胸腺基质细胞促进骨髓移植小鼠免疫功能重建

作者研究胸腺基质细胞对骨髓移植小鼠免疫功能重建的作用，经致死剂量照射的ＢＡＬＢ／ｃ小鼠，移植２×１０７个同基因骨髓细胞和１×１０￣６个胸腺基质细胞。免疫功能检测发现：该组小鼠胸腺内ＣＤＣＤＴ细胞比例恢复明显加快；其脾细胞对刀豆蛋白Ａ的增殖能力、对异型细胞的混合淋巴细胞反应、白细胞介素２（ＩＬ－２）诱生能力、活化的脾细胞对外源性ＩＬ－２的反应性及脾细胞对ＳＲＢＣ产生抗体的能力等多项免疫功能的恢复均较单纯骨髓移植小鼠明显加快，但对细菌脂多糖的增殖反应无明显变化。提示胸腺基质细胞可能通过促进胸腺微环境重建，促进Ｔ细胞在胸腺内发育成熟，从而促进Ｔ细胞及其介导的细胞免疫功能的早期重建。     

白血病细胞膜相关因子（MAF—J6—1）单克隆抗体的制备及性质

用部分纯化的白血病细胞膜相关因子（ＭＡＦ－Ｊ６－１）对ＢＡＬＢ／Ｃ小鼠脾细胞进行体外免疫，然后与ＳＰ２／０小鼠骨髓瘤细胞融合获杂交瘤，经３次再克隆得到能产生ＭＡＦ－Ｊ６－１单克隆抗体（单抗）的杂交瘤细胞系。用ＡＢＣ免疫酶标法检测，该单抗在Ｊ６－１细胞上产生强阳性反应，并与ｒｈ－Ｍ－ＣＳＦ单抗对ＭＡＦ－Ｊ６－１及ｒｈ－Ｍ－ＣＳＦ产生交叉中和反应。ＭＡＦ－Ｊ６－１单抗还对Ｊ６－２、ＪＣＬ、Ｋ５６２等细     

淋巴因子激活的骨髓细胞或脾细胞对小鼠移植物抗宿主病的预…

根据淋巴因子激活的骨髓细胞（ＬＡＢＭＣ）或脾细胞（ＬＡＳＣ）具有介导否决（Ｖｅｔｏ）效应和自然抑制（ＮＳ）效应的特性，实验分别以新生小鼠和亚致死剂量照射的成年小鼠为受鼠，腹腔注射同种脾细胞制备移植物抗宿主病（ＧＶＨＤ）模型，实验组同时注射ＬＡＢＭＣ或ＬＡＳＣ。结果显示：与受鼠带相同Ｈ－２单型的ＬＡＢＭＣ可明显减轻新生小鼠ＧＶＨＤ（Ｐ＜０．００１）；与受鼠带相同Ｈ－２单型的ＬＡＳＣ可有效预防致死性Ｇ     

淋巴因子诱导的细胞毒细胞小鼠体内外抗肿瘤作用的研究

采用Ｃ５７ＢＬ／６或ＤＢＡ／２小鼠脾脏制备淋巴因子诱导的细胞毒细胞（ＬＩＣＣ）并其肿瘤活性，实验结果表明ＬＩＣＣ在体外对小鼠黑色素瘤Ｂ１６和白血病Ｌ１２１０耐药株（ＭＴＸ）细胞杀伤活性比对照组显著增强（Ｐ〈０．０１），且具有逆转耐药株（ＭＴＸ）的作用，我们建立了小鼠黑色素瘤Ｂ１６细胞肺转移的模型，并给荷瘤小鼠尾静脉注射ＬＩＣＣ，发现治疗组小鼠肺表现肿瘤转移结节数显著减少（Ｐ〈０．０１）。本文又将２     

