双特异抗体导向骨髓中活化T细胞清除骨髓中肿瘤细胞

先用抗CD3单抗和IL-2激活肺癌患者骨髓中的T淋巴细胞,然后按不同比例将人肺癌细胞株PC84045细胞掺入到骨髓细胞中,再加入能同时识别CD3抗原和人肺癌细胞表面抗原的双特异抗体CD3-ALTO4,在IL-2和IL-3存在下共同培养3d后测试.在T细胞与PC84045癌细胞之比为8:1时,对PC84045肺癌细胞净化效率为4Logs,16;1时为5Logs,并在NC裸鼠体内证实清除效果是确切的.净化后的骨髓CFU-GM、BFU-E收获率均>85%(P>0.05;P>0.05);而且净化后的骨髓中的T细胞仍保持着对PC84045肺癌细胞很强的杀伤能力(P<0.01).双特异抗体CD3-ALTO4能有效地导向骨髓中活化T细胞清除污染的肿瘤细胞,造血干细胞无明显损伤.本文方法处理的骨髓细胞还保持着高度特异的细胞毒活性,移植这样的骨髓细胞将会在体内进一步清除化疗残存的肿瘤细胞,加速移植后免疫功能再建,降低复发.     

Suppressive Activity of Interleukin 4 on the Induction of Antigen-specific Cytotoxic T Cells in Humans

The effect of interleukin 4 (IL 4) on the induction of cytotoxic T cells (CTL) was studied by using human peripheral blood lymphocytes in vitro. IL 4 suppressed the induction of CTL specific for allogeneic antigens in a concentration-dependent manner. However, IL 4 did not suppress proliferative responses induced with allogeneic antigens or mitogens. The suppressive effect of IL 4 on CTL induction was observed when IL 4 was added at the early period of the CTL induction culture, but not at the later period. Furthermore, IL 4 did not suppress the effector function of CTL to target cells. IL 4 suppressed the production of IL 1 by monocytes/macrophages and the production of IL 2 and the expression of IL 2 receptors on T cells. Moreover, IL 4 suppressed the induction of lymphokine-activated killer cells. These results suggest that IL 4 has a suppressive activity on the induction of killer cells in humans.     

调节性T细胞与恶性肿瘤的过继免疫治疗

过继免疫治疗利用自身免疫细胞进行体外扩增培养,然后回输到体内,它通过增强机体免疫力来对抗肿瘤,对肿瘤细胞具有靶向杀伤性作用,是最有可能彻底清除手术或放化疗后残存肿瘤细胞的治疗手段.但是,调节性T细胞(Regulatory T cells,Tregs)在多种恶性肿瘤患者外周血及微环境中普遍存在着比例上调的情况,这不仅会导致机体固有免疫的负性平衡,还会影响过继免疫治疗的疗效,使其不能发挥最大的抗肿瘤作用.通过对Tregs日渐深入的了解,人们已经研究出多种抑制及消除Tregs的治疗策略.     

Quercetin Effects on Cell Cycle Arrest and Apoptosis and Doxorubicin Activity in T47D Cancer Stem Cells

Backgrounds: Targeting breast cancer stem cells with the CD44+/CD24- phenotype is critical for complete eradication of cancer cells due to its Self-renewal, differentiation, and therapeutic resistance ability. Quercetin is a popular flavonoid with lower adverse effects and has anti-tumor properties. Therefore, we assessed the anticancer activity of Quercetin and Doxorubicin alone and in combination in the T47D cells of human breast cancer and their isolated Cancer stem cells (CSCs). Materials and Methods: The human breast cancer cell line T47D was used for this experiment. T47D CSCs were isolated by magnetic bead sorting using the MACS system. The anticancer activity of Quercetin and Doxorubicin alone and in combination were evaluated using MTT cytotoxicity assay and cell cycle distribution and apoptosis induction by flow cytometry analysis. Results: We have shown that almost 1% of T47D cell populations are made up of CD44+/CD24- cells, which considered as cancer stem cells. Quercetin and Doxorubicin alone or in combination inhibited cell proliferation and induced apoptosis in breast cancer T47D cells and in lower extent in CD44+/CD24- cells. Quercetin significantly strengthened Doxorubicin’s cytotoxicity and apoptosis induction in both cell populations. Quercetin and Doxorubicin and their combination induced G2/M arrest in the T47D cells and to a lesser extent in isolated CSCs. A value of p < 0.05 was considered as indicating a statistically significant difference. Conclusion: These outcomes suggested that CSCs are a minor population of cancer cells, which play a significant role in drug resistance by being quiescent, slow cycling and resistance to apoptosis. Furthermore, our data showed that adding Quercetin to Doxorubicin is an effective approach for the treatment of both CSCs and bulk tumor cells.     

