雌二醇对BG-1卵巢癌细胞增殖及侵袭的影响

目的:探讨雌二醇(E2)对卵巢癌细胞BG-1增殖、侵袭的影响及作用机制。方法:MTT法检测10-9~10-5mol/L E2作用于BG-1细胞72h后的细胞增殖率的变化,并筛选最适作用浓度。将BG-1分为4组:E2组、E2+ICI(ICI182,780 ER阻断剂)组、E2+PPT(特异性ERα激动剂)组和E2+DPN(特异性ERβ激动剂)组。MTT法检测不同药物作用72h后的细胞增殖率;RT-PCR、Western blot法分别检测药物作用6、24h和48h后各组细胞中cyclin(细胞周期素)D1和p21表达水平;ELISA检测10-9~10-5mol/L E2作用后,BG-1细胞中MMP-9水平的变化;Transwell侵袭实验检测E2及ER调节剂作用后BG-1细胞的侵袭力变化。结果:10-9~10-5mol/L E2作用于BG-1细胞72h后,细胞增殖率均高于对照组(P0.05),并且E2浓度为10-7mol/L时BG-1细胞的增殖率最高。不同药物作用后,E2+PPT组的细胞增殖率显著高于E2组(P0.05),E2+ICI组的细胞增殖率显著低于E2组(P0.05),而E2+DPN组细胞增殖率无显著变化(P0.05)。与E2组相比,E2+ICI组中cyclin D1表达明显降低,p21表达显著升高(P0.05);E2+PPT组中cyclin D1与p21的表达水平与E2+ICI组大致相反(P0.05);E2+DPN组中cyclin D1和p21的表达均无明显变化(P0.05)。10-9~10-5mol/L E2均可促进MMP-9分泌(P0.05),其中10-8mol/L E2作用3h后MMP-9分泌最多。E2组的侵袭力显著高于空白对照组,而E2+ICI组的侵袭力显著低于空白对照组(P0.05)。结论:E2通过ERα信号通路促进BG-1细胞增殖;E2还能促进MMP-9分泌,增强BG-1细胞的侵袭力。     

Treg细胞/Th17细胞比例失衡在卵巢癌的生长以及微血管、淋巴管生成中的作用

目的:探讨Treg细胞/Th17细胞比例失衡对移植瘤生长及微血管和淋巴管生成的影响。方法:构建卵巢癌裸鼠皮下和原位移植瘤模型,用磁珠分选人外周血CD3+淋巴细胞,加曲古抑菌素A(TSA)刺激使其Treg/Th17细胞比例失衡,采用流式细胞仪检测Treg/Th17细胞比例。分别将Treg/Th17细胞比例较高和较低的淋巴细胞与人卵巢癌细胞系SKOV3按1∶20比例注射入裸鼠皮下和卵巢包膜下,分别于注射后28天和35天处死称重,观察其对移植瘤生长的影响;免疫组化CD31和LYVE-1染色检测微血管(MVD)和淋巴管(LVD)的表达。结果:Treg高组能促进皮下和原位卵巢癌的生长。皮下模型:注射后第21和28天,Treg高组的肿瘤总荧光强度值均大于Th17高组和对照组;Treg高组的肿瘤重量为(0.60±0.07)g,Th17高组(0.35±0.24)g,对照组(0.25±0.22)g,Treg高组肿瘤重量较Th17高组和对照组重。原位模型:注射后第28天Treg高组肿瘤总荧光强度值均大于其他两组;Treg高组(0.34±0.20)g,Th17高组(0.06±0.03)g,对照组(0.17±0.05)g,Treg高组肿瘤重量比Th17高组重。Treg高组的MVD和LVD表达高于Th17高组和对照组(P0.05)。结论:Treg细胞/Th17细胞比例失衡在卵巢癌裸鼠移植瘤生长中起着重要作用,且Treg高组能影响卵巢癌的生长、微血管和淋巴管的形成。     

Hypoxia limits differentiation and up-regulates expression and activity of prostaglandin H synthase 2 in cultured trophoblast from term human placenta

