应用SCID和NOD/SCID小鼠构建急性髓系白血病模型成瘤率的比较

目的:比较应用SCID和NOD/SCID小鼠构建急性髓系白血病模型成瘤率的不同。方法:分别给SCID和NOD/SCID小鼠腹腔注射HL-60细胞,观察小鼠的一般情况、应用流式细胞术检测骨髓细胞CD33阳性率,病理学检查鉴定动物模型。结果:应用SCID小鼠构建急性髓系白血病模型成瘤率为30%,而应用NOD/SCID小鼠构建急性髓细胞白血病模型成瘤率为100%。结论:接种HL-60的NOD/SCID小鼠相对于SCID小鼠成瘤率高,小鼠发病率稳定,更适宜应用于白血病机制研究。     

8-溴-7-甲氧基白杨素的合成及其抗人急性细胞性白血病作用

目的 观察8-溴-7-甲氧基白杨素对人急性粒细胞性白血病(HL-60)细胞增殖抑制作用。方法 MTT比色法测定8-溴-7-甲氧基白杨素对人急性粒细胞性白血病(HL-60)细胞增殖抑制作用。HL-60细胞小鼠肾囊膜下移植瘤模型观察8-溴-7-甲氧基白杨素对人急性粒细胞性白血病的治疗作用。结果 8-溴-7-甲氧基白杨素对人急性粒细胞性白血病(HL-60)细胞增殖具有抑制作用，呈浓度依赖性。8-溴-7-甲氧基白杨素对人急性粒细胞性白血病细胞小鼠肾囊膜下移植瘤生长具有抑制作用，呈剂量依赖性。结论 8-溴-7-甲氧基白杨素是一种急性粒细胞性白血病治疗新候选药物。     

131I-GM-CSF在人白血病SCID小鼠模型体内的生物学分布

目的 观察131I-GM-CSF在人急性髓系白血病SCID小鼠模型体内的生物学分布.方法 用SCID小鼠建立人白血病异种移植模型,用氯胺-T法制备131I-GM-CSF,观察131I-GM-CSF在白血病小鼠体内的生物学分布.结果①静脉接种的HL-60细胞在SCID小鼠体内植活,4周后发生白血病;②131I-GM-CSF主要滞留于模型小鼠的脾脏、骨髓及瘤体组织中,且前两者对131I-GM-CSF摄取于注入后30 min达峰值,组织摄取率分别为(442.9±86.4)%ID/g和(4283.8±252.8)%ID/g,滞留24h后高于其他脏器.而131I呈非特异性分布.结论 131I-GM-CSF集中分布于人白血病SCID小鼠模型脾脏、骨髓及白血病细胞浸润组织,具有组织器官相对特异性.     

SCID小鼠-人类白血病模型及其应用

本文综述了人类急性白血病细胞在SCID小鼠内生长、浸润特点和SCID小鼠-人类白血病模型的研究进展,并对该模型在研究白血病发病学、白血病细胞生物学和临床诊疗措施等方面的可能应用进行了探讨。     

VEGF及其受体在恶性血液细胞系HL-60及Raji的表达

为了探讨血管内皮生长因子(VEGF)在恶性血液病中的作用，以恶性血液细胞系HL-60和Raji为实验对象，应用RT-PCR及ELISA法检测肿瘤细胞。VEGF基因的表达，并采用免疫组织化学染色法测定细胞表面VEGF受体Fit-1的表达。结果显示：髓性白血病细胞系HL-60和淋巴瘤细胞系Raji中均存在VEGF—mRNA的表迭。在培养48小时后，HL-60细胞和Raji细胞的培养上清液中VEGF含量明显高于正常人外周血单个核细胞。VEGE-R(Flt-1)在两种肿瘤细胞的胞膜上均有表达，而正常人外周血单个核细胞上无表迭。这一结果表明，白血病和淋巴瘤细胞具有产生VEGF的能力，并可将VEGF分泌到细胞外基质微环境中。VEGF可作用于白血病和淋巴瘤细胞本身，在恶性血液肿瘤细胞中存有VEGF的自分泌途径，而VEGF的自分泌成为血液肿瘤细胞高侵袭性生物学特性的基础。结论：抑制VEGF的分泌将有助于阻断其与内皮细胞间的互动环，降低其增殖、抗凋亡和侵袭性能．对提高肿瘤细胞对化疗的敏感性也将十分有意义。     

