肿瘤相关成纤维细胞高表达Twist1增强卵巢癌侵袭能力

目的:检测肿瘤相关成纤维细胞Twist1基因表达,探讨肿瘤相关成纤维细胞高表达Twist1对卵巢癌侵袭能力的影响。方法:CSIOVDB数据库检索Twist1基因在卵巢各种组织类型中的表达水平。分离原代正常成纤维细胞(NOF),以及卵巢癌患者的原代肿瘤相关成纤维细胞(CAF),免疫荧光实验验证Twist1基因在NOF和卵巢癌CAF细胞中的表达差异。TCGA数据库489例卵巢癌中从mRNA水平验证Twist1与CAF经典活化指标FAP的相关性,以及炎症因子IL-6的相关性。GSEA(Gene Set Enrichment Analysis)软件探究CAF高表达Twist1与IL-6通路富集的关系。Western blot法验证下调Twist1基因对IL-6表达的影响。Transwell实验探究卵巢癌CAF-肿瘤细胞共培养体系中IL-6对卵巢癌细胞系SKOV3侵袭能力的影响。结果:Twist1基因在卵巢癌CAF中的表达水平明显高于肿瘤细胞和正常成纤维细胞。TCGA数据库中Twist1基因和FAP及IL-6基因呈显著正相关(P0.0001)。GSEA软件分析结果示,卵巢癌患者中CAF高表达Twist1的同时,肿瘤上皮表现为IL-6通路明显富集(P0.05,FDR25%)。Western blot法结果表明,Twist1基因下调后,IL-6基因表达量明显降低。在共培养体系中加IL-6中和抗体,卵巢癌细胞侵袭力明显下降。结论:肿瘤相关成纤维细胞高表达Twist1,可能通过提高IL-6水平增强卵巢癌侵袭能力。     

FGF9在卵巢癌发生中的作用

成纤维细胞生长因子9(fibroblast growth factor 9,FGF9)是成纤维细胞生长因子家族成员之一,作为细胞间信号分子在胚胎发生和分化过程中起重要作用。近年研究表明,FGF9与卵巢上皮癌(epithelial ovarian cancer,EOC)有着密切联系。FGF9作为Wnt信号通路的下游靶点,通过参与细胞增殖、促进上皮细胞恶性转化、促进上皮细胞和内皮细胞浸润,在卵巢癌发生发展过程中发挥重要作用。现就FGF9的生物学特征、FGF9诱发卵巢癌的作用机制及卵巢癌动物模型做一综述。     

二甲双胍通过下调IL-6抑制卵巢癌肿瘤相关成纤维细胞活性的研究

目的:探讨二甲双胍对卵巢癌肿瘤相关成纤维细胞(CAF)活性的影响,以及可能的调控机制。方法:RT-PCR法检测二甲双胍作用于SKOV3细胞系及原代CAF后炎症因子(IL-6、OPN、IL-1b、COX-2、Cyr61)mRNA水平变化;将MRC5于SKOV3-CM(condition medium)培养7~10天得到活化的MRC5即MRC5-CAF,原代CAF及MRC5-CAF经免疫荧光鉴定α-SMA表达;Western blot检测上述炎症因子蛋白水平。在TCGA数据库557例卵巢癌中验证炎症因子与CAF属性相关基因的相关性。免疫荧光验证活化的MRC5中二甲双胍对α-SMA及IL-6表达的影响。在MRC5-CAF细胞系中进一步验证了二甲双胍或IL-6对CAF属性相关基因的影响。增殖实验和Transwell实验比较二甲双胍或IL-6对MRC5-CAF促进SKOV3增殖及迁移能力的影响。胶原回缩试验比较二甲双胍或IL-6对MRC5-CAF收缩细胞外基质ECM的影响。结果:二甲双胍下调CAF中炎症因子尤其是IL-6,同时下调CAF中α-SMA水平。TCGA数据库中IL-6 mRna与CAF属性相关基因(FAP、PDGFRB、FN1、COLL6A6)呈显著正相关(P0.0001);二甲双胍、IL-6共刺激组较IL-6组,STAT3通路和α-SMA下调;二甲双胍能逆转IL-6对卵巢癌CAF促肿瘤增殖转移及收缩胶原的能力。结论:卵巢癌中IL-6可能参与维持了间质CAF细胞活性和间质属性,二甲双胍可能通过下调CAF中IL-6/P-STAT3通路从而抑制卵巢癌CAF对肿瘤细胞的支持作用。     

