抗独特型抗体免疫治疗卵巢癌的动物实验研究

目的：研究抗独特型抗体对卵巢癌的免疫治疗作用，为临床试验提供动物实验依据。方法：用人卵巢癌抗独特型抗体（Ａｂ２）免疫杂交一代小鼠ＢＣＦ１，为实验组；用正常小鼠ＩｇＧ免疫小鼠ＢＣＦ１，为对照组。两组均免疫３次，于末次免疫后１周，将经裸鼠传代的人卵巢癌细胞系移植于ＢＣＦ１小鼠肾脏包膜下，分别于移植后第２、４、６、８和１０天处死小鼠，取血清进行抗独特型抗体（Ａｂ３）分析。移植瘤行组织学检查，观察宿主淋巴细胞浸润情况及瘤细胞可见率。结果：实验组小鼠肾脏包膜下人卵巢癌细胞系很快受到排斥，在移植后第６天，淋巴细胞浸润即达高峰，而对照组在第１０天才达高峰；瘤细胞可见率，在移植第６天后即明显降低，而对照组呈逐渐下降。结论：Ａｂ２作为抗原免疫小鼠后，能够排斥瘤细胞的生长。     

鼠肾包膜下人卵巢癌移植瘤体积变化及病理学对照研究

人卵巢癌细胞系（ＳＫＯＶ＿３）经裸鼠传代后，移植于正常免疫功能Ｃ５７ＢＬ／６纯系小鼠肾包膜下，双侧肾脏多位点移植，３２只小鼠共移植瘤块１２９个，分别在移植术后４、６、８、１０、１２和１４天切取移植瘤，测量体积变化并观察病理组织学特征。结果：移植瘤在术后６～８天体积增加较快；１２～１４天增加幅度明显降低（Ｐ＜０．０５）；宿主淋巴细胞浸润，在８～１２天时达高峰，移植术后１４天，移植瘤几乎全部纤维化；光镜下瘤细胞可见率，术后４天为７７．２３％，以后逐渐下降，至１２～１４天，明显降低。提示：双侧肾脏多位点移植法简便易行。经裸鼠传代的人卵巢癌细胞可在正常免疫功能小鼠肾包膜下生存８～１０天。移植瘤体积变化并不能准确反映瘤细胞生长状况，尚需结合组织病理学检查。     

鼠肾包膜下人卵巢癌移植癌体积变化及病理学对照研究

人卵巢癌细胞系经裸鼠传代后，移植于正常免疫功能Ｃ５７ＢＬ／６纯系小鼠肾包膜下，双侧肾脏多位点移植，３２只小鼠共移植瘤块１２９个，分别在移植术后４、６、８、１０、１２和１４天切取移植瘤，测量体积变化并观察病理组织学特征。     

卵巢癌独特型疫苗免疫接种方案的探讨

目的：探讨卵巢癌抗独特型抗体（６Ｂ１１ ）疫苗免疫接种的理想方案。方法：用６Ｂ１１ 免疫ＢｃＦ１ 小鼠,观察基础免疫和加强免疫诱导的免疫应答情况,探讨加强免疫间隔时间、剂量及免疫途径对诱导免疫应答的影响,基础免疫为６Ｂ１１ １００μｇ—ＫＬＨ＋ ＣＦＡ,加强免疫为６Ｂ１１—ＫＬＨ＋ ＩＦＡ（或ＰＢＳ）,免疫部位为小鼠尾根及足底皮下或腹腔。观察指标：体液免疫为免疫鼠血清中抗原特异结合性抗独特型抗体（Ａｂ３ ）滴度,细胞免疫为免疫鼠脾脏淋巴细胞ＣＤ４＋ 、ＣＤ８＋ 亚群的变化。结果：（１）６Ｂ１１ 基础免疫后,Ａｂ３ 阳性率为８０．００％ ,Ａｂ３ 滴度高峰在第１４～２１天（１：３２０）,ＣＤ４＋ 亚群增加,ＣＤ＋８ 亚群变化不明显;（２）间隔４ 周加强免疫诱导的Ａｂ３ 滴度较２ 周者高,ＣＤ４＋ 亚群增加明显;６Ｂ１１５０μｇ 加强免疫诱导的免疫应答强于３０、１５μｇ;皮下途径加强免疫诱导的免疫应答略强于腹腔注射;（３）６Ｂ１１３ 次免疫后,Ａｂ３ 产生快（第３天１：３２０）,滴度高（最高１：６４０）,持续时间长（第４９天为１：４０）,ＣＤ＋４ ／ＣＤ８＋ 比值明显增加。结论：卵巢癌抗独特型抗体６Ｂ１１可作为肿瘤疫苗诱导ＢｃＦ１小鼠?     

