缺氧调控滋养细胞MMP-9/TIMP-1表达及侵袭能力的体外研究

目的:探讨缺氧对滋养细胞的生长、MMP-9/TIMP-1基因表达以及侵袭能力的影响。方法:滋养细胞在正常及二氯化钴(CoCl2)所致化学缺氧条件下的培养。MTT法检测正常及缺氧条件下滋养细胞的生长状况。细胞爬片确定MMP-9/TIMP-1在滋养细胞中的定位及缺氧前后的蛋白表达。荧光定量PCR检测滋养细胞中MMP-9和TIMP-1基因表达。Transwell侵袭模型检测滋养细胞的侵袭能力变化。结果:(1)MMP-9和TIMP-1均表达于滋养细胞的包膜和胞浆。在缺氧条件下,MMP-9和TIMP-1蛋白表达均呈上调趋势,且在缺氧48h时最明显(P0.01);(2)150、300μmol/L CoCl2均可显著抑制滋养细胞的增殖,其中300μmol/L抑制作用更明显,与150μmol/L比较,差异有统计学意义(P0.01);(3)缺氧条件下,MMP-9和TIMP-1的基因表达均增加,以48h最为显著;但MMP-9/TIMP-1比值降低,以72h最显著(P0.01);(4)缺氧后滋养细胞侵袭力显著降低(P0.05)。结论:缺氧能抑制滋养细胞增殖、促进滋养细胞分泌MMP-9和TIMP-1,但MMP-9/TIMP-1比值降低,使滋养细胞的侵袭力减弱。     

sgp190与白血病抑制因子对滋养层细胞金属蛋白酶-9及抑制因子-1表达的影响

目的:探讨sgp190与白血病抑制因子(LIF)在妊娠维持中的作用。方法:体外培养滋养层细胞,分别施加LIF和/或sgp190处理后,用RT-PCR法检测金属蛋白酶-9(MMP-9)与金属蛋白酶组织抑制因子(TIMP-1)mRNA表达情况,免疫印迹法检测细胞MMP-9与TIMP-1蛋白表达水平,ELISA法测定培养上清中MMP-9与TIMP-1的浓度。结果:LIF抑制体外培养滋养层细胞中MMP-9与TIMP-1中mRNA和蛋白表达(P<0.05),而sgp190可抑制LIF的这种作用。sgp190促进MMP-9mRNA与蛋白的表达(P<0.05),但对TIMP-1mRNA与蛋白表达无影响。ELISA分析结果基本与RT-PCR和免疫印迹分析结果相同。结论:LIF和sgp190可能通过调节MMPs和TIMPs表达来影响滋养层细胞的侵袭,而sgp190在LIF功能活性调节中扮演着一定角色。     

甲氧滴滴涕对人绒毛外滋养细胞侵袭能力的影响

目的:探讨甲氧滴滴涕(MXC)对绒毛外滋养细胞侵袭能力的影响。方法:用胰蛋白酶消化后再经流式细胞仪分离得到人绒毛外滋养细胞进行原代培养及鉴定。细胞分为MXC(1μmol/L,3μmol/L、5μmol/L、7μmol/L)处理组及空白对照组,培养12h、24h和48h。用侵袭小室检测每组各时间点的侵袭能力,Western blot检测各组各时间点MMP-2、MMP-9、TIMP-2、TIMP-1表达,RT-PCR法检测MMP-9 mRNA、MMP-2 mRNA表达。结果:MXC处理组滋养细胞侵袭能力呈时间剂量依赖型下降,MMP-9 mRNA、MMP-2 mRNA表达下降,MMP-9、MMP-2表达下降,TIMP-2、TIMP-1表达增高。MMP-2及其mRNA下降程度与MXC有时间剂量效应。结论:MXC可通过改变基质金属蛋白酶及其抑制物的比例降低绒毛外滋养细胞侵袭能力。     

