p27蛋白在子宫内膜癌组织中的表达及其临床意义

目的 :探讨p2 7在子宫内膜癌组织中的表达及其临床意义。方法 :采用免疫组化S -P法测定 16份正常子宫内膜、18份子宫内膜不典型增生及 50份子宫内膜癌组织中的p2 7蛋白表达。结果 :p2 7蛋白在子宫内膜癌、子宫内膜不典型增生及正常子宫内膜中的表达率分别为 34%、6 6 .6 7%和 93.75% ,正常子宫内膜及不典型增生组均显著高于子宫内膜癌组 ,差异有显著性 (P <0 .0 5)。p2 7蛋白表达与子宫内膜癌组织学分级、肌层浸润程度及患者预后显著相关 (P <0 .0 5) ,但与临床分期无关。结论 :p2 7蛋白表达下降或缺失可能在子宫内膜癌的发生、发展中起重要作用 ,并可能提示患者的预后     

Skp2和p27在宫颈癌组织的表达及临床意义

目的:探讨Skp2、p27在宫颈癌的表达、两者的相关性,以及与临床病理因素和预后的关系。方法:用免疫组化SP法检测65例宫颈鳞状细胞癌(SCC),20例高级别宫颈上皮内瘤变(CIN)中Skp2、p27的表达,以60例正常宫颈组织(NCT)为对照。结果:(1)Skp2在SCC组的阳性表达率为55.4%,明显高于高级别CIN组的25.0%(P0.05)及NCT组的6.7%(P0.01);(2)p27在SCC组的阳性表达率为52.3%,明显低于高级别CIN组的85.0%(P0.01)及NCT组的98.3%(P0.01);(3)半定量分析表明SCC中Skp2、p27表达与年龄、肿瘤大小、临床分期无明显关系(P0.05),而与肿瘤的病理分级、浸润深度、脉管浸润、淋巴结转移密切相关(P0.05);(4)宫颈癌中Skp2阳性表达组的5年生存率(52.78%)明显低于Skp2阴性组(68.93%),Kaplan-Meier生存率分析,Log-rank=8.60,P=0.0034。而p27阳性表达组的5年生存率(70.59%)明显高于阴性表达组(58.38%),Log-rank=8.33,P=0.0039;经Spearman等级相关分析表明,宫颈癌中Skp2和p27的表达呈显著负相关,相关系数r=-0.311,P=0.015。结论:Skp2表达增强与宫颈癌的发生、发展密切相关,其表达水平越高提示患者预后越差;p27的表达正相反,并且两者的表达呈负相关。联合检测Skp2和p27的表达对判断宫颈癌的恶性潜能和预后具有重要的参考价值;两者可作为宫颈癌的预后指标,为宫颈癌的治疗提供新的靶点。     

黄芪注射液联合顺铂辅助免疫化疗对子宫内膜癌细胞株HEC-1-B抑制作用的研究

子宫内膜癌化疗的目的是术后清除微小及无法切除的病灶,以及用于术后复发或晚期失去手术机会的患者,以期延长生存期,获取冉治疗的机会.化疗的毒副反应和多药耐药(multidrug resistance,MDR)是影响化疗疗效的主要原因[1].     

p16基因与细胞周期素D1在子宫内膜癌中的表达及其临床意义

目的　研究p16和细胞周期素D1在子宫内膜癌中的表达及其临床意义。方法　采用免疫组化LSAB法检测 42例子宫内膜癌中 p16、细胞周期素D1的表达。 结果　 42例子宫内膜癌中 2 0例 p16表达阳性 ,占47 6 % ,p16与子宫内膜癌的细胞分级、临床分期、肌层浸润深度有关 (P <0 0 5 ) ;17例子宫内膜癌细胞周期素D1表达阳性 ,占 40 5 % ,细胞周期素D1与子宫内膜癌的细胞分级、临床分期、淋巴结转移有关 (P <0 0 5 ) ;p16、细胞周期素D1协同表达 15例 ,均为晚期或低分化癌。结论　p16、细胞周期素D1作为细胞周期调节因子参与子宫内膜癌的发生、发展 ,其协同作用促进子宫内膜癌的发展、且预后不良     

