经阴道彩色多谱勒对不孕症患者卵巢基础状态的评价

目的 探讨卵巢基础状态与卵巢储备之间的关系，评价彩色多谱勒对卵巢储备功能预测的价值。方法 32例不孕症妇女于自然月经周期第三天测定血促卵泡素、促黄体生成素、雌二醇浓度，同时经阴道彩色多谱勒测定双侧卵巢体积、最大平面的平均直径、窦卵泡数、间质血流收缩期最大峰值(PSV)、搏动指数(PI)、阻力指数（RI)，并且行克罗米芬(CC)激动实验。B超监测卵泡发育，应用hCG诱导成熟卵泡排卵。结果 依据CC激动实验结果将患者分为卵巢储备正常组和低下组。卵巢储备下降者窦卵泡总数少(P＜0．05)；PSV降低(P＜0．01)；注射hCG日所获优势卵泡数少；PSV与注射hCG日优势卵泡数正相关(P＜0．05)。结论 应用经阴道彩色多谱勒能简便、有效、无创、快速的预测卵巢储备功能。     

ESPL1基因在卵巢癌中的表达及意义

目的 探讨分离酶(ESPL1)基因在卵巢癌中的表达及意义。方法 利用GEPIA数据库,对比相应正常组织,分析ESPL1在泛癌及卵巢癌中的表达。运用The Human Protein Atlas数据库,分析ESPL1蛋白在卵巢及卵巢癌组织中表达及定位;应用String数据库绘制ESPL1基因的相关蛋白网络图,并进行基因功能富集分析。使用Kaplan-Meier Plotter网站,对ESPL1表达影响不同临床特征患者预后指标及化疗药物疗效进行分析。结果 ESPL1在14种恶性肿瘤中表达明显上调,而在食管癌中表达量下调;与正常卵巢组织相比,在卵巢癌中ESPL1基因在mRNA及蛋白质水平的表达显著升高,在卵巢癌中表达定位于细胞膜或浆及核中。与ESPL1基因相互作用蛋白有SMC3、KIF11、PTTG1等10个,主要富集在细胞周期、卵母细胞减数分裂等信号通路。Kaplan-Meier生存分析显示,ESPL1高表达的卵巢癌患者总生存时间明显短于ESPL1低表达者,且临床I+II期患者总生存期较短(P<0.05),其它期别及病理分级的患者预后无差异;应用多西他赛单药组中,ESPL1高表达组较...     

miR-218抑制卵巢癌细胞A2780对顺铂耐药性的实验研究

目的研究miR-218抑制卵巢癌细胞A2780对顺铂耐药性的影响及可能机制。方法实时定量PCR检测A2780及顺铂耐药细胞株A2780/DDP中miR-218的表达,转染miR-218 inhibitors和mimics改变A2780及A2780/DDP细胞中miR-218的表达,MTT法检测转染前后细胞对顺铂的敏感性,并分析Wnt2B蛋白表达的改变。结果与A2780细胞相比,miR-218在A2780/DDP中的表达明显降低。A2780细胞转染miR-218 inhibitors后,细胞对顺铂的敏感性降低,Wnt2B蛋白表达明显增强;而A2780/DDP细胞转染miR-218mimics后,细胞对顺铂的敏感性增强,Wnt2B蛋白表达明显减少。结论 miR-218可能通过靶向Wnt2B抑制卵巢癌细胞A2780对顺铂的耐药性。     

Silencing MTA1 by RNAi Reverses Adhesion,Migration and Invasiveness of Cervical Cancer Cells （SiHa） via Altered Expression of p53, and E-cadherin/β-catenin Complex

It has been reported that metastasis-associated gene 1 (Mta1) is overexpressed in many malignant tumors with high metastatic potential. In addition, some studies indicated that MTA1 participated in invasion, metastasis, and survival of cancer cells by regulating cell migration, adhesion and proliferation. But the role of MTA1 is unclear in vitro in the development of cervical cancer cells. This study investigated whether and how MTA1 mediated cell proliferation, migration, invasion and adhesion in cervical cancer. MTA1 expression level was detected by Western blot in two cervical cancer cell lines of different invasion potentials. The effects of MTA1 expression on SiHa cell apoptosis, cycle, proliferation, migration, invasion and adhesion were tested by flow cytometry, MTT, wound-healing assay, Transwell assay and adhesion assay, respectively. The expression levels of p53, E-cadherin, and β-catenin activity were evaluated in untreated and treated cells. The results showed that MTA1 protein expression was significantly higher in SiHa than in HeLa, which was correlated well with the potential of migration and invasion in both cell lines. Furthermore, the cell invasion, migration and adhesion capabilities were decreased after inhibition of MTA1 expression mediated by Mta1-siRNA transfection in SiHa. However, no significant differences were found in cell apoptosis, cycle, and proliferation. In addition, E-cadherin and p53 protein levels were significantly up-regulated, while β-catenin was significantly down-regulated in SiHa transfected with the siRNA. These results demonstrated that MTA1 played an important role in the migration and invasion of cervical cancer cells. It was speculated that the decreased migration and invasion capability by inhibiting the MTA1 expression in the SiHa cell line may be mediated through the altered expression of p53, and E-cadherin/β-catenin complex. MTA1 could serve as a potential therapeutic target in cervical cancer.     

miR-106b对宫颈癌SiHa细胞运动侵袭功能的影响

目的 研究miR-106b对宫颈癌SiHa细胞转移侵袭功能的影响及作用机制.方法 SiHa细胞转染miR106b inhibitors和mimics,实时定量PCR检测转染效率,Transwell及划痕法检测细胞运动侵袭功能,分析E-cadherin 、PTEN、p-AKT(Ser473)蛋白表达情况.结果 miR-106b inhibitors和mimics改变SiHa细胞中miR-106b表达.转染miR-106b inhibitors后,细胞迁移数、移动距离减少(P＜0.05);PTEN及E-cadherin表达增强,p-AKT表达降低.转染miR-106b mimics后,细胞迁移数、移动距离增加(P＜0.05);PTEN及E-cadherin表达降低,p-AKT表达增强.结论 miR-106b可能通过E-cadherin/AKT、PTEN/AKT信号通路影响SiHa细胞运动侵袭功能.     

