人绒毛膜促性腺激素对绒癌细胞系TIMP表达的影响

目的：研究人绒毛膜促性腺激素(hCG)对滋养层细胞或绒癌细胞侵袭性的影响。方法：采用RT—PCR方法,观察不同浓度hCG对JEG—3绒癌细胞系表达金属蛋白酶组织抑制因子TIMP—1和TIMP—2的影响。结果：用不同浓度hCG处理48h后,JEG—3细胞中TIMP—1 mRNA的表达略降低,而TIMP—2 mRNA的表达则被诱导增加。结论：人绒毛膜促性腺激素(hCG)可能通过改变绒癌细胞中TIMP的表达,影响绒癌细胞的侵袭性。     

丹参单体IH764-3通过下调H2O2刺激的肝星状细胞FAK水平影响MMP-13和TIMP-1的表达

目的：研究丹参单体IH764—3对H2O2刺激的肝星状细胞（HSC）基质金属蛋白酶-13（MMP-13）、基质金属蛋白酶组织抑制因子-1（TIMP-1）表达的影响以及此过程中粘着斑激酶（FAK）的变化。方法：应用RT-PCR方法检测MMP-13及FAKmRNA表达,原位杂交方法检测TIMP-1mRNA水平,Western blotting技术检测FAK及TIMP-1蛋白表达。结果：IH764—3干预组的MMP-13mRNA在2h的表达强度明显上调,而TIMP-1mRNA表达明显受抑,FAKmRNA表达强度明显下调;IH764—3干预24h组FAK及TIMP-1蛋白表达受抑制。结论：丹参单体IH764—3可以诱导MMP-13表达,抑制TIMP-1表达,下调FAK表达是其中的机制之一。     

环孢素A对人早孕期滋养细胞MMP-9与MMP-2表达的调控作用

本研究的目的是探讨环孢素A对人早孕期滋养细胞侵袭能力及基质金属蛋白酶9与2 (matrix metalloproteinase 9 and 2,简称MMP-9与MMP-2)表达的调节作用,为治疗反复自然流产等妊娠疾患提供新的线索。侵袭试验观察CsA对人早孕期滋养细胞侵袭能力的调节作用;RT-PCR与明胶酶谱分析CsA对滋养细胞MMP-9与MMP-2 mRNA及蛋白水平表达的影响;In-cell West- ern检测CsA作用后滋养细胞ERK1/2磷酸化水平。结果发现,1．0μmol/L CsA明显增强滋养细胞侵袭能力,MEK激酶抑制剂U0126可抑制CsA对滋养细胞的促侵袭作用;1．0μmol/L CsA可诱导MMP- 9与MMP-2基因的转录与蛋白分泌;该诱导效应同样可被U0126所阻滞;1．0μmol/L CsA以时间依赖方式促进ERK1/2的磷酸化。结果表明,CsA可激活ERK1/2,通过MAPK/ERK1/2途径促进滋养细胞MMP-9与MMP-2基因的转录与蛋白分泌,从而增强滋养细胞的侵袭能力,对滋养细胞生物学功能具有良性调节作用。     

纤黏连蛋白重组多肽CH50抑制黑色素瘤MMP-2和MMP-9表达、激活的研究

目的 研究纤黏连蛋白重组多肽CH50对黑色素瘤B16细胞侵袭能力的影响,探讨CH50多肽抑制肿瘤生长、侵袭的机制。方法 体外培养小鼠黑色素瘤B16细胞、小鼠腿部皮下注射B16细胞建立肿瘤动物模型。采用明胶电泳法检测B16细胞和黑色素瘤组织中基质金属蛋白酶MMP-2和MMP-9的表达和激活;以CH50多肽体外处理B16细胞或体内表达CH50,观察CH50下调MMPs表达以及对肿瘤细胞侵袭能力的抑制作用。结果 B16细胞在体外培养条件下主要表达MMP-2,而在肿瘤微环境中则同时表达MMP-2和MMP-9。肿瘤组织中MMPs的表达明显高于体外培养B16细胞。CH50多肽对体外培养B16细胞的MMPs表达和激活无明显抑制作用,但处理后的B16细胞进入体内后表达MMPs的能力受到明显抑制。体内转染表达的CHSO多肽亦可明显抑制肿瘤表达MMPs、并抑制肿瘤侵袭能力。结论 纤黏连蛋白重组多肽CHSO可以抑制肿瘤微环境中基质金属蛋白酶MMP-2和MMP-9的表达和激活,从而抑制黑色素瘤生长及侵袭能力。     