粒细胞源性造血抑制活性

正常成熟外周血粒细胞提取物含有多种抑制因子(granulocyte-derived inhi-bitor,简称G-INH)。G-INH抑制骨髓CFU-GM及白血病细胞系(HL-60、K562、CEM、Raji-Li和Molt4)体外集落生长。形态学及DNA电泳技术证实:G-INH亦可诱导正常骨髓细胞、外周血粒细胞、白血病细胞系HL-60、K562和CEM细胞进入程序性死亡,但Molt4(一种T细胞白血病细胞系)则例外。集落抑制活性存在于分子量<3kD、3-100kD以及>100kD各组分中;而细胞程序性死亡诱导活性则存在于>3kD组分,主要存在于分子量>100kD组分。生物活性及ELISA检测提示:G-INH不含有TNF-α、IFN-α、GM-CSF、IL-3、乳铁蛋白以及前列腺素活性;热处理60℃,30min不影响活性。癌基因表达水平检测表明:G-INH显著降低HL-60细胞内bcl-2表达而p53无显著改变;同样实验条件下,CEM细胞并未显示bcl-2表达差异而表现为细胞内p53表达明显提高。提示这些癌基因在细胞程序性死亡之机理中具有重要调控作用,推测这些细胞死亡与基因调控有关。正常骨髓和外周血细胞并无bcl-2及p53阳性表达。G-INH各成分尚在分离纯化中。     

南瓜蛋白抗人慢性髓系白血病细胞系K562的作用研究

本研究观察南瓜蛋白(Cucurmosin,CUS)在人慢性髓系白血病(CML)K562细胞-NOD/SCID小鼠的皮下移植瘤和白血病模型体内抗肿瘤活性。采用建立K562-NOD/SCID小鼠皮下移植瘤和白血病模型,分别使用二种模型评价CUS在小鼠体内的抗K562活性。结果表明,给药剂量为0.5 mg/kg和1 mg/kg的CUS对人CML皮下移植瘤的抑瘤率分别为53.45%和59.43%;而剂量为0.25 mg/kg和0.5 mg/kg的CUS给药组小鼠的生存时间分别为39.8±5.5 d和43.4±6.6 d,抑瘤率分别为24.9%和36%。结论:CUS对小鼠体内人CML细胞系K562具有显著的抑制作用。     

K562/NOD-SCID小鼠白血病模型的建立

本研究探讨人CML急性变的白血病细胞在NOD—SCID小鼠体内建立白血病模型的方法并研究其生长特性。首先将K562细胞接种于全身受照射后的裸鼠，待皮下成瘤后取出局部瘤块，选取无坏死的瘤组织制成瘤细胞悬液，再腹腔接种于全身受照射后的NOD—SCID小鼠。结果表明：成功建立全身播散的白血病模型。4周时外周血涂片可见白血病细胞，晚期浸润肝、脾、骨髓等造血器官，白细胞上升到接种前的8—10倍，血涂片中白血病细胞达20％-30％。腹腔局部出现瘤块，多位于腹腔内或大网膜，较少累及其他器官。结论：腹腔接种K562瘤细胞于全身照射后的NOD—SCID小鼠能建成全身播散的白血病模型，该模型较好地反映白血病在体内的演变过程，是进行新药疗效试验、生物导向治疗及基因治疗的理想工具。     

经裸鼠体内多次传代所致的高致瘤HL-60细胞模型的建立及其细胞生物学特征

本研究探讨裸鼠体内多次传代的高致瘤性HL-60细胞的生物特性的变化,探索高致瘤性HL-60细胞致瘤率提高的机制。用裸鼠体内和体外传代方法建立高致瘤性白血病细胞系HL-60模型,并对其生物特性改变进行比较。用台盼蓝染色法检测HL-60细胞生长水平,流式细胞术分析细胞周期,电子显微镜观察HL-60细胞的超微结构和荧光水平。结果表明:细胞生长速度加快,细胞群体倍增时间缩短;S期细胞比率有上升的超势。细胞线粒体数目显著下降(p0.01),且多数内室扩张,嵴数目减少,有的空泡化;细胞微丝荧光减弱,差异显著(p0.01);克隆形成率有明显的增加。结论:裸鼠体内多次传代的HL-60细胞高致瘤性与致瘤细胞增殖能力增强,细胞中线粒体数目与结构的改变和细胞骨架微丝的变化有关。     

