Augmented anti-metastatic efficacy of a selective matrix metalloproteinase inhibitor,MMI-166, in combination with CPT-11

The anti-metastatic efficacy of MMI-166, which is a selective matrix metalloproteinase (MMP) inhibitor, in combination with CPT-11 was examined using two metastasis models of human gastrointestinal cancer cells. In the liver metastasis model, C-1H human colon cancer cells were injected into the spleen of athymic BALB/c nude mice. Daily oral (p.o.) dosing of MMI-166 at 200 mg/kg starting 1 day after tumor inoculation led to a significantly prolonged survival effect by inhibiting liver metastasis of C-1H tumor cells. CPT-11 (5 or 20 mg/kg) was administered intraperitoneally (i.p.) three times on day 3, day 7 and day 11 and also improved the survival of tumor-inoculated mice compared with the vehicle control. When MMI-166 was combined with CPT-11, the anti-metastatic efficacy was significantly augmented. Moreover, long tumor-free survival was noted in two of eight mice that were given the combination therapy but not either MMI-166 or CPT-11 monotherapy. In the peritoneal dissemination model, TMK-1 human gastric cancer cells were injected i.p. into nude mice. While both MMI-166, administered daily p.o. from day 1 at 200 mg/kg, and CPT-11, administered intravenously (i.v.) three times, inhibited the tumor dissemination and growth, the combination therapy of MMI-166 plus CPT-11 showed a greater inhibitory effect than each monotherapy. A hematotoxicity study demonstrated that CPT-11 alone significantly decreased the number of white blood cells (WBC) and bone marrow cells (BMC) in the mice during treatment, while the daily administration of MMI-166 alone had no such effect. More importantly, the combination therapy of MMI-166 with CPT-11 did not augment the hematotoxicity caused by CPT-11. An in vitro cytotoxicity study showed that MMI-166 itself neither has direct cytotoxicity in C-1H and TMK-1 tumor cells, nor does it augment the cytotoxicity of SN-38, an active form of CPT-11. The findings indicate that the augmented anti-metastatic efficacy in combination treatment was not simply due to the augmentation of direct cytotoxic activity, but was rather an additive or synergistic effect of anti-metastatic activities with different mechanisms. In conclusion, we demonstrated that the anti-metastatic efficacy against C-1H colon cancer and TMK-1 gastric cancer were augmented by the combination therapy of MMI-166, an orally active MMP inhibitor, with CPT-11. However, the hematotoxicity caused by CPT-11 was not augmented in the combination with MMI-166. Thus, the combination therapy of MMI-166 and CPT-11 exhibited potent anti-metastatic efficacy without increased hematotoxicity. These results point to the clinical advantage of using MMI-166 in combination with CPT-11. This revised version was published online in July 2006 with corrections to the Cover Date.     

Growth and dissemination of human malignant melanoma cells in mice with severe combined immune deficiency.

BACKGROUND: The severe combined immune deficiency (SCID) mouse is lacking mature B and T lymphocytes and may be permissive for human tumor growth and metastasis. EXPERIMENTAL DESIGN: SCID mice received human melanoma cells of diverse origins including: 2 established cell lines, 4 early passage cell lines, and fresh or cryopreserved cells obtained directly from 9 patient biopsies. They were introduced into SCID mice via intraperitoneal, subcutaneous and intravenous injections. RESULTS: Tumor growth occurred with each of the 15 melanoma specimens for a take rate of 100% considering cell source. In addition, 60% of the 102 total mice injected displayed tumor growth in at least one site. The most consistent tumor growth (77%) occurred after intraperitoneal injection. Tumors developed in 41 and 48% of mice injected subcutaneously and intravenously, respectively. The mice developed both local tumor growth with palpable tumor nodules at injection sites and hematogenous and/or lymphatic dissemination to multiple sites in the abdominal and thoracic cavities. The number of metastases per animal averaged 16.3 and the number per organ ranged from 1 to 38. Melanotic and amelanotic tumor nodules obtained from a single patient retained their original characteristics with regard to melanin production after passage in the SCID mouse. The appearance of the human melanoma cells in SCID mouse tissues ranged from implants on the organ capsule to frank parenchymal organ involvement and vascular invasion. Some small foci of tumor were only detected using immunohistochemistry with monoclonal antibodies against the S-100 and HMB-45 to melanoma-related antigens. CONCLUSIONS: We conclude that the SCID mouse consistently supports growth, invasion, and metastatic spread of human melanoma cells, including specimens obtained from fresh patient biopsies. The SCID mouse will serve as a relevant in vivo model for studying the biology of human malignant melanoma and screening new therapeutic agents.     

