SCID小鼠-人白血病模型在白血病研究中的应用新进展

本文综述了白血病细胞在重症联合免疫缺陷(severe combined immunodeficiency,SCID)小鼠体内的增殖、分化及发病机制的研究进展,并介绍了SCID小鼠-白血病模型在白血病发病学、白血病细胞生物学、临床诊疗措施和病人预后判断等方面的可能性应用价值,SCID小鼠-人白血病模型为白血病的深入研究提供了新的手段。     

SCID小鼠-人白血病模型在白血病研究中的应用新进展

本文综述了白血病细胞在重症联合免疫缺陷（severe combined immunodetlciency,SOD）小鼠体内的增殖、分化及发病机制的研究进展，并介绍了SCID小鼠．白血病模型在白血病发病学、白血病细胞生物学、临床诊疗措施和病人预后判断等方面的可能性应用价值，SCID小鼠-人白血病模型为白血病的深入研究提供了新的手段。     

人源化NOD/SCID小鼠的细胞免疫重建

目的探讨人脐带血CD34+细胞移植于非肥胖性糖尿病/重症联合免疫缺陷(NOD/SCID)小鼠后的免疫重建作用。方法免疫磁珠法分选人脐血CD34+细胞,经外侧尾静脉移植于亚致死量照射的NOD/SCID小鼠。移植后4、6、8、10周流式细胞术动态监测小鼠外周血人源CD45+、CD3+、CD56+细胞的数量;10周后PCR检测小鼠骨髓人ALU基因的表达,免疫组织化学染色检测小鼠脾脏人源CD3+、CD56+细胞的表达。结果 NOD/SCID小鼠经照射后骨髓腔内有核细胞及巨细胞数量均明显减少或消失,达到理想的清髓预处理效果;移植后4、6、8、10周,移植组所有存活小鼠外周血均可检测到人源性CD45+、CD3+、CD56+细胞的表达,人源淋巴细胞的数量随时间的延长而变化,8周时达到高峰,10周仍有较高的比例。10周时移植组所有存活小鼠骨髓细胞均检测到人ALU基因的表达,脾脏均检测到人CD3+、CD56+细胞;未移植组小鼠照射后2周内全部死亡。结论经照射后的NOD/SCID小鼠通过移植人脐血CD34+细胞成功地建立了hu-SRC-NOD/SCID模型,并有效地重建了小鼠细胞免疫系统。     

人重组TFAR19蛋白对白血病细胞株HL-60的促凋亡效应

目的：对一种新发现的凋亡相关基因ＴＦＡＲ１９进行蛋白质水平的促凋亡效应研究,并对其作用机理进行初步的探讨。方法：利用离子交换层析对大肠杆菌表达的人重组ＴＦＡＲ１９蛋白进行纯化,将其加入到培养的白血病细胞株ＨＬ－６０,通过ＤＮＡ片段化,ＰＩ及Ａｎｎｅｘｉｎ Ｖ标记进行流式细胞仪分析,观察ＴＦＡＲ１９蛋白的促凋亡效应。利用ＦＩＴＣ标记ＴＦＡＲ１９蛋白,分析其与细胞的结合及定位。     

雷公藤多甙诱导人前骨髓白血病细胞的凋亡

探讨雷公藤多甙诱导人前骨髓白血病细胞凋亡的作用。方法将ＨＬ－６０与雷公藤多甙、泼尼松共同培养,观察细胞的细胞凋亡形态学变化,如细胞膜泡样变、细胞浆及细胞核内染色质固缩、凋亡小体出现以及ＤＮＡ规律性断裂。（２）细胞周期分析提示作用首先影响增殖期细胞,并且呈剂量、时间依赖性。（３）雷公藤多甙诱导ＨＬ－６０细胞凋亡的作用较泼尼松强;两种药物间无协同作用。结论体外实验发现雷公藤多甙具有诱导细胞凋亡的作用,     

人M5型白血病-SCID小鼠模型的建立及白血病病理变化的分析

目的:建立人M5型白血病细胞系SHI-1/SCID(重症联合免疫缺陷)小鼠模型,探讨白血病细胞的接种密度与SCID小鼠成瘤率及病理变化的关系。方法:将不同密度的人白血病细胞SHI-1注射至SCID小鼠腹腔。定期尾静脉取血并以流式细胞术(FCM)监测肿瘤细胞表面标志CD14及CD137L的变化。通过病理组织学检查和FCM分析SCID小鼠的骨髓、肝脏、脾脏和肾脏中白血病细胞的浸润及病理变化。结果:将不同密度的SHI-1细胞移植至小鼠腹腔,均可发现瘤块生长。同时中、高剂量组小鼠的主要组织器官呈现不同程度的肿瘤细胞的浸润。最早在移植3周后即可在尾静脉检测到肿瘤细胞,并与注射细胞量相关。结论:建立的SHI-1/SCID小鼠模型为人M5型白血病的研究提供了有价值的动物模型。     