抗独特型抗体免疫治疗卵巢癌的动物实验动物实验研究

目的：研究抗独特型抗体对卵巢癌的免疫治疗作用，为临床试验提供动物实验依据。方法用人卵巢癌抗独特型抗体免疫杂交一代小鼠ＢＣＦ１，为实验组；用正常小鼠ＩｇＧ免疫小鼠ＢＣＦ１，为对照组。     

卵巢癌抗独特型抗体诱导特异性迟发过敏反应的实验研究

本研究应用卵巢癌抗原内影像型单克隆抗独特型抗体６Ｂ１１和１Ｈ１２代替肿瘤抗原，诱导同系小鼠产生卵巢癌特异性细胞免疫反应－迟发性过敏反应（ＤＴＨ），从而证明了６Ｂ１１和１Ｈ１２具有抗原模拟作用。经过６Ｂ１１或１Ｈ１２免疫的小鼠，用卵巢癌细胞系ＳＫＯＶ３进行脚掌注射攻击。而后，测量脚掌水肿厚度作为ＤＴＨ反应强度。６Ｂ１１诱导的ＤＴＨ平均水肿厚度为０．９３ｍｍ，卵巢癌组织抗原ＯＣ１６６－９诱导的阳性对照     

构建卵巢上皮性癌原位移植瘤动物模型的新方法

构建卵巢上皮性癌（卵巢癌）的动物模型方法包括：裸鼠皮下移植瘤模型，腹腔移植瘤模型，裸鼠网膜移植瘤模型，原位移植-转移瘤模型等。原位移植因为可以提供给被移植物类似于人体的微环境所以有广阔的应用前景。目前的原位移植技术主要是用细胞株构建裸鼠皮下移植瘤作为供瘤体，切成小块后在解剖显微镜下打开卵巢的包膜，将组织块植入卵巢实质。该技术只能间接反应卵巢癌细胞株的特点；此外难度高，需要解剖显微镜等特殊仪器。本研究在实验过程中巧妙地使用了一种新方法，将细胞悬液直接注射到小鼠的卵巢实质内，成功地建立了糖尿病合并重症联合免疫缺陷（NOD／SCID）小鼠卵巢癌模型，现报道如下。     

卵巢癌可溶性抗原和抗CD3单克隆抗体诱导细胞毒性T细胞抗人卵巢癌的实验研究

目的探讨卵巢癌过继细胞免疫治疗新方法。方法提取卵巢癌细胞（ＣＯＣ１和ＣＯＣ２ｎ）可溶性抗原（ＴＳＡ），用ＴＳＡ和抗ＣＤ３单克隆抗体（ＣＤ３ＭｃＡｂ）共同诱导正常人外周血单个核细胞（ＰＢＭＣ）产生细胞毒性Ｔ细胞（ＣＴＬ），用ＣＴＬ于体外杀伤ＣＯＣ１细胞和裸鼠体内抑制ＣＯＣ２ｎ移植瘤的生长，体外和淋巴因子激活杀伤细胞（ＬＡＫ）、淋巴因子和抗ＣＤ３单抗激活的杀伤细胞（ＣＤ３ＡＫ）细胞进行比较，体内和ＣＤ３ＡＫ细胞进行比较。结果ＣＴＬ、ＣＤ３ＡＫ和ＬＡＫ对ＣＯＣ１细胞的细胞毒作用分别为７９．４％、５２．１％和５１．７％（Ｐ＜０．０１）。在体内，ＣＴＬ与ＣＤ３ＡＫ和未经处理的对照组比较，卵巢癌细胞移植到裸鼠体内的第９天，肿瘤平均体积分别为４４．４±２４．２ｍｍ３、１１８．８±４０．０ｍｍ３和４４３．０±１５８．７ｍｍ３（Ｐ＜０．０１），裸鼠平均生存期分别为２８．５天、２５．５天和１７天（Ｐ＜０．０１）。结论本方法诱导产生的ＣＴＬ，在体内、体外对卵巢癌细胞均有较高的细胞毒作用，能明显抑制肿瘤生长，为卵巢癌治疗提供了新的思路。     