Regional administration of oncolytic Echovirus 1 as a novel therapy for the peritoneal dissemination of gastric cancer

The dissemination of malignant gastric cells to the peritoneum occurs frequently, usually as an early event in disease, and results in poor patient prognosis. Surgery and chemotherapy offer limited therapeutic success. The low-pathogenic human enterovirus, Echovirus 1 (EV1), is an oncolytic virus that selectively targets and destroys malignant prostate and ovarian cancer xenografts in vivo. Lytic EV1 infection requires the cell surface expression of α₂β₁, an integrin involved in the dissemination of gastric cancer cells to the peritoneum. Herein, we evaluated the capacity of EV1 for anti-neoplastic cell action in gastric peritoneal carcinomatosis. Flow cytometric analysis demonstrated that α₂β₁ was abundantly surface expressed on a panel of gastric cancer cell lines, rendering the majority of lines highly susceptible to in vitro lytic EV1 infection and supportive of efficient viral progeny production. A bioluminescent MKN-45-Luc SCID mouse model of peritoneal dissemination was developed to allow real-time non-invasive monitoring of peritoneal tumor burden. Employing this mouse model, we demonstrated a therapeutic dose-response for escalating oncolytic EV1 doses. Taken together, these results emphasize the exciting potential for EV1 as a single or adjunct therapy for the control of the peritoneal dissemination of gastric cancer.     

右旋柠烯乳剂对人胃癌移植瘤生长和转移的抑制作用

目的　研究并探讨右旋柠烯乳剂 (D limonene)对人胃癌生长和转移的抑制作用及其机制。　方法　采用人胃癌BGC 82 3细胞株经反复传代成实体瘤的完整组织块 ,建立人胃癌裸小鼠原位移植瘤模型。将 4 0只荷瘤裸小鼠随机分成 4组 ,移植后第 5d开始分别用生理盐水灌胃、5 氟尿嘧啶 (5 FU)腹腔注射、D limonene灌胃、D limonene灌胃 +5 FU腹腔注射共 7周。第 8周处死动物 ,测量原位肿瘤瘤重并计算抑瘤率 ,检测肿瘤细胞凋亡指数 (AI)、肿瘤微血管密度 (MVD)及免疫组织化学测定肿瘤组织血管内皮生长因子 (VEGF)的表达变化 ,光镜与电镜观察组织细胞超微结构变化 ;观察荷瘤鼠腹膜、肝、其他脏器肿瘤转移及腹水情况。　结果　D limonene组、联用组分别与对照组相比 ,胃癌的原位肿瘤瘤重与抑瘤率、AI、MVD、VEGF下调 ,差异有显著性 (P <0 0 5 )。腹膜、肝转移受到明显抑制 (P <0 0 5 )。　结论　D limonene通过抑制肿瘤微血管形成 ,诱导胃癌细胞凋亡 ,抑制了体内胃癌的生长和转移。     