Induction of MicroRNA-9 Mediates Cytotoxicity of Curcumin Against SKOV3 Ovarian Cancer Cells

Background: Curcumin, a phenolic compound extracted from the rhizomes of Curcuma longa, has showncytotoxic effects against a variety of cancers. The aim of this study was to identify potential microRNA (miRNA)mediators of the anticancer effects of curcumin in ovarian cancer cells. Materials and Methods: SKOV3 ovariancancer cells were treated with curcumin (10-60 μM) and miR-9 expression, cell proliferation, and apoptosiswere assessed. The effects of miR-9 depletion on curcumin-mediated growth suppression were also examined.Phosphorylation of Akt and forkhead box protein O1 (FOXO1) was measured in cells with miR-9 overexpressionor curcumin treatment. Results: Curcumin caused a significant and dose-dependent increase of miR-9 expressionin SKOV3 cells, while significantly impeding cell proliferation and stimulating apoptosis. Depletion of miR-9significantly (p<0.05) attenuated the growth-suppressive effects of curcumin on SKOV3 cells, coupled withreduced percentages of apoptotic cells. In contrast, overexpression of miR-9 significantly enhanced the cleavageof caspase-3 and poly(ADP-ribose) polymerase and promoted apoptotic death in SKOV3 cells. Western blotanalysis showed that both miR-9 overexpression and curcumin similarly caused a significant (p<0.05) declinein the phosphorylation of Akt and FOXO1, compared to untreated cells. Conclusions: The present studyprovided evidence that curcumin exerts its cytotoxic effects against SKOV3 ovarian cancer cells largely throughupregulation of miR-9 and subsequent modulation of Akt/FOXO1 axis. Further studies are needed to identifydirect targets of miR-9 that mediate the anticancer effects of curcumin in ovarian cancer cells.     

Augmentation of Murine Lymphokine-activated Killer Cell Induction by a Factor Produced by Nocardia rubra Cell Wall Skeleton-stimulated T Cells

Four-hour exposure of C3H/HeN mouse spleen cells to Nocardia rubra cell wall skeleton (N-CWS) before 4-day culture with a suboptimal dose of human recombinant interleukin 2 (rIL 2) augmented the induction of lymphokine-activated killer (LAK) cell activity, whereas the treatment with N-CWS alone induced no cytotoxicity. In accordance with this, the IL 2 binding activity of spleen cells was augmented by combined stimulation with N-CWS and rIL 2. The augmented cytotoxicity was mediated by Thy-1.2⁺, Lyt-1.1⁻, Lyt-2.1⁻ and asialo GM₁⁺ cells. Cell cultures in diffusion chambers revealed that N-CWS-treated spleen cells produced a LAK cell induction-helper factor (LAK-helper factor, LHF) when cultured with rIL 2. The LHF production required Thy-1.2⁺, Lyt-1.1⁺, Lyt-2.1⁺ and asialo GM₁⁻ cells, and the coexistence of unstimulated accessory cells was also essential for the LHF production. Cells responding to both LHF and rIL 2 to generate LAK activity were Thy-1.2⁻, Lyt-1.1⁻, Lyt-2.1⁻ and asialo GM₁⁺. The culture fluid of spleen cells stimulated with both N-CWS and rIL 2 contained no tumor necrosis factor (TNF) activity, and the additional stimulation with N-CWS caused no production of either IL 2 or interferon (IFN). Murine recombinant interleukin la (Mu-rIL 1α) could not replace the augmentative effect of N-CWS on LAK cell induction. These results suggest that in the presence of rIL 2, N-CWS stimulates murine T cells to produce LHF that is probably distinct from IL 1, IL 2, TNF and IFN.     