神经元特异烯醇化酶单克隆抗体的制备

用ＮＳＥ粗制品免疫雌性ＢＡＬＢ／Ｃ小鼠，经免疫小鼠脾细胞和鼠骨髓瘤细胞ＳＰ２／０的融合，ＥＬＩＳＡ法筛选，建立了四株持续分泌抗ＮＳＥ单抗的杂交瘤细胞。免疫沉淀法，单抗亲和柱纯化抗原鉴定等方法证实所分泌单抗对ＮＳＥ具有良好的特异性，注入同系小鼠腹腔可诱生含较高效价的抗ＮＳＥ的腹水。这四株细胞分泌ＩｇＧ１亚类抗体、且检查其染色体数目均大于９０条。     

白血病细胞膜相关因子（MAF－J6－1）单克隆抗体的制备及性质

用部分纯化的白血病细胞膜相关因子（ＭＡＦ－Ｊ６－１）对ＢＡＬＢ／Ｃ小鼠脾细胞进行体外免疫，然后与ＳＰ２／０小鼠骨髓瘤细胞融合获杂交瘤，经３次再克隆得到能产生ＭＡＦ－Ｊ６－１单克隆抗体（单抗）的杂交瘤细胞系。用ＡＢＣ免疫酶标法检测，该单抗在Ｊ６－１细胞上产生强阳性反应，并与ｒｈ－Ｍ－ＣＳＦ单抗对ＭＡＦ－Ｊ６－１及ｒｈ－Ｍ－ＣＳＦ产生交叉中和反应。ＭＡＦ－Ｊ６－１单抗还对Ｊ６－２、ＬＣＬ、Ｋ５６２等细胞系呈膜阳性反应，对白血病患者骨髓中部分原始和幼稚的单核、粒细胞呈阳性反应。在２４例被检测的白血病患者中，有三例呈强阳性反应，ＭＡＦ－Ｊ６－１可用作研究ｍ－Ｍ－ＣＳＦ与白血病关系的工具。     

白细胞介素6基因转移的白血病细胞体内诱导小鼠免疫功能的…

作观察了人白细胞介素６（ＩＬ－６）基因转移的、能高分泌ＩＬ－６的ＦＢＬ－３红白血病细胞对机体抗肿瘤免疫功能的影响。结果发现，高分泌ＩＬ－６的红白血病细胞接种到小鼠体内后，能显提高小鼠脾脏的细胞毒性Ｔ淋巴细胞、ＮＫ活性及ＩＬ－２诱导的ＬＡＫ活性，小鼠脾细胞诱生的ＩＬ－２、肿瘤坏死因子和粒－巨噬细胞系集落刺激因子水平亦增高，腹腔巨噬细胞杀伤活性明显增强。结果表明，高分泌ＩＬ－６的ＦＢＬ－３红白血病     

磁场对SP2/0瘤鼠抗体形成细胞的作用研究

磁场对ＳＰ２／０瘤鼠抗体形成细胞的作用研究高美华冯献启李波清邱世翠作者单位：２５６６０３山东省滨州医学院材料和方法取ＢＡＬＢ／Ｃ纯系小鼠２４只，体重１８～２２ｇ，雌雄各半。鼠背部用脱毛剂脱毛，取ＳＰ２／０骨髓瘤细胞３×１０６接种于鼠背部皮下（脾区）。...     

GM-CSF基因修饰、抗原预激的树突状细胞体内诱导白血病细胞分化的实验研究

目的：探讨树突状细胞在肿瘤免疫治疗过程中的作用及机制。方法：以小鼠ＦＢＬ３红白血病细胞皮下接种Ｃ５７ＢＬ／６小鼠建立荷瘤模型，采用流式细胞仪分析和透射电镜等技术，观察了粒巨噬细胞集落刺激因子（ＧＭＣＳＦ）基因修饰的抗原预激的树突状细胞体内治疗作用。结果：采用ＧＭＣＳＦ基因修饰的抗原预激的树突状细胞治疗，可以明显抑制白血病细胞生长；白血病细胞表达ＣＤ１４水平增加，同时ＭＨＣⅡ、Ｂ７１、Ｂ７２、ＶＣＡＭ１表达水平亦显著升高；组织学观察显示瘤组织内具有不同分化阶段的单核细胞，电镜下可见白血病细胞体积减小、细胞器较成熟、胞浆中出现较多的溶酶体、核／浆比值减小及白血病细胞的凋亡。肿瘤内部及外周血中，ＣＤ８＋细胞百分率亦较各对照组明显升高。结论：采用ＧＭＣＳＦ基因修饰的抗原预激的树突状细胞治疗，可以体内诱导白血病细胞向成熟单核细胞方向分化     

