表达精子抗原受精素β的重组沙门氏菌疫苗株的免疫性避孕效应研究

目的 :探讨表达精子抗原受精素β胞外区的重组沙门氏菌疫苗株用作避孕疫苗的可行性。方法 :应用表达精子抗原受精素β胞外区的两种重组沙门氏菌疫苗株 X46 3 2 (p FEC)和 X45 5 0(p FEC)口服接种雌鼠 ,通过 ELISA方法检测其激发雌鼠产生抗重组受精素 β蛋白的血清 Ig G抗体和阴道分泌物 Ig A抗体滴度变化 ,并观察其诱导的雌鼠免疫性避孕效应情况。结果 :重组 X46 3 2(p FEC)和 X45 5 0 (p FEC)疫苗株不但可激发雌鼠产生高水平血清抗重组受精素 β蛋白和抗菌体LPS Ig G抗体 ,而且在阴道分泌物中可出现一定程度抗重组受精素 β蛋白和抗菌体 LPS Ig A抗体。同时 ,口服免疫后的雌鼠生育能力明显降低 ,两实验组雌鼠妊娠率降低 3 0～ 3 5 %,一胎产仔数量平均降低 6 4 .76～ 6 5 .6 4 %。结论 :表达精子抗原受精素β的重组沙门氏菌疫苗株可在小鼠中诱导免疫避孕效应。     

小鼠富含半胱氨酸分泌蛋白-1DNA疫苗免疫避孕效果的评价

目的:观察小鼠富含半胱氨酸分泌蛋白-1(Crisp-1)DNA避孕疫苗免疫雌、雄性BALB/c小鼠后特异性抗体的表达及免疫避孕效果。方法:将雌、雄性BALB/c小鼠随机分为实验组和对照组,每组雌鼠12只,雄鼠8只。每只动物经肌肉接种重组质粒pcDNA3.1-Crisp-1或pcDNA3.1空载体3次,ELISA测定小鼠血清中抗Crisp-1抗体水平的变化,Western blotting检测抗体的特异性。免疫结束后2周,将实验小鼠分别与未接种的正常雌、雄性小鼠合笼,记录每组雌鼠的妊娠率与每窝产仔数。结果:重组质粒pcDNA 3.1-Crisp-1可以在小鼠体内诱发特异性抗Crisp-1免疫应答,免疫后雌、雄性小鼠妊娠率和平均每窝产仔数均显著降低(P<0.05)。结论:重组质粒pcDNA3.1-Crisp-1具有一定的抗生育潜能。     

hCGβ-C3d3融合蛋白免疫BALB/c小鼠及其抗血清对hCG诱导鼠子宫增重的抑制效应

目的:通过分子佐剂C3d3增强hCGb避孕疫苗的免疫原性。方法:采用分子生物学技术以phCMV1为载体分别构建分泌型、带有6个组氨酸纯化标签的真核表达质粒phCMV1-6His-hCGb-C3d3和phCMV1-6His-hCGb,在CHO细胞中获得稳定、高效表达的重组蛋白,并用镍柱和凝胶过滤层析对其进行分离、纯化。分别用hCGb-C3d3融合蛋白和单用hCGb间隔4周两次免疫生育期雌性BALB/c小鼠,ELISA测定血清中抗hCGb抗体滴度,并对各组小鼠产生的抗血清拮抗hCG诱导的小鼠子宫增重效应进行比较。结果:C3d3使hCGb蛋白疫苗的免疫原性增强1 995倍,hCGb-C3d3融合蛋白免疫小鼠产生的抗血清具有很强的抑制小鼠子宫增重作用。结论:通过分子佐剂C3d3可以大幅提高机体对hCGb的体液免疫应答能力,hCGb-C3d3融合蛋白免疫小鼠产生的抗血清对hCG的生物学作用具有更强的抑制效应。     