端粒酶在高三尖杉酯碱诱导HL-60细胞凋亡中的作用

目的 研究高三尖杉酯碱（homoharringtonine,HHT)对HL－60细胞端粒酶活性的影响及诱导凋亡作用。方法 采用端粒重复序列扩增法－酶联免疫吸附试验检测了HL－60细胞的端粒酶活性变化；用细胞形态学观察、DNA琼脂糖凝胶电泳和DNA片段原位末端标记法检测了细胞凋亡现象。结果 0.005-0.50μg/ml HHT处理HL－60细胞0－48小时，HL－60细胞端粒酶活性呈剂量依赖性和时间依赖性下降。同时，HL－60细胞发生了凋亡。结论 HHT有效抑制HL－60细胞的端粒酶活性，诱导细胞凋亡。     

人急性髓系白血病HL-60来源的树突样细胞诱导的CTL体内外作用研究

目的 观察人急性髓系白血病HL－60来源的树突样细胞（HL－60DC）对细胞毒T淋巴细胞（CTL）的免疫调节作用。方法 采用PKC激活剂佛波酯（PMA）及细胞因子GM－CSF、IL－4、TNF－α在体外诱导培养HL－60细胞，获得具有树突状细胞形态、表型（CD1a、CD80、CD86、RelB阳性表达）的HL60DC，观察HL－60DC诱导的CTL体外抗瘤效应，并建立人白血病HL－60／SCID（严重联合免疫缺陷）小鼠异种移植模型进行过继治疗。结果 联合GM－CSF、TNF－α及PMA处理7－11d，获得的HL－60DC体外激活的CTL在体外对HL－60细胞有显著的细胞毒活性。CTL以5：1效靶比多次腹腔回输，能够明显延长荷瘤鼠存活时间，降低瘤块重量，减轻肝、脾、骨髓受肿瘤浸润程度，但流式细胞检测发现部分受治疗的存活小鼠有微小病灶的存在。结论 人急性髓系白血病HL－60来源的树突样细胞诱导的CTL在体内外有一定的抗瘤效应。     

急性髓系白血病干细胞NOD/SCID小鼠白血病模型的建立

目的探讨用非肥胖型糖尿病/严重联合免疫缺陷（NOD/SCID）小鼠和经流式细胞仪分选出的KG1a中CD34＋CD38-的干细胞亚群建立白血病干细胞（LSCs）动物模型,为靶向治疗LSCs的药物筛选奠定体内实验基础。方法 18只雌性NOD/SCID小鼠,体质量18～20 g,鼠龄6～8周龄。实验组12只,应用流式细胞仪分选KG1a细胞中具有LSCs特性的CD34＋CD38-亚群,以2×106个/只尾静脉注射经全身X射线照射2Gy的NOD/SCID小鼠;正常对照组6只,只注射磷酸盐缓冲溶液。观察两组小鼠的一般情况和白血病发生情况,应用形态学、组织病理检查、流式细胞术、骨髓染色体检查等检测实验组小鼠的外周血、骨髓、肝脏、脾脏的白血病细胞标志。结果接种2周后实验组小鼠外周血可见白血病细胞,接种30 d实验组小鼠的白血病发病率为100%,无自发缓解;外周血、骨髓、肝、脾中均可发现大量的白血病细胞浸润;实验组小鼠骨髓细胞中CD13抗原阳性率15.47%～23.66%,并可见KG1a细胞的核型特征。结论尾静脉接种流式细胞仪分选后的CD34＋CD38-KG1a细胞于全身亚致死量X射线照射后的NOD/SCID小鼠,能成功建成全身播散的白血病模型,为进一步研究LSCs的靶向治疗药物奠定实验基础。     

Bcl-2反义硫代磷酸寡脱氧核苷酸抑制HL-60细胞在重度联合免疫缺陷鼠中生长的研究

目的 分析bcl-2反义硫代磷酸寡脱氧核苷酸(ASPO)抑制HL-60细胞在重度联合免疫缺陷(SCID)小鼠体内致白血病作用,探讨应用bcl-2 ASPO体外净化白血病可行性。方法 应用终浓度为10μmol/L ASPO和正义硫代磷酸寡脱氧核苷酸(SPO)与HL-60细胞共孵育,7d后,1×10~7个活细胞腹腔接种SCID鼠;在35d后处死2组动物,用半巢式逆转录聚合酶链反应(RT-PCR)检测SCID小鼠外周血、骨髓、肝脏、脾脏的HL-60细胞,组织病理观察它们浸润分布情况。结果 ASPO处理组HL-60细胞bcl-2表达下降,体外增殖抑制,凋亡,在SCID鼠中不会产生白血病,且未有残留;SPO组HL-60细胞不受影响,仍可在SCID鼠体内广泛增殖浸润,产生白血病。结论 bcl-2反义硫代磷酸寡脱氧核苷酸体外处理白血病细胞后可以抑制其体内增殖作用,可达到体外净化目的。     