OBJECTIVE: We determined the effect of hypoxic conditions on cellular differentiation and prostaglandin H synthase expression in cultured human term trophoblast. STUDY DESIGN: Cytotrophoblasts isolated from term placentas were cultured for 24-72 hours in a standard (20% oxygen) or hypoxic (1% to 2% oxygen) atmosphere. Trophoblast biochemical differentiation was determined by release of human chorionic gonadotropin. Morphologic differentiation was evaluated by epifluorescent and confocal microscopic examination of cultures after dual cytochemical staining for surface membrane desmosomes and intracytoplasmic nuclei. Prostaglandin H synthase 2 expression was determined by Western blot analysis and correlated with enzyme activity by immunoassay of 2 prostaglandin H synthase 2 products, prostaglandin E2 and thromboxane. RESULTS: Human chorionic gonadotropin levels in media and syncytiotrophoblast formation were markedly diminished in trophoblast cultured in hypoxia, compared with trophoblast in control cultures, whereas viability was unchanged. Hypoxia up-regulated expression of trophoblast prostaglandin H synthase 2 but not of prostaglandin H synthase 1 and increased prostaglandin E2 and thromboxane release. CONCLUSIONS: Hypoxia limits differentiation and enhances prostaglandin H synthase 2 expression in cultured villous trophoblast. These responses may account for the cytotrophoblast prominence characteristic of placentas exposed to attenuated oxygen delivery.     

Angiopoietin-1, 2 and Tie2 expressions in endometrial adenocarcinoma--the Ang2 dominant balance up-regulates tumor angiogenesis in the presence of VEGF

We investigated Ang1, Ang2 and Tie2 expressions including balance and intratumoral vessels in the role of angiogenesis of endometrial adenocarcinoma. Immunohistochemical staining was performed on 133 patients with endometrial (endometrioid) adenocarcinoma, including 73 with G1, 34 with G2, and 26 with G3. The levels of Ang1, Ang2 and Tie2 expressions were expressed as staining score. Total vessel count (TVC), microvessel count (MVC) and mean vessel diameter (VD) in the CD34-stained tissues were measured in five hot spot areas at x 200 magnification by image cytometry. These results were compared with high and low vascular endothelial growth factor (VEGF) expressions. Ang1, Ang2, Tie2 and CD34 were expressed in the cytoplasm of tumor cells. A significant correlation was found among Ang1, Ang2 and Tie2 expressions. In high VEGF cases, Ang1 expression was correlated negatively with TVC and MVC, but positively with VD, and the Angl < Ang2 group was significantly higher in TVC and MVC and tended to be smaller in VD than the Ang1 > Ang2 group. VD was significantly larger in G3 than in G1. The Ang1 < Ang2 balance may be one of the key factors for angiogenesis of endometrial carcinoma in the presence of high VEGF expression.     

The effects of oral administration of prostaglandin E2 on the human ejaculate

A single 1 mg dose of prostaglandin (PG) E2 was given orally to 19 men. Ejaculates were obtained 90 minutes and 24 and 48 hours thereafter. Before treatment, each man delivered another three semen samples with the same time intervals as during the study period. PGE2 was also administered to seven men during naproxen treatment and ejaculates were sampled as above. PGE2 did not influence the 90 minutes' posttreatment ejaculates, but after 24 hours there was a significant (P less than 0.05) decrease in sperm counts as compared to the control samples. The change in sperm count was suggested to be due to an effect of PG on the contractile elements in the deferent duct. Sperm motility, viability, and morphology as well as semen volume and adenosine triphosphate (ATP) content remained unchanged. The total semen PGE content was increased 24 hours after treatment from 169 micrograms/ejaculate to 213 micrograms/ejaculate (P = 0.02). In the combined PGE2/naproxen treatment the PGE levels were significantly (P less than 0.05) elevated in the ejaculate 48 hours after treatment. The increase may indicate an increased de novo synthesis of prostaglandins. Based on the results from the analysis of the composition of the 19-hydroxy PGF-isomers with and without naproxen treatment, it is speculated that oral PGE2 influences the cyclo-oxygenase activity.     