大肠癌患者手术前后T细胞及其活化抗原表达的变化与临床意义

研究大肠癌患者围手术期的免疫状态。应用流式细胞仪检测３５例大肠癌患者手术前后Ｔ细胞表面６种抗原标志，并与良性病变患者进行对比分析。结果大肠癌患者术前ＣＤ３、ＣＤ４、ＣＤ４／ＣＤ８、ＣＤ１６、ＣＤ６９均低于对照组，ＣＤ８高于对照组（Ｐ＜０．０５），ＣＤ３＋／ＨＬＡ－ＤＲ＋与对照组相同；术后ＣＤ８降低，ＣＤ３、ＣＤ４、ＣＤ４／ＣＤ８、ＣＤ１６、ＣＤ６９、ＣＤ３＋／ＨＬＡ－ＤＲ＋均升高（Ｐ＜０．０５），活化Ｔ细胞ＣＤ３＋／ＨＬＡ－ＤＲ＋明显高于对照组（Ｐ＜０．０１）。大肠癌患者术前免疫功能低下，术后活化Ｔ细胞明显增多，切除肿瘤有益于改善患者细胞免疫功能，故减瘤手术是必要的。     

胃癌组织中嗜银和亲银内分泌细胞及意义

应用嗜银和亲银染色技术研究50例胃癌组织中内分泌细胞。50例胃癌伴内分泌细胞阳性22例,其中4例胃癌内分泌细胞阳性率≥50%;高分化、癌细胞浸润深度Ⅰ级和未见淋巴结转移胃癌内分泌细胞阳性病例率,明显低于未分化、癌细胞浸润深度Ⅲ级和淋巴结转移胃癌;22例内分泌细胞阳性病例免疫组化发现hcG阳性6例、GN阳性5例和SM阳性3例。结果提示伴内分泌细胞胃癌恶性度高和预后差,部分病例内分泌细胞可分泌一些肽类激素。     

参芪抑癌液与细胞因子治疗晚期肝癌、胰腺癌临床观察

目的观察比较参芪抑癌液、细胞因子对晚期肝、胰腺癌治疗效果。方法不能手术切除的晚期肝、胰腺癌８２例随机分为３组,分别给予参芪抑癌液、细胞因子、常规化疗药物治疗,进行疗效对比。结果参芪抑癌液、细胞因子组在延长患者生存时间（参芪组１１３月;细胞因子组９５月;化疗组８５月）、改善症状等方面均优于常规化疗组。结论晚期肝、胰腺癌治疗中为改善患者生存质量、延长生存时间,可采用参芪抑癌液、细胞因子进行治疗。     

Suppressor T cells in B-cell chronic lymphocytic leukaemia: relationship to clinical stage

In 32 patients with B-cell chronic lymphatic leukaemia (CLL), OKT8+ suppressor T cells were increased in relative (mean 54 +/- 14%; normal mean 34 +/- 6%) and absolute (1.8 +/- 1.6 X 10(9)/1; normal range 0.3-0.6 X 10(9)/1) numbers. OKT4+ helper cells were reduced in relative (mean 53 +/- 15%; normal mean 65 +/- 7%) but not absolute (1.7 +/- 1.4 X 10(9)/1; normal range 0.6-1.4 X 10(9)/1) numbers. Essentially identical results were obtained in treated and untreated patients. There was no significant association between T-cell subset numbers and clinical stage, whether assessed by the Rai classification or the more recent Binet system, although the OKT4+/8+ ratio was slightly lower in advanced disease. The study suggests that the immunoparesis so characteristic of CLL may be attributable to increased suppressor T-cell activity.     

Expression and Clinical Significance of Myeloid Derived Suppressor Cells in Chronic Hepatitis B Patients

We here document discovery of expression profile of myeloid derived suppressor cells (MDSCs) in chronichepatitis B (CHB) patients and changes in the course of disease. The study population was composed of 75outpatient HBV cases and 15 healthy control cases. Peripheral blood samples were collected for separationof mononuclear cells. Levels of MDSCs labeled with Lin-DR-CD11b+CD33+ obtained from peripheral bloodmononuclear cells (PBMC), were revealed to have significant differences between the CHB and other groups.They were 0.414% for health control cases and 0.226% for CHB cases (Z=-2.356, p=0.0189). It also observed thatthe group of HBeAg positive cases had significant difference in MDSCs/ PBMC median (X2=11.877, p=0.003),compared with group of HBeAg negative cases and the healthy control group. It suggested considerable MDSCsmight be involved in HBeAg immune tolerance. In addition, negative correlations between MDSCs/PBMC andparameters of ALT, AST and TBil, while positive correlation between MDSCs/ PBMC and ALB parameter werefound. Multiple comparisons between the four phases and health control phase again, there was a statisticallysifnificant difference (X2=17.198, p=0.002). Taken together, these findings may provide a new immunotherapystrategy for reduced the expression levels of MDSCs in CHB patients, through induction of an autoimmuneresponse to virus removal.     

