Establishment of Xenotransplantation Model of Human CN-AML with FLT3-ITD^(mut)/NPM1 in NOD/SCID Mice

Summary： Patients with FLT3-ITD^mmutt/NPM1- cytogenetically normal acute myeloid leukemia （CN-AML）, as high-risk molecular group in CN-AML, are associated with a worse prognosis than other CN-AML patients. It is beneficial to generate xenotransplantation model of FLT3-ITD^mut/NPM1- CN-AML to better understand the pathogenesis and therapeutic strategies of such AML subtype. The purpose of present study was to establish the xenotransplantation model in NOD/SCID mice with FLT3-ITD^mut/NPM1- CN-AML primary cells. The FLT3-ITD^mut/NPM1- CN-AML primary cells from 3 of 7 cases were successfully transplanted into NOD/SCID mice, and human CD45 positive cells were detected in the peripheral blood, spleen and bone marrow of mice by using flow cytometry. Infiltration of human leukemia cells in various organs of mice was observed by using immunohistochemistry. Gene analysis confirmed sustained FLT3/ITD mutation without NPM1 mutation in mice. By performing serial transplantation, it was found that characteristics of the leukemia cells in secondary and tertiary genera- tion models remained unchanged. Moreover, in vivo cytarabine administration could extend survival of NOD/SCID mice, which was consistent with clinical observation. In conclusion, we successfully estab- lished xenotransplantation model of human FLT3-ITD^mut/NPM1- CN-AML in NOD/SCID mice. The model was able to present primary disease and suitable to evaluate the curative effects of new drugs or therapy strategies.

Establishment of Reproducible Xenotransplantation Model of T Cell Acute Lymphoblastic Leukemia in NOD/SCID Mice

T cell acute lymphoblastic leukemia(T-ALL) is an aggressive leukemia.However the poor prognosis and low morbidity restrict further analysis of the disease.Therefore there is an increasing demand to develop animal models for identifying novel therapeutic approaches.In this study,we inoculated the anti-mouse CD122 monoclonal antibody conditioned NOD/SCID mice with the leukemia cells from 9 T-ALL patients and 1 cell line via the tail vein.Four of the 9 patients and the cell line were successfully engrafted.Flow cytometry detected high percentage of human CD45 + cells in recipient mice.Immunohistochemistry showed infiltration of human CD45 + cells in different organs.Serial transplantation was also achieved.In vivo drug treatment showed that dexamethasone could extend survival,which was consistent with clinical observation.These results demonstrated that we successfully established 5 xenotransplantation models of T-ALL in anti-mCD122 mAb conditioned NOD/SCID mice,which recapitulated the characteristics of original disease.

ESTABLISHMENT OF INTRAPERITONEAL TRANSPLANTATION MODEL OF CISPLATIN-RESISTANT OVARIAN CARCINOMA CELL IN SCID MICE

Objective： to develop an intraperitoneal transplantation model of human ovarian carcinoma SKOV3/CDDP cell in severe combined immunodeficiency （SCID） mouse and to study its biologic characteristics. Methods： Sixteen qualified C.B 17/SCID mouse were divided into two groups randomly. Human ovarian carcinoma SKOV3 or SKOV3/CDDP cells were injected intraperitoneally into the SCID mouse at the amount of 1×10^7 cells （0.5 mL） per mouse. The behaviors of mice, tumor growth and morphology were analyzed. The expression of cancer antigen 125 （CA125）, GST-π and Topo-Ⅱ were examined by immunohistochemical method. Results： In this experimental study, transplanted tumors are formed in 100% SCID mice in the two groups. The morphology, growth pattern and CA125 secretion of SKOV3/CDDP group were as same as those of SKOV3 group. It shows that the tumors of the two groups kept the characteristics of ovaries serosity papillary adenocarcinoma. Compared with SKOV3 group, the expression of GST-π and Topo-Ⅱ gene in SKOV3/CDDP group were significantly higher （P〈0.05）. Conclusion： An intraperitoneal transplantation model of human ovarian carcinoma SKOV3/CDDP in SCID mice has been developed successfully. It may be an ideal animal model for biotherapy research of ovarian carcinoma as it can simulate the biological behavior of peritoneal metastasis of human ovarian carcinoma and the drug tolerance is maintained.