小鼠精子Sp17与IL-5融合蛋白的免疫性避孕效应和安全性研究

目的:研究小鼠精子抗原Sp17与佐剂IL-5融合蛋白避孕疫苗的避孕效应及毒性作用。方法:重组蛋白疫苗Sp17-IL-5经鼻内免疫途径免疫小鼠后,观察其妊娠率、每胎产仔数,对免疫后小鼠的重要脏器进行病理学检查,并对免疫后小鼠及其子代的骨髓有核红细胞的微核及精子进行检测。结果:蛋白疫苗Sp17-IL-5鼻内免疫雌性Balb/c小鼠可明显降低受孕率和每胎产仔数,免疫后小鼠的重要脏器病理学检查未见明显异常改变,免疫后小鼠及其子代的骨髓有核红细胞微核率及精子畸形率均无明显变化。结论:重组蛋白疫苗Sp17-IL-5有较明确的抗生育作用,对免疫后小鼠及其子代未产生明显的毒副作用。     

全酿酒酵母HPV16-E7疫苗的构建及免疫原性研究

目的构建人乳头状瘤病毒16型（HPV16）E7重组全酿酒酵母疫苗,评价疫苗的免疫效应。方法将HPV16 E7 cDNA亚克隆人酵母表达载体pYES2／NT,重组质粒转化酿酒酵母,经诱导表达、热灭活制备全酵母HPV16-E7疫苗,免疫C57BL／6小鼠。ELISA、MTT、LDH法分别检测疫苗诱导的细胞因子、脾淋巴细胞增殖和细胞毒性T淋巴细胞（CTL）应答水平。结果全酵母疫苗能够有效表达HPV16-E7蛋白;疫苗小鼠免疫,能够刺激脾淋巴细胞增殖,并诱导出Th1型细胞因子和特异性CTL杀伤效应;免疫效果优于单纯HPV16-E7蛋白免疫（P〈0．01）。结论全酵母疫苗既能表达HPV16-E7蛋白,又能有效诱导特异性CTL免疫应答。研究结果为进一步进行疫苗抗肿瘤疗效研究,提供了实验基础。     

人巨细胞病毒pp65原核表达载体构建及表达产物对特异性免疫应答的诱导

目的：构建人巨细胞病毒pp65原核表达载体，研究纯化后的重组pp65蛋白诱导特异性免疫应答的作用及其特异性。方法：采用体外DNA重组技术，构建pp65原核表达载体并诱导其表达，通过亲和层析对表达产物进行纯化和经Westen blot鉴定；用MTT法检测重组蛋白对人外周血单个核细胞（peripheral blood mononuclear cell,PBMC)的激活、增殖作用，检测激活淋巴细胞对受HCMV感染的人胚肺成纤维细胞（human embryo lung fibroblast,HEL)的杀伤作用。结果：成功构建pp65原核表达载体并诱导其高效表达，纯化后的pp65蛋白能促进PBMC激活和增殖，增殖的细胞可特异性地杀伤受HCMV感染的HEL细胞。结论：经重组pp65蛋白激活增殖的淋巴细胞，可特异性杀伤受HCMV感染细胞，对HCMV感染的免疫治疗具有十分重要的作用。     

含编码人精子膜多肽cDNA和乙肝表面抗原基因的重组痘苗病毒

合成编码一种人精子膜蛋白YWK-Ⅱ胞外区的一段多肽片段的双链寡核苷酸链,HSD-2a.将HSD-2a和3’端乙肝表面抗原主S基因连接并与pUC18质粒重组.经鉴定的重组质粒用BamH Ⅰ和EcoRl酶切后,可分离纯化得到HSD-2a和HBsAg的主S基因连接片段,将该连接片段插入痘苗病毒通用表达载体pGJP-5的痘苗病毒启动子P_(7.5)下游,构建成重组质粒pGJP-HSD/HBs.将该重组质粒传染已感染了天坛痘苗病毒的猴肾细胞CV-1,pGJP-5中含有的TK基因与出发株病毒基因组的TK基因发生体内同源重组,构成重组痘苗病毒vv-HSD/HBs.HuTK~-细胞经vv-HSD/HBs感染后,在5-溴脱氧尿苷存在下进行空斑分析,筛选出TK~-重组病毒.应用ELISA方法对已感染了重组病毒的HuTK~-细胞培养上清和细胞裂解液测定,表明有HSD-2a编码的多肽片段的表达.Western-blot法分析呈现28和30Kd两条显色带.重组痘苗病毒vv-HSD/HBs有可能作为抗生育疫苗用途.     