胚胎停育绒毛组织中TGF-β1、MMP-9及TIMP-1的表达及意义

目的:探讨TGF-β1、MMP-9及TIMP-1 mRNA的表达在胚胎停育发病机制中的作用。方法:用半定量逆转录聚合酶链反应(RT-PCR)技术检测正常人工流产(20例)和胚胎停育(25例)患者绒毛组织中TGF-β1、MMP-9与TIMP-1 mRNA的表达量。结果:(1)与对照组相比,实验组绒毛中TGF-β1 mRNA和TIMP-1 mRNA的表达量降低(P<0.05),而MMP-9 mRNA表达量升高(P<0.05);(2)绒毛组织中TGF-β1 mRNA与MMP-9 mRNA的表达呈负相关(r=-0.82,P<0.05)。结论:胚胎停育患者绒毛组织TGF-β1、MMP-9以及TIMP-1 mRNA表达有明显改变;TGF-β1表达的降低可能上调MMP-9表达,通过破坏MMPs/TIMPs动态平衡,最终导致胚胎停育发生。     

邻苯二甲酸二(2-乙基己)酯可抑制人原代培养早孕绒毛滋养细胞浸润能力

目的研究邻苯二甲酸二(2-乙基己)酯[di-(2-ethylexyl)phthalate,DEHP]对人原代培养早孕绒毛滋养细胞浸润能力及基质金属蛋白酶(MMP)-2、MMP-9、基质金属蛋白酶抑制因子(TIMP)-1和TIMP-2表达的影响,探讨DEHP对妊娠的影响及其毒性机制。方法2006年12月于北京军区总医院采用不同浓度DEHP预处理人原代培养早孕绒毛滋养细胞,采用Transwell体外浸润实验检测滋养细胞浸润能力的改变。RT-PCR法检测滋养层细胞侵袭性相关基因MMP-2、MMP-9、TIMP-1和TIMP-2的mRNA表达,Western blot法检测MMP-2、MMP-9、TIMP-1和TIMP-2蛋白表达水平。结果DEHP作用后,滋养细胞浸润能力明显下降。当DEEP为50、100μmol/L时,MMP-2、MMP-9、TIMP-1的mRNA表达量分别为0.35±0.07、0.26±0.03、0.97±0.18和0.30±0.10、0.20±0.05、1.02±0.20,与DEEP为0时MMP-2、MMP-9、TIMP-1的mRNA的表达量(0.77±0.15、0.45±0.04、0.80±0.20)比较P<0.05;当DEEP为50、100μmol/L时,MMP-2、MMP-9、TIMP-1的蛋白表达量分别为0.35±0.03、0.23±0.05、0.69±0.08和0.24±0.02、0.17±0.02、0.86±0.10,与DEEP为0时MMP-2、MMP-9、TIMP-1的mR-NA的表达量(0.69±0.04、0.57±0.03、0.24±0.12)比较P<0.05。可见DEHP剂量≥50μmol/L时,滋养细胞MMP-2和MMP-9表达下降、TIMP-1表达增加,且呈剂量依赖性。结论DEHP可通过抑制MMP-2、MMP-9的表达并促进TIMP-1表达,影响滋养细胞浸润能力。     

过氧化物酶体增殖物激活受体γ及其配体对早期细胞滋养细胞MMP-2、MMP-9表达的调控

目的:探讨过氧化物酶体增殖物激活型受体γ(PPARγ)及其配体(15-脱氧-前列腺素J2,15-d-PGJ2)对细胞滋养细胞表达基质金属蛋白酶-2(MMP-2)和MMP-9的调控作用。方法:采用免疫荧光细胞化学方法检测细胞滋养细胞中PPARγ的表达;利用免疫荧光共聚焦技术观察15-d-PGJ2作用前后细胞滋养细胞MMP-2和MMP-9表达强度的变化;通过荧光定量PCR(Real-timePCR)和Westernblot方法定量检测MMP-2和MMP-9mRNA和蛋白的表达变化。结果:在细胞滋养细胞中有PPARγ蛋白表达,且主要定位在细胞滋养细胞核中;15-d-PGJ2作用后细胞滋养细胞中MMP-2和MMP-9的表达明显下降,与对照组相比差异显著(P<0.01);15-d-PGJ2对MMP-2的作用强于MMP-9。结论:PPARγ及其配体15-d-PGJ2调节滋养细胞浸润作用可能是通过调节MMP-2和MMP-9的表达实现的。     