子宫肌瘤及子宫平滑肌PA/PAI差异表达与腹腔镜子宫动脉阻断术治疗子宫肌瘤相关性的研究

目的:观察纤溶酶原激活因子及纤溶酶原激活物抑制因子(PA/PAI)在子宫肌瘤及子宫平滑肌组织及原代细胞中表达的差异,为腹腔镜子宫动脉阻断术治疗子宫肌瘤提供理论依据。方法:组织学:采用荧光定量PCR检测子宫肌瘤及子宫平滑肌组织中组织型纤溶酶原激活因子(t-PA)和尿激酶型纤溶酶原激活因子(u-PA)以及纤溶酶原激活物抑制因子(PAI-1)的表达;用免疫组织化学方法检测3种蛋白的表达;细胞学:选取8例子宫肌瘤患者,分别培养子宫肌瘤及子宫平滑肌细胞并传代,应用实时定量PCR及免疫细胞检测u-PA及PAI-1。结果:组织学:(1)肌瘤组织中u-PA mRNA相对表达为(1.34±2.18)%,显著低于平滑肌组织的(2.84±2.97)%,差异有统计学意义(P0.05);t-PAmRNA在肌瘤组织中表达为(1.46±1.64)%也低于平滑肌组织的(1.65±3.08)%,但差异无统计学意义(P0.05)。PAI-1 mRNA在肌瘤组织中的表达为(2.67±1.82)%,显著高于平滑肌组织的(1.27±1.99)%,差异有统计学意义(P0.05);(2)肌瘤组织u-PA蛋白表达显著低于平滑肌组织,差异有统计学意义(P0.05);肌瘤组织PAI-1蛋白表达显著高于平滑肌组织(P0.01);两种组织t-PA蛋白表达差异无统计学意义(P0.05)。细胞学:(1)肌瘤细胞中u-PA mRNA相对表达为0.123±0.189,显著低于平滑肌细胞的0.331±0.306,差异有统计学意义(P0.05);PAI-1 mRNA在肌瘤细胞的表达为0.091±0.036,显著高于平滑肌细胞的0.016±0.020,差异有统计学意义(P0.05);(2)肌瘤细胞u-PA蛋白表达8.805±1.645显著低于平滑肌细胞的22.173±4.381,差异有显著的统计学意义(P0.05);肌瘤细胞PAI-1蛋白表达44.765±1.090显著高于平滑肌细胞35.928±5.351(P0.05)。结论:PA/PAI系统在子宫肌瘤及平滑肌细胞中表达存在差异,并与组织中的差异基本一致,提示该系统差异表达机制可以为腹腔镜子宫动脉阻断术治疗子宫肌瘤提供理论依据。     

卵巢癌细胞与成纤维细胞相互作用对明胶酶释放与活化的影响

明胶酶-A(MMP-2)和明胶酶-B(MMP-9)具有降解细胞外基质的主要成分——Ⅳ型胶原和明胶的功能，参与恶性肿瘤的侵袭与转移。有研究发现，明胶酶免疫组织化学(免疫组化)着色重的区域集中在癌巢的边缘，提示卵巢癌细胞与成纤维细胞的相互作用，对明胶酶的释放和活化产生影响。我们观察了卵巢癌细胞与成纤维细胞在非接触性和     