骨桥蛋白通过调控MMP-2和VEGF参与肝癌细胞侵袭的机制研究

目的：骨桥蛋白（Osteopontin,OPN）在肝癌细胞侵袭中的作用机制。方法：采用siRNA干涉的方法处理人肝癌细胞,用PCR和Western-blot法检测OPN的表达;用transwell小室检测不同处理后的HepG2和MHCC97H细胞的侵袭能力;采用Western．b1．ot和ELISA方法检测基质金属蛋白酶-2（matrixmetalloproteinase-2,MMP-2）和血管内皮生长因子（vascularendothelialgrowthfactor,VEGF）蛋白表达和活力的变化情况。结果：在不同肝癌细胞系中,随着肝癌细胞系侵袭能力的增强,OPN的表达逐渐增高。siRNA可以降低HepG2和MHCC97H细胞中OPN的表达,并且能够降低HepG2和MHCC97H细胞的侵袭能力;抑制OPN的表达能够降低MMP-2和VEGF蛋白表达和蛋白活性。结论：OPN在肝癌侵袭过程中起着重要作用,其作用机制可能是通过调控MMP-2和VEGF蛋白表达和活性来参与肝癌的侵袭,OPN可作为肝癌侵袭转移治疗的新靶点。     

缺氧对肺动脉内皮细胞MMP-2、MMP-9表达的影响

目的观察缺氧对培养的猪肺动脉内皮细胞基质金属蛋白酶-2(MMP-2)、基质金属蛋白酶-9(MMP-9)的表达的影响.方法采用RT-PCR、Western blot、底物胶电泳(酶谱图)、免疫细胞化学等方法.结果肺动脉内皮细胞缺氧24h可使MMP-2的mRNA表达和蛋白分泌减少,酶活性减弱,与常氧组比较有显著性差异, MMP-9无明显变化.结论缺氧时MMP-2降低,溶解细胞外基质的能力降低,可能是缺氧性肺血管构型重组的机制之一.     

二氢杨梅素抑制人乳腺癌细胞侵袭和下调MMP-2/-9蛋白表达研究

藤茶活性成分二氢杨梅素(3, 5, 7, 3′, 4′, 5′-六羟基-2, 3-二氢黄酮醇,DMY)体外对几种癌细胞具有抗增殖作用,但机制尚未完全清楚．本文研究DMY对人高转移型乳腺癌MDA-MB-231细胞侵袭的影响,并探讨可能的机制．用MTT法检测DMY对MDA-MB-231细胞的增殖抑制率;明胶酶谱分析明胶酶活力;基质金属蛋白酶(MMP-2/-9)的基因表达水平和蛋白质表达水平分别利用实时定量PCR和Western blot分析进行检测．Transwell模型检测DMY对肿瘤细胞侵袭的影响．结果显示,DMY以剂量依赖方式抑制MDA-MB-231细胞的增殖,作用48 h的IC50为73.6 mg/L．DMY显著抑制明胶酶活性和MMP-2/-9蛋白表达,并抑制MMP-2/-9 的mRNA表达水平．此外,DMY不依赖细胞毒作用和以剂量依赖方式抑制MDA- MB-231细胞的侵袭．这些结果提示：DMY能显著抑制人乳腺癌MDA-MB-231细胞的侵袭和增殖, 其侵袭抑制的机制可能与其下调MMP-2/-9蛋白表达水平相关．     