生长激素对小鼠子宫内膜VEGF、LIF、MMP-9及TIMP-1表达的影响

目的:探讨生长激素(GH)对子宫内膜容受性的作用。方法:将150只小鼠随机平均分成3大组:A组(动情后期组)、B组(假孕组)、C组(妊娠d4组);每大组再分为2亚组:第1亚组皮下注射GH1.5mIU/g,第2亚组注射等体积生理盐水;用免疫组化技术检测子宫内膜VEGF、LIF、MMP-9及TIMP-1的表达。结果:B组子宫内膜中,注射GH者LIF、VEGF、MMP-9及TIMP-1的表达呈阳性,强于其对照组,二者比较差异有显著性(P均<0.05);C组子宫内膜中,注射GH者LIF、VEGF、MMP-9及TIMP-1的表达呈强阳性,强于其对照组,二者比较差异有显著性(P均<0.05)。结论:GH可以提高小鼠着床期子宫内膜VEGF、LIF、MMP-9及TIMP-1的表达,从而改善子宫内膜容受性,提高临床妊娠率。     

蜕膜基质细胞条件培养液对滋养细胞浸润功能的影响

目的:探讨蜕膜基质细胞条件培养液(DSCM)对滋养细胞浸润力的影响。方法:体外培养人早孕正常蜕膜基质细胞,收集DSCM处理早孕滋养细胞系(B6Tert)。应用浸润实验分析B6Tert细胞浸润力的改变,采用RT-PCR与明胶酶谱技术检测B6Tert细胞中基质金属蛋白酶(MMPs)的表达变化。结果:DSCM浓度较低时(占细胞培养液5%),可促进B6Tert细胞的浸润力与该细胞MMP-2mRNA及pro-MMP-2的表达;反之,高浓度的DSCM(20%)则抑制滋养细胞的浸润力与MMP-2的表达,差异有统计学意义(P<0.05)。DSCM不影响B6Tert细胞中MMP-9的表达。结论:早孕DSCM可能通过调节滋养细胞中MMP-2的表达影响滋养细胞的浸润能力。     

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。     

瘦素对妊娠早期绒毛滋养细胞MMP-2/TIMP-2表达的调控作用

目的:观察体外模拟早孕微环境(雌激素、孕激素)条件下瘦素(leptin)对妊娠早期绒毛滋养细胞MMP-2/TIMP-2表达的影响,初步探讨其调控机制及意义.方法:选取正常妊娠妇女人工流产绒毛组织(6～9周),按常规方法分离滋养细胞,分为雌激素加孕激素(E+P组)、瘦素组(leptin组)、雌激素加孕激素加瘦素组(E+P+leptin组)、空白对照组(N组).置37℃、5%CO2培养24小时,收集细胞及培养上清,流式细胞仪检测瘦素受体(leptin R)表达;ELISA检测上清中可溶性瘦素受体(sleptin-R)表达,RT.PCR检测MMP-2、TIMP-2 mRNA;酶谱检测上清中MMP-2表达.结果:流式细胞仪检测结果显示,E+P组滋养细胞leptin R表达显著上调(P<0.05),上清中未检测到sleptin-R;E+P组、leptin组和E+P+leptin组MMP.2均显著上调,TIMP-2的表达下调(P<0.05).E+P+leptin组的调节效应强于E+P组和leptin组(P<0.05).结论:leptin-leptin R途径参与滋养细胞MMP-2的上调,并增强雌、孕激素联合对MMP-2的上调作用.该调节途径可能通过调控MMP-2/TIMP-2的平衡来调节滋养细胞侵袭性.     

