L615小鼠白血病脾脏中存在抗肿瘤的L3T4~ T细胞

L615细胞是一株表型为L3T4~ 的体外培养的能表达一定程度肿瘤特异性移植排斥抗原的T淋巴细胞系.但它在移植的同系宿主体内并不能激发有效的抗肿瘤反应.本研究试图阐明L615细胞移植后患鼠体内是否存在着抗肿瘤的T细胞及其特性,L615小鼠预先经照射灭活的L615细胞接种,然后再攻击具有可供选择性杀灭标志的L615细胞(L615HPRT~-).于活瘤细胞攻击后7天左右取脾,制成单细胞悬液,经含低浓度HAT培养液的选择性培养,再给以照射灭活的L615细胞和正常的L615鼠脾细胞致敏.得到体外可长期培养的细胞系.分析其细胞表型并测定细胞毒性,探     

转输正常小鼠脾细胞悬液对小鼠白血病（L615）保护作用机理的研究

本室过去的工作表明转输正常小鼠脾细胞悬液对小鼠白血病L615有明显的保护作用,并且可以用普通条件下及SPF(SpecificPathogen free)条件下饲养的小鼠重复。本实验在此基础上探讨了诱导保护作用的机制。材料和方法 (一)动物:所用动物均为615近交系小鼠,体重20-26克,自中国医学科学院血液学研究所引入,在本实验室饲养繁殖。 (二)瘤株:L615,系615系小鼠的T淋巴细胞性白血病,移植成功率为100％,无     

ehCGβ肿瘤基因疫苗诱生的效应脾细胞过继免疫抗肿瘤作用的研究

目的:探讨ehCGβ肿瘤基因疫苗诱生的效应脾细胞过继免疫在抗肿瘤中的作用.方法:通过转染建立稳定表达ehCGβ和HBV-preS2/S的细胞株Sp2/0-ehCGβ和Sp2/0-preS2/S.以TR421-hCGβ质粒实施基因免疫,免疫后检测小鼠脾细胞,特异性细胞毒活性;同期将脾细胞过继免疫给正常小鼠,以过继TR421-hCGβ质粒免疫小鼠脾细胞为实验组、过继TR421质粒免疫小鼠脾细胞为对照组,检测脾细胞杀伤效应.结果:效应脾细胞对SP2/0-ehCGβ的杀伤率明显高于Sp2/0-preS2/S(P＜0.05).Sp2/0-ehCGβ在实验组小鼠仅2只形成实体瘤,TR421-hcGβ质粒基因免疫抗肿瘤任用有肿瘤特异性,两组有显著性差异(P＜0.05),而在对照组小鼠均形成实体瘤.Sp2/0-preS2/S在所有的小鼠均形成了实体瘤,其瘤重无显著性差异.结论:ehCGβ肿瘤基因疫苗诱生的效应细胞可特异性杀伤肿瘤,过继免疫效应细胞能使正常小鼠获得明显的特异性抗肿瘤能力.     

异源性CTL的诱导及细胞毒性测定

脑胶质瘤是目前难治的恶性肿瘤之一,寻找有效的效应细胞是目前研究的方向.我们根据文献报道的方法,用自己研制的瘤苗,给昆明种小鼠体内致敏,取其脾细胞,体外再次用胶质瘤细胞刺激,获得特异性异源性CTL.具体方法是用榄香烯和丝裂霉素共同处理等量G422胶质瘤培养18小时,可得胶质瘤特异性瘤苗(榄香烯/丝裂霉素瘤苗),配制成单细胞悬液2×10~6/ml.取上述瘤苗1ml给昆明鼠多点注射体内致敬,2周后重复一次.第二次致敏10天后,取其脾细胞.在RPMI1640培养液1ml中加入脾细胞2×10~6、G422瘤细胞4×10~5和rIL-2400U,共同培养10天,收获特异性源性CTL.分别做正常脾细胞、体外经rIL-2培养的正常脾细胞(LAK)、     

