HER-2/neu胞外区的基因工程表达及其免疫避孕作用

目的:研究HER-2/neu胞外区部分片段重组蛋白疫苗在免疫避孕中的作用。方法:构建原核表达重组体pET-hECDu,在大肠杆菌中表达人HER-2/neu胞外区部分片段,经Hi-Trap亲和层析柱纯化得到重组蛋白(hECDu),以ELISA法检测其结合活性后免疫雌性小鼠。用ELISA法检测抗HER-2/neu体液免疫应答,3H-TdR掺入法检测细胞免疫应答。结果:经原核表达系统成功纯化得到与理论分子量77 ku(1 u=1 Da)相符的hECDu,并证实其可与抗HER-2/neu胞外区特异性单克隆抗体结合。该重组蛋白疫苗可诱导雌鼠产生较高水平体液及细胞免疫应答。其生育力(平均产仔数4.0±1.8)与对照组(7.8±1.3)相比明显降低,但免疫鼠产后4 d子宫及卵巢均无明显病理改变。结论:经原核表达系统成功纯化得到HER-2/neu胞外区部分片段重组蛋白,该蛋白疫苗可在小鼠中诱导产生免疫避孕效应。     

HER2/neu蛋白过度表达与卵巢癌研究进展

HER2/neu蛋白是原癌基因HER2/neu编码的一种具有酪氨酸激酶活性的跨膜糖蛋白,在卵巢癌的发生、转移及恶性表型维持中起着重要作用.约15%～30%的卵巢癌存在HER2/neu蛋白过度表达.HER2/neu蛋白过度表达与卵巢癌患者较短存活期相关,可作为预后因素.HER2/neu蛋白表达高低与卵巢癌对化疗药物敏感性有关,对临床化疗药物的选择起指导作用.但以HER2/neu蛋白为靶点的抗卵巢癌治疗效果需进一步临床实验验证.     

卡培他滨联合卡铂治疗局部晚期宫颈癌5例的临床观察

宫颈癌的治疗以手术或放疗为主，但近年来随着化疗药物的不断发展，化疗在宫颈癌尤其是局部晚期宫颈癌中的地位显得越来越重要。卡培他滨（Capecitabine，希罗达）是一种新型口服氟尿嘧啶氨基甲酸酯，体内通过酶的活化在肿瘤组织中转化成5-Fu发挥抗肿瘤作用。由于卡培他滨转化过程中的关键酶-胸苷磷酸化酶在肿瘤组织中的活性大大高于正常组织，因此这种药物对肿瘤细胞的细胞毒作用具有较高的选择性。目前关于卡培他滨在宫颈癌辅助化疗中的研究，国内外的资料均不多。现将我科在2005年4月～8月采用卡培他滨联合卡铂作为辅助化疗方案治疗局部晚期宫颈癌5例患者的临床观察报道如下。     

HER-2/neu及细胞角蛋白在卵巢上皮性肿瘤原发灶和转移灶中表达的检测及临床意义的研究

目的：了解卵巢上皮性肿瘤原发灶和转移灶中癌基因ＨＥＲ－２／ｎｅｕ和细胞角蛋白表达状况及其临床意义。方法：用免疫组化法检测卵巢上皮肿瘤原发灶６９份及转移灶３８份（５１处）标本中ＨＥＲ－２／ｎｅｕ和细胞角蛋白的表达，并与术后组织学分级及患者生存期分析相结合。结果：原发灶中二者阳性表达率与术后患者生存期无相关性（Ｐ＞０．０５）。角蛋白与组织学分级有关（Ｐ＜０．０５），而且在转移灶癌细胞中表达较稳定。在转移灶中ＨＥＲ－２／ｎｅｕ的表达明显低于原发灶中者（Ｐ＜０．０５），在原发灶和转移灶中都呈阳性表达的患者预后较差（Ｐ＜０．０５）。结论：在卵巢上皮性肿瘤原发灶和转移灶中ＨＥＲ－２／ｎｅｕ的表达有显著差别，其原因有待进一步研究。原发灶和转移灶中ＨＥＲ－２／ｎｅｕ都表达的患者提示预后不良。角蛋白与卵巢上皮性肿瘤分化程度有关，在转移灶中表达较稳定     