子宫肌瘤细胞凋亡及增殖状况研究

目的 探讨细胞凋亡与凋亡调控基因在子宫肌瘤的发生、发展中的作用。方法 2000～2001年应用原位末端脱氧核苷酸转移酶标记(terminal deoxynucleotity transferase mediated dUTP nick end labeling,TUNEL)技术和免疫组织化学染色观察子宫肌瘤和正常子宫肌层组织中细胞凋亡和凋亡调控基因增殖细胞核抗原(PCNA的表达。结果 子宫肌瘤组织中细胞凋亡指数显著高于正常子宫肌层组织(P<0.01)。子宫肌瘤组织中PCNA蛋白阳性率高于正常子宫肌层组织(P<0.01)。凋亡细胞多分布在PCNA蛋白阴性区,阳性区仅有少量分布,且PCNA阴性区细胞凋亡指数高于阳性区。结论 细胞凋亡失控在子宫肌瘤生成过程中起重要作用,PCNA蛋白表达能比较准确地反映子宫平滑肌瘤的细胞增殖信息,子宫肌瘤的发生与增殖和(或)凋亡失衡密切相关。     

Dicer1在卵巢癌间质成纤维细胞中调控DNA损伤修复相关基因

目的:检测Dicer1在卵巢癌间质肿瘤相关成纤维细胞中的表达情况,探讨其对DNA双键断裂损伤(DSB)修复相关基因的影响。方法:应用基因集合富集分析(GSEA)方法分析卵巢癌间质和正常卵巢间质基因表达公共数据库GSE40595,探索Dicer1对DSB修复相关通路及关键基因的调控作用,并筛选出相关差异基因。应用重组人转化生长因子β1(h TGFβ1)细胞因子诱导成纤维细胞系MRC5细胞活化成为肿瘤相关成纤维细胞(CAFs),即MRC5-CAFs。以靶向Dicer1基因的siRNA转染MRC5-CAFs,下调其Dicer1表达水平。qRT-PCR、Western blot法分别检测Dicer1及分析所得相关基因,如XRCC6、TP53BP1、H2AFX、BRCA1、ATR、RAD51 mRNA水平及Dicer1和DSB损伤修复关键基因γ-H2AX(磷酸化H2AX)、RAD51蛋白水平。荧光共聚焦检测细胞核蛋白γ-H2AX表达;采用CCK8试验检测细胞活性。结果:GSEA分析结果显示,Dicer1表达水平与DSB(NES=1.7942109,FDRq=0.0067545176)及DNA损伤修复(NES=2.4433048,FDRq=0)显著正相关,进一步通过相关分析筛选出在上述通路中与Dicer1表达正相关的显著基因集。沉默MRC5-CAFs细胞中Dicer1表达后,DSB相关基因,如XRCC6、TP53BP1、H2AFX及损伤修复相关基因BRCA1、ATR、RAD51均较对照组显著下降,与GSEA分析结果一致。荧光共聚焦显微镜检测到沉默Dicer1 MRC5-CAFs细胞内聚集较少的DSB。CCK8试验结果显示,MRC5-CAFs细胞在20μmol/L顺铂作用下,Dicer1-si组不同时间梯度的细胞活性率均低于NC-si组。结论:Dicer1在卵巢癌间质成纤维细胞中正向调控DNA损伤修复相关基因,抑制Dicer1表达后其对顺铂敏感性显著增加。     

米非司酮对离体子宫肌瘤细胞PR、PCNA、Bcl-2表达的影响

目的　探讨米非司酮治疗子宫肌瘤的可能机制。并证实孕激素在子宫肌瘤发病中的作用及子宫肌瘤与细胞调亡的关系。方法　体外原代培养子宫肌瘤细胞并传代后 ,加入不同浓度的米非司酮 ,继续培养 ,观察细胞增殖情况 ,并以免疫细胞化学方法检测孕激素受体 (PR)、增殖细胞核抗原 (PCNA)及凋亡抑制基因(Bcl- 2 )蛋白的表达。结果　成功的进行了子宫肌瘤细胞的原代培养 ,加入 1 0 - 4mol L米非司酮后 ,细胞增殖活动减少 ,PR、PCNA、Bcl- 2在子宫肌瘤细胞中的表达均明显下降 (P <0 0 5 ,P <0 0 5 ,P <0 0 1 )。结论　米非司酮治疗子宫肌瘤的机制可能是 :①米非司酮可直接抑制肌瘤细胞生长。②米非司酮通过抗孕激素作用抑制肌瘤细胞生长。③米非司酮可促进子宫肌瘤细胞调亡。反证了孕激素是子宫肌瘤的生长因素之一 ,亦说明子宫肌瘤与细胞调亡有关     