GM—CSF基因修饰,抗原预激的树突状细胞体内诱导白血病细胞分化？…

目的：探讨树突状细胞在肿瘤治疗过程中的作用及机制。方法：以小鼠ＦＢＬ－３红白血病细胞皮下接种Ｃ５７ＢＬ／６小鼠建立荷瘤模型，采用流式细胞仪分析和透射电镜等技术，观察了粒－巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ）基因修饰的抗原预激的树突状细胞体内治疗作用。结果：采用ＧＭ－ＣＳＦ基因修饰的抗原预激的树突状细胞治疗，可以明显抑制白血病细胞生长；白血病细胞表达ＣＤ３４水平增加，同时ＭＨＣ－Ⅱ，Ｂ７－１，Ｂ７－     

Gene transfer of GM-CSF, CD80 and CD154 cDNA enhances survival in a murine model of acute leukemia with persistence of a minimal residual disease

Gene transfer of various cytokines and co-stimulatory molecules has been reported to induce a potent antileukemic immunity in murine models, however, the relative efficiency and possible synergistic effects between candidate genes have not been extensively investigated. We analyzed in a murine model of BCR/ABL acute leukemia whether gene transfer of CD154, CD80 or GM-CSF as a single agent or combination of CD154 + GM-CSF, CD80 + CD154 and GM-CSF + CD80 in leukemic cells could enhance survival. We observed that CD154 gene transfer induced a marked inhibition of leukemogenicity, and also that CD154 and combination of GM-CSF and CD80 gene transfer protected mice against subsequent challenge with leukemic cells and had a therapeutic effect for a pre-established leukemia disease. We also found minimal residual leukemic disease by RT-PCR for 6 to 12 months in 0 to 25% of animals injected with transduced leukemic cells and surviving the challenge without evidence of disease, except in the control empty plasmid group where very few mice survived the challenge but all of those were positive by RT-PCR. These findings suggest that leukemic cell vaccination by gene transfer can induce a tumor dormancy phenomenon compatible with long-term survival.     

Cell therapy with preimmunized effector cells mismatched for minor histocompatible antigens in the treatment of a murine mammary carcinoma

Cell therapy with allogeneic donor cells mismatched for minor histocompatible (MiHC) antigens was applied to a murine mammary carcinoma (4T1) model to test the feasibility of graft versus tumor (GVT) effect against metastatic epithelial tumor cells. BALB/c mice bearing a 4T1 tumor of BALB/c origin were given syngeneic or MiHC-mismatched splenocytes. GVT effects were determined in secondary recipients of adoptively transferred lung cells derived from primary hosts who had previously been inoculated intravenously with 4T1 cells, and injected with one of the following: 1) naive BALB/c splenocytes, 2) naive DBA/2 splenocytes, 3) 4T1-immune DBA/2 splenocytes, or 4) DBA/2 splenocytes immunized with host-derived BABL/c spleen cells. Naive DBA/2 splenocytes inhibited tumor growth only slightly and only slightly prolonged the survival of secondary recipients, in comparison with fully matched tumor/host BALB/c spleen cells. An efficient GVT reaction was demonstrated in vitro and in vivo with MiHC-mismatched DBA/2 splenocytes from mice presensitized by multiple injections of irradiated tumor or BALB/c-derived spleen cells. All 30 mice adoptively inoculated with lung cells from primary hosts that had previously been treated with these presensitized effector cells were tumor free for >250 days. Secondary recipients inoculated with lung cells from mice given naive BALB/c or DBA/2 spleen cells died of metastatic tumors within 33 to 46 days. These results suggest that preimmunized donor cells represent an effective tool against metastatic disease; hence, the next goal should be to control graft-versus-host disease while exploiting the GVT potential.     

