PINK1/Parkin通路介导的线粒体自噬对宫腔粘连纤维化的影响及机制

目的:研究PINK1/Parkin介导的线粒体自噬在宫腔粘连纤维化中的影响及机制。方法:分离培养人子宫内膜基质细胞,分为空白组、H_2O_2组、siRNA-PINK1组、过表达PINK1组、siRNA对照组、空质粒组。除空白组外,其余各组均采用100μmol/L H_2O_2进行处理,建立氧化应激模型。分别采用RT-qPCR、Western blot和流式细胞术检测细胞中α-SMA、ColⅠ、FN、TIMP-1、MMP-9、p-NF-κB、NF-κB、TGF-β1、PINK1、Parkin、LC3Ⅱ和p62 mRNA和蛋白的表达及ROS水平和线粒体膜电位。结果:相较于空白组,H_2O_2组细胞中α-SMA、ColⅠ、FN、TIMP-1、p-NF-κB、NF-κB、TGF-β1和p62的表达以及ROS水平均显著升高,MMP-9、PINK1、Parkin和LC3Ⅱ的表达及线粒体膜电位均显著降低(P0.05);相较于H_2O_2组,过表达PINK1组细胞中α-SMA、ColⅠ、FN、TIMP-1、p-NF-κB、NF-κB、TGF-β1和p62的表达及ROS水平均显著降低,MMP-9、PINK1、Parkin和LC3Ⅱ的表达及线粒体膜电位均显著升高(P0.05),而siRNA-PINK1组的结果与之相反;空质粒组、siRNA对照组与H_2O_2组结果相比,差异无统计学意义(P0.05)。结论:上调PINK1/Parkin介导的线粒体自噬可通过调控ROS/NF-κB/TGF-β1信号通路,进而抑制子宫内膜基质细胞纤维化。     

细胞自噬在甲氨蝶呤耐药绒癌中的保护性作用

目的：通过检测耐药绒癌细胞内自噬的变化,以及评估抑制自噬或敲除自噬相关基因后细胞凋亡情况,探讨细胞自噬在耐药绒癌中的作用。方法：以人绒毛膜上皮癌JEG-3细胞及本课题组前期构建的甲氨蝶呤（MTX）耐药绒癌JEG-3／MTXR细胞为研究对象。采用透射电镜、免疫荧光法观察MTX处理后细胞内自噬的变化;Westernblot法检测自噬相关蛋白LC3、Bec]in-1的表达变化。RNA干扰法沉默自噬相关基因LC3后,采用Westernblot法和Annexin-V／PI双染法流式细胞仪检测MTX作用后JEG-3／MTXR耐药绒癌细胞凋亡的变化。结果：透射电镜和荧光显微镜下均可观察到MTX处理后JFG-3、JEG-3／MTXR细胞内自噬小体的产生;Icm浓度MTX处理后,两种细胞内自噬相关蛋白LC3、Beclin-1表达水平上调。相同浓度MTX处理后,JEG-3细胞内LC3、Beclin-1表达下调。沉默自噬相关基因LC3后,沉默LC3联合MTX组的JEG-3／MTXR和JEG-3细胞内凋亡相关蛋白caspase-9表达上调,凋亡率显著增多。结论：MTX可诱导耐药绒癌产生保护性自噬。     

顺铂诱导卵巢癌SKOV3细胞自噬和凋亡的研究

目的:研究顺铂诱导卵巢癌SKOV3细胞自噬和凋亡的发生情况,并探讨可能的发生机制。方法:顺铂干预卵巢癌SKOV3细胞后,用MDC染色荧光显微镜观察自噬囊泡及流式细胞仪检测自噬率;AnnexinV-FITC/PI双染色流式细胞仪检测凋亡率及TUNEL法检测凋亡指数;Western blot检测自噬蛋白Beclin1,LC3-Ⅱ和凋亡蛋白caspase9,caspase3的表达。结果:顺铂干预SKOV3细胞后,MDC阳性细胞数明显增多;自噬率提高(P<0.05);凋亡率提高(P<0.05);凋亡指数提高(P<0.05);自噬蛋白Beclin1,LC3-Ⅱ和凋亡蛋白caspase9,caspase3表达均增强(P<0.05)。结论:顺铂不仅可诱导卵巢癌SK-OV3细胞凋亡,而且可诱导卵巢癌SKOV3细胞自噬性死亡。     