子宫内膜癌顺铂耐药细胞株的建立和鉴定及耐药机制研究

目的 建立子宫内膜癌顺铂耐药细胞株ISH/DDP,鉴定其生物学特性,并探讨其顺铂耐药的机制.方法 采用顺铂浓度逐步递增联合大剂量顺铂冲击方法,在体外连续诱导、培养子宫内膜癌细胞株Ishikawa细胞,建立子宫内膜癌顺铂耐药细胞株ISH/DDP.非放射性细胞增殖法(MTS法)测定ISH/DDP细胞对顺铂的耐药指数;绘制细胞生长曲线,计算细胞群体倍增时间;流式细胞术检测ISH/DDP细胞的细胞周期比例;免疫细胞化学法检测ISH/DDP细胞中耐药蛋白--乳腺癌耐药蛋白(BCRP)、P糖蛋白(P-gp)以及谷胱甘肽S转移酶π(GST-π)蛋白的表达水平,以综合积分表示.结果 Ishikawa细胞在体外采用顺铂浓度逐步递增联合大剂量顺铂冲击方法 连续诱导、培养,历时10个月,成功建立了子宫内膜癌顺铂耐药细胞株ISH/DDP,其耐药指数为3.77.Ishikawa细胞和ISH/DDP细胞的群体倍增时间分别为34.1 h和40.4 b,两者比较,差异有统计学意义(P<0.05).与Ishikawa细胞相比,ISH/DDP细胞的S期细胞比例[分别为(44.2±6.1)%和(55.3±8.4)%]增加,G2/M期细胞比例[分别为(11.9±0.7)%和(5.7±2.4)%]减少,差异均有统计学意义(P<0.05);G0/G1期细胞比例[分别为(44.3±5.7)%和(39.0±10.7)%]减少,但差异无统计学意义(P>0.05).ISH/DDP细胞中BCRP蛋白的表达水平为(16.3±2.0)分,高于Ishikawa细胞的(13.4±1.5)分,差异有统计学意义(P<0.05);而Ishikawa和ISH/DDP细胞中P-gp蛋白的表达水平分别为(16.1±1.0)和(15.5±1.2)分,GST-π蛋白的表达水平分别为(14.9±1.1)和(15.2±1.9)分,分别比较,差异均无统计学意义(P>0.05).结论 ISH/DDP细胞具有耐药表型及耐药细胞的生物学特性;其耐药机制可能与细胞中BCRP蛋白的过度表达相关.

Abstract：

Objective To establish the cisplatin(DDP)-resistant cell line from human endometrial cancer cell line Ishikawa and to investigate its resistant mechanism to DDP. Methods A resistant endometrial cancer cell line ISH/DDP was established by gradually increasing dose of cisplatin and high-dose stimulation. The resistant index was estimated by 3-(4,5-dimethylthiazol-zyl)-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl)-2H-tetrazolium,inner salt (MTS) assay. Cell growth curve, doubling time and cell cycle phase distribution were measured; drug-resistant protein of breast cancer resistance protein (BCRP),P-glycoprotein (P-gp) and glutathione-S-transferase-π (GST-π) were examined by immuocytochemistry. Results The DDP-resistant cell line ISH/DDP was established with the resistant index of 3. 77. The proliferation of ISH/DDP got slow, doubling time prolonged, which were 40. 1 hours, while it was 34. 1 hours in Ishikawa (P<0.05); and the cell number of G0/G1 phase [(44. 3 ±5. 7)% and (39. 0 ± 10. 7)% ,P >0. 05] and G2/M phase [(11.9 ±0.7)% and (5. 7 ±2. 4)% ,P <0. 05] decreased, while S-phase [(44. 2 ±6.1)% and (55. 3 ± 8. 4) % , P < 0. 05] increased compared with parent cells. The comprehensive score of the expression of BCRP in ISH/DDP was 16. 3 ± 2. 0, while it was 13.4 ± 1. 5 in Ishikawa (P < 0. 05). The score of the expression of P-gp in ISH/DDP and Ishikawa were 15. 5 ± 1. 2 and 16. 1 ± 1. 0 (P > 0. 05) ,respectively. The score of the expression of GST-π in ISH/DDP and Ishikawa were 15. 2 ± 1. 9 and 14. 9 ± 1.1 (P > 0. 05) . Conclusion ISH/DDP cell line showed a typical resistant phenotype and biological characteristics, which may be accounted for high BCRP expression.     