结肠肿瘤干细胞巢研究—从潘氏细胞谈起

 近年来,对肿瘤干细胞巢的深入研究开启了肿瘤研究的“巢”时代,结直肠癌是实体肿瘤的典型代表,有较为典型的发生发展机制,是肿瘤干细胞巢研究的可靠模型。潘氏细胞是正常肠上皮的重要组成细胞,对肠干细胞具有重要的支持、保护等作用,是肠干细胞巢的关键成分之一。然而目前在结肠上皮恶性转化中,以潘氏细胞为代表的干细胞巢成分的作用如何尚有待研究,进一步研究潘氏细胞和肠干细胞的相互关系,从而推导其在结肠肿瘤不同发展阶段的可能作用,将有助于对肿瘤干细胞巢的进一步了解,同时也可为结直肠癌临床治疗提供潜在的新靶点。     

Apoptotic Effects of psiRNA-STAT3 on 4T1 Breast Cancer Cells in Vitro

Background: The aim of this study was to investigate the effect of a Lipofectamine2000 (Life2000) Transfection Reagent transfected psiRNA-STAT3 plasmid on 4T1 breast cancer cells. Materials and Methods: MTT was usedto detect the cell proliferation of breast cancer 4T1 cells at different periods (0h, 6h, 8h, 10h); the cell cycle was assessed by flow cytometry; variation of apoptosis and mitochondrial membrane potential was observed under a fluorescence microscope; immunohistochemical staining was used to determine the expression of caspase-3 and cyclin-D1 protein. Results: An obvious effect of inhibition to 4T1 cancer cells could be observed at 8h after the psiRNA-STAT3 was transfected. Typical alterations of apoptotic morphological features were visible in the psiRNA-STAT3 treatment group. Mitochondrial membrane potential decreased significantly, the number of cells was increased in G0/G1 phase, and the number of cells was decreased in S phase, and the data were statistically significant (p<0.05), compared with the Scramble and Mock groups. Expression of caspase-3 protein was increased significantly, while that of cyclin D1 was significantly decreased. Conclusions: Life2000 transfected psiRNA-STAT3 plasmid can inhibit 4T1 tumor cell proliferation and promote apoptosis of 4T1 tumor cells, which process depends on the regulation of expression of cyclin D1 and caspase-3 protein.     

Tumor-specific T Cells which Form Clusters with Dendritic Cells and Tumor Cells and Deliver Macrophage-activating Factors

T cells prepared from tumor (Meth A)-bearing mice were cocultured with homologous tumor cells and splenic dendritic cells to enrich tumor-specific T cells by the separation of clusters. T blasts generated from clusters were capable of inhibiting the in vivo tumor cell growth. The culture supernatant of clustering cells (CLSN) was effective in activating macrophages (MØ) to be cytostatic and cytocidal against tumor cells. Moreover, it was found that CLSN contains at least 3 distinct factors; one was identified as interferon-γ (IFN-γ), and the others are so far unidentified, but one acts synergistically with IFN-γ, possibly as the second signal, and the other cooperates with lipopolysaccharide but not with IFN-γ. We propose that the tumor-specific T cells secrete soluble mediators which cooperate with each other in MØ activation against tumor cells.     

Expression of the Breast Cancer Metastasis Suppressor 1 (BRMS1) maintains in vitro chemosensitivity of breast cancer cells

The Breast Cancer Metastasis Suppressor 1 (BRMS1) belongs to an expanding category of proteins called metastasis suppressors that demonstrate in vivo metastasis suppression while still allowing growth of the orthotopic tumor. Since BRMS1 decreases either the expression or function of multiple mediators implicated in resistance to chemotherapy (NF-κB, AKT, EGFR), we asked whether breast carcinoma cells expressing BRMS1 could be sensitized upon exposure to commonly used therapeutic agents that inhibit some of these same cellular mediators as BRMS1. In this report, we demonstrate that chemosensitivity of breast cancer cells is preserved in the presence of BRMS1. Further, BRMS1 does not change expression of AKT isoforms or PTEN, implicated in chemoresistance to common drug agents. Overall, our data with two different metastatic breast cancer cell lines indicates that BRMS1 expression status may not interfere with the response to commonly used chemotherapeutic agents in the management of solid tumors such as breast cancer. Since tumor protein expression analysis increasingly guides therapy decisions, our data may be of clinical benefit in disease management including profiling for BRMS1 expression before start of therapy.     