CD3AK细胞对白血病细胞杀伤活性的实验研究

目的应用CD3单克隆抗体(CD3McAb)和重组人白细胞介素-2(rhIL-2)共同诱导外周血单个核细胞制备CD3AK细胞,研究其对白血病细胞的杀伤作用。方法用台盼蓝活细胞计数法计算细胞扩增倍数,MTT法检测CD3AK细胞杀伤活性。结果正常人CD3AK细胞对各型急性白血病细胞及K562细胞均有明显的杀伤活性,且无显著性差异(P>0.05);急性白血病完全缓解期CD3AK细胞与正常人CD3AK细胞对白血病细胞的杀伤活性,亦无显著性差异(P>0.05);急性白血病未缓解期CD3AK对白血病细胞杀伤活性明显减弱,与正常人CD3AK细胞比较,差异非常显著(P<0.01)。结论正常人与急性白血病完全缓解期CD3AK细胞具有明显的抗白血病作用,急性白血病未缓解期CD3AK细胞杀伤活性明显减弱。     

表达绿荧光蛋白的多药耐药微小残留白血病细胞的小鼠模型

为了方便研究多药耐药微小残留白血病的病理生理和治疗,采用携带绿荧光蛋白（EGFP）基因标记的小鼠多药耐药白血病细胞系P388/VCR-G和DBA小鼠建立一个检测多药耐药微小残留白血病的动物模型.结果显示,P388/VCR-G细胞腹腔接种DBA小鼠,白血病的发病率为100%,无自发缓解.P388/VCR-G白血病模型小鼠对环磷酰胺（Cy）敏感,有较好的药物剂量-生存时间关系,接种细胞的对数与Cy剂量间有良好回归相关关系.白血病诱导缓解后残留白血病细胞在肝、脾、胸腺及骨髓等脏器呈不均匀分布.冰冻切片荧光显微镜下观察EGFP＋细胞是检测小鼠体内微小残留白血病细胞最敏感的方法.结论：采用P388/VCR-G细胞和DBA小鼠可建立表达EG-FP的多药耐药小鼠微小残留白血病模型.     

CD3单克隆抗体联合IL-2激活的单个核细胞对白血病细胞体外杀伤作用

抗CD3单克隆抗体(CD3 McAb)对T细胞具有强烈的丝裂原作用。作者研究证实CD3McAb联合IL-2激活的杀伤细胞(CD3 AK)对人急性白血病新鲜细胞和K562细胞均有明显的杀伤作用。正常人CD3 AK细胞对各型急性白血病细胞杀伤活性无差别;正常人与白血病缓解期的CD3 AK细胞杀伤力类同,且自体与异体亦无区别;但复发期CD3 AK细胞杀瘤作用明显减弱。血型不影响CD3 AK细胞的杀伤作用。     

CD34+ cord blood DC-induced antitumor lymphoid cells have efficacy in a murine xenograft model of human ALL