p16基因与细胞周期素D1在子宫内膜癌中的表达及？…

研究ｐ１６和细胞周期素Ｄ１在子宫内膜癌中的表达及其临床意义。方法 采用免疫组化ＬＳＡＢ法检测４２例子宫内膜癌中ｐ１６、细胞周期素Ｄ１的表达。结果 ４２例子宫内膜癌中２０例ｐ１６表达阳性,占４７．６％,ｐ１６与子宫内膜癌的细胞分级、临床分期、肌层浸润深度有关（ｐ〈０．０５）;１７例子宫内膜癌细胞周期素Ｄ１表达阳性,占４０．５％,细胞周期素Ｄ１与子宫内膜癌的细胞分级、临床分期、淋巴结转移有关（ｐ〈０．     

Skp2基因沉默对子宫内膜癌HEC-1A细胞p27蛋白表达及细胞增殖的影响

RNA干扰(RNAi)是一项新的使基因表达阻断的技术,一种广泛使体外基因失活或沉默的方法,是在转录水平抑制基因表达的一种研究基因功能的有力工具,不仪可用于研究基因功能,同时也为基因治疗提供了一个新的重要技术.     

顺铂对人子宫内膜癌RL-952细胞系Matriptase,uPA-mRNA表达的影响

目的探讨顺铂对人子宫内膜癌RL-952细胞系Matriptase,uPA-mRNA表达的影响。方法体外培养人子宫内膜癌RL-952细胞,采用反转录聚合酶链反应(RT-PCR)检测顺铂对人子宫内膜癌RL-952细胞Matriptase,uPA-mRNA表达的影响。结果 Matriptase,uPA-mRNA在人子宫内膜癌RL-952细胞系呈阳性表达。顺铂可下调Matriptase,uPA-mRNA在人子宫内膜癌RL-952细胞系的表达,具有浓度依赖性。8mg/L顺铂溶液作用不同时间后,4h后RL-952细胞Matriptase-mRNA表达量即明显下降(P0.05),而uPA-mRNA表达量于作用8h后才开始下降(P0.05),具有时间依赖性。结论顺铂可抑制人子宫内膜癌RL-952细胞系Matriptase,uPA-mRNA的表达,并具有浓度依赖性。8 mg/L的顺铂作用于人子宫内膜癌RL-952细胞后,Matriptase-mRNA比uPA-mRNA更早出现表达下调的变化。     

子宫内膜癌Dickkopf1表达及对内膜癌侵袭力的影响

目的:研究Dickkopf1在子宫内膜癌组织和Ishikawa细胞系的表达及对子宫内膜癌侵袭力的影响。方法:应用免疫组化和免疫荧光法检测子宫内膜癌组织和Ish-ikawa细胞系中Dickkopf1的表达定位;向子宫内膜癌Ishikawa细胞系施加外源性Dickko-pf1,应用Transwellchamber法进行体外侵袭试验,检测它对内膜癌侵袭能力的影响。结果:Dickkopf1在子宫内膜癌腺上皮和基质组织中均有表达,腺上皮的表达高于基质;Dickkopf1主要表达于内膜癌细胞浆和细胞膜中;施加外源性Dickkopf1后,子宫内膜癌细胞的侵袭能力下降。结论:Dickkopf1能减弱子宫内膜癌的侵袭能力,它有望作为内膜癌的治疗靶点。     

P27，P73和CyelinD1蛋白在子宫内膜癌中表达的相关性

目的：探讨P27,P73和CyclinD1在子宫内膜癌组织中的表达及其相关性。方法：采用免疫组化SP法对14例正常增殖期子宫内膜、19例不典型增生内膜以及43例子宫内膜癌组织中P27,P73和CyciinD1蛋白的表达进行研究。结果：在子宫内膜癌中。P27表达降低或缺失,P73和CycJinD1表达明显增高,与正常增殖期子宫内膜和不典型增生内膜相比差异有统计学意义（P〈0．05或P〈0．01）。P27的表达缺失、P73的过表达与组织学分级有关,CyclinD1的高表达与手术一病理分期有关。有肌层浸润者P27,P73和CyclinD1的表达与无肌层浸润者差异均有统计学意义（均P〈0．05）。有淋巴结转移者P73和CyclinD1均明显高表达。子宫内膜癌中,P27与CyclinD1呈负相关（R=-0．3627,P〈0．05）,P73与CyclinD1呈正相关（r=0．3923,P〈0．05）。结论：子宫内膜癌中P27表达缺失,P73和CyclinD1过度表达,联合检测可能成为子宫内膜癌高危人群早期筛查、判断内膜癌的生物学行为及评估预后的参考指标。     