Preliminary evaluation of safety of conditionally replication adenovirus M4

Conditionally replication adenovirus M4, which was constructed in our lab, was proved to have good clinical application prospect for its good antitumor and antimetastasis effect. However, clinically applying M4 faces many problems. One of the most important is the safety of M4. In this study, we investigated the safety of M4 by comparing with Adv-TK, which was proved to be safe in Ⅰ-Ⅲ phase clinical trials. M4 and Adv-TK were injected into mice via the tail vein separately, and the mice were sacrificed at the indicated time. Blood was collected for biochemical tests, the liver was harvested for hematoxylin and eosin (H&E) staining and viral quantification, and splenic lymphocytes were separated for adenovirus specific cellular immune response. Our results showed that M4 had no obvious effect on mouse general symptoms. A transient reversible infiltration of inflammatory cells in collect abbacy was only observed in M4 group, and a transient slight increase in Cr level was detected both after M4 and Adv-TK injection. The adenovirus specific cellular immune response induced by M4 was similar to that by Adv-TK, and the distribution and metabolism of M4 in the mouse liver were also similar to those of Adv-TK. It was concluded that conditionally replication adenovirus M4 had the same safety as Adv-TK. The study provides safety basis for the coming clinical trials of M4.     

应用siRNA技术下调Wnt2B抑制乳腺癌细胞恶性表型的研究

目的:通过体外实验探讨Wnt2B在乳腺癌发生发展中的作用.方法:应用免疫组织化学的方法检测正常乳腺组织和乳腺癌组织中Wnt2B的表达；采用RNA干扰技术,在MCF7细胞中沉默Wnt2B的表达,并经Western blot检测其沉默效率；应用流式细胞术和激光共聚焦显微镜检测、观察Wnt2B沉默后对MCF7细胞凋亡、细胞骨架构成的改变.同时在蛋白水平上检测沉默Wnt2B后对细胞P53/P21通路的影响.结果:Wnt2B蛋白在乳腺癌中的表达明显高于在正常乳腺组织中的表达；第2对Wnt2B siRNA(2号siRNA)对Wnt2B的沉默效果最为明显；沉默Wnt2B后细胞早期凋亡率和晚期凋亡率相对于对照细胞均明显增高并伴有细胞骨架明显的改变,表现为细胞骨架溶解,正常形态消失.同时,细胞内P53及P21表达水平随Wnt2B沉默而明显下降.结论:Wnt2B基因可以改变乳腺癌细胞的凋亡水平和骨架结构,在乳腺癌的发生发展中起着重要的作用,为乳腺癌的治疗提供了新的靶点.     

BRMS1对卵巢癌细胞c13*转移侵袭的影响及机制

目的探讨乳腺癌转移抑制基因1(BRMS1)对c13*细胞转移侵袭、粘附的影响及机制。方法由质粒PC-MV-HA-BRMS1中扩增目的基因BRMS1,将其插入pcDNA3.1(+)质粒中,构建pcDNA3.1(+)-BRMS1质粒。经酶切及测序鉴定正确后,转染c13*细胞,PCR、Real-time PCR、Western blot检测BRMS1的表达;划痕实验、Transwell实验、粘附实验检测c13*细胞运动、侵袭、粘附能力;Western blot检测整合素β1、p-AKT、AKT表达。结果成功构建了pcDNA3.1(+)-BRMS1质粒;转染目的基因的c13*细胞中,BRMS1的mRNA和蛋白表达均显著增加,细胞运动、侵袭、粘附能力降低,整合素β1及p-AKT表达均下降,AKT表达无改变。结论成功构建pcDNA3.1(+)-BRMS1质粒。BRMS1可能通过抑制整合素β1/AKT通路,抑制卵巢癌c13*细胞的运动、侵袭和粘附能力。     

MTA1通过β-catenin、MMP-9调节HeLa细胞粘附、侵袭、转移

目的观察转染转移相关基因1(metastasis-associated 1,MTA1)基因对人宫颈癌细胞HeLa迁移、侵袭、粘附能力以及β-catenin、基质金属蛋白酶-9(MMP-9)表达的影响。方法 Western blot法检测HeLa、SiHa细胞中MTA1表达;以脂质体LipofectamineTM2000介导的方法将含MTA1全长基因的质粒pEGFP-C1-MTA1转染HeLa细胞,Western blot法检测目的基因表达;划痕、侵袭、粘附试验分别检测HeLa细胞迁移、侵袭、粘附能力变化;Western blot法检测β-catenin、MMP-9表达变化。结果与SiHa细胞相比,MTA1在HeLa细胞中表达较低(P<0.05);MTA1质粒转染的HeLa细胞中,MTA1表达、划痕愈合、Matrigel侵袭、Matrigel粘附能力均明显高于未转染及转染空载体对照组(均P<0.05);β-catenin、MMP-9表达在转染后均上调(均P<0.05)。结论 MTA1基因过表达可促进HeLa细胞转移、侵袭和粘附,上调β-catenin、MMP-9表达,MTA1基因可能成为肿瘤治疗靶点。