三种人卵巢癌动物模型的生物学特性比较

目的:建立人卵巢癌裸小鼠皮下移植瘤,腹水瘤和原位移植瘤模型,观察比较其生物学特性,为卵巢癌的理论研究和选择临床模型选择提供参考。方法:利用卵巢癌细胞株SW626先制备裸小鼠皮下移植瘤模型,再将瘤组织条植入裸小鼠一侧卵巢内,建立人卵巢癌的原位移植模型,将该细胞悬液注入小鼠腹腔建立腹水瘤模型。用组织病理学,电镜和流式细胞仪DNA含量及染色体核型分析鉴定模型,并比较3种模型在生物学特性上的差异。结果:3种模型的瘤细胞与细胞株在形态和结构上一致,腹水瘤模型自然生存率明显短于其它两种模型,后两者无明显差异。在生物学特性上各具特点,其中原位移植瘤模型中肿瘤的生长和转移完全模拟人卵巢癌的临床过程。结论:3种模型从不同层面展现了人卵巢癌的生物学特点,其中原位移植瘤模型是研究卵巢癌的转移基础和评定抗癌药物药效更客现的模型。     

顺铂耐药卵巢癌细胞鼠肾包膜下动物模型的建立及其临床意义

目的 :建立顺铂耐药卵巢癌细胞鼠肾包膜下动物模型 ,探讨其临床意义。方法 :采取肾包膜下纤维蛋白细胞凝块移植法 ,用环磷酰胺免疫抑制小鼠建立顺铂耐药卵巢癌模型。结果 :移植后 7d肿瘤体积明显增大 ,顺铂耐受细胞组与非耐受细胞组体积无明显差异 (P >0 .0 5) ;移植后细胞形态学特征无改变 ;在用顺铂治疗后 ,耐药细胞组肿瘤体积较非耐药细胞组体积大 (P <0 .0 5)。结论 :该动物模型可作为短期体内实验模型 ,能保持细胞的耐药性     

C57BL/6小鼠上皮性卵巢癌模型的建立及其生物学特性

目的在免疫功能正常的C57BL/6小鼠体内建立上皮性卵巢癌腹腔转移瘤模型及皮下瘤模型,为卵巢癌的诊断、治疗及预防的相关研究提供基础。方法体外培养近交系C57BL/6小鼠卵巢上皮低分化腺癌细胞株ID-8,将对数生长期的ID-8细胞按1×10^7、5×10^6、1×10^6和1×10^5个细胞/只的剂量,分别接种于6～8周雌性SPF级C57BL/6小鼠腹腔及左侧肩部皮下,共8组,每组6只。观察腹腔瘤及皮下瘤的成瘤时间、成瘤率、腹水、腹腔肿瘤转移情况及小鼠生存期;处死小鼠时留取主要脏器、腹腔肿瘤及皮下肿瘤标本,行病理学检查。另外6只小鼠接种5×10^6个ID-8细胞,分别在4、8、16周后处死进行系统解剖,做常规病理学检查。结果将不同数量的ID-8细胞接种C57BL/6小鼠腹腔及皮下后,成瘤率均为100%,腹腔瘤模型组：腹腔注射1×10^5,1×10^6,5×10^6,1×10^7个ID-8细胞,平均生存时间分别为（141±6.7）d、（122.8±4.5）d、（83.4±7.2）d和（74.4±4.5）d,随着肿瘤细胞接种负荷增加,动物生存期明显缩短（P〈0.05）。皮下瘤组：1×10^7细胞组和5×10^6细胞组,1周左右成瘤;1×10^6细胞组,3周左右成瘤;1×10^5细胞组,6周左右成瘤。随着肿瘤接种负荷的增加,肿瘤直径和体积明显增大（P〈0.05）。结论 C57BL/6小鼠腹腔瘤模型类似人类Ⅲ、Ⅳ期卵巢上皮癌患者的临床特点。皮下瘤模型更易于观察免疫治疗或药物治疗的疗效。在免疫功能正常的C57BL/6小鼠建立的ID-8细胞卵巢癌肿瘤模型,是适合于卵巢癌分子和免疫治疗研究的模型。     