榄香烯瘤苗抗原冲激的树突状细胞对HCA-F的免疫保护作用及其机制的研究

树突状细胞(Dendritic cell,DC)在肿瘤主动特异性免疫治疗中起重要作用,肿瘤抗原冲激的DC作为一种新型瘤苗颇受研究者的重视.本实验中我们选择小鼠肝癌H22的高淋巴道转移亚系HCa-F(H-2~-)为主要肿瘤模型,采用榄香烯瘤苗可溶性上清冲激小鼠脾脏DC,研究抗原冲激的DC对HCa-F的免疫保护作用及机制.主要实验方法和结果如下:①从未处理的HCa-F细胞及其榄香烯瘤苗中提取可溶性上清冲激正常小鼠脾脏DC,所得DC分别称为HCa-F上清冲激的DC和瘤苗上清冲激的DC(以下简称HDC和TDC).②转输HDC或TDC(5×10~5个细胞/只)给正常615系小鼠,7d后用HCa-F(5×10~5个细胞/只)攻击,观察小鼠瘤重及存活时     

抗CD3单抗和rIL-2共刺激法诱生的小鼠CD3AK对Ca761乳腺癌的治疗作用

采用抗小鼠CD3单抗(145-2Cll)和rIL-2共刺激小鼠脾细胞,可制备出具有增殖能力和体外抗瘤活性的抗CD3单抗激活的杀伤细胞(CD3AK).在本实验中我们以615系小鼠的自发乳腺癌Ca761为模型,研究了小鼠CD3AK对Ca761的免疫治疗作用和继承性化学免疫治疗作用.于第0天给小鼠接种1×10~6个Ca761细胞,第4天时局部已长出瘤结,第7天平均瘤体积达2.3±0.46cm~3,第10天达4.07±1.63cm~3,第14天活杀时平均瘤重为3.88±2.41g.如果于第4天起每日注射(ip)rIL-2(1000 U/只/日)达7天,无抑瘤作用,活杀时瘤重为4.4±2.59g;给同样大rIL-2的基础上,于第7天瘤内注射1×10~7CD3AK细胞,也无抑瘤作用,活杀时瘤重为4,81±1.57g,抑瘤率为-16.86%;如果注射2×10~7CD3AK细胞,则有一定抑瘤作用,活杀时瘤重为2.82±2.72     

抗B-CLL-IgM Id×抗CD3双功能抗体对LAK细胞杀伤活性的影响

抗肿瘤双特异性抗体(bispecific antibody,BsAb)由于具有同时与效应细胞及靶瘤细胞两种不同抗原特异性结合的双重特异性,从而提高了效应细胞对靶瘤细胞的特异性杀伤活性,因此,其在肿瘤靶向生物治疗中的应用日益受到重视.本研究通过体外实验,观察了抗人慢性B淋巴细胞白血病LgM独特型×抗CD3双特异性抗体(抗B-CLL-LgM Id×抗CD3 BsAb)对LAK细胞杀伤靶瘤细胞活性的影响,现报道如下.     

泰素耐药相关基因-3细胞毒性T淋巴细胞表位负载树突状细胞疫苗的体外免疫效应研究

目的探讨泰素耐药相关基因(TRAG)3来源的细胞毒性T淋巴细胞(CTL)表位负载树突状细胞疫苗的体外免疫效应,为临床开展基于TRAG3表位的特异性免疫治疗奠定基础。方法采用标准Fmoc方案合成表位肽,以反相高压液相色谱仪(RPHPLC)纯化、分析多肽,质谱鉴定多肽;从外周血单个核细胞(PBMC)分离培养树突状细胞(DC),应用相差显微镜和电镜从形态学上鉴定DC,采用流式细胞仪(FACS)分析DC表型;将TRAG3CTL表位肽(50μg/ml)负载的DC体外刺激PBMC3周,应用51Cr释放分析检测效应细胞CTL活性,同时应用酶联免疫吸附实验(ELISA)检测效应细胞IFNγ的分泌。结果Fmoc方案合成的表位肽纯度>90%,质谱鉴定的分子量与预计值一致;PBMC分离培养的DC在光镜、电镜下显示了其特殊形态,FACS分析证实其具有成熟DC的表型;经TRAG3表位肽负载的DC在体外能够有效活化特异性抗瘤CTL反应,并能显著刺激效应细胞内IFNγ的释放。结论TRAG3CTL表位肽负载的DC疫苗在体外能够有效激发抗瘤CTL免疫反应,可开展进一步的临床实验。     