Radioprotective effects of the expression of FLT3 ligand regulated by Egr-1 regulated element on radiation injury of SCID mice

Objective: In order to explore the radioprotective effects of the expression of hematopoietic growth factors regulated by radio-inducible promoter on radiation injury. Methods:The human FL (Flt3 ligand) cDNA and EGFP (enhanced green fluorescent protein) cDNA were linked together with IRES and then inserted into the eukaryotic expression vector pCI-Egr, which was constructed by substituting CMV promoter in pCIneo with the Egr-1 promoter (Egr-EF). The vector was transferred into human bone marrow stromal ...

Establishment of Retinoblastoma Model in NOD-SCID Mice and Study of Metastasis

Purpose: To establish model of retinoblastoma subcutaneously in NOD-SCID mice and study rules of formation and distribution of retinoblastoma metastasis.Methods: Retinoblastoma cells SO-RB50 were inoculated subcutaneously in NOD-SCID mice. Animal acts and tumor formation, growth and metastasis in NOD-SCID mice were observed. Primary and metastatic tumors were studied pathohistologically by HE and immunohistochemical staining.Results: The latent periods of tumor growth were 12～19 days and the taken rate of tumor was 100%. 32 days later, 5 NOD-SCID mice were found with tumors that had metastasized to areas mainly located in the abdominal cavity and the side of the kidney; the metastatic time of tumors in the mice also differed. The tumor cells of the primary nodules and the metastasis were similar with human retinoblastoma cells and positive in immunohistochemical staining of NSE.Conclusion: The subcutaneous model of retinoblastoma in NOD-SCID mice showed a high taken rate and a short latent period of tumor, which had a high metastatic rate and was the best model in research of behaviors of retinoblastoma at present.

Xeno-repopulation of Fah^(-/-)Nod/Scid mice livers by human hepatocytes

Functional human hepatocytes xenografted into the liver of mice can be used as a model system to study pharmacokinetics,infection of hepatitis viruses,and the efficacy of hepatitis vaccines.Significant levels of liver xeno-repopulation have been reported in Fah-/-Rag2-/-Il2rg-/-mice.However,the high mortality and low breeding rate of this model may hinder its application.A new model,termed Fah-/-Nod/Scid mice,which combines the advantages of liver repopulation in Fah-/-mice with the ease of xenotransplantation in Nod/Scid mice was obtained by gradual cross-breeding.Fah-/-Nod/Scid mice were easily maintained in breeding colonies and in adult animal care facilities.FK506 treatment combined with gradual withdrawal of NTBC before cell transplantation ensured that Fah-/-Nod/Scid mice were susceptible to liver xeno-repopulation by human hepatocytes;the proportion of engrafted human hepatocytes reached 33.6%.The function of the expanded human hepatocytes within the chimeric liver was confirmed by weight curve analysis,the expression of characteristic proteins,and the biochemical analysis of liver function.These results show that Fah-/-Nod/Scid mice are an ideal humanized liver mouse model with many useful applications.

SLAM family predicting the initiation potential of human acute lymphoblastic leukemia in NOD/SCID mice