具生殖系嵌合能力的C57BL/6J小鼠胚胎干细胞系的建立

目的:建立能实现种系传递的C57BL/6J小鼠胚胎干细胞系。方法:从C57BL/6J小鼠3.5d的囊胚中分离培养内细胞团。经体外适宜培养建系后,将C57BL/6J胚胎干细胞(ES)注入ICR小鼠受体囊胚腔,制备嵌合体小鼠。结果:成功地建立了3个C57BL/6J小鼠胚胎干细胞系,该C57BL/6JES细胞呈集落状生长,正常稳定的核型率>80%,具高水平的磷酸酶活性,并表达ES细胞特殊性细胞表面标记SSEA-1,不表达SSEA-3和SSEA-4;体内外的分化实验也证实mC57ES1具向三胚层组织分化的能力。经显微注入ICR小鼠囊胚腔中,产生4只嵌合体小鼠,经证实其中1只为种系嵌合小鼠。结论:建立了能实现种系传递的C57BL/6J小鼠胚胎干细胞系,该系的ES细胞可用于今后制备转基因动物和基因敲除动物。     

Development of a syngeneic mouse model of epithelial ovarian cancer

Background

Most cases of ovarian cancer are epithelial in origin and diagnosed at advanced stage when the cancer is widely disseminated in the peritoneal cavity. The objective of this study was to establish an immunocompetent syngeneic mouse model of disseminated epithelial ovarian cancer (EOC) to facilitate laboratory-based studies of ovarian tumor biology and preclinical therapeutic strategies.

Methods

Individual lines of TgMISIIR-TAg transgenic mice were phenotypically characterized and backcrossed to inbred C57BL/6 mice. In addition to a previously described line of EOC-prone mice, two lines (TgMISIIR-TAg-Low) were isolated that express the oncogenic transgene, but have little or no susceptibility to tumor development. Independent murine ovarian carcinoma (MOVCAR) cell lines were established from the ascites of tumor-bearing C57BL/6 TgMISIIR-TAg transgenic mice, characterized and tested for engraftment in the following recipient mice: 1) severe immunocompromised immunodeficient (SCID), 2) wild type C57BL/6, 3) oophorectomized tumor-prone C57BL/6 TgMISIIR-TAg transgenic and 4) non-tumor prone C57BL/6 TgMISIIR-TAg-Low transgenic. Lastly, MOVCAR cells transduced with a luciferase reporter were implanted in TgMISIIR-TAg-Low mice and in vivo tumor growth monitored by non-invasive optical imaging.

Results

Engraftment of MOVCAR cells by i.p. injection resulted in the development of disseminated peritoneal carcinomatosis in SCID, but not wild type C57BL/6 mice. Oophorectomized tumor-prone TgMISIIR-TAg mice developed peritoneal carcinomas with high frequency, rendering them unsuitable as allograft recipients. Orthotopic or pseudo-orthotopic implantation of MOVCAR cells in TgMISIIR-TAg-Low mice resulted in the development of disseminated peritoneal tumors, frequently accompanied by the production of malignant ascites. Tumors arising in the engrafted mice bore histopathological resemblance to human high-grade serous EOC and exhibited a similar pattern of peritoneal disease spread.

Conclusions

A syngeneic mouse model of human EOC was created by pseudo-orthotopic and orthotopic implantation of MOVCAR cells in a susceptible inbred transgenic host. This immunocompetent syngeneic mouse model presents a flexible system that can be used to study the consequences of altered gene expression (e.g., by ectopic expression or RNA interference strategies) in an established MOVCAR tumor cell line within the ovarian tumor microenvironment and for the development and analysis of preclinical therapeutic agents including EOC vaccines and immunotherapeutic agents.

     

Prevention of graft rejection by donor type II CD8(+) T cells (Tc2 cells) is not sufficient to improve engraftment in fetal transplantation