成纤维细胞生长因子在宫颈癌中的表达及对细胞增殖影响的研究

目的 探讨酸性成纤维细胞生长因子(aFGF)和碱性成纤维细胞生长因子(bFGF)在宫颈癌发生发展中的意义.方法 应用逆转录-聚合酶链反应技术(RT-PCR)对2000年9月至2006年10月中国医科大学第一医院等3家医院的10例正常宫颈组织、10例宫颈上皮内瘤变组织和50例宫颈癌组织aFGF mRNA和bFGF mRNA的表达进行分析;通过四甲基偶氮唑蓝(MTT)比色法,观察aFGF和bFGF对宫颈癌细胞株Hela细胞增殖的影响.结果 aFGF mRNA和bFGF mRNA在宫颈癌组织中的半定量检测结果分别为1.226±0.049和1.323±0.096,与正常宫颈组织相比,差异有统计学意义(P<0.05),且在Ⅲ-Ⅳ期宫颈癌中的表达水平明显高于I-II期(P<0.05);aFGF mRNA及bFGF mRNA在宫颈上皮内瘤样病变中也有过度表达,与正常宫颈相比,其差异有统计学意义.加入外源性aFGF和bFGF后.Hela细胞株增殖比明显增加,其增殖程度与aFGF和bFGF浓度呈显著正相关,当aFGF浓度为1.2ug/mL,Hela细胞增殖比为对照组的1.55倍,bFGF浓度为75ng/mL, Hela细胞增殖比为对照组的1.91倍.结论 aFGF和bFGF表达与宫颈癌的发生、发展、浸润呈正相关,与宫颈癌细胞增殖密切相关,可作为宫颈癌生物学行为的重要指标之一.     

碱性成纤维细胞生长因子在EMs中研究进展

子宫异位内膜的种植和发展必须依赖足够的血供,新生血管形成是子宫内膜异位症(EMs)发生的基本条件.在众多与血管生成相关的因子中,碱性成纤维细胞生长因子(bFGF)起到了关键调节因子的作用.在正常子宫内膜组织中,该因子及其受体有表达,且受卵巢激素的调节.在EMs中,该因子及其受体的异常表达与异位内膜的增殖、黏附和血管生成有一定关系.     

ET-1、ET_A-R、c-fos原癌基因mRNA在子宫肌瘤中的表达

目的 :探讨ET 1及ETA R表达在子宫肌瘤发生中的病理生理作用及其与肌瘤中c fos原癌基因之间的关系。方法 :应用半定量RT PCR方法检测 30例子宫肌瘤组织及邻近正常子宫肌组织中ET 1、ETA R、c fosmRNA的表达强度 ,对照其差异。结果 :( 1)子宫肌瘤组织中ET 1mRNA的表达强度 ( 2 .717± 0 .5 2 0 )与正常子宫肌组织 ( 2 .4 83± 0 .62 3)差异有显著性 (P <0 .0 5 ) ;ETA RmRNA在子宫肌瘤组织中表达为 2 .2 17± 0 .4 68,明显高于正常子宫肌组织的 1.617± 0 .4 86(P <0 .0 0 1) ;c fosmRNA在子宫肌瘤组织中表达为 4 .90 0± 0 .4 0 3,也明显高于正常子宫肌组织的 4 .183± 0 .5 94 ,(P <0 .0 0 1) ;( 2 )相关分析表明 :肌瘤组织中ETA RmRNA与c fosmRNA之间呈正相关 ,而ET 1mRNA与ETA RmRNA、c fosmRNA之间无相关性。结论 :子宫肌瘤组织中ETA R的表达上调对子宫肌瘤细胞的分裂增殖起促进作用 ,ET 1/ETA R系统可能是子宫肌瘤发生发展的机理之一     

Tumor-stromal cell contact promotes invasion of human uterine cervical carcinoma cells by augmenting the expression and activation of stromal matrix metalloproteinases