MMP-9在绒毛外滋养细胞诱导内皮细胞凋亡中作用的研究

目的:探讨绒毛外滋养细胞基质金属蛋白酶-9(MMP-9)的表达对血管内皮细胞凋亡的影响。方法:MMP-9的siRNA转染绒毛外滋养细胞株(TEV-1),利用实时定量逆转录-聚合酶链反应(real time RT-PCR)和ELISA法检测转染前后细胞中MMP-9、FasL基因及蛋白表达的变化;通过trans well共培养技术,研究滋养细胞对内皮细胞凋亡的影响。结果:(1)MMP-9 siRNA转染后24h,滋养细胞MMP-9 mRNA的表达较对照组明显降低(P0.05),而FasL mRNA表达与对照组无明显差异(P0.05);转染后48h,细胞培养液中MMP-9蛋白及FasL蛋白表达均较对照组显著降低(P0.05);(2)与未转染者比较,转染MMP-9 siRNA的滋养细胞与内皮细胞共培养48h后,内皮细胞凋亡率显著降低,差异有显著性(P0.05)。结论:绒毛外滋养细胞表达MMP-9的水平影响其诱导内皮细胞凋亡的作用,此作用可能与MMP-9调节FasL蛋白的分泌表达有关。     

自噬在卵巢癌失巢凋亡抵抗中的作用

目的 探讨自噬在卵巢癌失巢凋亡抵抗中的作用。分析自噬和整合素相关蛋白在卵巢癌细胞株A2780不同培养方式下的表达情况,并通过siRNA和化学抑制剂来明确其调控机制。方法 2012年6月至2013年6月在上海交通大学医学院附属仁济医院以卵巢癌细胞株A2780在常规培养、Matrigel铺胶培养和悬浮培养这3种培养方式中用Western blot检测自噬和整合素相关蛋白的表达,流式细胞技术检测细胞凋亡情况。结果 卵巢癌细胞株A2780在常规培养、Matrigel铺胶培养和悬浮培养这3种培养方式中自噬相关分子LC3、Beclin1和整合素CD51(αv),CD61(β3),CD29(β1)的表达情况发现,CD51和CD61的表达逐渐下降（[WTBX]P[WTBZ]< 0.05）,而自噬相关分子LC3的表达却为递增（[WTBX]P[WTBZ]<0.05）,Beclin1蛋白表达差异无统计学意义（[WTBX]P[WTBZ]>0.05）。在3种不同培养方式中,RGD使用后自噬相关分子LC3的蛋白表达增强（[WTBX]P[WTBZ]<0.05）;RGD阻断整合素信号途径并没有促进细胞的凋亡（[WTBX]P[WTBZ]>0.05）,但是RGD作用下同时加入自噬抑制剂3-MA可以促进细胞的凋亡（[WTBX]P[WTBZ]<0.05）。结论 自噬在卵巢癌失巢凋亡中主要通过整合素信号αvβ3途径来调控,可以为失巢细胞早期提供能量支持,在一定条件下抵抗失巢凋亡,这也是造成卵巢恶性肿瘤转移的重要原因。     