人成纤维样基质细胞系对HL-60细胞增殖和VEGF表达的影响

为了观察正常人骨髓成纤雏样细胞系(HFCL)对白血病细胞系HL-60细胞增殖和血管内皮生长因子(VEGF)表达的影响，用免疫组织化学对HFCL细胞进行组织学鉴定；建立HL-60细胞和HFCL细胞共培养体系，台盼蓝柜柒法测定生长曲线；既染色和瑞-姬姆萨染色后进行分裂指数(MI)测定；流式细胞术检测细胞周期的变化；Western blot检测增殖细胞核抗原(PCNA)表达；ELISA双抗体夹心法检测HL-60细胞表达VEGF水平。结果发现。与HFCL细胞共培养后HL—60细胞生长受抑，且与HFCL细胞直接接触组的抑制作用大于用跨室共培养组，其分裂指数表现为单独HL-60细胞组大于用跨室共培养组，更大于与HFCL细胞直接接触组。同时发现HL-60细胞与HFCL细胞共培养后G1期细胞增多，S期细胞减少，且PCNA表达下调，以直接接触组为甚。HL-60细胞与HFCL细胞共培养后，培养上清的VEGF水平减低，直接接触组和跨室共培养组下降程度一样，而与HL-60细胞共培养，HFCL细胞增殖无明显变化。结论：正常人骨髓成纤维样细胞能抑制白血病细胞系HL-60的增殖，抑制细胞周期，降低VEGF因子的表达。     

SUDHL-4细胞体外培养和小鼠肿瘤模型的建立

本研究探讨人弥漫性大B细胞淋巴瘤(DLBCL)细胞株SUDHL-4的体外培养和小鼠成瘤条件。在不同条件下培养SUDHL-4细胞,对细胞生长形态及生物学特性进行观察分析;对肿瘤相关抗原表达进行免疫学分析;采用SCID小鼠皮下接种,对肿瘤生长情况和组织学形态进行研究。结果显示:培养液中加入10%胎牛血清,SUDHL-4细胞在体外生长良好,并表达多数重要的B细胞和肿瘤相关标记。采用SCID小鼠皮下接种107细胞可成功地建立人DLBCL移植瘤模型,成瘤率70%,肿瘤的组织学表现类似于人DLBCL。结论:适当条件下培养的SUDHL-4细胞的生物学和免疫学特性符合人DLBCL的特征,并具有良好的致瘤性,可为人DLBCL的相关研究提供良好的模型。     

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。     

Experimental systems in CML biology]

It has been established that the BCR-ABL oncogene produced on the Philadelphia chromosome in human chronic myeloid leukemia(CML) directly causes leukemic transformation of multipotential progenitor cells. In order to study the molecular basis of this process, many convenient and useful biological assays have been found, including transformation of mouse bone marrow cells in primary culture and of Rat1 fibroblasts, cytokine-independent growth of dependent cell lines, retrovirus-mediated murine CML model, and transgenic mice model. New biological activities such as anti-apoptosis, anti-DNA repair, differentiation of ES cells may further give supportive explanations for clinical manifestation of CML. NOD/SCID transplantation model and conditional transgenic model may be the current best animal system by which to investigate cell dynamics in the most strict and natural circumstances.     