独特型蛋白致敏的树突细胞瘤苗治疗多发性骨髓瘤

多发性骨髓瘤(MM)有独特型蛋白,为肿瘤特异性抗原.大量研究表明,使用负载独特型蛋白的树突细胞(DC)瘤苗进行主动免疫治疗,有助于消除(MM)微小残留病变,延长患者的无病生存期.近年来在抗肿瘤免疫治疗中的应用已成为肿瘤主动特异性免疫治疗的研究热点.     

IL—2/LAK细胞在白血病、淋巴瘤治疗中的应用

自1982年Grimm等首先描述了淋巴因子激活的杀伤细胞(Lymphokine activated killer cells,LAK)现象以来,对LAK细胞的研究日益深入。LAK细胞不仅有广谱的体外杀瘤效应,临床应用也具有抗肿瘤作用。LAK细胞和白细胞介素2(Interleukin—2,IL—2)疗法已成为临床上肿廇的继承性免疫治疗的主要方法之一。本文对近年来LAK细胞的研究及在血液病中的临床应用进展做一简要的概述。一、LAK细胞的生物学特性及其制备方法 LAK细胞是淋巴细胞在含有IL—2条件下经短期体外液体培养后能杀伤对自然杀伤(NK)敏感和不敏感的瘤细胞系及原代肿瘤细胞的一类功能细     

STUDY ON AUTOINHIBITORY ACTIVITY OF LYMPHOID LEUKEMIA

Autoinhibitory activity has been discovered in murine T lymphocyte leukemia models derived from 615 mice in our lab. It was designated 615 mice leukemia associated inhibitor (LAI-615) . To further confirm whether LAI activity could be found in human leukemia, 6 ALL cases and 2 AML cases were examined. The results showed that 5/6 of ALL and 1/2 of AML cases had detectable LAI activity. The different sensitivities of LAI activity were also found between autologous bone marrow cells and human leukemic cell lines, which indicate that autoinhibitory activity might have individual specificity.     

淋巴细胞白血病的自身抑制活性研究

我们由615小鼠诱发的T淋巴细胞白血病发现了白血病相关自身抑制活性LAI—615。为探索人类白血病有无类似的自身抑制活性,对6例ALL和2例AML病人骨髓上清进行了Sephadex G-200凝胶过滤分析,结果5/6ALL和1/2AML的病例有自身抑制活性。这些抑制活性呈现个体特异性。     

负载胃癌新抗原肽和CD73抑制剂纳米粒子的中性粒细胞疫苗的制备和抗肿瘤效果评价

目的:制备负载胃癌细胞新抗原肽和CD73抑制剂的中性粒细胞（NEs）疫苗并表征,初步评价其在荷瘤615小鼠中的抗肿瘤效果。方法:通过Percoll梯度密度离心法分离纯化小鼠骨髓来源的NEs。分别制备仅负载胃癌细胞来源新抗原肽疫苗（DP-Ag-MFC-NEs）,以及同时负载新抗原肽和CD73抑制剂纳米粒子的联合疫苗（DP-Ag-MFC-NEs+antiCD73+CpG）,观察疫苗对荷瘤小鼠的治疗效果。结果:一只10周龄615小鼠可分离纯化到平均约1.5×106个NEs,细胞活性在95%以上,纯度在90%以上。和对照组相比,DP-Ag-MFC-NEs可显著抑制小鼠皮下肿瘤生长,使部分小鼠痊愈（CR）,小鼠淋巴结中CD4、CD8阳性细胞比例显著增高（P<0.05）。CR小鼠再次接种MFC胃癌细胞,没有小鼠肿瘤复发。联合疫苗的治疗效果确切,小鼠存活率最高（75%,P=0.038）。结论:负载胃癌细胞来源的新抗原肽和CD73抑制剂的中性粒细胞疫苗对小鼠无明显毒性,可显著抑制肿瘤生长,甚至治愈小鼠。同时,该疫苗可在小鼠体内激活记忆型免疫应答。     