Acute lymphocytic leukemia (ALL) patients who relapse after transplantation have few therapeutic options. An immunotherapeutic approach that enhances the graft versus leukemia effect may improve their survival. We postulate that cytotoxic T lymphocytes (CTLs) generated from total RNA loaded cord blood CD34+-derived dendritic cells can control the kinetics of leukemic growth in a nonobese diabetic/severe combined immunodeficient (NOD-SCID) mouse model of human ALL. CD34+-derived dendritic cells electroporated with total RNA from an ALL xenograft generate antileukemic CTL with specificity for the ALL xenograft while sparing autologous cord blood mononuclear cells. The CD3+ T-cell compartment of the CTL was dominated by CD4+ T cells, although CD8+ T cells accounted for an average of 30% of the CD3+ T cells present. Expansion of both CD4+ and CD8+ memory and terminal effector memory subsets from predominantly naive cells was evident. Natural killer (NK) cells accounted for an average of 13% of the final antitumor lymphoid cells produced. Blocking experiments confirmed that the CD8+ T-cell compartment was responsible for the antileukemic activity of the polyclonal CTL pool. Administration of antileukemic CTL to NOD-SCID mice bearing ALL xenograft cells was able to delay, but not prevent the growth of ALL in vivo. Coadministration of antigen-loaded antigen-presenting cells did not further improve upon the delay in ALL engraftment kinetics observed with CTL alone. The efficacy of adoptively transferred polyclonal CTL can be improved with coadministration of recombinant human interleukin-2. However, in NOD-SCID mice, the efficacy of these adoptively transferred cells is masked by interleukin-2 stimulation of murine NK cells, which facilitate killing of ALL cells. Our data highlights the role for NK cells in antileukemic responses posttransplant. Collectively, our results support the notion that ALL-specific adoptive immunotherapy could be used clinically and provide an alternative strategy for preventing and treating disease relapse posttransplant and that the success of this therapy is likely to be maximized if given in the setting of minimal residual disease.     

Correlation between enhancement of graft-versus-leukemia effects following allogeneic bone marrow transplantation by rIL-2 and increased frequency of cytotoxic T-lymphocyte precursors in murine myeloid leukemia

A model of mouse acute myeloid leukemia (mAML) was used to study the effector mechanism mediating the graft-versus-leukemia (GVL) effects in recipients of allogeneic bone marrow cells (BMC). mAML-bearing SJL/J (H-2s) mice were lethally irradiated and then transplanted with a mixture of BMC and spleen cells (SC) derived from normal syngeneic or allogeneic mice. To augment the GVL effect, recipients were injected intraperitoneally with recombinant human interleukin-2 (rIL-2) (1.2 x 10(5) IU) for 3 consecutive days, starting one day post BMC + SC transplantation. Spleen cells from treated recipients were adoptively transferred to untreated secondary SJL/J mice to test for the existence of residual tumor cells. All the secondary recipients of SC from mAML-bearing SJL/J mice rescued with syngeneic (SJL/J) or allogeneic (B10.S) BMC+SC (H-2s) differing at minor antigens of the histocompatibility complex (MiHC) developed leukemia and died. In sharp contrast, none of the secondary recipients of SC obtained from identical mAML-bearing mice rescued with B10.S BMC + SC but activated in vivo with IL-2 developed leukemia. Adoptive recipients of SC obtained from mAML-bearing recipients of major histocompatibility complex (MHC)-disparate (C57BL/6, H-2b) cells remained free of leukemia regardless of the use of rIL-2. In parallel with the in vivo findings, a 4-day in vitro exposure of splenocytes to 6 x 10(3) IU/ml rIL-2 resulted in a 5- to 20-fold increase in the frequency of alloreactive cytotoxic T-lymphocyte (CTL) precursors (CTLp) across MiHC and MHC barriers and a 2- to 6-fold increase in their cytotoxic activity. Our data suggest that augmentation of GVL effects by rIL-2 may be due to CTL activation by rIL-2, not excluding the potential beneficial role of rIL-2-activated allogeneic natural killer cells and MHC non-restricted killer cells. Cumulatively, our results suggest potentially beneficial effects of rIL-2, when used jointly with bone marrow transplantation or allogeneic cell therapy, on eradication of leukemia.     