人肺癌细胞LALU血管生成过程的形态学观察及意义

目的：观察人肺癌细胞血管生成过程的病理形态学、超微结构特点及意义。方法：采用人肺癌细胞LALU皮下移植瘤模型,以病理动态形态学及透射电镜不同时期肿瘤血管生成状态。结果：光镜显示,人肺癌移植瘤第2天至第10天之间可分为血管生成前期和血管形成期,在肿瘤血管生成的第20天出现肺转移灶。电镜显示,人肺癌移植瘤第2天出现成血管细胞,第4－10天,不成熟血管内皮细胞逐渐形成务管腔伴有较完整的新生基底板,内皮细胞趋向成熟发展,第20天肿瘤血管内皮细胞更成熟,部分区域新生毛细血管基底板发育不全或缺陷。在以上全过程中癌细胞突起直接与成血管细胞、血管内皮细胞及血管壁相连。结论：人肺癌细胞可诱导自身肿瘤血管生成,病理形态学及电镜有其特征性形态学改变,且与转移密切相关,为肺癌的血管导向治疗提供了重要依据。     

Antitumor efficacy of viral therapy using genetically engineered Newcastle disease virus [NDV(F3aa)-GFP] for peritoneally disseminated gastric cancer

Peritoneal dissemination is a common and fatal clinical manifestation of gastric cancer with few effective therapies available. Natural Newcastle disease virus (NDV) has been shown to be an effective oncolytic agent, and recent advances now allow genetic manipulation of this virus to improve cancer killing and safety. This study was designed to investigate the effectiveness of a genetically engineered NDV in the treatment of peritoneally disseminated gastric carcinoma. NDV mutant virus containing a modified F cleavage site and insertion of enhanced green fluorescent protein (GFP), NDV(F3aa)-GFP, was tested in vitro against human gastric cancer cells by standard cytotoxicity at different multiplicities of infection. To test NDV(F3aa)-GFP in vivo in a peritoneal carcinomatosis gastric tumor model, MKN-74 human gastric cancer cells were injected intraperitoneally (IP) in severe combined immunodeficient mice. Mice were treated with NDV(F3aa)-GFP either once or multiple times after tumor challenge. Effective killing of MKN-74 cells by NDV(F3aa)-GFP was found in vitro. This cancer killing was dose-related and correlated with viral replication. GFP expression was a good marker of infection. The virus was also effective as an antitumor therapy in a peritoneal cancer model that simulates clinical disease. Half the animals treated with virus had no evidence of disease. Genetically engineered NDV [NDV(F3aa)-GFP] administered IP is an effective antitumor therapy against peritoneal carcinomatosis from human gastric cancer in a xenograft model, without significant toxicity. These data provide further rationale for clinical trials involving NDV for peritoneal carcinomatosis from gastric cancer.     

The SCID mouse: an experimental model for endometriosis

The purpose of this study was to validate the suitability of the severe combined immunodeficient (SCID) mouse as an experimental model for endometriosis, by defining the morphological and histological features of induced endometrial implants, and characterizing specific biochemical properties of these implants. Human secretory endometrial tissues were injected into the peritoneal cavity of SCID/SCID CB17 mature female mice. Successful peritoneal implantation was observed in 55 of 57 (96.5%) SCID mice and consisted of circumscribed elevated nodules. Haematoxylin-eosin staining of implanting lesions demonstrated the presence of endometrial glandular tissue in a mixed background of stromal and inflammatory cells. When progesterone was administered to mice, epithelial glands underwent well-defined secretory changes. Immunohistochemical analysis using polyclonal human pan-cytokeratin antibodies demonstrated selective positive staining in the glandular epithelium of the human implants with none in the surrounding stroma. In-situ hybridization analysis using complement component 3 cDNA radiolabelled riboprobes yielded significantly more intense signals in glands compared to stroma. As human endometrial implants in SCID mice were shown to retain specific histological, functional and biochemical properties, we conclude that the SCID mouse is an attractive animal model for the study of endometriosis.     