骨化三醇对人宫颈癌SiHa细胞维生素D受体及S期激酶相关蛋白的影响

目的：检测骨化三醇对人宫颈癌SiHa细胞维生素D受体（VDR）及S期激酶相关蛋白2（Skp2）表达的影响,探讨其抗癌机制。方法：选用人宫颈癌SiHa细胞进行体外培养。CCK-8法测定骨化三醇对细胞生长抑制率的影响;流式细胞仪进行细胞周期分析;Western blot法检测VDR及Skp2表达的变化。结果：（5×10^-8～10^-6）mol／L骨化三醇作用于SiHa细胞1～4天,细胞生长增殖均受到显著抑制;且随着作用浓度增加,细胞增殖能力逐渐下降。10^-7mol／L骨化三醇作用于SiHa细胞后,G0／G1期细胞比例增加,S、G2／M期细胞比例降低,细胞增殖指数（PI）降低（P〈0.05）;VDR蛋白表达增加（P〈0.05）,Skp2蛋白表达显著减少（P〈0.01）。结论：骨化三醇对人宫颈癌SiHa细胞的增殖具有显著的抑制作用,其机制可能是通过上调VDR的表达,参与Skp2基因转录的调控,降低Skp2表达。     

Curcumin down-regulates Ets-1 and Bcl-2 expression in human endometrial carcinoma HEC-1-A cells

OBJECTIVE: Curcumin has been demonstrated to have an anti-tumor activity but the underlying molecular mechanisms are not fully uncovered. The present study was undertaken to determine the effect of curcumin on the expression of the proto-oncogene Ets-1 and the anti-apoptotic molecule Bcl-2 in human endometrial adenocarcinoma HEC-1-A cells. METHODS: Confluent HEC-1-A cells were treated with curcumin at various doses for 16 h or at 60 microM for various time points. At the end of the designated treatments, changes in cell morphology, DNA fragmentation and protein contents of Ets-1 and Bcl-2 were determined, respectively, by light microscopy, DNA laddering assay and Western blot analysis. As an initial step towards understanding whether Ets-1 was a possible up-stream regulator of Bcl-2 expression in HEC-1-A cells and if so, whether curcumin could attenuate the Ets-1-induced up-regulation of Bcl-2 expression, cells were transiently transfected with an Ets-1/GFP (Green Fluorescence Protein) fusion construct and the transfectants were treated with 60 microM curcumin for 16 h, followed by whole cell lysate preparation for Western blot analysis of Bcl-2 protein contents. RESULTS: Curcumin induced apoptosis-like morphological changes and DNA degradation and decreased basal levels of Ets-1 and Bcl-2 protein contents in HEC-1-A cells in a time- and dose-dependent manner. Overexpression of Ets-1 in the cell resulted in an increase in Bcl-2 protein contents and that increase was attenuated by curcumin treatment. CONCLUSIONS: Curcumin down-regulates Ets-1 and Bcl-2 expression and induces apoptosis in HEC-1-A cells, suggesting a novel molecular mechanism for the anti-tumor activity of curcumin.     

p27、cdk4在子宫内膜癌中的表达

目的 :探讨子宫内膜癌中p2 7、cdk4蛋白的表达及临床意义。方法 :采用免疫组化S P法检测 42例子宫内膜癌子宫内膜中p2 7及cdk4蛋白的表达。结果 :(1 )子宫内膜癌p2 7蛋白核表达率为 1 6 .7% ,明显低于对照组 (正常子宫内膜及增生子宫内膜为51 .4% ) ,cdk4的核表达率为 76.2 % ,明显高于对照组的 2 9.7% ;(2 )p2 7或cdk4蛋白核表达均与子宫内膜癌的组织学分级有关 ,分化差的细胞p2 7核表达率低 ,cdk4核表达率则高 ;(3)p2 7核阴性表达合并cdk4核阳性表达与组织学分级及手术病理分期相关 ,分化差及手术病理分期晚的病例p2 7核阴性表达合并cdk4核阳性表达的出现率明显增高 ;(4)p2 7与cdk4蛋白表达之间无相关性 ;(5)内膜癌中p2 7蛋白胞浆表达率明显增高 ,cdk4蛋白胞浆表达则有降低趋势。结论 :p2 7蛋白核表达缺失及cdk4蛋白核表达增高与内膜癌的发生发展相关 ,联合检测p2 7、cdk4蛋白有助于估计预后、指导治疗     