人源化NOD/SCID小鼠免疫细胞的动态变化与鉴定

目的： 比较脐血干细胞与单个核细胞移植NOD/SCID鼠所建立的人源化SCID模型，分析人源化淋巴细胞重建。方法： 磁珠分选法分离脐血中CD34⁺细胞，淋巴细胞分层液分离脐血单个核细胞，分别经尾静脉输入NOD/SCID小鼠。每隔2周采血至10周，流式细胞术动态检测人源淋巴细胞CD45、CD19、CD3抗原。第10周处死小鼠收集外周血、骨髓、胸腺组织，RT-PCR检测模型鼠组织中人β₂M基因及RAG2基因。结果： 两种类型细胞移植均可重建人源免疫细胞，人源淋巴细胞表达水平均在第8周达高峰。骨髓中人源淋巴细胞表达水平明显高于外周血。RT-PCR在外周血与骨髓检测到人β₂M基因及RAG2基因标志。结论： CD34⁺细胞移植重建人源化NOD/SCID免疫系统模型效果要好于脐血单个核细胞。人源T淋巴细胞在模型鼠骨髓中分化成熟。     

Spontaneous Abortion in Immunodeficient SCID Mice

PROBLEM: Immunodeficient SCID mice on the CB-17 have been used to test the role of “rejection” in a xenogeneic blastocyst transfer model of recurrent miscarriage, but interpretation of the data requires knowing syngeneic within-species matings have a high success rate and do not require immunotrophic factors expected only in immunocompetent non-T-cell deficient mice. METHOD: Resorption rates were studied in a SCID CB-17 barrier facility that provided the mice used to test the role of immunology in the resorption model. RESULTS: Spontaneous resorption in syngeneically mated immunodeficient SCID mice on the CB-17 background occurred at an unexpectedly high rate and could not be prevented by treatment with anti-asialo GM1 antibody or GM-CSF, both of which are effective in ameliorating abortion in DBA/2J-mated CBA/J mice. Immunocompetent CB-17 +/+ mice showed an even higher rate of loss. The latter was also not affected by treatment with anti-asialo GM1 antibody or by GM-CSF and was not prevented by tetracycline (which is effective in the DBA/2-CBA/J system) or progesterone treatment. Mating experiments showed a scid/+ × scid//+ cross gave the highest rate of loss, and it appeared that the presence of +/+-type embryos in the uterus could be augmenting abortion with selective discrimination against scid/scid embryos. High abortion rates were associated both with appearance of a coagulase-negative Staphylococcus sp. in feces and with loss of one component of the SPF flora. Decidual tissue from mated CB-17 +/+ mice showed premature release of TNF-cc in absence of TGF-β2-related suppressor activity, and vascular lesions (fibrinoid necrosis), varying in extent, were associated with both scid/scid × scid/scid and +/+ × +/+ pregnancies. TNF-α also appeared prematurely in pregnant scid/scid mice, but the levels were lower (and areas of necrosis smaller than in +/+ × +/+ pregnancies). Outcrossing onto a C57B1/6 background dramatically reduced the abortion rate, indicating an important genetic effect on susceptibility with heterogeneity protecting against abortion. CONCLUSIONS: SCID mice on the CB-17 background do not have a high rate of successful syngeneic pregnancies, and a TNF-α induced vasculopathy may be responsible. Abortion was not caused by immunodeficiency leading to loss of immunotrophism because immunocompetent non-SCID CB-17 mice had a higher rate of loss. Factors augmenting the abortion rate included the presence of embryos of the +/+ genotype in the uterus and treatment with anti-asialo GM1 antibody. Abortion rates were not reduced by treatments effective in the DBA/2-mated CBA/J mouse model but were reduced by re-establishing a new colony with defined flora (a temporary effect) and by outcrossing mice with a different (C57B1/6) background. Together, the data suggest an infectious trigger (identity uncertain) of the vasculopathy and an important genetic influence on susceptibility with heterozygosity and a SCID mouse mutation providing against abortion a degree of protection.     