OBJECTIVES: Tc2 cells, a subset of CD8(+) T cells, are able to facilitate engraftment in a murine model of postnatal allogeneic bone marrow transplantation. The purpose of this study was to evaluate whether Tc2 cells could improve engraftment in fetal transplantation. METHODS: Gestational day 13 C57BL/6 (H-2(b)) fetal mice were used as recipients, adult B6D2F(1) mice (C57BL/6 x DBA/2, H-2(b/d)) as donors, and splenocytes from B6C3F(1) (C57BL/6 x C3H/He, H-2(b/k)) mice were used as stimulators in cultures used to generate the Tc2 cells from B6D2F(1) mice. Peripheral blood chimerism was examined monthly for 3 months. Thereafter, recipients were sacrificed to evaluate the levels of peritoneal, splenic and bone marrow chimerism. The T-cell responses of recipient splenocytes to cells of host origin were measured as a proliferative response in mixed lymphocyte cultures. RESULTS: Low levels of peripheral blood cell chimerism (<0.3%) were observed at 1 month of age, which declined further by 3 months of age. The levels of donor cells in the spleen, bone marrow and peritoneal cavity were usually not more than 0.05%. The peritoneal cavity tended to have higher levels of donor cells with 1 recipient sustaining as high as 25.03% at the age of 3 months. Higher peritoneal chimerism correlated with a lower donor-specific T-cell response. CONCLUSIONS: Transplantation of Tc2 cells was insufficient to improve bone marrow engraftment in utero, suggesting that graft rejection is not the major barrier to successful in utero transplantation. Donor cells can persist in the peritoneal cavity and might play an important role in inducing immune tolerance in fetuses.     

Tumor-infiltrating lymphocytes contain higher numbers of type 1 cytokine expressors and DR+ T cells compared with lymphocytes from tumor draining lymph nodes and peripheral blood in patients with cancer of the uterine cervix

OBJECTIVE: The aim of this study was to compare the phenotype and function of lymphocytes collected from the peripheral blood (PBL), tumor draining regional lymph nodes (LND), and infiltrating tumor tissues (TIL) of patients with stage IB-IIA cervical cancer. METHODS: Leukocytes from peripheral blood (n = 35), tumor draining lymph nodes (n = 33), and tumor tissues (n = 15) of cervical cancer patients were evaluated for the relative proportions of lymphocyte subsets including CD3+, CD4+, CD8+, CD19+, CD56, and the early (CD25) and late (HLA-DR) activation markers on CD3+ T cells, as well as the ability to synthesize type 1 cytokines (IFN-gamma and IL-2) and a type 2 cytokine (IL-4) by flow cytometry. RESULTS: In all patients, T cells (CD3+) were the major leukocyte population detected in each tissue, with CD4+ T cells being dominant in PBL and LND, while CD8+ T cells predominated in TIL (CD4:CD8 ratios, 2.4 vs 4.0 vs 0.7, respectively). CD19+ lymphocytes (B cells) were significantly higher in LND compared to PBL and TIL (P > 0.01) while CD56+ lymphocytes were higher in PBL compared to LND (P > 0.01) and TIL (P > 0.05). The early activation marker CD25 was significantly up-regulated in LND, while TIL had a higher proportion of T cells expressing the late activation marker HLA-DR. Type 1 cytokines were the dominant type produced by in vitro stimulated T cells for each population, with a greater proportion of IFN-gamma+ CD4+ and CD8+ T cells (i.e., Th1 and Tc1) and IL-2+ CD8+ T cells (Tc1) seen in TIL, as compared with LND and PBL (P > 0.01). Low percentages of IL-4+ T cells (i.e., Th2 and Tc2) were detected only in PBL. CONCLUSIONS: This study demonstrates significant differences in the phenotype and activation state of lymphocyte subsets from different anatomical sites, as well as differences in their ability to synthesize immunostimulatory cytokines. The recruitment and accumulation of high concentrations of antigen-experienced T lymphocytes in the cervical tumor tissue may represent an important local barrier to neoplastic dissemination.     

Immunization with a syngeneic regressor tumor causes resorption in allo-pregnant mice

The purpose of our studies is to establish experimental systems in which one can deliberately disrupt the apparent maternal tolerance toward the semiallogeneic fetuses. Bases on the hypothesis that immunization against tumor-associated antigens may lead to a subsequent immune response directed against cross-reacting fetal antigens, we have immunized C57BL/6J female mice with a syngeneic regressor tumor. Mice were subsequently mated to B6D2F1, DBA/2, CBA/J or C57BL/6J males. We show that a high proportion of embryos sired by either B6D2F1 or DBA/2 males undergo resorption whereas those engendered by CBA/J or C57BL/6J males remain fully protected.     