Establishment and characterization of endometrial clear cell carcinoma cell line (TMCC-2)

A new cell line, designated TMCC-2, has been established from operation material from a woman with endometrial clear cell carcinoma. TMCC-2 was successively subcultured 40 times in about 1 year. The monolayer culture cell showed a pavement-like arrangement of polygonal and short spindle-shaped cells, and had a tendency to pile-up without contact inhibition. Since PAS positive and Alcian-Blue negative substance could be seen in the cytoplasm, the cells were found to produce glycogen. The population doubling time, the saturation density and plating efficiency of the 25th passage cells were 24 hours, 1.8 x 10(5) and 23%, respectively. The nuclear DNA histogram obtained by flow cytometry showed two peaks at 2.1C and 4.1C. Therefore, the DNA index was 1.05. A tumor maker assay of the culture medium revealed significantly high values for TPA, CA125, CA19-9, and SLX compared with the control medium. The TMCC-2 cells produced the tumors in nude mice after subcutaneous transplantation. In addition, the histological findings were similar to those in the original tumor. As mentioned above, the TMCC-2 cell line derived from endometrial clear cell carcinoma will be very valuable in basic research on clear cell carcinoma of the endometrium.     

Effect of 1,25(OH)2 vitamin D3 on cytokine production by endometrial cells of women with repeated implantation failure

Repeated implantation failure (RIF) is a worldwide health problem that imposes a great deal of cost on patients and health care system. Vitamin D₃ has been proposed to have positive impact on the process of implantation. The present study was performed to compare the effect of 1,25-dihydroxy vitamin D₃ (1,25(OH)₂D₃) on cytokine production by endometrial cells of women with RIF and healthy fertile controls. Whole endometrial cells (WECs) and endometrial stromal cells (ESCs) from RIF and normal fertile women were treated with 1,25(OH)₂D₃. The levels of IL-10, TGF-β, IFNγ, Il-6, IL-8 and IL-17 in culture supernatants were assayed by ELISA. Also, ability of the cells from both groups to produce 1,25(OH)₂D₃ was evaluated and compared. 1,25(OH)₂D₃ down-regulated cytokine production in WECs from both groups except for IL-8 which was upraised. Similar trends were also observed in ESCs except up-regulation of TGF-β in RIF group. Endometrial cells of both groups had comparable capacity to produce 1,25(OH)₂D₃. Based on the minimal differential immunoregulatory effect of vitamin D₃ on endometrial cells from RIF and control women, it may be suggested that circulating levels of maternal vitamin D₃ be the subject of further investigation.     

microRNA-142-3p对TET2基因的调控及其对卵巢癌细胞SKOV3增殖的影响

目的:研究miR-142-3p慢病毒载体对TET2的调控作用及其对SKOV3细胞增殖作用的影响。方法:构建pSicoR-miR-142-3p及pMIR-Report-TET2过表达载体,包装重组慢病毒,应用实时荧光定量PCR、Western blot及双荧光素酶报告基因系统检测方法验证miR-142-3p与TET2的靶向作用关系,应用MTT法检测miR-142-3p慢病毒颗粒对SKOV3细胞增殖的影响。结果:成功构建了pSicoR-miR-142-3p和pMIR-Report-TET2重组质粒,以及可携带miR-142-3p的慢病毒颗粒。miR-142-3p过表达明显抑制TET2蛋白及mRNA表达水平(P0.05);抑制miR-142-3p表达则明显上调TET2蛋白及mRNA表达水平(P0.05);MTT试验证实,miR-142-3p可显著抑制SKOV3细胞增殖。结论:过表达miR-142-3p可有效抑制SKOV3细胞中TET2 mRNA和蛋白水平表达,并抑制SKOV3细胞增殖。     