人急性B淋巴细胞白血病-NOD/SCID/IL2rγnull新生小鼠异种移植模型的建立

本研究旨在应用NOD/SCID/IL2rγnull新生小鼠建立人急性B淋巴细胞白血病(B-ALL)的异种移植模型。NOD/SCID/IL2rγnull新生期(出生48 h之内)小鼠经亚致死剂量(100 cGy)137Cs全身照射后,由面静脉输注FACSAria流式细胞仪分选纯化的B-ALL患者骨髓CD3-CD4-CD8-CD34+CD19+细胞,于移植后8-12周用流式细胞术检测受鼠外周血、骨髓和脾脏中人源细胞的植入水平及其免疫表型,并通过HE染色评价人源B-ALL细胞在受鼠各组织器官中的迁移浸润能力。结果表明,在接受B-ALL患者CD34+CD19+细胞移植的受鼠外周血、骨髓和脾脏细胞中,不仅检测到了不同程度的人源B-ALL(huCD45+CD19+)细胞的植入〔(83.36±10.05)%,(93.88±5.05)%,(88.31±5.01)%〕,而且植入细胞具有与B-ALL患者相似的细胞形态和免疫表型特征。此外,人源B-ALL细胞广泛迁移浸润到受鼠的肝脏、肺脏、肾脏和脑等组织器官中。结论:NOD/SCID/IL2rγnull新生小鼠模型能够支持B-ALL患者CD34+CD19+细胞的高水平植入,是对人B-ALL进行体内功能性研究的新型异种移植模型。     

SCID小鼠腹腔内人B细胞非霍奇金淋巴瘤模型的建立

目的 为探讨人B细胞非霍奇金淋巴瘤诊治策略提供动物模型.方法 采用4-5周龄的SCID小鼠,经腹腔接种7.5×106 个Namalwa细胞.观察小鼠的发病情况及生存时间.取瘤组织进行HE及免疫组化染色检测CD20,CD79a,Ki-67和CD3抗原的表达.结果 Namalwa细胞在SCID小鼠腹腔发病的主要表现是腹腔出现肿块,时间（18.67±1.94） d,伴竖毛、精神萎靡、行动迟缓,一般情况随时间的延长而不断恶化.荷瘤SCID小鼠中位存活时间（28.00±6.36） d.瘤细胞主要浸润肠壁、肠系膜,胃、胰、肝、脾、肾、肺的周缘、盆腔以及邻近肌组织和皮肤.CD20在瘤组织表达为散在阳性,CD79a和Ki-67为弥慢阳性,CD3为阴性.结论 成功建立SCID小鼠腹腔人Namalwa细胞株B细胞非霍奇金淋巴瘤模型,为探索淋巴瘤提供载体.     

人恶性胰岛细胞瘤SCID鼠原位移植瘤免疫组织化学和超微结构的研究

目的　为探讨人恶性胰岛细胞瘤转移机制和抗转移治疗提供实验工具。方法　将人恶性胰岛细胞瘤组织植入SCID胰腺被膜下 ,观察移植瘤的侵袭转移及形态学特征 (光镜、免疫组织化学、电镜 )。结果　在SCID鼠体内成功地建立了一株人恶性胰岛素瘤原位移植模型HMI HMN 1,已传 2 1代 ,和一株人恶性胰多肽瘤原位移植模型HPPT HMN 2 ,已传2 5代 ,共移植SCID鼠共 14 7只 ,其移植生长率和自发转移率达 10 0 %。表现为肿瘤广泛侵袭胃十二指肠并有淋巴结和肝转移。病理组织学、免疫组织化学和电镜观察结果证明移植瘤细胞与瘤源人胰岛细胞瘤细胞完全一致。结论　人恶性胰岛细胞瘤SCID鼠原位移植模型的建立为研究人恶性胰岛细胞瘤转移机制和抗转移治疗提供了理想的动物模型。     

Use of a SCID mouse/human lymphoma model to evaluate cytokine-induced killer cells with potent antitumor cell activity