人乳头状瘤病毒16型多肽疫苗的制备及体内外效应观察

目的 探讨人乳头状瘤病毒(HPV)16型多肽疫苗的制备,并观察HPV16多肽疫苗的体内外效应.方法 (1)针对抗原加工相关转运子(TAP)设计HPV16 E7蛋白的主要组织相容性复合物Ⅰ类分子(MHC-I)的抗原结合表位,利用生物信息学分析平台筛选出一致性较高、特异性及亲和力较强的HPV16 E7多肽作为研究对象制备HPV16多肽疫苗用于以下研究,本研究共筛选出3段多肽,分别命名为E7Pa、E7Pb、E7Pc.(2)C57BL/6小鼠注射鼠肺上皮细胞株TC-1细胞(为鼠源性的HPV16阳性的肿瘤细胞株)后,采用等额抽取的随机方法分为5组,ETPa+二核苷胞嘧啶(CpG)、E7Pb+CpG、E7Pc+CpG[均为实验组,分别加入终浓度为50μg/ml的E7Pa、E7Pb、E7Pc和终浓度为12 mg/L的刀豆蛋白(ConA)]、CpG(为阳性对照,加入终浓度为12 mg/L 的Con A)和空白对照组(不做任何处理).采用四甲基偶氮唑蓝(MTT)比色法检测各组作用不同时间后小鼠脾T淋巴细胞的体外增殖效应,乳酸脱氢酶(LDH)释放法检测小鼠脾T淋巴细胞在不同效靶比下的体外细胞毒T淋巴细胞(CTL)活性,实时荧光定量RT-PER技术检测小鼠肿瘤组织中γ干扰素(IFN-γ)、白细胞介素2(IL-2)mRNA的表达水平,酶联免疫吸附试验(ELISA)检测小鼠外周血中IFN-γ、IL-2的表达水平,通过定期测量比较各组小鼠接种HPV16多肽疫苗后体内肿瘤体积的变化.结果 (1)本研究筛选出了一致性较高、特异性及亲和力较强的3段HPV16 E7多肽作为研究对象制备HPV16多肽疫苗,分别命名为E7Pa、E7Pb、E7Pc.(2)MTT比色法检测显示,在接种疫苗24、48、72、96 h后,以E7Pa+CpG组的增殖效应最明显,其细胞增殖率分别为(131±32)%、(302±15)%、(552±28)%、(731±24)%,明显高于空白对照组的(72±15)%、(120±57)%、(176±41)%、(288±29)%(P<0.01);E7Pb+CpG、ETPc+CpG、CpG组的细胞增殖率也均明显高于空白对照组(P<0.05);但E7Pb+CpG、E7Pc+CpG、CpG组间比较,差异则无统计学意义(P>0.05).LDH释放法榆测显示,效靶比为100:1时,E7Pa+CpG、E7Pb+CpG、E7Pc+CpG、CpG和空白对照组CTL 活性分别为(85.9±3.0)%、(55.9±2.5)%、(60.2±1.5)%、(41.0±1.7)%和(4.1±1.0)%,E7Pa+CpG组与空白对照组比较,差异有统计学意义(P<0.01);E7Pb+CpG、E7Pc+CpG、CpG组分别与空白对照组比较,差异也有统计学意义(P<0.05);但E7Pb+CpG、E7Pc+CpG、CpG组间比较,差异则无统计学意义(P>0.05).在肿瘤组织及外周血中,小鼠IFN-γ、IL-2的表达水平,E7Pa+CpG组与空白对照组比较,差异均有统计学意义(P<0.01);E7Pb+CpG、E7Pc+CpG和CpG组分别与空白对照组比较,差异也均有统计学意义(P<0.05);但E7Pb+CpG、E7Pc+CpG、CpG组问比较,差异则无统计学意义(P>0.05).小鼠体内的肿瘤体积,各实验组肿瘤生长均明显被抑制,接种后第60大,E7Pa+CpG组与空白对照组比较,差异有统计学意义(P<0.01);E7Pb+CpG、E7Pc+CpG和CpG组分别与空白对照组比较,差异也均有统计学意义(P<0.05);但 E7Pb+CpG、E7Pc+CpG、CpG组间比较,差异则无统计学意义(P>0.05).结论 在动物模型中,针对TAP筛选的HPV16 E7多肽联合CpG制备的HPV16多肽疫苗,可以有效治疗HPV16 E7阳性的肿瘤.     