OBJECTIVE: The augmentation of the expression and activation of matrix metalloproteinases (MMPs) is associated with tumor invasion and metastasis. In addition, tumor-stromal cell contact provides a crucial signal for regulating the pericellular proteolysis for the progression of tumor invasiveness. The present study evaluates the regulation of the expression and activation of MMPs and tissue inhibitors of metalloproteinases (TIMPs) by tumor-stromal cell contact in an in vitro co-culture model of human uterine cervical carcinoma cells and human uterine cervical fibroblasts. METHODS: When human uterine cervical carcinoma SKG-II cells were co-cultured with human uterine cervical fibroblasts (HUCFs), the invasive activity of SKG-II cells was analyzed using an in vitro invasion assay using Matrigel. The production, mRNA expression and activation of MMPs and TIMPs were monitored by Western blot and Northern blot analyses and gelatin zymography. RESULTS: SKG-II cells, which constitutively produced membrane-type 1 MMP (MT1-MMP) and a trace of proMMP-2 but neither TIMP-1 nor TIMP-2, showed poor invasiveness in vitro. Upon co-culturing with HUCFs, SKG-II cells were found to transform to the invasive phenotype by enhancing the production and mRNA expression of tumoral MT1-MMP. In addition, a sequential increase in the activation of fibroblast proMMP-2 was observed along with the formation of an MT1-MMP-TIMP-2-proMMP-2 complex on the tumor cell surface. Furthermore, the production and gene expression of fibroblast proMMP-1 and proMMP-3 were augmented under co-culture conditions, whereas mRNA expression of proMMP-2, TIMP-1 and TIMP-2 was unchanged. Moreover, we demonstrated the partial involvement of tumor-cell-derived soluble factors in the augmentation of the production of proMMP-1 and proMMP-3 in HUCFs. However, anti-integrin beta1 and beta3 antibodies failed to abolish the augmentation of fibroblast proMMP-3 production and proMMP-2 activation in the co-culture. CONCLUSION: Cell-cell contact between cervical carcinoma cells and peripheral stromal fibroblasts augments the production and activation of MMPs, and therefore the subsequent imbalance between MMPs and TIMPs may result in the progression of invasiveness of cervical carcinoma cells in vivo.     

Transforming growth factor-beta3 is expressed at high levels in leiomyoma where it stimulates fibronectin expression and cell proliferation

OBJECTIVE: To investigate the expression of TGF-beta3 in leiomyoma and myometrium as well as the effect of TGF-beta3 on the expression of fibronectin and on the proliferation of leiomyoma and myometrial cells. DESIGN: Observational and in vitro experimental study. SETTING: University medical center.Patient(s): Women with (n = 18) leiomyoma. INTERVENTION(s): First TGF-beta3 mRNA and protein levels in myometrium and leiomyoma were measured, and then myometrial and leiomyoma cells in culture were treated with TGF-beta3. MAIN OUTCOME MEASURE(s): TGF-beta3 and fibronectin mRNA were evaluated by Northern analysis. Myometrial and leiomyoma cell proliferation was assessed with use of [(3)H]thymidine incorporation. RESULT(s): The TGF-beta3 mRNA level in the leiomyoma samples was 3.5-fold higher than in the myometrial samples. The highest TGF-beta3 mRNA level was observed in leiomyoma samples from midsecretory phase and was 5-fold higher than in proliferative phase samples. Fibronectin mRNA expression also was higher in the leiomyoma than in the myometrium. TGF-beta3 induced fibronectin expression in leiomyoma cells and directly stimulated myometrial and leiomyoma cell proliferation in cultures. CONCLUSION(s): These findings suggest that TGF-beta3 may be mediating the growth-promoting effects of sex steroids on leiomyomas by playing a role in the fibrogenic process and cell proliferation that characterize these tumors.     