Background The SLAM family recently has been reported to show an important biological role in lymphocyte development and immunological function, and it is efficient to highly purify hematopoietic stem cells using a simple combination of SLAM family members. To elucidate the presence of this family on acute lymphoblastic leukemia （ALL）,as well as its relationship with the leukemia-initiating potential, we analyzed the expression pattern of this family members on human ALL progenitor cells, combined with serial xenotransplantation assay.Methods Expression analysis was carried out by flow cytometry. We combined the expression pattern of human CD150,CD244 and CD48 with serial xenotransplantation of B-ALL progenitor cells to indicate their relationship.Results CD48 and CD244 were expressed on most B-ALL progenitor cells, the percentage being （93.08±6.46）% and （63.37±29.31）%, respectively. Interestingly, the proportion of CD150＋ cells declined obviously in engrafted cases （（24.94±7.32）%） compared with non-engrafted cases （（77.54±5.93）%, P 〈0.01）, which indicated that only blast cells with low percentage of CD150＋ population were able to reconstitute leukemia into primary, secondary and tertiary NOD/SCID mice.Conclusions SLAM family members are present on B-ALL progenitor cells and the leukemia-initiating potential of leukemic blasts is correlated negatively with the proportion of CD150＋ cells, the percentage of which can serve as a useful predictor for engraftment success of B-ALL to immune deficient mice.

Experimental study on ex vivo expanded hematopoietic stem/progenitor in the two step culture from human umbilical cord blood transplanted into NOD/SCID mice

The effects of hematopoietic stem/progenitor cells(HSPCs)expanded in the two step coculture with human bone marrow mesenchymal stem cells(hMSCs)on the hematopoietic reconstruction of irradiated NOD/SCID mice were studied.Mononuclear cells(MNCs)were isolated from human umbilical cord blood(UCB)and cultured in the non-coculture scheme of rhSCF+rhG−CSF+rhMDGF combination and the coculture scheme of rhSCF+rhG−CSF+rhMDGF+hMSCs.Sublethally-irradiated NOD/SCID mice were transplanted with ex vivo expanded HSPCs with the dose of 8.5×10^(6) cells per mouse.After transplantation,the dynamics of WBC in the transplanted mice was measured periodically,and the Alu sequence fragment special for human in the transplanted mice was inspected by PCR.Results showed that the coculture scheme increased proliferation of UCB-derived HSPCs.After transplantation with expanded HSPCs,the population of WBC in the transplanted mice increased in 12 d and reached the first peak in 25 d,then showed the second increasing of WBC in 45~55 d.Expanded cells from the coculture scheme appeared to be favorable for the second increasing of WBC in the transplanted mice.After 85 d,the Alu sequence fragment was detected in the probability of 87.5%(7/8)for the non-coculture scheme and 88.9%(8/9)for the coculture scheme.

NOD-Scid小鼠播散性弥漫大B细胞淋巴瘤模型的构建

目的利用免疫缺陷的NOD-Scid小鼠建立播散性弥漫大B细胞淋巴瘤(diffuse large B-cell lymphoma,DLBCL)小鼠模型,为动物模型的构建提供新思路。方法先将人源DLBCL细胞OCI-Ly8导入荧光素酶基因,使其长期稳定表达荧光素酶。再将OCI-Ly8-luc^(+)细胞经尾静脉注射到NOD-Scid小鼠体内,利用生物发光成像系统每隔3天观察1次小鼠体内肿瘤细胞播散情况。结果实验第7天监测到OCI-Ly8-luc^(+)细胞在小鼠体内发生播散,并且进展迅速,20天时出现小鼠死亡。结论利用OCI-Ly8-luc^(+)细胞尾静脉注射成功构建了NOD-Scid小鼠DLBCL模型,为DLBCL的动物模型构建提供新方法。

小剂量K562细胞NOD/SCID小鼠动物模型的建立

【目的】探索建立小剂量(1×106)人急性红白血病K562细胞株的NOD/SCID小鼠动物模型及通过流式细胞仪对NOD/SCID小鼠的肿瘤负荷情况进行评价的可行性。【方法】实验组NOD/SCID小鼠分别经尾静脉接种K562细胞1×106、5×106,比较不同剂量接种实验组小鼠的生存时间、组织病理改变及通过流式细胞术对NOD/SCID小鼠体内的肿瘤标记进行检测。【结果】1×106及5×106K562细胞接种的NOD/SCID小鼠的生存时间分别为(30.3±4.3)d和(22.2±3.7)d;其外周血、骨髓及肝、肺组织匀浆中均可发现不同比例的肿瘤细胞;通过流式细胞术检测5×106K562细胞接种组NOD/SCID小鼠外周血、肝匀浆中人CD13表达水平显著高于1×106接种组,而肺匀浆的CD13表达水平两组无显著性差异。【结论】小剂量(1×106)K562细胞NOD/SCID小鼠动物模型的建立是完全可行的,这有助于降低实验成本。