Class I major histocompatibility complex antigen expression on early murine trophoblast and its induction by lymphokines in vitro. II. The role of gamma interferon in the responses of primary and secondary giant cells

The induction of paternal Class I and II MHC antigens by crude lymphokine preparations or purified recombinant gamma interferon was investigated on (C57BL/6J X CBA/H)F1 primary and secondary trophoblast giant cell outgrowths from 3.5-day post-coital (pc) blastocyst and 7.5-day pc ectoplacental cone preparations, respectively, using sensitive immunogold labelling techniques and electron microscopy. Class I MHC (but not Class II) antigens could readily be induced on secondary trophoblast giant cells, by incubation in vitro with gamma interferon for 40 h. However, repeated attempts to induce detectable MHC antigens on primary trophoblast giant cells failed. Mock-treated (C57BL/6J X CBA/H)F1 secondary trophoblast giant cell control preparations failed to express detectable MHC antigens. These findings suggest that, at the time of implantation, there is a time window during which MHC antigens are neither expressed constitutively nor are inducible by soluble factors which normally modulate cell surface MHC antigen concentration.     

Experimental transplantation study for possible transformation of bone marrow cells in the mouse placenta

The aim of the present study is to establish a mouse model of the transplantation of bone marrow cells into the placenta in mid-gestation. The mononuclear fraction of bone marrow cells was isolated by Ficoll gradient centrifugation from the femur bones of C57BL/6 green fluorescent protein (GFP) gene transgenic (Tg) mice. After intraperitoneal injection of pentobarbital sodium, the abdominal cavities of pregnant non-Tg (C57BL/6 or ICR) mice were opened at 9.5 days postcoitum (dpc). The mononuclear fraction of bone marrow cells from Tg mice (3-5 x 10(5)cells/3 microl) was directly injected into the placental portion of the pregnant uterus, at a depth of approximately 3 mm, using a 31-gauge injector. The placenta was sampled at 14.5 dpc. Confocal laser scanning microscopic analysis of the serial sections of the sampled placenta (150-250 sections/placenta) was carried out to detect GFP-positive cells and to assess immunostaining for cytokeratin, CD34, p57(Kip2) and prolactin. Most pregnant mice survived until sampling of the placenta at 14.5-18.5 dpc (88.9% for C57BL6 and 100% for ICR). The survival rate of fetuses from mice in which the placenta was transplanted with GFP-positive bone marrow cells was approximately 50%. A small population (0.154%) of injected bone marrow cells was retained in the placental tissue. Immunohistochemically, cytokeratin, CD34 and p57(Kip2) were positively stained in 0.062%, 4.5% and 2.1% of GFP-positive cells, respectively, while prolactin was not positive in any of the cells examined. GFP-positive bone marrow cells were successfully transplanted to the murine placenta. Future investigations of the specific antigens in bone marrow cells retained in the placenta may enable a better understanding of the local regulation of placental development.     

葡萄球菌肠毒素激活肿瘤浸润淋巴细胞及外周血淋巴细胞体外抗人卵巢癌的对比研究

目的:探讨葡萄球菌肠毒素A(SEA)对卵巢癌肿瘤浸润淋巴细胞(TIL)及其外周血淋巴细胞(PBL)抗瘤活性的诱导作用。方法:取10例卵巢癌伴腹水患者实体瘤、腹水及外周血标本,分离TIL和PBL。在SEA及IL-2作用下培养,定时计数,了解其增殖情况;流式细胞仪检测其CD3、CD4、CD8表达;噻唑蓝(MTT)比色法测定其对K562及自体肿瘤细胞的细胞毒活性;酶联免疫吸附试验(ELISA)测定培养上清液中TNF-a和IFN-γ浓度。结果:10例中8例成功分离实体瘤TIL、腹水TIL及PBL。(1)SEA刺激的实体瘤TIL、腹水TIL及PBL增殖速率明显较IL-2诱导组快(P<0.05),但增殖高峰后出现下降趋势,IL-2组未出现此现象;(2)CD3+CD4+及CD3+CD8+T表达率均明显上升,其中SEA诱导组比IL-2组增加比例明显(P<0.05),以SEA作用的CD3+CD8+T比例增加最快;(3)TIL对自体肿瘤细胞的杀伤活性明显高于对K562细胞的杀伤活性(P<0.05),PBL对自体肿瘤细胞的杀伤活性则明显低于对K562细胞的杀伤活性(P<0.05),SEA激活组比IL-2组杀伤率高(P<0.05);(4)各效应细胞分泌的TNF-a、IFN-γ分别在培养的第2天和第4天达到高峰,高峰后迅速下降,SEA诱导组在前10天明显高于IL-2诱导组(P<0.05)。结论:SEA可高效、迅速诱导卵巢癌TIL的抗瘤活性。