HPV58型E2蛋白表达纯化与多克隆抗体制备

目的:表达纯化重组人乳头瘤病毒(human papillomavirus,HPV)58型E2蛋白,制备多克隆抗体。方法:构建重组质粒p ET-28b-E2,转化至BL21(DE3)p Lys S中诱导表达,包涵体洗涤后经镍柱亲和层析分离得到纯化目的蛋白。用纯化的重组E2蛋白免疫新西兰白兔,制备兔抗HPV58型E2蛋白多克隆抗体,ELISA分析多克隆抗体的效价,免疫印迹检测抗体的特异性。结果:表达纯化了重组HPV58型E2蛋白,制备了高滴度和高特异性的多克隆抗体。结论:制备的多克隆抗体可用于对HPV58型E2蛋白进行精细B细胞线性表位鉴定。     

《生殖与避孕》2004年总目次

2004年总目次特约述评卷期页精子功能检测对选择IVF或ICSI治疗不育症的临床意义………………………………………………………244193实验研究HER-2/neu胞外区的基因工程表达及其免疫避孕作用………………………………………………………2411雄激素致不孕大鼠胰腺、下丘脑及卵巢雄激素受体mRNA表达………………………………………………2416转化生长因子β受体Ⅱ在大鼠实验性隐睾中的表达及与生精细胞凋亡的关系………………………………2419干预CD86协同刺激信号对母胎界面Th1/Th2型细胞因子转录调控及妊娠结局的影响………………………     

溶脲脲原体蛋白UreG抗体对小鼠体外受精的影响

目的:探索与人精子膜存在交叉反应的溶脲脲原体蛋白UreG抗体对小鼠体外受精的影响。方法:提取溶脲脲原体总DNA,用自行设计的引物扩增UreG全序列,PCR产物经TA克隆后,用BamHⅠ和HindⅢ双酶切,克隆到表达载体pET-28a(+)中。在E.coliBL21中,异丙基-β-硫代半乳糖苷(IPTG)诱导下表达His融合蛋白。用亲和层析纯化的重组蛋白免疫BALB/c小鼠,获取多克隆抗体。观察该抗体对小鼠体外受精的影响。结果:对表达重组蛋白的质粒进行DNA测序以及十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析,证实获取了目的蛋白。ELISA检测证实,免疫小鼠产生了高效价的抗体。该抗体能够抑制小鼠精卵的融合。结论:与人精子膜存在交叉反应的溶脲脲原体蛋白UreG抗体,可导致小鼠精卵结合率降低。     

HER-2/neu expression in Paget disease of the vulva and the female breast

OBJECTIVES: Paget disease of the vulva is a rare lesion that accounts for <1% of vulva neoplasms. A 12% prevalence of invasive Paget carcinoma and a 4% prevalence of associated adenocarcinomas are described. Furthermore, a high recurrence rate of 30% after surgical therapy is observed. This study aims to search for therapeutic strategies for recurrent Paget disease, which are less mutilating and less aggressive than reexcision, x-ray therapy, or chemotherapy. Trastuzumab (Herceptin) is a recombinant monoclonal antibody against HER-2/neu, approved by the U.S. FDA for the treatment of patients with HER-2/neu-positive metastatic breast carcinomas. The results of recent studies indicate that HER-2/neu oncoprotein may play a role in the pathogenesis of extramammary Paget disease. METHODS: Using HercepTest, we analyzed HER-2/neu overexpression in seven noninvasive Paget lesions, two invasive lesions, and one Paget disease of the vulva with underlying adenocarcinoma. In addition, we investigated five mammary Paget diseases. RESULTS: Overexpression of HER-2/neu oncoprotein labeling exclusively the membranes of Paget cells was demonstrated in 8 out of 10 cases. One noninvasive and one with underlying adenocarcinoma stained negatively. Overexpression of HER-2/neu was demonstrated in all five cases of mammary Paget disease. CONCLUSION: Using HercepTest as a standardized detection system, overexpression of HER-2/neu can be demonstrated in a majority of both noninvasive and invasive Paget disease of the vulva. The use of Trastuzumab should be considered for the treatment of patients with recurrent Paget disease of the vulva with overexpression of HER-2/neu.     