COX2促进卵巢癌细胞迁移及相关机制的研究

目的:探讨COX2对卵巢癌细胞株增殖及迁移的影响及其相关机制。方法:构建慢病毒载体Lenti-COX2-EGFP,转染卵巢癌细胞株SKOV3和ES2。PCR及Western blot法鉴定转染效率,CCK-8实验检测细胞增殖,划痕实验观察细胞迁移能力,qRTPCR法检测E-cadherin、Vimentin、Snail和Slug表达。结果:与对照组相比,过表达COX2后卵巢癌细胞的增殖和迁移能力增强,差异均有统计学意义(P0.05);E-cadherin表达下调,Snail、Slug、Vimentin表达上调。COX2抑制剂塞来昔布处理则可抑制COX2过表达细胞的增殖及迁移能力,与对照组比较差异有统计学意义(P0.05)。结论:COX2可促进卵巢癌细胞增殖及上皮间质转化(EMT)继而增强肿瘤细胞迁移能力;塞来昔布可发挥阻断作用。推测COX2可能是卵巢癌临床治疗的一个潜在靶点。     

Markers and modulators of angiogenesis in ovarian cancer

Understanding of the role played by angiogenesis in the growth of ovarian malignancies and an attempt to link this process with the clinical outcome of the disease call for an employment of reliable quantitative and qualitative measures--markers and modulators of angiogenesis. These tumor phenotypic qualities can be assessed with the use of immunohistochemical and immunoenzymatic assays, what reflects angiogenesis intensity, as well as its specific neoplastic properties. Supplementing clinical and histopathological data with the information on markers and modulators of angiogenesis enables a notable increase in the prognosic accuracy of the clinical outcome in ovarian cancer.     

Inhibition of human ovarian cancer cell growth by prostaglandin D2

The effects of prostaglandin D2 (PGD2) on human ovarian tumor growth were examined in vitro and in vivo by using a cell line, designated HR, derived from patients with serous cystadenocarcinoma of the ovary. The cell proliferation was dose-dependently inhibited by PGD2 between concentrations of 0.1 and 4.0 micrograms/ml after 24 hrs, 48 hrs and 72 hrs of contact time. Concentrations of PGD2 required for 50% inhibition of the cell proliferation were 2.0, 1.1 and 0.55 micrograms/ml with 24, 48 and 72 hrs of contact time, respectively. From the results of 51Cr-release assay, the inhibition of cell proliferation by PGD2 was considered to result from the direct cytotoxic effects. The incorporations of 3H-thymidine, 3H-uridine and 3H-valine were inhibited in a dose-dependent fashion with more than 1.0 micrograms/ml of PGD2. Tumor growth in nude mice treated with 0.3 mg/mouse PGD2 was significantly inhibited, compared to that of untreated nude mice. In untreated nude mice the tumor growth curve was parallel to the changes in the plasma alpha-hydroxybutyrate dehydrogenase (HBD). In both PGD2-treated groups with 0.1 mg/mouse and 0.3 mg/mouse, the HBD activity markedly decreased on the 14th and the 21st day after inoculation. The 50% survival time in untreated mouse, 0.1 mg/mouse and 0.3 mg/mouse PGD2-treated groups was 52 days, 55 days and 67 days, each respectively.     

Epigenetic silencing of the LDOC1 tumor suppressor gene in ovarian cancer cells

Purpose

To establish a model of ovarian cancer with highly lymphatic metastasis in immunocompetent rats.

Methods

Thirty-two female Fischer 344 rats were divided randomly and equally into two groups: footpad group and intraperitoneal (i.p.) group. At 8 weeks after injection with NuTu-19 ovarian cancer cells, lymphatic metastasis were analyzed by pathohistology; body weight was monitored per week, Survival curves were determined by Kaplan–Meier analysis.

Results

Footpad injection could efficiently generate the lymphatic metastasis; specifically, the incidences of metastasis in the ipsilateral popliteal, inguinal and para-iliac lymph nodes were 100 % (8/8), 75 % (6/8), and 37.5 % (3/8), respectively. The mean volume and weight of the ipsilateral popliteal lymph nodes were 0.405 ± 0.096 cm³ and 0.418 ± 0.118 g in footpad group. However, no lymphatic metastasis lesions were found in i.p. group. Moreover, Kaplan–Meier analysis showed that the average survival time of the footpad group was significantly longer than that of the i.p. group (18.429 ± 1.112 vs. 10.286 ± 0.505 weeks).