Absence of host-secreted protein acidic and rich in cysteine (SPARC) augments peritoneal ovarian carcinomatosis

The matricellular glycoprotein SPARC (secreted protein acidic and rich in cysteine) possesses multifaceted roles in modulation of cell-matrix interactions, as well as tumor growth and metastasis. To investigate the influence of host-derived SPARC on peritoneal dissemination of ovarian cancer, we established a murine model that faithfully recapitulates advanced human disease by intraperitoneal injection of syngeneic ID8 ovarian cancer cells into SPARC-null and wild-type mice. Compared to wild-type mice, SPARC-null mice showed significantly shorter survival and developed extensive nodular peritoneal dissemination with hemorrhagic ascitic fluid accumulation. Ascitic fluid collected from SPARC-null mice showed significantly augmented levels and activity of vascular endothelial growth factor and gelatinases. Immunohistochemical analysis of tumor nodules from SPARC-null mice revealed higher proliferation and lower apoptosis indices with minimal staining for major extracellular matrix constituents. In vitro, SPARC significantly suppressed adhesion to and invasion of various peritoneal extracellular matrix constituents by murine and human ovarian cancer cell lines. Our findings suggest that SPARC ameliorates ovarian peritoneal carcinomatosis through abrogation of the initial steps of disease pathogenesis, namely tumor cell adhesion and invasion, inhibition of tumor cell proliferation, and induction of apoptosis. Thus, SPARC represents an important therapeutic candidate in ovarian cancer.     

Adoptive immunotherapy of mucin1 expressing adenocarcinomas with mucin1 stimulated human peripheral blood mononuclear cells

Mucin1 stimulated hematopoietic mononuclear cells (M1SHMC) from patients with breast cancer, adoptively transferred to non-obese diabetic, severe combined immunodeficient (NOD SCID) mice, extended survival in a therapy model of gross adenocarcinoma and prevented tumor growth in a model of minimal disease. M1SHMC exhibited specific lysis of a human breast adenocarcinoma cell line expressing mucin1, MCF-7 and produced interferon gamma. M1SHMC were injected intraperitoneally (IP) in NOD SCID mice after gross, palpable tumors appeared after MCF-7 were injected subcutaneously (SC). Survival was increased as compared to no M1SHMC controls. However tumors eventually regrew in all mice. To determine whether minimal disease (MD) could be controlled, NOD SCID were injected with MCF-7 cells, and on the same day, injected IP with M1SHMC. The M1SHMC injected mice were protected from tumor growth. These results imply that M1SHMC can prolong survival, but not cure NOD SCID mice bearing gross palpable adenocarcinomas. However in a minimal disease model tumor growth was prevented.     

Inhibitory effects of a specific phage-displayed peptide on high peritoneal metastasis of gastric cancer

Peritoneal dissemination in gastric cancer is the most frequent cause of the noncurative resection and recurrence after curative resection. We, therefore, evaluated the feasibility of a peptide, which was obtained by screening a random phage display library, in the treatment of peritoneal metastases of gastric cancer. In this study, a novel cell line, GC9811-P, with a high potential peritoneal metastasis of gastric cancer derived from its parental cell line, GC9811, was established. Using a phage display library, we isolated a specific peptide that selectively bound to GC9811-P cells rather than its parental GC9811cells. The isolated phage-displaying peptide, SMSIASPYIALE (named peptide PIII), was obtained after four rounds of selection, showing a tendency to preferentially bind to GC9811-P cells compared with a panel of other gastric cancer cell lines, and preferentially accumulate in peritoneal metastasis tumor tissue in comparison with control organs, peritoneum, liver, pancreas, spleen, lung, and kidney. Further study showed that synthetic peptide PIII could significantly inhibit adhesive and invasional ability of GC9811-P cells and could effectively block the corresponding phage binding to the GC9811-P cells, whereas, exposure of the cells to various concentrations of peptide PIII showed no obvious cell growth inhibition. Furthermore, a highly reproducible animal experimental model of gastric cancer with peritoneal dissemination was established in nude mice by injecting a suspension of the cell line into the gastric wall of nude mice. Animals intraperitoneally treated with peptide PIII in this model or another animal model of gastric cancer with peritoneal dissemination established using MKN45 cells showed suppressed tumor metastasis to peritoneum and significantly prolonged survival. In conclusion, the selected peptide PIII was a biologically active peptide and could effectively inhibit peritoneal dissemination of gastric cancer.     