Anticollagen antibody responses in DBA/1 (H-2q) mice associated with type II collagen-induced arthritis and the effect of pregnancy

We have previously shown that DBA/1 mice immunized with heterologous type II collagen showed remission of the subsequent collagen-induced arthritis (CIA) when pregnant, but experienced exacerbation postpartum. Measurement of anticollagen antibody (aCa) responses by ELISA in primiparous mice immunized at day 1 of pregnancy revealed no significant difference compared to aCa titres in virgin animals, apart from slightly increased titres following the primary immunisation. When mice received collagen challenge during early pregnancy, however, the date at which maximal antibody titres was reached was delayed by 5 days. Pregnancy initiated following the intraperitoneal boost caused a ten-fold suppression in aCa titres with a rise post-partum. Measurements of aCa levels in individuals which showed fetal resorption indicated that suppression of humoral responses was dependent on the presence of a viable conceptus. Antibody titres declined in all animals after a period of time, which was more prolonged in multigravidae where aCa titres were higher than in nulliparous and primiparous mice. The results show that although pregnancy alters aCa responses during the course of gestation, no long-term modification of humoral immunity occurs, an observation in agreement with the clinical findings in these mice and in humans.     

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。     

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.     

Inoculation of Lactobacillus expressing hCG beta in the vagina induces an anti-hCG beta antibody response in murine vaginal mucosa

OBJECTIVES: To test the possibility of vaccination with lactobacillus expressing hCG beta antigen administered by vaginal mucosal immunization. METHODS: A plasmid pIlac-hCG beta was constructed and then transfected into Lactobacillus casei CECT5276, which stably expressed hCG beta protein. RIA was used to detect hCG beta in the culture supernatant and cell lysate. Western blotting was performed to evaluate the expressed protein of interest. Female BALB/c mice aged 6-8 weeks received inoculations in the vagina of the recombinant L. casei CECT5276. ELISA was used to determine the anti-hCG beta IgA antibody in vaginal lavage fluid from the BALB/c mice after vaginal mucosal immunization. RESULTS: The pIlac alone appeared to have a higher efficiency than pIlac-hCG beta, and the highest transfection efficiency of both plasmids was at pulse voltages of 1200 V and 1500 V. About 78.5% of the hCG beta protein was excreted into the culture supernatant. Excretion of hCG beta was most efficient when the pH of the culture medium was adjusted to around 7.0 and the concentration of lactose was around 1%. The hCG beta protein in the vaginal lavage fluid of these BALB/c mice was positive on the third day after vaginal inoculation. Anti-hCG beta IgA antibody continued to be found in the vaginal lavage fluid for 2 weeks following a booster vaginal inoculation. The splenic lymphocytes of the mice immunized with hCG beta through the vagina underwent a proliferative reaction to hCG antigen restimulation in vitro. Interferon gamma (IFN-gamma) and interleukin (IL)-4 were secreted at higher levels after vaginal mucosal immunization of L. casei expressing hCG beta than after vaginal mucosal immunization of L. casei alone. CONCLUSIONS: Vaginal immunization of lactobacillus expressing hCG beta induced an anti-hCG beta antibody response in the murine vaginal mucosa. Induction of the antigen-specific antibodies in the reproductive tract following vaginal inoculation of recombinant lactobacillus will lead to the development of a safe, efficient, and easy-to-use form of immunocontraception.     