吉西他滨联合奥沙利铂腹腔热灌注治疗复发性卵巢癌的效果分析

目的 探讨吉西他滨联合奥沙利铂腹腔热灌注治疗复发性卵巢癌的效果。方法 选择88例复发性卵巢癌患者为研究对象,按照随机数字表法分为对照组和研究组,每组44例。对照组采用吉西他滨联合奥沙利铂治疗,研究组采用吉西他滨联合奥沙利铂腹腔热灌注治疗,比较两组治疗效果、血管内皮生长因子(VEGF)、糖类抗原125(CA125)、人附睾蛋白4(HE₄)水平与不良反应发生情况。结果 研究组总有效率高于对照组(P<0.05);治疗前,两组VEGF、CA125及HE₄水平比较,差异无统计学意义(P>0.05);治疗后,研究组VEGF水平低于对照组,CA125、HE₄水平均高于对照组(P<0.05);研究组不良反应发生率低于对照组(P<0.05)。结论 吉西他滨联合奥沙利铂腹腔热灌注治疗复发性卵巢癌的效果较好,能有效控制患者的病情,降低不良反应发生率,对提高患者的生存质量有一定的意义,值得临床推广与应用。     

c-erbB-2与c-raf-1反义寡核苷酸联合转染对裸鼠人卵巢癌皮下移植瘤形成的抑制作用

目的 探讨c-erbB-2与c-raf-1反义寡核苷酸(antisense oligodeoxynucleotides,ASODN)联合转染,对人卵巢癌细胞株SKOV3,在裸鼠皮下移植瘤形成过程中的抑制作用。方法 共分7组：Ⅰ组(正常对照)、Ⅱ组[c-erbB-2正义寡核苷酸(SODN)]、Ⅲ组(c-raf-1 SODN)、Ⅳ组(c-erbB-2 ASODN)、V组(c-raf-1 ASODN)、Ⅵ组(全剂量联合)、Ⅶ组(半剂量联合)。根据不同分组条件将相应寡核苷酸导入SKOV3细胞株内,然后接种于裸鼠皮下,观察肿瘤出现的时间和肿瘤体积的变化,并计算抑瘤率。结果 Ⅱ、Ⅲ组与对照组成瘤能力比较,差异无显著性,Ⅳ、V组的成瘤能力明显降低,Ⅵ组与Ⅶ组的成瘤能力最低,Ⅵ组与Ⅶ第1次出现肉眼可见肿瘤的时间分别为13．7d和15．2d,最大抑瘤率分别为61．1％和71．3％。结论 反义寡核苷酸联合转染,对卵巢癌细胞的成瘤能力和肿瘤生长抑制作用更明显。     

Ad.Ela对SKOV3细胞的转染效率及体外杀伤作用

目的：研究腺病毒Ela质粒对HER-2/neu高表达卵巢癌细胞系SKOV3的转染效率和体外治疗作用。方法：脂质体介导腺病毒载体重组,腺病毒扩增、纯化、滴度测定,不同滴度病毒转染卵巢癌细胞的效率分析,以20：1的Ad.Ela抑制SKOV3细胞生长。结果：体外20：1 AdRSVlacZ感染SKOV3细胞1次,基因转染效率732%。此后增加病毒量,转染效率曲线接近平台。103*"SKOV3细胞分别以2×104 PFU的Ad.Ela+、Ad.Ela-和PBS感染1次,结果显示,Ad.Ela+治疗后卵巢癌细胞生长明显抑制,FACS检测转染后的SKOV3细胞,P185蛋白的表达明显下降。结论：Ad.Ela对HER-2/neu高表达细胞SKOV3的生长有显著抑制作用。     

