportunity to develop correlations between a successful or unsuccessful fertilization and the maturity of the oocytes or the quality of the sperm. Furthermore, it provides information concerning the origin of chromosomal defects.     

Induction of the human sperm acrosome reaction by human oocytes

The acrosome reaction of human spermatozoa incubated in the presence or absence of vested human oocytes was investigated. All gametes were obtained from human in vitro fertilization (IVF) cases. Spermatozoa were collected after incubation in insemination medium only and following removal of the oocytes from insemination medium during the IVF procedure. After 16 hours of incubation 18.5% of the spermatozoa in insemination medium alone were acrosome-reacted compared to 31.5% for spermatozoa incubated in medium containing oocytes. The acrosome reaction of spermatozoa incubated with fertilized or unfertilized oocytes was also investigated. The percentage of acrosome reaction did not differ (P greater than 0.05) between the two groups (29.7% in the fertilized cases versus 30.7% in the unfertilized cases). Completion of oocyte nuclear maturation did not affect the proportion of acrosome-reacted spermatozoa observed with unfertilized eggs. A similar (P greater than 0.05) percentage of acrosome reacted spermatozoa were observed regardless of whether the unfertilized oocytes had (29%) or had not (35%) reached metaphase II. These findings indicate the acrosome reaction of human spermatozoa is enhanced in the presence of vested human oocytes. Furthermore, there is no apparent correlation between the percentage of the population of spermatozoa that acrosome react in the medium and the potential of an oocyte for fertilization.