卡培他滨对高表达HER-2/neu卵巢癌裸鼠的治疗作用

目的:探讨卡培他滨对高表达HER-2/neu卵巢癌裸鼠的治疗作用。方法:66只雌性健康裸鼠皮下接种人卵巢癌SKOV3细胞,建立试验模型。根据用药情况将裸鼠分成两组:对照组(塞来昔布灌胃治疗,33只)和观察组(塞来昔布联合卡培他滨灌胃治疗,33只)。比较两组SKOV3细胞的生长抑制率、肿瘤体积及肿瘤抑制率,同时观察两组裸鼠的p53标记指数、HER-2/neu标记指数。结果:观察组的细胞生长抑制率为(65.11±0.86)%,高于对照组(40.38±0.58)%,差异有统计学意义(P0.01)。观察组的肿瘤抑制率高于对照组,差异有统计学意义(P0.01)。观察组裸鼠的p53标记指数、HER-2/neu标记指数均低于对照组,差异有统计学意义(P0.01)。结论:卡培他滨在高表达HER-2/neu卵巢癌裸鼠的治疗中作用显著,能有效抑制肿瘤细胞的生长,作用机制可能是卡培他滨能调控p53、HER-2/neu基因表达。     

Influence of chemotherapy on the expression of p53, HER-2/neu and proliferation markers in ovarian cancer.

OBJECTIVE: Mutated p53 and HER-2/neu play a role in the etiology of ovarian cancer. It is important to know whether the expression of these proteins is affected by platinum-containing chemotherapy. STUDY DESIGN: Together with the cell proliferation markers Ki-67 and PCNA, the expression of p53 and HER-2/neu was assessed before and after chemotherapy. Paraffin-embedded tumor sections from 20 patients with ovarian cancer and four patients with benign disorders of the ovaries (controls) were analyzed. The expression of p53 was determined by the antibodies DO-1 and BP53-12. In addition to HER-2/neu and PCNA specific antibodies, MIB-1 was used to detect Ki-67. RESULTS: The expression of all markers was higher in ovarian cancer patients than in non-malignant controls. MIB-1 showed a significant increase of expression after chemotherapy (P=0.002). HER-2/neu, p53 and PCNA also showed a clear increase after treatment, but this was not statistically significant. HER-2/neu is of prognostic relevance with respect to the response to chemotherapy (P=0.005) and survival (P=0.0002). CONCLUSION: The different markers tested all increase after chemotherapy, but the differences are not statistically significant. Low HER-2/neu expression correlates with good outcome at second look.     

Immunological response of female macaques to the PH-20 sperm protein following injection of recombinant proteins or synthesized peptides

Because of its location on the sperm surface and its multiple functions during fertilization, the PH-20 protein is a potential target for contraceptive vaccines. Cynomolgus macaques were immunized using four different adjuvants together with synthesized peptides or recombinant proteins representing selected regions of macaque PH-20. The synthesized peptide (amino acids 387-412, designated Peptide 4) was used as a linear molecule in a 1:1 ratio with a peptide sequence of tetanus toxoid, as well as a multiple antigenic peptide (MAP) matrix held together by scaffolding lysine residues. In the MAP construct, the ratio of Peptide 4 to tetanus peptide was 4:1. To circumvent the poor production of recombinant PH-20 in bacterial cells, two truncated forms of the molecule were expressed in Escherichia coli, G18 (encoding amino acids 143-510) and E10 (encoding amino acids 291-510). The adjuvants were Montanide ISA 51, Titermax Gold, Syntex adjuvant formulation (SAF), and QS-21. All of the antigen/adjuvant combinations produced significant immune responses as measured by ELISA. The circulating antibodies from immunized animals recognized macaque sperm surface PH-20 on Western blots and were shown by indirect immunofluorescence to bind to the surface of macaque sperm. Montanide and Titermax were associated with higher titers of anti-PH-20 antibodies than QS-21 and SAF adjuvants. Immunization with Titermax, however, resulted in sterile abscesses in 4 of 8 animals injected. We conclude that antigens derived from synthesized peptides and recombinant proteins representing selected regions of the PH-20 molecule can be used as vaccine components in combination with the adjuvant Montanide to elicit a significant sperm-directed antibody response in immunized macaques.