Conclusions

Our experiments suggest that footpad injection is a very efficient method to generate ovarian cancer with lymphatic metastasis in an immune-competent animal, and we believe that this model will be very helpful for shedding light on the mechanism of lymphogenous metastasis and developing novel therapeutic targets for ovarian cancer patients.     

Mechanical stretching increases prostaglandin E2 in cultured human amnion cells

Mechanical stretching increases synthesis and release of prostaglandin E2 (PGE2) in cultured amnion cells. The maximum level of PGE2 in stretched amnion cells is three times higher than that in nonstretched amnion cells. The maximum level of PGE2 in stretched cell medium is nine times higher than the maximal level of PGE2 in nonstretched cell medium. 3H-thymidine incorporation into DNA is 211 +/- 25 cpm/10(5) cells in nonstretched groups; that in stretched groups is 582 +/- 94 cpm/10(5) cells. There is a significant difference between the two groups. These results suggest that stretch stimulation facilitates prostaglandin production in fetal membranes, which may contribute to uterine contraction in labor.     

光动力学治疗后的肿瘤细胞裂解物对卵巢上皮性癌大鼠的抗肿瘤作用

目的 探讨光动力学治疗(PDT)后的肿瘤细胞裂解物对卵巢上皮性癌(卵巢癌)大鼠的抗肿瘤作用.方法 选择6-8周龄Fischer344雌性大鼠,实验分为PDT组(腹腔注射PDT后的肿瘤细胞裂解物)、冻融组(腹腔注射冻融后的肿瘤细胞裂解物)、盐水组(腹腔注射生理盐水),间隔1周,3组均腹腔注射大鼠卵巢痛细胞系NuTu19细胞105个;设仅腹腔注射生理盐水者为空白对照组.采用酶联免疫斑点(ELISPOT)实验定量检测经PTD后的肿瘤细胞裂解物和冻融后的肿瘤细胞裂解物刺激后各组大鼠脾淋巴细胞中分泌肿瘤特异性γ干扰素(IFN-γ)的细胞数;乳酸脱氢酶(LDH)释放试验检测各组大鼠脾淋巴细胞中细胞毒T淋巴细胞(CTL)的杀伤活性;观察各组大鼠的生存时间以了解肿瘤细胞裂解物的抗肿瘤效果.结果 经PDT后的肿瘤细胞裂解物刺激后,PDT、冻融、盐水、空白对照组大鼠脾淋巴细胞中分泌肿瘤特异性IFN-γ的细胞数分别为(448.8+34.2)、(211.2±47.9)、(43.3±11.1)、(16.1±2.4)个,PDT组细胞数最多,分别与其他3组比较,差异均有统计学意义(P均<0.05);经冻融后的肿瘤细胞裂解物刺激后,上述4组大鼠脾淋巴细胞中分泌肿瘤特异性IFN-γ的细胞数分别为(151.7±22.6)、(188.7±53.0)、(18.2±12.2)、(8.8±7.7)个,PDT组与冻融组比较,差异无统计学意义(P>0.05).PDT组脾淋巴细胞中CTL的杀伤活性较其他3组明显增强(P<0.05).PDT、冻融、盐水组大鼠中位生存时间分别为234、171、168 d,空白对照组观察至截止时间全部存活,PDT组大鼠中位生存时间长于冻融和盐水组,差异有统计学意义(P<0.05).结论 PDT后的肿瘤细胞裂解物在大鼠体内诱发了特异性的抗肿瘤免疫反应,并可延长卵巢癌大鼠的生存时间,具有较好的抗肿瘤作用.     