紫杉醇加卡铂与吉西他滨为基础联合化疗序贯用药对降低上皮性卵巢癌复发的临床研究

目的 探讨紫杉醇加卡铂与吉西他滨为基础的联合化疗序贯用药对预防上皮性卵巢癌耐药的临床价值。方法 分析2005年4月至2011年6月山东省肿瘤医院经手术病理分期确定为Ⅲc期的按标准纳入的初治手术后上皮性卵巢癌患者的临床病理及随访资料,共121例,其中59例应用紫杉醇加卡铂化疗4个周期,然后调整为以吉西他滨为基础的联合化疗用药2～4个周期（序贯化疗组）,62例常规应用紫杉醇加卡铂持续化疗6~8个周期（常规化疗组）,观察两组患者的反应率、复发率、化疗毒副反应、无进展生存期（PFS）、复发后生存期和总生存率。结果　截至2011年12月31日,序贯化疗组中位随访时间为39个月（4~73个月）,常规化疗组为41个月（3~69个月）。序贯化疗组的完全缓解率（CR）、部分缓解率（PR）和总缓解率（CR+PR）分别为58.4%、24.3%和82.7%,常规化疗组为59.5%、27.1%和86.6%,组间比较,差异无统计学意义（P>0.05）。序贯化疗组中位无进展生存期28个月（6~62个月）,明显高于常规化疗组的18个月（4~57个月）,差异有统计学意义(P=0.037)。序贯化疗组复发率44.6%（25/56）,常规化疗组复发率55.2%（32/58）,两组比较差异无统计学意义 (P=0.322)。复发后生存期两组分别为24个月（8~38个月）及19个月（6~33个月）,组间比较差异无统计学意义（P=0.114）。序贯化疗组患者1年、3年无进展生存率分别为87.5%和35.7%,明显高于常规化疗组（72.4%和24.1%）,差异具有统计学意义（P=0.022）;序贯化疗组5年总生存率32.2%,显著高于常规化疗组的18.6%（P=0.014）。但5年无进展生存率及1年、3年总生存率两组间差异均无统计学意义（P>0.05）。血液系统化疗主要毒性反应两组均为骨髓抑制,两组发生率差异无统计学意义(P>0.05);非血液系统化疗毒性反应主要为神经系统毒性反应,序贯化疗组发生3～4级神经系统毒性发生率显著低于常规化疗组（3.3% vs. 14.5%,P=0.016）。结论　上皮性卵巢癌减瘤术后紫杉醇加卡铂用药4个周期后调整为吉西他滨为主的化疗方案,可提高患者的PFS、延缓复发、提高近期无进展生存率及远期总生存率,且毒性反应少,有临床推广价值。     

RNA干扰技术抑制卵巢癌细胞系SKOV3.ip1细胞内Her-2/neu基因表达的研究

目的 探讨卵巢癌细胞株SKOV3．ip1细胞转染表达短发卡状RNA（shRNA）的质粒后，SKOV3．ip1细胞中Her-2／neu基因表达的变化，和对SKOV3．ip1细胞凋亡的影响。方法 将针对Her-2／neu基因的shRNA表达载体转染SKOV3．ip1细胞，从mRNA水平、蛋白表达水平和细胞凋亡的变化三个方面评价shRNA表达载体对Her-2／neu基因表达的抑制作用。结果shRNA表达载体可以有效的抑制SKOV3．ip1细胞Her-2／neu基因的表达，使Her-2／neu基因的mRNA和蛋白表达量明显下降，细胞凋亡增多。结论靶向Her-2／neu的shRNA表达载体可以有效抑制Her-2／neu基因的表达。     