过表达外源FUT8增加乳腺癌细胞的迁移和侵袭能力

核心岩藻糖的修饰被认为是对糖蛋白进行转录后加工和功能调控的一种重要方式, 与肿瘤的发生发展密切相关, 但其在乳腺癌恶性转化过程中所发挥的生物学功能尚不明确. 本文构建了α1,6-岩藻糖基移转酶（FUT8）基因真核表达载体,将其转染人乳腺癌细胞Bcap-37, 实时PCR及Western印迹检测FUT8 mRNA和蛋白质表达, 通过细胞划痕实验和Transwell小室观察FUT8过表达对细胞体外迁移、侵袭能力的影响, 免疫共沉淀和Western印迹检测肿瘤细胞整合素、基质金属蛋白酶（matrix metalloproteinases, MMPs）家族相关蛋白质表达水平变化. 结果显示,外源FUT8基因在mRNA水平和蛋白质水平的表达均显著增加, FUT8过表达组细胞迁移和侵袭能力明显增强;上调FUT8基因表达可使Bcap-37细胞中核心岩藻糖基化的整合素-α3β1、MMP-2和MMP-9在蛋白质水平呈不同程度升高. 上述研究表明,FUT8可通过核心岩藻糖化修饰正性调控整合素-α3β1的功能, 诱导MMP-2、MMP-9的表达, 促进乳腺癌细胞的迁移和侵袭.     

重楼皂苷Ⅶ抑制肺癌H460细胞增殖和迁移能力研究

为研究重楼皂苷Ⅶ(polyphyllin Ⅶ)抑制人肺癌H460细胞增殖、迁移能力和诱导凋亡的作用和机制。本实验采用MTT法检测重楼皂苷Ⅶ处理后H460细胞生长抑制率,Hoechst 33258染色观察细胞形态,细胞集落形成实验考察细胞的增殖能力,划痕实验和Transwell小室实验研究H460细胞迁移和侵袭能力的改变,并通过western blot法检测在重楼皂苷Ⅶ处理前后细胞蛋白表达变化情况。结果发现,重楼皂苷Ⅶ可显著抑制H460细胞增殖,影响其集落形成,并使细胞形态发生变化,抑制细胞体外的迁移和侵袭能力,并可诱导凋亡的发生。重楼皂苷Ⅶ处理后,H460细胞中基质金属蛋白酶MMP-2和MMP-9显著降低,凋亡相关蛋白剪切型caspase-3、Bax表达增加,ICAD、Bcl-2表达降低,提示重楼皂苷Ⅶ体外可能通过调节基质金属蛋白酶抑制H460细胞迁移和侵袭,同时诱导凋亡的发生。     

Differential regulation of hCG alpha and beta subunit mRNAs in JEG-3 choriocarcinoma cells by 8-bromo-cAMP

The coordinate regulation of human chorionic gonadotropin (hCG) subunit synthesis by JEG-3 choriocarcinoma cells was studied at the pretranslational level. The responses of the hCG alpha and beta mRNAs were measured during stimulation with the potent cAMP analog 8-bromo-cAMP (8-Br-cAMP) using 32P-labeled hCG alpha and beta cDNA probes. The hCG alpha mRNA (850 bases) and beta mRNA (1050 bases) from JEG-3 cells were identical in size to that of their respective mRNAs from placenta, by Northern blot analysis. After 48 h of stimulation with 2 mM 8-Br-cAMP, production of immunoreactive alpha and beta subunits increased 25- and 52-fold, respectively; corresponding levels of the alpha and beta mRNAs increased 36- and 43-fold, respectively, in a dot blot hybridization assay. Total cellular protein, DNA content, and messenger RNA pools were not altered by treatment with 8-Br-cAMP. The temporal coordination of the expression of the hCG alpha- and beta-subunit genes was examined by comparing the time course of stimulation of the respective mRNAs and the production of immunoreactive subunits. The kinetic responses of the alpha and beta mRNAs differed: the increase in hCG alpha mRNA preceded the increase in hCG beta mRNA, while levels of free alpha subunit and intact hCG increased in parallel with the increase in beta mRNA. hCG alpha mRNA levels increased rapidly between 8 and 24 h after the addition of 8-Br-cAMP, and approached a plateau by 48 h. The levels of hCG beta mRNA increased steadily throughout the 8-48 h period. These results demonstrate that the cAMP analog 8-Br-cAMP differentially regulates hCG subunit biosynthesis in JEG-3 cells at a pretranslational level, and that the stimulation by 8-Br-cAMP in this system appears to be relatively selective for hCG subunits.     