HMGI-C和HMGI(Y)基因在子宫肌瘤中的表达

目的:研究HMGI-C、HMGI(Y)基因在子宫肌瘤及子宫肌层中的表达。方法:采用Real-timePCR、Westernblot、免疫组化方法检测HMGI-C、HMGI(Y)基因在30例子宫肌瘤患者的肌瘤组织和子宫肌层组织中的表达。结果:HMGI-CmRNA及蛋白在肌瘤组织中的表达显著高于肌层组织(P<0.01)。HMGI(Y)mRNA在肌瘤组织与肌层组织中的表达无统计学差异(P>0.05),而HMGI(Y)蛋白在肌瘤组织中的表达显著高于肌层组织(P<0.05)。结论:HMGI-C、HMGI(Y)基因的异常表达与子宫肌瘤的发生有关。     

表皮生长因子对子宫肌瘤细胞MAPK信号通路的活化作用

目的：研究表皮生长因子（EGF）对子宫肌细胞（HM-SMCs）和子宫肌瘤细胞（HL．SMCs）中有丝分裂原激活的蛋白激酶（MAPK）信号转导通路的活化作用。方法：收集30例子宫肌瘤患者的子宫肌瘤组织和正常子宫肌组织进行原代细胞培养。采用激光扫描细胞法（LSC法）检测BrdU渗入率;Western blot法检测p44／42MAPK及其磷酸化水平和p44／42MAPK特异性底物ELK-1的表达,MAPK特异性抑制剂PD98059和EGFR受体抑制剂AG1478对p44／42MAPK表达的抑制作用,以及p44／42MAPK的时间依赖性表达以及细胞周期相关蛋白p27的表达。结果：EGF诱导p-p44／42MAPK和ELK-1水平显著上调,HM．SMCs中的表达量显著高于HL-SMCs。AG1478和PD98059可抑制EGF对MAPK的诱导作用。HM-SMCs中p44／42MAPK对EGF的刺激呈持续平稳的激活,伴随着p27的显著上调;HL．SMCs中则呈快速短暂的激活,伴随着p27低水平上调。结论：EGF通过活化MAPK信号通路促进细胞增殖可能是子宫肌瘤发病的-个重要的机制,为肌瘤的非手术治疗提供了可能。     

Interleukin 8 production and interleukin 8 receptor expression in human myometrium and leiomyoma

OBJECTIVE: Interleukin 8 is a potent chemoattractant cytokine that is expressed in a variety of human tumors and is known to induce mitogenesis. We aimed to investigate the production of interleukin 8 and the expression of its receptor in myometrium and leiomyoma, in which we hypothesized that interleukin 8 may contribute to cellular proliferation. STUDY DESIGN: Myometrial and leiomyoma tissue pairs (n = 14) were obtained from human uteri after hysterectomy conducted for leiomyomatous uterus. Expression of interleukin 8 and interleukin 8 receptor type A was identified in the leiomyomatous myometrium by means of specific antibodies directed against interleukin 8 and interleukin 8 receptor type A for immunohistochemical detection. Interleukin 8 production by cultured cells was measured by enzyme-linked immunosorbent assay. The regulation of interleukin 8 messenger ribonucleic acid expression was assessed by means of the Northern blot analysis after treatment of myometrial cells with interleukin 1alpha and tumor necrosis factor alpha. Myometrial cell proliferation was determined by means of colorimetric assay after cells were treated with interleukin 8 and antihuman interleukin 8 neutralizing antibody. RESULTS: Immunostaining for both interleukin 8 and interleukin 8 receptor type A was stronger in the myometrium adjacent to leiomyoma compared with leiomyoma itself (2-fold, P <.05). Compared with samples from nonusers, samples from patients who had used gonadotropin-releasing hormone agonists revealed a trend for decreased staining for both interleukin 8 and interleukin 8 receptor type A. Interleukin 1alpha and tumor necrosis factor alpha caused a time- and dose-dependent increase in interleukin 8 production by myometrial cells (P <.001). There was a dose-dependent inhibition of cell proliferation with antihuman interleukin 8 antibody to 55% of the control (P <.001). CONCLUSION: Our demonstration of high levels of interleukin 8 and its receptor in myometrium immediately surrounding leiomyoma and the inhibition of cell proliferation when interleukin 8 is blocked by a neutralizing antibody suggest a potential role for interleukin 8 in the growth of myometrial tissue surrounding leiomyomatous tissue. This study could lead to a better understanding of potential involvement of cytokines in leiomyoma growth and in gonadatropin-releasing hormone agonist-induced regression.