血管生成在上皮性卵巢肿瘤中的意义

目的：探讨上皮性卵巢肿瘤内微血管密度及其与患者预后的意义。方法：对４３例卵巢上皮性肿瘤（交界性６例、恶性３７例）石蜡组织切片采用抗Ⅷ因子相关抗原抗体、免疫组化ＳＡＢＣ法染色,显微镜下寻找微血管密集区,通过计算机图象分析系统计数微血管（４０×视野）,以平均微血管数／４０×视野为肿瘤内微血管密度（ｉｎｔｒａｔｕｍ ｏｒｍ ｉｃｒｏｖｅｓｓｅｌｄｅｎｓｉｔｙ,ＩＭＤ）。结果：４３例中交界性瘤６ 例的平均ＩＭＤ为２４．３３±３．２７,上皮性癌３７ 例的平均ＩＭＤ为３５．２７±１７．２４,二者差异不显著（Ｐ＞ ０．０５）;ＩＭＤ随临床期别增加而增加,亦随细胞分化不良而增加;高ＩＭＤ组（≥３０）较低ＩＭＤ组（＜ ３０）死亡风险高２．４倍,复发风险高４．４ 倍,Ｋａｐｌａｎ Ｍｅｉｅｒ法描记两组生存率曲线差异亦有显著性（Ｐ＝ ０．０３）。结论：ＩＭＤ在上皮性卵巢肿瘤中具有预后意义,是预示患者生存和肿瘤复发有意义的参数。     

血小板反应素-1在卵巢上皮癌组织的表达及与p53肿瘤抑制蛋白和肿瘤血管形成的关系

目的：检测血小板反应素-1（TSP-1）和p53肿瘤抑制蛋白在卵巢上皮癌的表达，并探讨其表达与肿瘤血管形成及患者预后的关系。方法：用免疫组化法检测57例原发性卵巢上皮癌组织和22例正常卵巢组织中TSP-1和p53蛋白的表达，并用CD34抗体免疫染色后对微血管密度（MVD）计数。结合随诊资料，回顾分析上述指标与卵巢上皮癌临床病理特征及3年存活率之间的关系。结果：卵巢上皮癌组织中TSP-1的阳性表达率低于正常卵巢组织（分别为47．4％和72．7％，P=0．042），且多为弱阳性表达；卵巢上皮癌组织中p53的阳性率为61．4％，正常卵巢组织未检测到p53蛋白。TSP一1表达和p53蛋白阳性与卵巢上皮癌的手，术病理分期、大网膜转移和癌性腹水相关。卵巢上皮癌组织的MVD高于正常卵巢组织（分别30．3土8．5和20．1±8．1，P=0．014）。TSP-1表达与MVD呈负相关（P〈0．001），p53蛋白阳性与MVD呈正相关（P〈0．001）。TSP-1表达与患者的3年存活率呈正相关（P=0．001），p53蛋白阳性与3年存活率呈负相关（P=0．002）。TSP-1阴性而p53蛋白阳性组患者的3年存活率明显低于TSP-1阳性而p53蛋白阴性组（分别为13．6％和78．6％，P〈0．001）。结论：卵巢上皮癌组织TSP-1和p53蛋白的表达与肿瘤血管形成有关，对上述分子的检测有助于临床判断卵巢上皮癌的生物学行为。     

Fn14在上皮性卵巢癌细胞转移中的作用

目的:探讨成纤维细胞生长诱导因子14(Fn14)在上皮性卵巢癌转移中的作用。方法:选取人上皮性卵巢癌细胞株HO8910及其配对高转移能力细胞株HO8910-PM。Western blot法检测HO8910、HO8910-PM细胞株中Fn14表达,以及Fn14过表达质粒转染HO8910-PM细胞株后Fn14、Slug、MMP-9蛋白的表达情况。Transwell实验检测细胞株体外迁移和侵袭能力。结果:HO8910-PM细胞的迁移和侵袭细胞数均多于HO8910细胞(P0.05)。HO8910-PM细胞中Fn14表达水平明显低于HO8910细胞(P0.05)。Fn14过表达质粒转染HO8910-PM细胞后,细胞内Fn14表达水平上调(5.4±0.314)倍(P0.05),细胞的迁移和侵袭能力明显降低,同时细胞中侵袭转移相关蛋白Slug和MMP-9表达明显下降(P0.05)。结论:Fn14可能通过下调Slug和MMP-9表达进而抑制上皮性卵巢癌细胞的侵袭转移,Fn14可能为临床治疗上皮性卵巢癌提供新靶标。