C.B-17 severe combined immune deficient (SCID) mice, which lack functional B and T lymphocytes, allow xenografts and, therefore, can be used to study the biology of human malignancies. Two different human B cell lymphoma cell lines, SU-DHL-4 and OCI-Ly8, which both harbor the t(14;18) chromosomal translocation, were injected into C.B-17 SCID mice. Mice injected intravenously or intraperitoneally developed tumors and died in a dose-dependent manner. The presence of tumor cells in various murine tissues could be demonstrated by a clonogenic tumor assay, staining of frozen sections with a monoclonal antibody (mAb) against a human B cell antigen (CD19), and with the polymerase chain reaction technique. A protocol using cytotoxic effector cells was developed and used to selectively deplete the tumor cells from bone marrow. These cells were developed by growing peripheral blood mononuclear cells in the presence of interferon gamma (IFN-gamma), anti-CD3 mAb, and interleukin 2 (IL-2). The timing of IFN-gamma treatment was critical and optimal if IFN-gamma was added before IL-2 treatment. The cells that were stimulated by IFN-gamma, followed by IL-2, could be expanded by treatment with a mAb directed against CD3. These cells could be further activated by IL-1, but not by tumor necrosis factor alpha. With this protocol, a tumor cell kill of 3 logs was obtained as measured by a clonogenic assay. Interestingly, despite their high cytotoxic activity against lymphoma cells, these cells had little toxicity against a subset of normal human hematopoietic precursor cells (granulocyte/macrophage colony-forming units). These cells were further tested by treating murine bone marrow contaminated with the human lymphoma cell line SU-DHL-4, and injecting these cells into SCID mice to assay for tumor growth in vivo. The animals injected with bone marrow contaminated with SU-DHL-4 cells had enhanced survival if the bone marrow was treated with the cytokine-induced killer cells before infusion. The SCID mouse provides a useful in vivo model for evaluation of new therapeutic approaches for lymphoma treatment. The cytokine-induced killer cells generated as described here could have an important impact on bone marrow purging for autologous bone marrow transplantation as well as for adoptive immunotherapy.     

SCID小鼠-人白血病模型在白血病研究中的应用

SCID小鼠-白血病模型是研究白血病细胞增殖、分化及其调控机制的重要手段和方法。本文综述了SCID小鼠-白血病模型的建立及研究进展，并对该模型在白血病发病学、白血病细胞生物、临床诊疗措施和判断病人预后等方面的可能应用进行了探讨。最后提出了此模型的应用局限性，以便更好地完善该模型，用于白血病的研究。     

Targeting of natural killer-like T immunologic effector cells against leukemia and lymphoma cells by reverse antibody-dependent cellular cytotoxicity

Recently, highly efficient natural killer-like T immunologic effector cells called cytokine-induced killer (CIK) cells have been described. Most interestingly, CIK cells have been shown to eradicate established human lymphoma cells in a severe combined immunodeficient (SCID) mouse xenograft model in vivo. The current study was aimed at increasing the sensitivity of leukemia and lymphoma cells to CIK cells. In particular, the authors wanted to target CIK cells to leukemia and lymphoma cells via reverse antibody-dependent cellular cytotoxicity. Binding of an anti-CD3 monoclonal antibody to CIK cell cultures derived from patients with lymphoma was shown using flow cytometric analysis. For the target side, several B-cell lines were found to express CD19 on the cell surface. There was an impressive increase in sensitivity to CIK-mediated lysis of various lymphoma and leukemia cell lines by preincubation of the targets with a monoclonal antibody against CD3. This increase could be partially blocked by preincubation with anti-CD16 (Fc receptor III) and anti-CD32 (Fc receptor II) antibodies. These data suggest that the increase in cytotoxic activity is caused by Fc receptor-mediated antibody binding. Cytotoxic activity could be further increased by adding an anti-CD28 antibody in addition to anti-CD3. Finally, there was a further increase in sensitivity to CIK-mediated lysis of CD19+ malignant cells using the bispecific OKT3xHD37 antibody with specificity against CD3 and CD19. Interestingly, preincubation of malignant cells with an anti-CD3 monoclonal antibody followed by addition of the bispecific OKT3xHD37 antibody led to a further increase of cytotoxic sensitivity compared with the addition of the bispecific antibody alone. In conclusion, these data suggest that cytotoxic activity of immunologic effector cells can be increased not only by using the bispecific antibody OKT3xHD37 in vitro but also by preincubation of CD19+ leukemia and lymphoma cells with a monoclonal antibody against CD3. In addition, the immunostimulatory effect of the bispecific antibody OKT3xHD37 can be further increased by adding a monoclonal antibody against CD3.