Smac基因转染对卵巢癌SKOV3/DDP裸鼠移植瘤顺铂敏感性及微血管生成的影响

目的:探讨转染Smac基因对卵巢癌SKOV3/DDP裸鼠移植瘤顺铂敏感性及微血管生成的影响。方法:建立卵巢癌SKOV3/DDP裸鼠移植瘤模型,随机分为5组,分别给予不同药物干预:(1)对照组:移植瘤内注射生理盐水;(2)DDP组:腹腔内注射顺铂;(3)空质粒+DDP组:移植瘤内注射空质粒,24h后腹腔内注射顺铂;(4)Smac组:移植瘤内注射Smac重组质粒;(5)Smac+DDP组:移植瘤内注射Smac重组质粒,24h后腹腔内注射顺铂。顺铂浓度为0.5mg/m1,给药剂量按3mg/kg计算。观察各组裸鼠移植瘤体积变化,计算抑瘤率。TUNEL法检测移植瘤组织标本的凋亡指数,免疫组化法检测CD34并计算微血管密度(MVD)。结果:Smac+DDP组的移植瘤体积最小,平均抑瘤率72.94%,与其他组比较差异有统计学意义(P0.05)。对照组、DDP组、空质粒+DDP组、Smac组、Smac+DDP组的凋亡指数分别为(19.90±3.18)%,(21.82±4.40)%,(22.64±3.81)%,(30.23±4.06)%,(48.59±4.47)%,MVD计数分别为(24.8±5.36),(21.2±3.11),(19.8±3.70),(14.4±3.85),(11.6±2.70)。其中,Smac+DDP组的凋亡指数最高,MVD最低,与其他组比较差异有统计学意义(P0.05)。结论:Smac基因转染可增强卵巢癌耐药移植瘤对顺铂的敏感性,同时减少微血管生成。     

Amplification of HER-2/neu oncogene in human ovarian cancer

Amplification and/or increased expression of the HER-2/neu oncogene has been reported to occur in ovarian tumors and possibly to correlate with biologic behavior and prognosis. The frequency with which amplification is reported to occur is quite variable ranging from 0–30% in different series and this variability is probably accounted for by technical and methodologic factors. The variability and lack of reproducibility has raised questions about the usefulness of assessing amplification of the HER-2/neu oncogene and in particular its clinical relevance. In this study by using strict criteria for amplification and using multiple controls we could demonstrate unequivocal amplification of the HER-2/neu oncogene by Southern blot analysis in only 11% of malignant ovarian tumors. The potential pitfalls with the techniques used to detect HER-2/neu oncogene amplification and overexpression are reviewed and possible ways to overcome some of the problems are suggested.     

Overexpression of HER-2/neu is not a risk factor in ovarian clear cell adenocarcinoma

OBJECTIVE: To evaluate the significance of the expression of HER-2/neu as a prognostic factor, we retrospectively examined its overexpression rates chiefly in ovarian clear cell adenocarcinoma (CCA) and their relationships with FIGO stage, lymph node metastasis, and prognosis. METHODS: In addition to 90 specimens of CCA (stage I, 52; stage II, 7; stage III, 28; stage IV, 3) obtained between 1987 and 2002, 19 specimens of serous adenocarcinoma (S), 19 specimens of mucinous adenocarcinoma (M), and 19 specimens of endometrioid adenocarcinoma (E) collected at initial surgery were studied. The expression of HER-2/neu was immunohistologically scored on a scale of 0 to 3+ according to the HercepTest scoring system, and 2+ and 3+ were regarded as overexpression. The relationship between HER-2/neu overexpression and outcome was evaluated by the Kaplan-Meier method and the log-rank test. The relationship with advanced-stage cancer was analyzed by Fisher's exact test. The relationships with lymph node metastasis and histologic types were compared by the t test. RESULTS: The rate of HER-2/neu overexpression in CCA was 45.6% (0, 1+, 2+, and 3+ in 14, 35, 36, and 5 cases, respectively), and no differences in the overexpression rate were noted among histologic types: 52.7% in S, 42.1% in M, and 26.4% in E. In CCA, no association was observed between HER-2/neu overexpression and cumulative survival rate (P = 0.5708), FIGO stage (P = 0.5147), or lymph node metastasis (P = 0.3624). CONCLUSION: The absence in CCA of an association between HER-2/neu overexpression and outcome, stage, or lymph node metastasis indicates that HER-2/neu overexpression is not a prognostic risk factor.     