Correlation between melanoma angiogenesis and the mesenchymal stem cells and endothelial progenitor cells derived from bone marrow

Endothelial progenitor cells (EPC) reportedly differentiate into endothelial cells and participate in angiogenesis, including neovascularization at sites of neoplastic development. Recently, we reported that Flk+/CD31-/CD34- mesenchymal stem cells (MSC) possess the potential of differentiating into both endothelial and hematopoietic cells. We hypothesized that these MSC contribute to tumor angiogenesis. This concept is controversial and this study was undertaken to address this controversy. We show that progeny of human MSC as well as differentiated endothelial cells possess the ability to participate in tumor angiogenesis. When human marrow-derived MSC were injected into tail veins of severe combined immunodeficient (SCID) mice engrafted with human malignant melanoma, human cells incorporated into tumor vessels. Moreover, human-derived endothelial cells were identified in the walls of mouse tumor vessels by immunohistology. We report for the first time that similar results are obtained when mice carrying malignant melanoma are injected with differentiated human endothelial cells. Thus, we demonstrate that both differentiated endothelial cells from tissue peripheral to that of a tumor as well as progeny of human MSC have similar capacities to participate in angiogenesis.     

Inhibition of cervical lymph node metastasis by marimastat (BB-2516) in an orthotopic oral squamous cell carcinoma implantation model

Activation of matrix metalloproteinase-2 (MMP-2) is a common event in head and neck squamous cell carcinoma. An OSC-19 cell line, derived from human oral squamous cell carcinoma and known to metastasize to cervical lymph nodes, was implanted into the lingual margin of mice. The effect of marimastat (BB-2516), a broad MMP inhibitor, on the suppression of regional cervical lymph node metastasis was evaluated with an orthotopic implantation nude mice model. Marimastat was given immediately after OSC-19 implantation and continuously administered by an osmotic pump. The mice were divided into three groups by marimastat dose; Group A; 0 mg/kg/day, Group B; 30 mg/kg/day, and Group C; 150 mg/kg/day. Twenty-one days after implantation, primary oral tumors and cervical lymph nodes were resected. Cervical lymph node status was microscopically examined. Activation of MMP-2 in primary oral tumor was examined by gelatin zymography. Both cervical lymph node metastasis and activation of MMP-2 were significantly suppressed in Group C (P<0.05). Moreover, the Group C mice had a significantly better survival than group A (P=0.0026). There was a significant difference between Group A and Group C in terms of proliferation of tumor cells by proliferating cell nuclear antigen immunostaining (P=0.0120). These results suggest a positive role for marimastat in the inhibition of MMP-2 activation and prevention of cervical lymph node metastasis in oral squamous cell carcinoma (OSCC). Improvement of survival in patients with OSCC could be expected using adjuvant therapy with marimastat. This revised version was published online in July 2006 with corrections to the Cover Date.     

在不同转移潜能人肺癌细胞移植瘤中血管性血友病因子的表达

目的探讨血管性血友病因子(vWF)在不同转移潜能人肺癌裸鼠移植瘤中的表达。方法体外培养低转移及高转移人肺癌细胞系A549和D95,Transwell小室法检测肿瘤细胞体外侵袭和迁移能力;10只Balb/c裸小鼠随机分为A549组和D95组(n=5)。0. 2 mL两种肿瘤细胞悬液(浓度约为1×10^7/mL)分别皮下注入A549组及D95组裸小鼠左上肢,构建人肺癌裸鼠移植瘤模型。观察vWF对裸小鼠移植瘤生长的影响;ELISA检测外周血vWF水平;免疫组化检测移植瘤vWF蛋白表达;免疫印记检测移植瘤、肺和肝vWF蛋白表达。结果 D95肺癌细胞体外侵袭和迁移细胞数多于A549肺癌细胞(P<0. 05);D95组裸小鼠于皮下接种后第5天全部出现瘤结节,A549组于第7天全部出现;与A549组相比,D95组裸小鼠移植瘤质量及肺转移瘤结节数显著增多(P<0. 05),D95组移植瘤、肺及肝组织vWF蛋白的表达显著高于A549组(P<0. 01)。结论 vWF与人肺癌细胞的转移潜能密切相关。     