Polyamines control human chorionic gonadotropin production in the JEG-3 choriocarcinoma cell

The effect of inhibition of ornithine decarboxylase with difluoromethylornithine (DFMO) and the resultant lowering of polyamine levels upon human chorionic gonadotropin (hCG) production in JEG-3 choriocarcinoma cells was investigated. DFMO (10 mM) totally inhibited ornithine decarboxylase activity. In DFMO-treated cells, cellular spermidine concentrations fell to nondetectable levels (less than 1% of control values) within 24 h and spermine concentrations were reduced to 41.9% of controls over 6 days. DFMO caused a 70-80% inhibition of hCG production. Levels of mRNA for both the alpha and beta subunits of hCG were also inhibited relative to mRNA for tubulin. Exogenous putrescine normalized hCG production in a dose-dependent manner. Other diamines, including cadaverine, 1,3-diaminopropane, 1,6-diaminohexane, and 1,7-diaminoheptane, were ineffective in reestablishing hCG production in DFMO-treated cells. Dibutyryl cAMP (1 mM) stimulated hCG production and increased levels of mRNA for the alpha and beta subunit 5-40-fold in both DFMO-treated and control cells. Polyamines appear to have a fundamental role in hCG production in JEG-3 choriocarcinoma cells. However, dibutyryl cAMP can partially overcome or circumvent the requirement for polyamines in hCG biosynthesis.     

血小板活化因子对大鼠黄体细胞孕酮分泌及血管内皮生长因子表达的作用

本文旨在研究血小板活化因子(platelet-activating factor,PAF)对大鼠黄体细胞孕酮分泌及血管内皮生长因子(vascularendothelial growth factor,VEGF)mRNA表达的作用.将未成年(25～28 d)Sprague-Dawley雌性大鼠颈部皮下注射50 IU孕马血清促性腺激素(pregnant mare serum gonadotrophin,PMSG),48 h后注射25 IU人绒毛膜促性腺激素(human chorionicgonadotrophin.hCG)诱导卵泡发育和黄体生成,第6天(hCG注射日为第1天)收集卵巢黄体细胞,体外培养24 h后,不加或加入不同剂量(0.1 μg/mL、1 μg/mL、10 μg/mL)PAF,37℃、5%CO2培养箱内培养24 h.用放射免疫方法测定培养液中孕酮的含量,流式细胞仪和RT-PCR方法检测黄体细胞凋亡以及VEGF mRNA的表达.结果显示,PAF促进黄体细胞孕酮分泌,1 μg/mL PAF作用最强(P<0.05);PAF促进黄体细胞凋亡无明显剂量依赖性,但10 μg/mL PAF显著促进大鼠黄体细胞凋亡(P<0.05):PAF刺激黄体细胞VEGF mRNA表达,1 μg/mL PAF效果最显著(P<0.01).结果提示,PAF可通过调节黄体细胞孕酮的分泌和VEGF mRNA的表达来促进黄体形成.     

Abnormal biantennary sugar chains are expressed in human chorionic gonadotropin produced in the choriocarcinoma cell line, JEG-3

Over the past two decades, sugar chain structures of human chorionic gonadotropin (hCG) produced in healthy people, three types of trophoblastic disease, and some types of cell lines have been analyzed. The abnormal biantennary structure of hCG is a good marker for the diagnosis of malignant choriocarcinoma. In spite of much research, hCG with an abnormal biantennary structure is only detected in the urine of choriocarcinoma or pregnant diabetic patients. We hypothesized that the formation mechanism of the abnormal biantennary sugar chain structure is mainly caused by high GnT-IV activity. To confirm this, we measured the N-acetylglucosaminyltransferase (GnT)-IV activity and hCG productivity in three choriocarcinoma cell lines, and selected JEG-3 cells. hCG samples were purified from medium conditioned by JEG-3 cells, and their sugar chain structures were analyzed. We detected an abnormal biantennary structure, and the proportions were different from those previously reported in the urine samples of choriocarcinoma patients. These findings proved our hypothesis and suggest the usefulness of JEG-3 cells for further analyses of abnormal biantennary structure formation. Published in 2004.     