不同白血病细胞对同器官侵袭能力的比较观察

Using patho-morphological method and transplantation bio-assay, the in vivo invasiveness of leukemia cells in three transplantable mouse T cell leukemia models was comparatively studied. The results showed that the invasion to the liver was consistent, but that to other organs was obviously different. L615 and L7212 leukemia cells preferred bone marrow and spleen to peritoneum but L7811 leukemia cells were just the opposite. Transplantation bio-assay demonstrated that leukemia cells were present in the bone marrow of L615 mice as early as 6 hours after leukemic cell inoculation, but no leukemia cells were detected in the bone marrow of L7811 mice 2 days after inoculation. In the terminal phase, L615 mice bone marrow became filled with leukemia cells, but L7811 mice bone marrow contained only a few leukemia cells. The difference in invasiveness of leukemia cells into organs is probably related to "homing" receptor. The same type of leukemia cells may possess multiple "homing" receptors.     

同基因混合G-CSF动员的异基因骨髓移植物诱导的抗白血病效应

Objective:To explore whether the graft-versus-leukemia (GVL) effects could be enhanced and acute graft-versus-host disease (aGVHD) could be relieved by syngeneic bone marrow mixed with G-CSF-primed H-2 haploidentical marrow grafting.Methods:Female L615 (H-2k) mice were recipient mice and male (615×BALB/c) F1 (6BF1) (H-2k×H-2d) mice were donors respectively.Donor mice were injected subcutaneously with G-CSF daily at 0.01 μg/g for 6 days,and splenocytes were harvested on day 7.A total of 615 mice were loaded with L615 tumor cells and received 8.5 Gy (60Co γ-ray) irradiation three days later,followed by a mixed bone marrow transplantation (MBMT).The allo-grafts consisted of a mixture of syngenetic plus G-CSF-mobilized (control diluents) H-2 haploidetical marrow cells.GVL effects were monitored by survival time and survival rate of recipient mice.GVHD was assessed by clinical signs of weight loss,ruffled fur,diarrhea and histological changes of skin,liver and small intestines.Allogeneic chimerism was detected using cytogenetic analysis.Enzyme-linked immunosorbent assay (ELISA) method was used to detect cytokines (IL-2,IL-4 and IFN-γ).Fluorescence-activated cell sorting (FACS) analysis was used to detect T-cell phenotype.Results:(1) The mice merely received L615 leukemia cells were all died of leukemia.The L615 mice of 3∶1 and 4∶1 MBMT groups survived longer than those post syngeneic BMT (P＜0.01).(2) The survival time of mice in the G-CSF-treated MBMT groups was longer than that of non-primed MBMT groups (P＜0.05).Administration of G-CSF-treated 6BF1 mice could markedly increase the survival rate of 3:1 and 4:1 MBMT mice (P＜0.01) with little or a little GVHD.(3) As the post-transplanted time prolonged,the rates of allogeneic chimerism were decreased.The chimerism rates decreased to zero when the mice died of leukemia relapse.(4) L3T4 cells and relative ratio in both subsets were significantly reduced in G-CSF-treated donor mice.After G-CSF-treated donors,splenocytes from recipients at day 14 post-MBMT showed an increased production of IL-4 and a decreased production of IL-2 and IFN-γ.Conclusion:Syngeneic bone marrow mixed with H-2 haploidentical marrow grafts had a potential way to increase GVL effects,and this GVL effects could be enhanced with little or a little GVHD by G-CSF preetreatment of donors.Improved survival in recipients of G-CSF-mobilized donors is associated with increased IL-4 production and decreased IL-2 and IFN-γ production.     