Glycosylation patterns of the human colon cancer cell line HT-29 detected byHelix pomatia agglutinin and other lectins in culture,in primary tumours and in metastases in SCID mice

Human colonic cancer cells (HT-29, 10 ⁷ cells/dose) were injected subcutaneously between the scapulae of 19 severe combined immunodeficient (SCID) mice. After 19 days, large tumours had developed in 18 out of the 19 animals and the mice were then killed. Metastases were detected in the lungs of 16 animals but not in other organs investigated. Surgical removal of the primary tumour in another group of five animals led to a prolonged survival and further growth of metastases in the lungs. HT-29 injection into the tail vein (n=5)resulted in colonization of the lungs. The tumours that developed in the animals were signet cell carcinomas; these forms are not seen in HT-29 cells in culture. Glycoconjugate expression of the tumours was assessed using several lectins. In many cases the results indicated a stability of lectin-binding patterns from cell culture conditions to implantation into the SCID mice. This was true for the lectin Helix pomatia agglutinin (HPA), the binding of which is associated with a high metastatic potential in some human tumours, including colon cancer. All the primary tumours and metastases were HPA positive. This xenograft tumour model seems to be a clinically relevant system for the study of glycoconjugate expression in human colon cancer cells and their metastases.     

Angiogenesis and metastasis marker of human tumors.

Tumor growth and metastasis are dependent on angiogenesis. Therefore, certain angiogenesis markers may be useful as metastasis markers and/or the targets for antiangiogenic therapy. We and others have been studying endoglin(EDG; CD105) for such purposes. EDG is a proliferation-associated antigen of endothelial cells and essential for angiogenesis. In addition, EDG is a component of the transforming growth factor(TGF)-beta receptor complex. Expression of EDG is up-regulated in tumor-associated angiogenic vasculature compared with normal tissue vasculature. Microvessel density detected for EDG expression in breast cancer tissues showed a statistically significant correlation with overall and disease-free survival. In addition, elevated serum EDG was associated with metastasis in patients with colorectal, breast, and other solid tumors. On the other hand, We have been targeting EDG on tumor vasculature to suppress tumor growth and metastasis by systemic(i.v.) administration of anti-EDG monoclonal antibodies(mAbs) and immunoconjugates(IMCs). To thid end, we have been using three animal models, i.e., severe combined immunodeficient(SCID) mouse model of MCF-7 human breast cancer, human skin/SCID mouse chimera model bearing MCF-7 tumor, and syngeneic metastasis model of colon-26 adenocarcinoma cells in BALB/c mice. In addition, antiangiogenic activities of anti-EDG mAbs and IMCs were evaluated in mice using the dorsal air sac assay. The IMCs were prepared by coupling deglycosylated ricin A-chain or 125I to individual anti-EDG mAbs. These anti-EDG IMCs and mAbs showed substantial antitumor efficacy and antimetastatic activities without showing severe toxicity. Recently, we generated a recombinant human/mouse chimeric anti-EDG mAb to facilitate clinical application of the mAb.     

Role of pancreatic stellate cells in pancreatic cancer metastasis

Pancreatic stellate cells (PSCs) produce the stromal reaction in pancreatic cancer (PC), and their interaction with cancer cells facilitates cancer progression. This study investigated the role of human PSCs (hPSCs) in the metastatic process and tumor angiogenesis using both in vivo (orthotopic model) and in vitro (cultured PSC and PC cells) approaches. A sex mismatch study (injection of male hPSCs plus female PC cells into the pancreas of female mice) was conducted to determine whether hPSCs accompany cancer cells to metastatic sites. Metastatic nodules were examined by fluorescent in situ hybridization for the presence of the Y chromosome. Angiogenesis was assessed by i) immunostaining tumors for CD31, an endothelial cell marker; and ii) quantifying human microvascular endothelial cell (HMEC-1) tube formation in vitro on exposure to conditioned media from hPSCs. Transendothelial migration was assessed in vitro by examining the movement of fluorescently labeled hPSCs through an endothelial cell monolayer. Human PSCs i) were found in multiple metastatic sites in each mouse injected with male hPSCs plus female PC cells; ii) increased CD31 expression in primary tumors from mice injected with MiaPaCa-2 and hPSCs and stimulated tube formation by HMEC-1 in vitro; and iii) exhibited transendothelial migration that was stimulated by cancer cells. Human PSCs accompany cancer cells to metastatic sites, stimulate angiogenesis, and are able to intravasate/extravasate to and from blood vessels.     