5-氟尿嘧啶在体外对卵巢癌细胞SKOV3自噬过程的诱导作用

目的:探讨化疗药物5-氟尿嘧啶(5-FU)对卵巢癌细胞SKOV3自噬的诱导作用及其机制。方法:应用吖啶橙染色、间接免疫荧光技术观察50μmol/L5-FU处理48 h对卵巢癌细胞SKOV3自噬过程的影响;并通过Western blotting检测5-FU对SKOV3细胞中微管相关蛋白轻链3(LC3)和自噬相关蛋白Beclin1表达水平的影响。结果:5-FU作用后SKOV3细胞产生明显的酸性区域,LC3定位的荧光亮点增多,细胞内Beclin1和LC3蛋白表达量也显著增加。结论:5-FU可能通过增加SKOV3细胞酸性区域的数量、促进细胞内Beclin1、LC3蛋白表达而诱导SKOV3细胞自噬。     

HLA-E介导孕激素对绒毛膜癌JEG-3细胞血管内皮生长因子表达的调节作用

目的:研究人类白细胞抗原E(HLA-E)是否参与孕激素对JEG-3细胞VEGF基因表达的调节作用。方法:JEG-3细胞分为5组,接受不同处理:空白对照(组1)、孕激素处理(组2)、转染HLA-E siRNA慢病毒后孕激素处理(组3),HLA-E siRNA慢病毒转染(组4)以及慢病毒阴性对照转染(组5)。48小时后收集细胞,分别采用Real-time PCR和Western blot方法检测JEG-3细胞血管内皮生长因子(VEGF) mRNA及蛋白表达水平。结果:组2 JEG-3细胞的VEGF mRNA和蛋白表达较组1增高,差异有统计学意义(P0.05);沉默HLA-E基因(组4)导致VEGF mRNA和蛋白表达降低,与组1比较,差异有统计学意义(P0.05)。组3 JEG-3细胞经HLA-E siRNA慢病毒转染、沉默HLA-E表达后再接受孕激素处理,VEGF mRNA及蛋白表达水平与组1比较差异无统计学意义(P0.05)。组5与组1比较,VEGF mRNA及蛋白水平比较,差异无统计学意义(P0.05)。结论:HLA-E介导孕激素对JEG-3滋养细胞VEGF表达的上调,孕激素对JEG-3细胞VEGF的上调作用被HLA-E基因的沉默消除。     

子痫前期孕妇母胎界面炎症介质的表达与线粒体自噬的相关性研究

目的:探讨子痫前期(PE)孕妇母胎界面炎症介质的表达量以及线粒体自噬的发生情况,并探究两者的相关性。方法:选取2018年6月—2019年2月于南方医科大学附属深圳妇幼保健院住院的35例正常孕妇(对照组)、33例轻度PE孕妇(轻度PE组)及37例重度PE(重度PE组)。检测胎盘剥离面血清IL-1β、IL-18、TNF-α的表达量,分析母胎界面炎症形态学变化,观察胎盘滋养细胞线粒体发生情况,检测自噬相关分子BNIP3及DRAM1 mRNA及蛋白的表达变化,并进行炎症介质与线粒体自噬的相关性分析。结果:PE孕妇胎盘剥离面血清IL-1β、IL-18、TNF-α表达量显著高于对照组(P0.01),重度PE组表达量明显高于轻度PE组(P0.01)。HE染色显示PE孕妇胎盘组织炎症细胞浸润程度高于对照组。透射电镜可见重度PE组孕妇胎盘滋养细胞线粒体自噬阳性率为(2.61±1.07)%,轻度PE组为(7.65±3.19)%,均明显低于对照组[(14.07±3.11)%,P0.01]。PE孕妇BNIP3及DRAM1 mRNA及蛋白表达均低于对照组(P0.01)。相关性分析表明PE孕妇IL-1β、IL-18、TNF-α等炎症介质的表达量与线粒体自噬阳性率呈负相关。结论:PE孕妇滋养细胞线粒体自噬活性下降,受损线粒体积累可能导致母胎界面IL-1β、IL-18、TNF-α等炎症介质升高,从而参与PE发病。     