Tim-3 Expression by Peripheral Natural Killer Cells and Natural Killer T Cells Increases in Patients with Lung Cancer - Reduction after Surgical Resection

Background: The purpose of this study was to investigate Tim-3 expression on peripheral CD3-CD56+natural killer (NK) cells and CD3+CD56+ natural killer T (NKT) cells in lung cancer patients. Materials andMethods: We analyzed Tim-3+CD3-CD56+ cells, Tim-3+CD3-CD56dim cells, Tim-3+CD3-CD56bright cells, and Tim-3+CD3+CD56+ cells in fresh peripheral blood from 79 lung cancer cases preoperatively and 53 healthy controlsby flow cytometry. Postoperative blood samples were also analyzed from 21 members of the lung cancer patientcohort. Results: It was showed that expression of Tim-3 was significantly increased on CD3-CD56+ cells, CD3-CD56dim cells and CD3+CD56+ cells in lung cancer patients as compared to healthy controls (p=0.03, p=0.03 andp=0.04, respectively). When analyzing Tim-3 expression with cancer progression, results revealed more elevatedTim-3 expression in CD3-CD56+ cells, CD3-CD56dim cells and CD3+CD56+ cells in cases with advanced stages(III/IV) than those with stage I and II (p=0.02, p=0.04 and p=0.01, respectively). In addition, Tim-3 expression wassignificantly reduced on after surgical resection of the primary tumor (p<0.01). Conclusions: Tim-3 expressionin natural killer cells from fresh peripheral blood may provide a useful indicator of disease progression of lungcancer. Furthermore, it was indicated that Tim-3 might be as a therapeutic target.     

Induction of Mouse Anti-melanoma Cytotoxic and Suppressor T Cells in vitro by an Artificial Antigen, GM3-lactone

We investigated the ability of GM3-lactone liposomes to induce anti-melanoma T cell responses in mice. GM3-lactone liposomes, like murine B16 melanoma cells, induced anti-melanoma cytotoxic T cells (CTL) and also suppressor T cells (Ts). A small dose of GM3-lactone (0.0003 μg/ml) was enough to generate CTL in the in vitro primary response, whereas relatively large amounts of the antigen (0.03–0.3 μg/ml) were required for anti-melanoma Ts induction. As the epitope for anti-melanoma Ts is NeuAc but not NeuGc residue on GM3, and anti-melanoma CTL are effectively induced by either GM3(NeuAc) or GM3(NeuGc)-lactone liposomes, GM3(NeuGc)-lactone or GM3(NeuGc) liposomes have potent activity as an artificial melanoma antigen to induce anti-melanoma CTL in vitro .     

紫杉醇诱导人膀胱癌EJ细胞凋亡及与Bcl-2基因表达的关系

目的探讨紫杉醇对人膀胱癌EJ细胞凋亡的作用及其机制。方法体外培养人膀胱癌EJ细胞,以不同浓度紫杉醇处理12～72 h后,应用流式细胞仪检测凋亡率,测定细胞周期;原位杂交法检测紫杉醇作用后Bcl-2基因表达的变化。结果流式细胞仪检测结果发现紫杉醇诱导后的膀胱癌EJ细胞周期阻滞于G2/M期,高浓度紫杉醇（100、1 000 nmol/L）更明显,出现凋亡峰,细胞凋亡率随紫杉醇浓度的增加而增高;紫杉醇作用后Bcl-2基因表达发生明显变化,随紫杉醇浓度的增加而降低。结论紫杉醇能够诱导EJ细胞凋亡,细胞凋亡率随紫杉醇浓度的增加而增高;紫杉醇通过抑制Bcl-2基因表达而诱导细胞凋亡,为其诱导凋亡的作用机制之一。