The development of a novel immunotherapy model of human ovarian cancer in human PBL-severe combined immunodeficient (SCID) mice.

The reported ability of SCID mice to accept xenografts of both human tumors and peripheral blood lymphocytes (PBL) provides the potential for the development of novel immunotherapy models in these animals. This study describes the development of a novel small animal model of human ovarian cancer. This was achieved by engrafting a human ovarian cancer cell line (Ovan-4) into the peritoneal cavity of immunodeficient SCID and immune reconstituted human PBL-SCID mice. When transplanted to SCID mice this cell line exhibited growth characteristics similar to the clinical disease observed in patients with implantation of metastatic nodules onto the interior surface of the peritoneal wall. Reconstituted human PBL-SCID mice challenged with identical numbers of Ovan-4 cells exhibited a significant increase in survival time, suggesting a role for cells of the human immune system in preventing the development of this type of malignancy in vivo. Furthermore, vaccination of human PBL-SCID mice against Ovan-4 produced tumour-specific human antibodies in the serum of these animals. Animals reconstituted with CD8-depleted PBL exhibited increased serum immunoglobulin levels and produced enhanced anti-Ovan-4 activity after vaccination. Subsequent challenge of these animals with Ovan-4 revealed a further increase in survival time. These results suggest that human antibodies may have a role in immunity against ovarian cancer and could be of therapeutic value in this type of disease.     

白藜芦醇诱导人胃癌原代细胞裸鼠移植瘤凋亡的研究

目的：探讨白藜芦醇诱导人胃癌原代细胞裸鼠移植瘤凋亡的机制。 方法： 建立人胃癌原代细胞裸鼠移植瘤模型，采用瘤旁注射的方法，用不同剂量的白藜芦醇进行治疗，并设对照，用透射电镜和TUNEL法检测肿瘤组织细胞凋亡情况，免疫组织化学法和RT-PCR法检测肿瘤组织的凋亡相关基因bcl-2和bax的表达情况。 结果： 白藜芦醇对胃癌原代细胞移植瘤有明显的抑制作用； 透射电镜和TUNEL法发现白藜芦醇导致移植瘤内大量细胞发生凋亡；免疫组织化学法发现白藜芦醇注射组的Bcl-2蛋白阳性细胞率（3.92％±0.85％、5.70％±0.84％和11.86％±0.97％）明显低于两个对照组（P<0.05），两个对照组（27.56％±1.40％和29.48％±0.51％）之间无显著差异；Bax蛋白阳性细胞率（52.30％±1.54％、45.72％±0.88％和40.02％±1.20％）明显高于两个对照组（P<0.05），两个对照组（20.88％±0.91％和19.34％±0.35％）之间无显著差异； RT-PCR法发现白藜芦醇注射组的bcl-2 mRNA条带强度随剂量的增大而递减，并明显低于两个对照组（P<0.05），两个对照组之间无显著差异（P>0.05）；bax mRNA条带强度递增，并明显高于两个对照组（P<0.05），两个对照组之间无显著差异（P>0.05）。 结论： 白藜芦醇对胃癌原代细胞移植瘤有明显的抑制作用，通过下调bcl-2 的表达和上调bax 的表达而诱导胃癌裸鼠移植瘤细胞发生凋亡，是其抗胃癌作用的机制之一。     

The fate of human peripheral blood lymphocytes after transplantation into SCID mice