Concentrations of MMP-1, TIMP-1, MMP-1/TIMP-1 and I CTP complexes in follicular fluid as related to fertilization rate in women treated with in-vitro fertilization

OBJECTIVES: Metalloproteinases and their inhibitors appear to control connective tissue remodelling during follicular rupture. The aim of the study was to establish if oocytes fertilisation rate after ovulation induction depends on the concentrations of MMP-1, its inhibitor TIMP-1, MMP-1/TIMP-1 complex and I CTP in follicular fluid (FF). MATERIAL AND METHODS: FF were collected from 37 infertile patients undergoing ovulation induction using either short or long protocol. FF was obtained 36 hours after administration of hCG (Pregnyl). The level of MMP-1, TIMP-1, MMP-1/TIMP-1 complex were measured using ELISA kits and I CTP, E2, FSH, LH, using RIA assay kits. RESULTS: Statistically significant (p < 0.05) difference was found in TIMP-1, E2, and FSH concentration, being higher in the group with more than 75% fertilisation rate: TIMP-1 728.8 + 100.1 ng/ml vs 666.3 + 94.5 ng/ml; E2 477.3 +/- 160.0 ng/ml vs 368.0 +/- 190.0 ng/ml and FSH 7.27 +/- 1.45 mIU/ml vs 6.24 +/- 1.6 mIU/ml. CONCLUSIONS: Statistically significant increase in TIMP-1 concentration observed among patients with fertilisation rate above 75% indicates an important role of this substance in ovulation process.     

低分子肝素对不同氧浓度下早孕绒毛滋养层细胞侵袭力的影响作用

目的:研究低分子肝素(LMWH)对不同氧浓度下早孕绒毛滋养层细胞侵袭力的影响。方法:取妊娠8~10周正常绒毛组织,酶消化法分离人早孕绒毛滋养层细胞,分别置2%和20%O_2分压的细胞培养箱培养。用不同浓度LMWH(0、0.1、5、10IU/ml)处理滋养细胞,观察滋养层细胞侵袭力的改变,检测细胞中HIF-1α、MMP-2、MMP-9、TIMP-2、TIMP-3表达。结果:低氧浓度下,早孕绒毛滋养层细胞的侵袭能力随着LMWH浓度(0,0.1,5IU/ml)升高而增强,但高浓度的LMWH(10IU/ml)对其有抑制作用;随着LMWH浓度增加,HIF-1α和MMP-2表达呈先上升后下降的趋势;TIMP-2、TIMP-3表达改变的趋势与MMP-2相同。正常氧浓度下的滋养层细胞,其侵袭能力未见显著增强,各细胞因子的表达无上调。结论:低氧环境下,一定浓度的LMWH可提高早孕绒毛滋养层细胞的侵袭能力,其发生机制可能是通过诱导、增强滋养层细胞HIF-1α和MMP-2基因表达。     

Differential expression of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 protein and mRNA in epithelial ovarian tumors

OBJECTIVE: Matrix metalloproteinase-9 (MMP-9) can degrade gelatin and type IV collagen and is known to play an important role in tumor cell invasion across the basement membrane. The tissue inhibitor of metalloproteinase-1 (TIMP-1) is able to prevent activation of pro-MMP-9 and forms a 1:1 complex with the active form of MMP-9. The aim of the present study was to investigate the expression of MMP-9 and TIMP-1 in benign, borderline, and invasive epithelial ovarian tumors. MATERIALS AND METHODS: A total of 90 patients with epithelial ovarian tumor were treated at the Brigham and Women's Hospital and were used as the study population. Immunohistochemistry and in situ hybridization were performed to detect protein and mRNA expression of MMP-9 and TIMP-1. RESULTS: In the 90 epithelial ovarian tumors tested, MMP-9 expression in tumor cells was found to be significantly enhanced in serous and mucinous ovarian carcinomas compared with benign and borderline tumors. We also observed the immunostaining of MMP-9 in stromal cells of benign, borderline, and invasive epithelial ovarian tumors. Moreover, the expression levels of TIMP-1 in tumor cells were significantly higher in borderline and invasive ovarian tumors than in benign tumors. CONCLUSION: Using an in situ hybridization technique, we disclosed a direct correlation between the presence of mRNA and protein expression for both MMP-9 and TIMP-1. The present data suggest that high levels of MMP-9 protein in invasive epithelial ovarian carcinoma are strongly associated with tumor cell invasion. Enhanced expression of TIMP-1 protein in borderline and invasive tumors indicates that endogenous TIMP-1 protein may play a paradoxical role in ovarian tumor progression.     