Characterization of 1,25-dihydroxyvitamin D3 receptors and in vivo targeting of [3H]-1,25(OH)2D3 in the sheep placenta

We sought to detect the presence of receptors for 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in placental tissues of five late gestational pregnant sheep and to quantitate their biochemical properties and abundance. Cytosol prepared from cotyledonary tissue was found to contain two [3H]-1,25(OH)2D3 binding macromolecules that sedimented at 3.2 S and 4.1 S, respectively, on linear (4-20 per cent) hypertonic sucrose gradients. The 4.1 S component cosedimented with serum that had been prelabelled with [3H]-25-hydroxyvitamin D3 (25-OHD3) and was present in cytosols despite extensive washing of the tissue prior to homogenization. Concurrent incubation of the cytosol with [3H]-1,25(OH)2D3 and a tenfold molar excess of radioinert 25-OHD3 resulted in complete resolution of the 3.2 S macromolecule and disappearance of the 4.1 S binding component. The binding of [3H]-1,25(OH)2D3 to the 3.2 S component was completely abolished by coincubation with a 100-fold molar excess of radioinert 1,25(OH)2D3 and was replaced by a well resolved peak in the 4.1 S region. Scatchard analysis of cytosol binding to [3H]-1,25(OH)2D3 in the presence of a tenfold molar excess of radioinert 25OHD3 revealed a single class of non-interacting saturable binding site in the cotyledon and the endometrium of high affinity and low capacity. The mean +/- s.e. of the dissociation constant of the cotyledonary receptor of 0.21 +/- 0.06 nM was not different from that of 0.16 +/- 0.03 nM for the endometrial receptor. However, the abundance of the cotyledonary receptor was fourfold higher than that in the endometrium (110 +/- 20 versus 28 +/- 7 fmol/mg protein). Since it is not possible to completely separate endometrial tissue from cotyledonary tissue, the low abundance of receptor in endometrial cytosols may merely represent contamination of endometrial tissue with cotyledonary tissue. Further analysis of the [3H]-1,25(OH)2D3 occupied receptor in cotyledonary cytosols showed that it bound to DNA cellulose and was eluted with 0.16 M KCl. This in vitro binding of [3H]-1,25(OH)2D3 to DNA was confirmed in vivo by the finding of preferential nuclear targetting of [3H]-1,25(OH)2D3 (56 per cent of total cellular activity), 4 h after fetal intravenous administration of [3H]-1,25(OH)2D3 to five chronically catheterized fetal sheep. Total placental uptake of [3H]-1,25(OH)2D3 at this time amounted to 3.7 +/- 0.9 per cent of the injected dose. Preliminary analysis of ovine placental cytosols revealed a calcium binding protein of similar molecular weight to that found in the ovine intestine and in the intestine and placenta of rodents.(ABSTRACT TRUNCATED AT 400 WORDS)     

蛋白酪氨酸磷酸酶1B对子宫内膜癌细胞增殖和凋亡的影响

目的:探讨蛋白质酪氨酸磷酸酶1B(protein tyrosine phosphatase 1B,PTP1B)对子宫内膜癌细胞增殖和凋亡的影响。方法:体外培养人子宫内膜癌细胞株(Ishikawa),并将其分为4组,即空白对照组(A组),凋亡组[B组,十二烷基硫酸钠(SDS)],空病毒载体凋亡组(C组,SDS+Lacz)和过表达凋亡组(D组,SDS+PTP1B)。利用Western blotting检测PTP1B和Bcl-2表达;四甲基偶氮唑蓝(MTT)检测细胞活力,吖淀橙/溴化乙啶(AO/EB)双染色观察过表达PTP1B对Ishikawa形态学的影响。结果:Western blotting显示Ishikawa无PTP1B表达,D组Ishikawa高表达PTP1B。A组、B组、C组及D组的Ishikawa均有Bcl-2的表达,且D组比B组高37.04%。MMT结果显示D组比B组的细胞存活率提高30.66%。AO/EB结果显示D组仅少量细胞有橙红色荧光,细胞形态较规则。结论:过表达PTP1B能在一定程度上促进子宫内膜癌Ishikawa细胞增殖并抑制其凋亡。