Human peripheral blood lymphocytes (hu-PBL) can be adoptively transferred by intraperitoneal injection into mice with severe combined immunodeficiency (SCID). The transplanted lymphocytes can produce immunoglobulin (Ig), respond to antigens, and survive for months in this chimeric model (hu-PBL SCED). However, whether the lymphocytes actually repopulate and reconstitute lymphoid structures and organs has been subject of some debate. To address this question and to characterize the hu-PBL SCID model better, we employed a novel technique for the identification of human cells in xenogeneic mice. We used fluorescence in situ hybridization (FISH) with a biotinylated DNA probe to all human centromeres. We demonstrated that FISH could be used to detect human cells when they accounted for less than 1 % of human/mouse cell mixtures; it could also be employed for the identification and localization of individual human cells in tissue sections. By using FISH, we studied 31 SCID mice injected with 1.5 × 10⁷ ?4 × 10⁷ hu-PBL via intravenous (i.v.) or intraperitoneal (i.p.) routes. In the 6 i.v -injected mice, we found that the human cells were removed from the circulation into the lung within 1 h. In 22 of 25 i.p -injected animals, 90–3716 μg/ml of human IgG was found in the sera at 3 to 13 weeks after transplantation (a.t.). Human cells colonized the peritoneal cavity and persisted for up to 13 weeks a.t. and, in the 12 mice studied, accounted for 4 % to 57 % of the cells in the peritoneal fluid. However, only rare, isolated human cells were found in the spleen, blood, bone marrow, lung or Peyer's patches. In 7 of 19 mice that received hu-PBL i.p. from Epstein-Barr virus-seropositive donors, we found masses of human cells usually beneath the peritoneal lining but sometimes infiltrating normal tissue. We conclude that FISH offers a simple means for accurate identification of human cells in the xenogeneic mouse. Although there is colonization of the peritoneal cavity in most mice, and development of lymphoid masses in some, there is no reconstitution of lymphoid structures and only minimal engraftment of lymphoid organs by human cells in conventionally-prepared hu-PBL SCID constructs.     

人直肠癌组织中脑特异性新生血管抑制因子1表达及该因子抗肿瘤血管生成作用的观察

 目的 探讨直肠癌组织中脑特异性血管生长抑制因子1（BAI1）基因的表达情况及BAI1对肿瘤血管生成和肿瘤生长的作用。 方法 提取20例直肠癌患者（II期6例，III期14例）手术切除的直肠癌、无转移淋巴结、有转移淋巴结组织和直肠腺癌细胞株CC95总RNA，应用逆转录聚合酶链反应方法检测BAI1的表达。将背部皮下接种CC95细胞（1×105个）的BALB/C裸鼠随机分成3组，每组10只，分别在接种后第1、3、5、7、9天于荷瘤部位注射RPMI1640、腺病毒载体（Ad-LacZ）或基因重组BAI1表达腺病毒载体（Ad-hBAI1），每次0.2 ml，载体含量2 μg。每组中5只裸鼠于接种后第15天断颈处死，取肿瘤组织行抗Ⅷ因子抗体染色,观察微血管密度（MVD）；另5只接种后每天观察肿瘤体积的变化，共观察21 d。 结果 20例患者中，7例（临床II期6例、III期1例）肿瘤组织BAI1表达呈阳性，13例无表达；无转移淋巴结均呈阳性表达，有转移淋巴结均无表达。CC95细胞均无BAI1表达。在BALB/C裸鼠肿瘤移植实验中，Ad-hBAI1组肿瘤组织内血管增生受到明显抑制，MVD（6.2±2.3）明显低于RPMI1640组（16.7±1.9）和Ad-LacZ组（13.4±4.3）（均为P＜0.05）；肿瘤生长速度明显低于RPMI1640组和Ad-LacZ组（均为P＜0.01）。 结论 BAI1在人直肠癌及转移的淋巴结组织中表达降低；局部注射Ad-hBAI1表达载体可抑制肿瘤血管生成并抑制肿瘤的生长。