可移植性人髓系白血病BALB/c小鼠模型建立

造血干/祖细胞移植已成为迄今为止治疗恶性血液病等最有效措施,而建立骨髓型可移植性白血病小鼠模型将为造血干细胞移植治疗白血病提供实验用动物模型。本实验用K562细胞接种BALB/c裸鼠产生红白血病的小鼠模型。将4-5周龄雌性BALB/c裸鼠,腹腔连续两天注射环磷酰胺(CTX)2mg,第3天腹腔或尾静脉直接接种K562白血病细胞2×105-2×106/只。定时取小鼠尾静脉外周血、骨髓细胞用流式细胞术和RT-PCR分别检测CD45,CD13,CD33抗原及bcr/abl融合基因。结果显示,4-5周龄BALB/c裸鼠无论通过腹部或尾静脉接种,无论有无CTX预处理,当接种K562细胞数大于2×105/只时,均可在BALB/c裸鼠身上产生可移植性人髓系白血病,荷瘤小鼠可存活30-60天。结论:腹腔或尾静脉接种大于2×105细胞/只均可产生人髓系白血病荷瘤小鼠模型,有无CTX2毫克/只的预处理不影响4-5周龄BALB/c裸鼠产生人白血病模型。     

Allogeneic cell therapy in murine B-cell leukemia (BCL1): 2. The role of non-activated and rIL-2-activated CD4+ and CD8+ T cells in immunotherapy for leukemia

Graft-versus-leukemia (GVL) effects play a key role in the elimination of residual leukemia cells in the course of allogeneic bone marrow transplantation (alloBMT). GVL effects can also be induced by donor lymphocyte infusion following alloBMT. We have investigated the role of CD4+ and CD8+ T cells in the development of GVL in mice with B-cell leukemia/lymphoma (BCL1) following allogeneic cell therapy. Sublethally irradiated (C57BL/6 x BALB/c)F1 mice were intravenously inoculated with 10(5) BCL1 cells and given untreated or recombinant human Interleukin-2 (rIL-2)-activated C57BL/6 spleen cells. Effective elimination of clonogenic BCL1 cells was confirmed by adoptive transfer of spleen cells obtained from treated mice into secondary BALB/c recipients. GVL effects were maintained after inactivation of CD4+ cells with monoclonal anti-CD4 antibodies in the inoculum, while inactivation of CD8+ cells with monoclonal anti-CD8 antibodies resulted in complete loss of GVL effects induced both by resting and rIL-2-activated allogeneic spleen lymphocytes. These results indicate that Thy-1 cells play the major role in the induction of GVL effects, mediated by C57BL/6 effector T cells in this model. Since the number of natural killer (NK) cells also increased during in vitro culture with rIL-2, their contribution, especially that of CD8+ NK cells, in GVL effects mediated by rIL-2-activated CD8+ cells cannot be ruled out.     

槲皮素增敏阿霉素抗白血病效应的实验研究

本研究采用槲皮素作用于P388白血病移植瘤裸鼠模型,探讨其在体内抗白血病机制及增强阿霉素化疗效果的机制。取对数生长期的P388白血病细胞注入BALB/c裸鼠皮下,建成白血病皮下移植瘤裸鼠模型;采用槲皮素、阿霉素及二者联合用药作用于白血病小鼠,观察小鼠生存期的变化;定期复查血象,计数外周血细胞数;通过流式细胞术测定细胞周期,观察槲皮素对细胞增殖的影响;通过ELISA法检测Caspase-3蛋白表达水平,并通过实时荧光定量PCR、Western-blot技术检测NF-κB、BCL-2、BAX基因及蛋白的变化。结果表明,槲皮素和阿霉素均能显著延长P388白血病移植瘤裸鼠的生存期,且两者联合作用于动物的生存期较单一药物组的延长更明显;槲皮素和阿霉素单独及联合应用均可使白血病小鼠瘤组织G0/G1期细胞比例减少,S期及G2/M细胞比例增加,且槲皮素、阿霉素联合用药组作用更显著;槲皮素可激活Caspase-3酶,诱导白血病小鼠白血病细胞的凋亡;同时,槲皮素可以下调抗凋亡基因BCL-2及NF-κB的表达并上调促凋亡基因BAX的表达。结论：槲皮素可在体内通过调控凋亡相关基因的表达来抑制白血病细胞增殖,促进癌细胞凋亡,并可增敏蒽环类药物阿霉素的化疗效果,具有进一步研发为高效抗白血病药物的潜能。