Expression of HER-2/neu oncoprotein, DNA-ploidy and S-phase fraction in advanced ovarian cancer

The immunohistochemical expression of HER-2/neu and cytofluorimetric data were retrospectively analyzed in a group of primary advanced ovarian cancers. Thirty-three out of 94 (35%) cases showed a specific p185/neu immunoreaction. No correlation between p185/neu expression and any of the clinico-pathologic parameters examined was observed. As far as cytofluorimetric data are concerned, 38 out of 69 (55%) of the tumors were diploid (DNA index = 1) while 31 (45%) were aneuploid (DNA index from 1.10 to 2.50 with a median value of 1.50). Ovarian tumors were defined as of low and high S-phase fraction in 68% and 32% of the cases, respectively. Tumor ploidy and S-phase fraction did not correlate with the clinico-pathologic characteristics or p185/neu oncoprotein expression. Aneuploid tumors had a higher S-phase fraction (mean: 15.81 ± 13.44) than diploid tumors (mean: 8.89 ± 7.98) ( P < 0.01). p185/neu expression failed to affect significantly both overall and progression free survival. On the other hand tumor ploidy was found to be related to the prognosis of advanced ovarian cancer patients although the difference was not statistically significant. As far as progression free survival is concerned, the median time to recurrence was not reached for diploid cases whereas it was 21 months for aneuploid cases ( P < 0.05). The 5-year survival for patients with a low S-phase fraction (58%) was significantly higher than for patients with high S-phase fraction tumors (28%) ( P < 0.01). Median time to recurrence was 48 and 17 months for low and high S-phase fraction tumor patients, respectively ( P < 0.05). However, in a multivariate analysis both tumor ploidy and S-phase fraction did not retain their prognostic value. The assessment of the role of the parameters examined in improving the prognostic characterization of ovarian cancer patients should be investigated in large multicenter clinical trials.     

卵巢子宫内膜样癌HER－2／neu的表达

目的研究卵巢子宫内膜样癌ＨＥＲ－２／ｎｅｕ的表达以及与临床病理特征和预后的关系。方法应用ＨＥＲ－２／ｎｅｕ单克隆抗体免疫组化技术对卵巢子宫内膜样癌２８例进行ＨＥＲ－２／ｎｅｕ癌基因蛋白测定。结果１３例细胞浆染色阳性，占４６．４％，Ⅰ期肿瘤阳性率为２０．０％，Ⅱ～Ⅳ期肿瘤阳性率为５２．２％，手术有残留肿瘤者ＨＥＲ－２／ｎｅｕ的表达率是６２．５％，无残留肿瘤者ＨＥＲ－２／ｎｅｕ表达率是２５．０％，差异有显著性（Ｐ＝０．０４９）。ＨＥＲ－２／ｎｅｕ表达与发病年龄、ＣＡ１２５水平、组织学分级及淋巴转移无关。术后随诊６～９６个月，平均３１．３个月，ＨＥＲ－２／ｎｅｕ阳性者术后死亡率是３８．５％，ＨＥＲ－２／ｎｅｕ阴性者术后死亡率是２１．４％。结论ＨＥＲ－２／ｎｅｕ阳性者与卵巢子宫内膜样癌的不良预后有一定的关系。     