IN VIVO COMPARATIVE OBSERVATION ON THE INVASIVENESS OF VARIOUS ORGANS BY DIFFERENT LEUKEMIA CELLS

Using patho-morphological method and transplantation bio-assay, the in vivo invasiveness of leukemia cells is three transplantable mouse T cell leukemia models was comparatively studied. The results showed that the invasion to the liver was consistent, but that to other organs was obviously different. L615 and L7212 leukemia cells preferred to the bone marrow and spleen than to the peritoneum while L7811 leukemia cells were just the opposite. Transplantation bio-assay demonstrated that leukemia cells were present in the bone marrow of L615 mice as early as 6 hours after leukemic cell inoculation, but no leukemia cells was detected in bone marrow of L7811 mice 2 days after inoculation. In the terminal phase, L615 mice bone marrow became filled with leukemia cells, but L7811 mice bone marrow contained only a few leukemia cells. The difference of invasiveness of leukemia cells among organs is probably related to "homing" receptor. The same type of leukemia cells may possess multiple "homing" receptor.     

Establishment and experimental study of a transplantable hepatocellular carcinoma model in 615-strain mice (H 615)

H 615, the first transplantable mouse liver carcinoma model established in China, was derived from a spontaneous hepatocellular carcinoma of an inbred 615 mouse and has been successfully propagated for 52 generations during the past 7 years and more. Its biologic and pathologic features are relatively stable. H 615 was a syngenic transplantable tumor model of 615-strain mice with a successful transplantation rate of 85.6% without spontaneous regression. The course of tumor growth after subcutaneous inoculation was divided into 4 stages: latent, slowly growing, rapidly growing and terminal stages. Cancer metastasis was rare. The tumor-bearing host would die of cachexia finally. The mean survival time was 62.2 +/- 11.0 days regardless of sex or age. Histologically and ultrastructurally, H 615 was a well-differentiated hepatocellular carcinoma, rather resembling human liver carcinoma. The short-term primary passage culture of H 615 showed that, in vitro, tumor tissue could easily grow into monolayer, the majority of which appeared as epithelioid cells in cytomorphology. Therapeutic tests of 15 anticancer drugs showed that H 615 was sensitive, in varying degrees, to 5 drugs, i. e. MMC, Thio-Tepa, 5FU, CPT and DACT. The inhibition rate of MMC and Thio-Tepa could be as high as 100%. These experimental results are similar to those of the human liver cancer chemotherapy. Hence, the authors believe that H 615 may be a useful model in experimental study of the liver cancer and screening of anticancer drugs.     

Purge of malignant cells from bone marrow by hematoporphyrin derivatives and light exposurein vitro

During autologous bone marrow graft in treatment of malignant diseases, it is critical to purge malignant cells from the marrow. In the present study, the sensitivity to photodynamic inactivation of 3 leukemic cell lines was compared with their counterpart normal hematopoietic cells. After mouse leukemic L₁₂₁₀ cells were treated with a preparation of hematoporphyrin derivatives, YHpD, 10 μg/ml for 1 hr. and irradiated with blacklight (peak wavelength 395 nm, light intensity 0.6 mW/cm²) for 5 minutes, the survival rate of clonogenic cells decreased to <10%, while that of bone marrow granulocyte-macrophage progenitor cells (CFU-GM) in DBA/2 mice remained at nearly normal level (>80%). Similar results were obtained when human leukemic HL-60 cells were compared with human CFU-GM and mouse leukemic L₆₁₅ cells with CFU-GM in 615 strain mice. It is suggested that hematoporphyrin photoradiation may be useful for selectively killing leukemic cells in bone marrow.     

亲代L7811及体外培养小鼠白血病细胞系L7811—85...

L7811 was an ascitic form of lymphocytic leukemia induced by myleran in 615 mice. Under electron microscope, cytoplasmic and intracisternal type A virus-like particles were observed. L7811-85 was a cell line established in vitro from the parental in vivo L7811 line. Instead of type A particles, mature and immature type C murine virus-like particles could be observed budding from the plasma membrane to extracellular space. With the in vitro subpassage of L7811-85 cells, more intracellular and extracellular type C particles appeared with an increase in its tumorigenicity. When L7811-85 cells, were inoculated IP to normal 615 mice, ascites tumor developed. The type A virus particles appeared again budding into cisterna of endoplasmic reticulum but not to extracellular space. The mechanism of virus particle type transformation remains to be studied.