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。

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。

人肝癌NOD-SCID小鼠原位移植模型的建立及其生物学特性的研究

目的：建立人肝癌NOD-SCID小鼠原位移植模型,并通过和裸小鼠原位移植模型生物学特性的比较,对两种不同类型免疫缺陷动物在人肝癌异种移植方面的应用价值进行初步探讨。方法：将组织学完整的SMMC-LTNM瘤块植入NOD- SCID小鼠和裸小鼠肝脏内,观察成瘤率、肿瘤体积和重量、脏器的转移情况以及血清甲胎蛋白(AFP)、γ-谷氨酰转肽酶同工酶Ⅱ(γ-GTⅡ)等。结果：NOD-SCID小鼠于移植后5周可扪及肿瘤的生长,成瘤率为100%,11周肿瘤体积为(4．48±0．93) cm^3,瘤重为(7．02±1．15)g,肺部转移率为53．85%；裸小鼠于移植后6～7周可扪及肿瘤的生长,成瘤率为100%,11周肿瘤体积为(1．02±0．70)cm^3,瘤重为(2．87±0．44)g,体内未见转移发生。NOD-SCID小鼠的瘤体积、瘤重、肺转移率均高于裸小鼠(P＜0．01)。两者均保持AFP高分泌和γ-GTⅡ同工酶阳性的特性。结论：建立了一个与临床相似的人肝癌动物模型,与裸小鼠相比,NOD-SCID小鼠在建立人肝癌异种移植模型方面有更大的应用价值。

NOD/SCID小鼠平台上的肿瘤实验研究进展

非肥胖糖尿病/严重联合免疫缺陷(non-obese diabetes-SCID mice,NOD/SCID)小鼠具有T、B淋巴细胞联合免疫缺陷、NK细胞活性低下、无循环补体、巨噬细胞和抗原提呈细胞功能损害等特性,近年已成为人类肿瘤移植瘤的最佳研究模型。NOD/SCID鼠的人类血液系统肿瘤模型能够研究造血微环境、白血病细胞的分化调控机制和潜在的治疗靶点,并且建立在此平台上的体内药敏实验,可以指导临床化疗方案设计,目前已显示出突出的疗效。乳腺癌等实体瘤原代肿瘤组织的移植瘤模型,也显示出可复制人类肿瘤特点的优势。此外,NOD/SCID鼠可进行肿瘤干细胞的筛选和鉴定,并寻找针对肿瘤干细胞的特异性治疗靶点。目前的研究已展现了NOD/SCID鼠在肿瘤研究领域的广阔前景。

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。

应用SCID鼠筛选肝癌转移性亚克隆及M-H7402亚细胞系的建立

本研究应用人肝癌细胞SCID鼠转移模型,筛选和建立肝癌转移细胞系,旨在为肝癌转移研究提供实验材料.采用人肝癌细胞H7402,腋后背部皮下接种SCID鼠,接种后66天时,在荷瘤鼠羸弱时引颈处死,取其肺组织,进行原代细胞培养和传代培养,获得肝癌细胞H7402的亚细胞克隆,命名为M-H7402.然后,对M-H7402细胞进行了生物学鉴定,检测了细胞形态、染色体、细胞动力学、细胞周期、甲胎蛋白表达、癌基因表达和转移相关基因表达等指标.结果显示,M-H7402细胞的生长、增殖和形态等与亲本细胞H7402十分相近,其染色体形态仍为人类核型,众数维持在72～80之间,占75.0%.RT-PCR检测结果显示,M-H7402细胞甲胎蛋白表达为阳性.Western blot检测结果显示,与亲本细胞H-7402相比,癌基因C-myc表达水平较高,转移抑制基因nm23的表达水平明显下调.从肺组织中获得的肝癌转移细胞系M-H7402,在体外可连续传代培养,细胞形态不变.应用SCID鼠筛选获得的亚细胞克隆,与亲本细胞形成配对关系,可为肝癌细胞转移的研究提供新的实验材料.