BALB/c和SCID小鼠人偏肺病毒感染特点的比较研究

目的 比较研究人偏肺病毒(hMPV)在BALB/c小鼠和SCID小鼠肺内复制动力学和肺病理特点.方法 GFP-rhMPV滴鼻感染BALB/c小鼠和SCID小鼠,于感染后3、5、7、9、14d处死小鼠并无菌获取心、肝、脾、肺、肾和脑用于病毒分离和病理检查,空斑形成法检测病毒滴度,RT-PCR和real-time PCR法检测hMPV mRNA表达.结果 GFP-rhMPV滴鼻感染BALB/c小鼠和SCID小鼠后第5天肺组织均分离到病毒.感染GFP-rhMPV的BALB/c和SCID小鼠在感染后第5天肺组织病毒滴度均达峰值,分别为(5.25±1.69)×104 PFU/g和(5.83±1.21)×105 PFU/g.SCID小鼠在感染后第14天肺组织内病毒滴度仍可达(4.25±1.04) ×101 PFU/g,此时BALB/c鼠肺内已不能分离到病毒,但可检测到GFP-rhMPV F蛋白mRNA表达.感染后第5天所有小鼠的心、肝、脾、肾和脑组织内均不能分离到病毒,也无法检测到hMPV F蛋白mRNA表达.肺组织病理改变在感染后第5天最明显,为典型的间质性肺炎改变,BALB/c小鼠组肺组织病理评分略低于SCID小鼠组,差异无统计学意义.结论 GFP-rhMPV滴鼻感染后只能在小鼠肺内复制.同BAL B/c小鼠相比,SCID小鼠感染GFP-rhMPV后肺内病毒滴度高,复制时间久,但病理损伤无明显差异.     

Decontamination of a Mycoplasma-infected Chlamydia pneumoniae strain by pulmonary passage in SCID mice

We describe a procedure to eliminate contaminating Mycoplasma from Chlamydia pneumoniae (C. pneumoniae) cultures by pulmonary passage in severe combined immunodeficiency mice (SCID). Four weeks after experimental infection only C. pneumoniae could be cultured from the lungs of the infected animals while Mycoplasma could not be detected any longer, as shown by PCR, culture and transmission electron microscopy (TEM).     

二十碳五烯酸联合视黄酸对HL-60细胞凋亡相关基因表达的影响

目的 研究二十碳五烯酸(EPA)是否协同视黄酸(RA)影响HL-60细胞的凋亡及相应分子机制。方法 流式细胞仪(FCM)检测细胞周期相分布以测定细胞增殖和凋亡功能；Western blot法分析bcl-2和caspase—3表达。结果 与单独应用相比，EPA和RA联合应用可增强HL-60细胞的调亡效应，以及下调节bcl—2和增强caspase-3基因表达。结论 EPA和RA联合应用对HL-60细胞凋亡的增强效应，可能与它们增强caspase-3和降低bcl—2基因表达相关。     

FMLP刺激HL—60细胞NADPH氧化酶激活的信号转导途径

目的 澄清中性粒细胞中[Ca^2 ]i及某些激酶在NADPH氧化酶激活中的作用和NADPH氧化酶激活的信号转导途径。方法 利用中性粒细胞样HL-60细胞,研究[Ca^2 ]i和某些激酶对fMLP刺激NADPH氧化酶激活的影响。结果 用10umol/L BAPTA-AM去除[Ca^2 ]i后使O2^-生成明显减少;8umol/L的PKC抑制物GF109203x显著地抑制了O2^-产生;50umol/L的p38抑制物SB203580、50umol/L的ERK抑制物PD098059和0.1umol/L的PI3K抑制物Wortmannin使O2^-产生受到不同程度抑制;PKC、PI3K、p38和ERK激活对[Ca^2 ]i升高没有影响;[Ca^2 ]i、PKC和PI3K对p38激活有一定作用,而ERK和Akt激活主要受PI3-K的调控。结论 试验说明[Ca^2 ]i依赖途径（PKC）和[Ca^2 ]i非依赖途径（PI3K、p38和ERK）对NADPH氧化酶激活都起着重要作用。     

Suppression of dsDNA-specific B lymphocytes reduces disease symptoms in SCID model of mouse lupus

Abstract

Self-specific B cells play a main role in the pathogenesis of lupus. This autoimmune disease is characterized by the generation of autoantibodies against self antigens, and the elimination of B and T cells involved in the pathological immune response is a logical approach for effective therapy. We have previously constructed a chimeric molecule by coupling a DNA-mimotope peptides to an anti-CD32 antibody. Using this protein molecule for the treatment of lupus-prone MRL/lpr mice, we suppressed selectively the autoreactive B-lymphocytes by cross-linking B cell receptors with the inhibitory FcγRIIb receptors. This approach was limited by the development of anti-chimeric antibodies in MRL mice. In order to avoid this problem, we established a murine severe combined immunodeficiency lupus model, allowing a long-term chimera therapy. Elimination of the double-stranded DNA-specific B cells by chimera therapy in MRL-transferred immunodeficient mice resulted in inhibition of T cell proliferation and prevented the appearance of IgG anti-DNA antibodies and of proteinuria.