Abnormal biantennary sugar chains are expressed in human chorionic gonadotropin produced in the choriocarcinoma cell line, JEG-3

Over the past two decades, sugar chain structures of human chorionic gonadotropin (hCG) produced in healthy people, three types of trophoblastic disease, and some types of cell lines have been analyzed. The abnormal biantennary structure of hCG is a good marker for the diagnosis of malignant choriocarcinoma. In spite of much research, hCG with an abnormal biantennary structure is only detected in the urine of choriocarcinoma or pregnant diabetic patients. We hypothesized that the formation mechanism of the abnormal biantennary sugar chain structure is mainly caused by high GnT-IV activity. To confirm this, we measured the N-acetylglucosaminyltransferase (GnT)-IV activity and hCG productivity in three choriocarcinoma cell lines, and selected JEG-3 cells. hCG samples were purified from medium conditioned by JEG-3 cells, and their sugar chain structures were analyzed. We detected an abnormal biantennary structure, and the proportions were different from those previously reported in the urine samples of choriocarcinoma patients. These findings proved our hypothesis and suggest the usefulness of JEG-3 cells for further analyses of abnormal biantennary structure formation.     

PACAP mRNA在大鼠妊娠黄体及离体细胞的表达与调节

目的:观察垂体腺苷酸环化酶激活肽(PACAP)mRNA在大鼠妊娠黄体中的表达及调节。方法:①于妊娠不同时期收集大鼠卵巢。用RT-PCR和原位杂交方法,观察妊娠过程卵巢PACAP mRNA表达的动态变化;②未成年雌性大鼠颈部皮下注射50IU孕马血清促性腺激素,48h后注射25IU人绒毛膜促性腺激素,第6天收集培养黄体细胞。用放免法测定给予不同处理后,培养液中孕酮的含量;用RT-PCR方法检测各组PACAP mRNA表达水平。结果:从妊娠11d起,PACAP mRNA表达逐渐增强,在妊娠19d达高峰;与对照组相比,血小板活化因子(PAF)、福司考林(forskolin)、佛波酯(PMA)均使培养黄体细胞孕酮分泌量及PACAP mRNA表达显著增高(P0.05)。结论:PACAP与中、晚期妊娠的维持密切相关;PAF可促进培养黄体细胞PACAP mRNA的表达,蛋白激酶C(PKC)和蛋白激酶A(PKA)途径都有可能参与了此过程。     

Vasoactive intestinal peptide increases intracellular cAMP and gonadotropin-alpha gene activity in JEG-3 syncytial trophoblasts. Constraints posed by desensitization.

Expression of the human chorionic gonadotropin (hCG)-alpha gene in placental trophoblasts is markedly stimulated by cAMP, a property preserved in a reporter plasmid containing its cAMP response elements (CREs) linked to the chloramphenicol acetyltransferase coding sequence (CRE alpha CAT). In search of a potential physiologic regulator of hCG gene expression via cAMP, we found that JEG-3 syncytial trophoblast cells have specific binding sites for vasoactive intestinal peptide (VIP) with dissociation constant of 1 nM. VIP maximally increased the transient expression of CRE alpha CAT and the expression of endogenous hCG-alpha mRNA in JEG-3 cells by 4- and 9-fold, respectively. Exposure of JEG-3 cells to 30 nM VIP increased cAMP levels 60-fold after 10-30 min, but cAMP rapidly declined thereafter. As a consequence of this desensitization, the effect of VIP on stimulation of both CRE alpha CAT and endogenous hCG-alpha and hCG-beta mRNA levels more closely resembled that of forskolin or 8-br-cAMP at time points much less than 24 h. Moreover, transient exposure to 8-br-cAMP was much less effective than 24 h of continuous incubation on CRE alpha CAT activity. We conclude that VIP rapidly increases cAMP content and activates hCG-alpha gene expression in JEG-3 cells, but sustained elevations in cAMP are necessary for maximal accumulation of this CRE-regulated gene product.     

Human glycoprotein hormone production in human-human and human-mouse somatic cell hybrids

The production of both protein and steroid hormones was studied utilizing somatic cell hybrids formed with human choriocarcinoma cells. The human JEG-3 cell line produced the species and organ-specific hormone human chorionic gonadotropin (hCG), the steroid hormone progesterone, and converted 19-carbon steroids to estrogens. Hybrids formed with human VA-2 cells, mouse Cl 1D cells and mouse 3T3-4EF cells had detectable hCG synthesis in 20 of 41 total clones. There was no detectable progesterone or 19-carbon aromatization to estrogens in any hybrids. These data demonstrate that the differentiated function of human protein hormone production can be retained in inter- and intra-specific somatic cell hybrids. These results also suggest that protein hormone production can occur independently of steroid production in these cells of placental origin.