Doxycycline alters the expression of matrix metalloproteases in the endometrial cells exposed to ovarian steroids and pro-inflammatory cytokine

Evidence suggests that doxycycline (Dox), acting through an anti-inflammatory mechanism, inhibits expression of matrix metalloproteinases (MMPs). Since the endometrial environment in contraceptive users experiencing breakthrough bleeding is characterized by elevated production of MMPs, we examined the effect of Dox on endometrial expression of MMPs using an in vitro model consisting of endometrial glandular epithelial cells (GEC), stromal (ESC) cells, and an endometrial surface epithelial cell line (HES). GEC, ESC and HES maintained under defined culture conditions expressed variable levels of MMP-2 and MMP-9, and Dox in a dose-dependent manner (1-50 microg/ml) reduced the production of proMMP-2 after 24h treatment (P<0.05). Dox (25 microg/ml), alone or in combination with 17beta estradiol (E2), medroxyprogesterone acetate (MPA) and E2+MPA (10(-8)M), as well as TNF-alpha (25 ng/ml) in cell- and time-dependent manners, moderately altered the expression of MMP-2 and MMP-9 mRNA in GEC, ESC and HES compared to untreated controls (P<0.05). Dox, either alone or in combination with ovarian steroids and TNF-alpha, reduced production of pro-MMP-2 and proMMP9, as well as TIMP-1 and TIMP-2, without affecting the level of active MMPs produced by these cells (P<0.05). In conclusion, the results indicate that Dox only moderately and in a cell-specific manner reduces expression of MMPs without influencing their activity, suggesting that Dox's therapeutic benefits in controlling irregular breakthrough bleeding in contraceptive users occurs site specifically and possibly through a mechanism involving MMPs and TIMPs expression.     

雌激素受体亚型α对子宫内膜癌细胞HEC-1B侵袭能力调控的研究

目的：特异性下调子宫内膜癌细胞中的ERα基因，探讨E胁亚型表达在子宫内膜癌侵袭中的作用。方法：将ERa的小干扰RNA（siRNA-small interfering RNA）转染子宫内膜癌细胞HEC-1B，通过RT-PCR和Western blot证实ERα基因的有效阻断。通过transwell小室法检测下调ERa基因表达前后HEC-1B细胞的侵袭能力；应用RT-PCR检测转染前后细胞MMP-2、MMP-9、TIMP-1和TIMP-2 mRNA表达水平的变化；Western blot及明胶酶谱分别检测细胞分泌TIMP-1、TIMP-2、MMP-2和MMP-9蛋白的水平。结果：（1）将ERα-siRNA转染HEC-1B细胞后，转染效率大于90％，ERα mRNA及蛋白的表达水平均明显下调（分别为72％，67％）；（2）下调ERα基因表达后，肿瘤细胞的侵袭能力下降（P〈0．05）；在mRNA水平和蛋白水平均可检测到ERα-siRNA组细胞的MMP-2、MMP-9表达下降（P〈0．05），TIMP-1、TIMP-2表达增加（P〈0．05）。结论：使用ERα-siRNA能够有效地阻断ERa基因表达；子宫内膜癌细胞中，17β-雌二醇对MMPs／TIMPs具有调节作用，这种作用可通过ERα介导；ERα表达水平影响子宫内膜癌细胞的侵袭能力。