NAC-1通过自噬介导卵巢癌细胞顺铂耐药的研究

目的:探讨NAC-1在自噬介导的卵巢癌细胞顺铂耐药中的作用。方法:Western blot及流式细胞术检测顺铂诱导的耐药卵巢癌细胞(SKOV3/DDP)自噬及凋亡过程中,NAC-1蛋白的表达情况。采用siRNA下调NAC-1表达,检测其对SKOV3/DDP细胞自噬及凋亡的影响,并检测其对顺铂耐药性的影响。结果:顺铂处理SKOV3/DDP细胞后,细胞自噬及凋亡水平升高,NAC-1蛋白表达升高。siRNA下调NAC-1基因后,顺铂处理SKOV3/DDP细胞的自噬水平降低,凋亡升高,细胞顺铂耐药性降低。结论:NAC-1基因可能通过调节自噬参与卵巢癌细胞顺铂耐药,降低NAC-1基因表达有助于提高顺铂对卵巢癌细胞的杀伤作用。     

活性氧影响SKOV3细胞间充质转化及其机制研究

目的:探讨活性氧(ROS)对卵巢上皮性癌细胞株SKOV3细胞的间充质转化的影响及其中可能存在的分子机制。方法:1通过采用ROS生成剂大黄素(Emodin)、ROS清除剂二硫苏糖醇(DTT)调节卵巢上皮性癌细胞株SKOV3细胞内ROS水平,采用PCR技术及蛋白印迹法测定3组(Emodin组、DTT组、对照组)细胞中间充质转化标志分子E钙黏蛋白(E-cadherin)mRNA及蛋白的表达,比较细胞株侵袭转移能力。2结合改变活性氧水平,构建缺氧诱导因子-1α(HIF-1α)的化学合成,小干扰RNA(SiRNA)沉默HIF-1α表达(HIF-1αSiRNA组、DTT组、对照组),比较HIF-1αmRNA的表达。3使用赖氨酰氧化酶(LOX)抑制剂β-氨基丙腈(β-APN)抑制LOX表达,观察各组(Emodin组、DTT组、β-APN组、Emodin+β-APN组、对照组)细胞HIF-1α、LOX、E-cadherin mRNA及蛋白表达。结果:1SKOV3细胞Emodin组ROS水平较对照组明显升高[(164.85±8.93)%vs(97.42±6.47)%,P0.01],E-cadherin mRNA及蛋白的表达均较对照组显著降低(P0.05),Emodin组的细胞侵袭率高于对照组[(19.48±0.49)%vs(10.74±2.38)%,P0.01],而DTT组结果与之相反,与对照组比较差异有统计学意义(P0.05)。2HIF-1αSiRNA作用下,HIF-1αSiRNA组的HIF-1αmRNA表达较对照组降低(0.20±0.09 vs 0.87±0.10,P0.05),LOX mRNA表达较对照组降低(0.40±0.08 vs 3.76±0.66,P0.05),而E-cadherin mRNA表达较对照组增高(1.01±0.15 vs 0.30±0.09,P0.05)。DTT组的效果较HIF-1αSiRNA组更为显著,两组比较,差异均有统计学意义(P0.05)。3β-APN作用下,β-APN组的HIF-1α蛋白表达较对照组无明显变化(0.92±0.19 vs 0.94±0.09,P0.05),LOX蛋白表达较对照组降低(0.35±0.11 vs 0.81±0.13,P0.05),而E-cadherin蛋白表达较对照组增高(4.65±0.90 vs 2.23±0.61,P0.05)。结论:使ROS升高,上调HIF-1α,使LOX过表达,进而下调E-cadherin,ROS可能介导卵巢癌细胞间充质转化,继而发生侵袭转移。     

PKC delta in preeclamptic placentas promotes Bax dissociation from 14-3-3 zeta through 14-3-3 zeta phosphorylation