三种酶联免疫吸附法筛选“渗漏”型SCID小鼠的比较

目的 选择简单的方法筛选“渗漏”型SCID小鼠。 方法 采用 3种酶联免疫吸附法 (双抗夹心ELISA、双抗夹心间接ELISA及生物素 亲和素夹心ELISA)测定SCID小鼠血清中IgG含量。 结果 生物素 亲和素夹心ELISA法灵敏度最高 ,用此法共检测了 4 1只 6～ 8周龄小鼠 ,其中 4 0只的IgG含量为 0 2 4± 0 16mg L ;只有 1只小鼠IgG含量为 2 3mg L ,渗漏比例为 2 4 %。 结论 用生物素 亲和素夹心ELISA法对SCID小鼠“渗漏”进行初筛 ,是简便、敏感。

NOD/SCID小鼠模型在实验血液学研究中的应用

NOD/SCID(非肥胖糖尿病/重症联合免疫缺陷)小鼠是在SCID(重症联合免疫缺陷)小鼠的基础上与非肥胖性糖尿病小鼠(NOD/Lt)品系回交的免疫缺陷鼠。NOD/SCID小鼠既有先天免疫缺陷,又有T和B淋巴细胞缺乏,各种肿瘤细胞可以植入,且较少发生排斥反应及移植物抗宿主病(GVHD),所以NOD/SCID小鼠逐渐成为血液学实验研究的有用工具。本文从NOD/SCID小鼠的生物学特性、建立人类白血病模型、干细胞移植、药物研究及NOD/SCID小鼠应用中存在的不足和改良等方面综合述评。

NOD/SCID雌性小鼠动情周期的观察

目的观察NOD/SCID小鼠动情周期及卵巢切除术后阴道涂片的变化。方法连续观察9 d NOD/SCID鼠,每日进行两次阴道涂片,计算动情周期时间及发生率。卵巢切除后,阴道涂片并观察阴道涂片变化。结果NOD/SCID小鼠动情周期为4-6 d。有规律动情的小鼠占80%。阴道口状态与动情周期无明显相关。卵巢切除术后,阴道涂片呈动情后期或动情间期改变。结论采用阴道脱落细胞涂片法可判断NOD/SCID雌性小鼠的动情周期及特点,NOD/SCID雌性小鼠卵巢切除手术有效。

视网膜母细胞瘤SO-RB_(50)瘤细胞NOD-SCID鼠皮下异位移植全身转移瘤模型的建立

目的:建立人视网膜母细胞瘤(RB)的严重联合免疫缺陷(severe combined immunodeficent)小鼠(NOD-SCID鼠)皮下移植模型,并探讨RB转移的发生、分布规律.方法:NOD-SCID小鼠皮下接种人视网膜母细胞SO-RB50细胞,定期观察小鼠的行为及肿瘤的形成、生长、转移.采用HE,免疫组织化学染色方法,对皮下肿瘤原发灶及远处转移灶行病理组织学研究.结果:皮下接种小鼠肿瘤生长的潜伏期为12～19d,成瘤为100%,5只鼠有远处转移灶,发生转移的时间在32d后,时间分布不一,肿瘤转移灶主要分布在腹腔和肾旁,小鼠的异种移植瘤和转移灶组织形态上与人类的RB一致,光镜下细胞胞质少、核深染、可见病理性核分裂,为未分化型.结论:NOD-SCID小鼠的荷瘤人视网膜母细胞瘤模型,成瘤率高,成瘤潜伏期短,较高转移率的特点.