卵巢子宫内膜样癌HER－2／neu的表达

目的研究卵巢子宫内膜样癌ＨＥＲ－２／ｎｅｕ的表达以及与临床病理特征和预后的关系。方法应用ＨＥＲ－２／ｎｅｕ单克隆抗体免疫组化技术对卵巢子宫内膜样癌２８例进行ＨＥＲ－２／ｎｅｕ癌基因蛋白测定。结果１３例细胞浆染色阳性，占４６．４％，Ⅰ期肿瘤阳性率为２０．０％，Ⅱ～Ⅳ期肿瘤阳性率为５２．２％，手术有残留肿瘤者ＨＥＲ－２／ｎｅｕ的表达率是６２．５％，无残留肿瘤者ＨＥＲ－２／ｎｅｕ表达率是２５．０％，差异有显著性（Ｐ＝０．０４９）。ＨＥＲ－２／ｎｅｕ表达与发病年龄、ＣＡ１２５水平、组织学分级及淋巴转移无关。术后随诊６～９６个月，平均３１．３个月，ＨＥＲ－２／ｎｅｕ阳性者术后死亡率是３８．５％，ＨＥＲ－２／ｎｅｕ阴性者术后死亡率是２１．４％。结论ＨＥＲ－２／ｎｅｕ阳性者与卵巢子宫内膜样癌的不良预后有一定的关系。     

Effect of HER-2/neu overexpression on chemoresistance and prognosis in ovarian carcinoma

AIM: To investigate the effect of HER-2/neu protein overexpression on chemoresistance and prognosis in patients with epithelial ovarian carcinoma. METHODS: A total of 141 ovarian carcinoma tissues surgically resected between 1987 and 2003 were assessed by immunohistochemistry (IHC). The characteristic of the patients and immunohistochemical results were compared by chi2-test. Survival analysis was performed by the Kaplan-Meier method and the log-rank test. RESULTS: HER-2/neu overexpression was detected in 18 cases (12.8%). There were no significant differences in histopathological subtypes (P = 0.3550), FIGO stages (P = 0.8858), or residual tumor size at first surgery (P = 0.6607) between the cases with HER-2/neu overexpression and the cases without HER-2/neu overexpression. Among the 58 cases which responded to chemotherapy, only five cases (8.6%) showed HER-2/neu overexpression. However, among the 38 cases which did not respond to chemotherapy, eight cases (21.1%) showed HER-2/neu overexpression. Overexpression of HER-2/neu had a tendency to relate with chemoresistance of epithelial ovarian carcinoma, but there were no statistically significant differences (P = 0.0817). No association was observed between HER-2/neu overexpression and cumulative survival rate (P = 0.4970). CONCLUSIONS: The results of the current study show that although HER-2/neu overexpression has a tendency to be associated with chemoresistance, it can not be a prognostic factor for the patients with epithelial ovarian carcinoma.     

HER-2/neu overexpression in uterine papillary serous cancers and its possible therapeutic implications

Uterine papillary serous carcinoma (UPSC) is a highly aggressive variant of endometrial cancer with features similar to high-grade ovarian cancer. Patients tend to be elderly, thin, have a high grade tumor with extensive extrauterine disease at the time of diagnosis. The transmembrane receptor encoded by the HER-2 cellular oncogene is amplified in several types of human carcinomas and provides an attractive therapeutic target. HER-2/neu, the transmembrane receptor encoded by the c-erbB2 gene, is overexpressed by immunohistochemistry in <25% of ovarian cancers and 20-30% of breast cancers, and <10% of endometrial cancer. There are prognostic and therapeutic implications associated with the overexpression of this transmembrane protein. Herceptin, a humanized murine monoclonal antibody directed against the extracellular domain of the HER-2/neu protein, is being used to treat breast cancer that overexpresses HER-2/neu. We reviewed all patients diagnosed with UPSC between 1999-2001. Twenty-six patients were identified, and 19 patients had specimens available for evaluation. We performed immunohistochemical analysis (Herceptest, Dako, Carpinteria, CA) on 19 paraffin embedded blocks of UPSC tumors looking for HER-2/neu over expression. Five out of 19 (26%) stained heavily (3+) for HER-2/neu receptor protein. Four of these five patients had advanced disease at diagnosis. Two of these patients were subsequently treated with Herceptin; one with complete response and one with stable disease based on CT scan and CA-125 findings. Targeting HER-2/neu may be beneficial for a select group of patients with UPSC. We are continuing to evaluate samples for HER-2/neu over expression by fluorescence in situ hybridization (FISH).