OBJECTIVE: We investigated placental apoptosis and the expression of and interactions between 14-3-3 and Bcl-2 family proteins during preeclampsia. In addition, we explored the mechanism of Bax dissociation from 14-3-3, hypothesizing that PKC-mediated phosphorylation of 14-3-3 results in dissociation of Bax from 14-3-3 proteins, and leads to apoptosis. METHODS: Placental samples from 10 women with preeclampsia and 10 normotensive control patients were analyzed using M30-specific immunohistochemistry to assess placental apoptosis. Biochemical markers of cellular apoptosis, such as cleaved caspase-3, Bax, Bcl-2, 14-3-3, and PKC were followed by Western blotting. Interaction of 14-3-3 proteins with Bax and with PKC was assessed by immunoprecipitation. RESULTS: M30-positive cells were widespread in the preeclamptic placentas. The levels of cleaved caspase-3, Bax, 14-3-3 zeta, phospho-(Ser)-14-3-3, and PKC delta were significantly higher in the preeclamptic placentas than in normal placentas. Preeclampsia was also associated with weaker interactions between 14-3-3 zeta and Bax and stronger interactions between 14-3-3 zeta and PKC delta. CONCLUSION: Our results suggest that PKC delta in preeclamptic placentas promotes Bax dissociation from 14-3-3 zeta through the phosphorylation of 14-3-3 zeta. This finding may at least in part explain the apoptosis-inducing activity of PKC delta, revealing the important role of PKC delta in the development of apoptosis-related diseases such as preeclampsia.     

3 classifications of gestosis

Three classifications: of the American College of Obstetricians and Gynecologists (ACOG), of the Organization of Gestosis (OG) and of the International Society for Study of Hypertension in Pregnancy (ISSHP) differ among one another in some essential points. According to ACOG the term "preeclampsia" means a state with hypertension and albuminuria or edema, according to ISSHP--hypertension and albuminuria. This may lead to serious misunderstanding as many obstetricians use this term to identify a directly threatening eclampsia attack. But the two classifications do not provide a term for such a condition whereas according to OG this state is identified as threatening eclampsia. The OG and ACOG classifications give a classifications according to the progression of the disease, the ISSHP does not give such a classification. ISSHP does not consider edema as a symptom, ACOG takes it into consideration if it is accompanied by hypertension according to OG the very edema not subsiding after relaxation is enough to diagnose gestosis E. ISSHP has introduced the term "hypertension in pregnancy", but also includes one-symptom form "pregnancy albuminuria" not accompanied by hypertension. Such terms suggest lack of etiological relation between those forms, which has not however been proved. The names of one-symptom forms according to OG "gestosis H" and "gestosis P" do not impose such a relation (it is a classification according to symptoms), but they do not exclude it. Contrary to ACOG and OG, ISSHP gives the level of diastolic blood pressure only.     

Analysis of SYCP3 encoding synaptonemal complex protein 3 in human aneuploidies

Objectives

To test the hypothesis that mutations of SYCP3 encoding synaptonemal complex protein 3, result in increased frequency of aneuploidies in humans.

Methods

Mutation analysis of the PCR-amplified 8 coding exons and exon–intron boundaries of the SYCP3 gene was done by direct sequencing of DNA isolated from 35 aneuploid fetuses of women having a potentially increased likelihood for an underlying genetic predisposition for chromosomal non-disjunction.

Results

Based on the results of conventional karyotyping, the 35 aneuploid fetuses of 33 women were divided into separate groups: 9 aneuploid conceptuses of couples with recurrent aneuploid conceptions (4 of the women 35 years or younger), 12 conceptuses with double/multiple aneuploidies (5 of the women 35 years or younger), and 14 conceptuses with single aneuploidies of women younger than 35 years (8 trisomies and 6 monosomies). No pathogenic mutations in the SYCP3 coding exons and the immediately flanking intronic sequences were found.

Conclusions

Under the assumption that genetic predisposition for chromosomal non-disjunction leading to aneuploidy is most likely polygenic in nature, our data suggest that SYCP3 mutations are not one of the common causes in humans.