人胸腺和脐血造血干/祖细胞联合移植对裸鼠细胞免疫功能的影响

背景：T细胞在抗感染、抗肿瘤和免疫调节等中起重要作用,但T细胞分化发育机制尚未完全阐明。 目的：观察人胸腺、人脐血联合移植后裸鼠体内T细胞分布及免疫功能的重建。 方法：Balb/c nu/nu裸鼠30 只,随机分为2组：实验组肾被膜下移植胸腺组织,2周后将新鲜分离的脐血CD34+细胞悬液经小鼠静脉输入,对照组不经胸腺移植直接给予CD34⁺细胞移植,两组小鼠饲养至60 d时检测免疫功能。 结果与结论：人胸腺在裸鼠肾被膜下存活并且表达CD3、HLA-DR分子,胸腺与CD34⁺细胞联合移植组小鼠脾脏可见点块状分布的CD3⁺细胞。实验组CD3⁺细胞、CD4⁺细胞、CD8⁺细胞及CD4⁺CD25⁺细胞比例均显著高于对照组。实验组裸鼠对移植人胃癌BGC823细胞有排斥作用,而对照组没有。结果显示胸腺和CD34⁺细胞联合移植能使裸鼠获得T细胞介导的细胞免疫功能,具有抗肿瘤能力。     

脐血体外同时扩增T、NK及干祖细胞移植治疗BALB/c小鼠白血病

目的观察体外同时扩增T、NK及干祖细胞脐血用于移植治疗BALB/c小鼠白血病效果.方法在不同的细胞因子组合作用下定向扩增脐血干祖细胞及T、NK免疫细胞.扩增7天后,5 × 106个/只脐血细胞经尾静脉接种X射线照射的白血病小鼠,观察各组小鼠存活时间及检查存活证据及微小残留白血病细胞.结果1.细胞因子SCF,IL-3,IL-6,IL-7,IL-2组合能显著扩增脐血中的CD34 、CD3 T、NK细胞.2.尾静脉接种2×106个/只K562产生可移植性白血病BALB/c裸鼠.3.BALB/c白血病小鼠移植6周后,实验组共存活鼠12只.而未移植者在60天内死于肿瘤.4.流式细胞分析移植30天后小鼠外周血中人CD3 细胞从最低新鲜脐血移植组(0.48±0.40)%,最高SCF IL-3,6,7组中为(3.01±1.25)%.RT-PCR 检测发现,在12只扩增后移植存活6周小鼠中,检测不到bcr/abl 基因.在3只新鲜脐血移植存活6周的小鼠中,1只呈现阳性,说明移植取得了造血与免疫重建.所有脐血移植小鼠均未出现明显临床GVHD 症状.结论这种扩增后含一定水平T、NK免疫细胞的脐血能重建可移植性BALB/c 白血病小鼠的造血和免疫.     

BALB/c鼠和裸鼠呼吸道合胞病毒感染比较研究

目的 比较普通BALB/c鼠和裸鼠呼吸道合胞病毒(RSV)感染免疫及炎症反应特点.方法 BALB/c鼠和裸鼠感染RSV后不同时间空斑形成试验检测肺组织病毒滴度,计数支气管肺泡灌洗液(BALF)白细胞总数和分类,HE染色分析肺组织病理学改变,免疫组化检测肺组织F4/80+细胞和CD49b+细胞.ELISA检测BALF中TNF-α、IFN-r、IL-12和IL-10浓度.结果 BALB/c鼠和裸鼠感染RSV后肺组织病毒滴度在第3天达峰值,感染裸鼠带毒时间更长,在感染后各天病毒滴度明显高于BALB/c鼠(P＜0.05),肺组织病理改变也更重.感染BALB/c鼠和裸鼠BALF白细胞总数明显升高,分类以淋巴细胞为主.感染裸鼠与感染BALB/c鼠比较,肺组织检测到更多的F4/80+巨噬细胞和CD49b+NK细胞(P＜0.05),BALF中TNF-α、IL-12和IL-10水平更高(P＜0.05).结论 RSV感染裸鼠与BALB/c鼠比较,病毒复制水平更高,时间更持久,炎症反应更重.单核巨噬细胞和NK细胞是RSV感染重要的免疫细胞和炎症细胞,炎症反应强度并不一定与T细胞免疫应答平行.     

细胞因子介导的人脐血单个核细胞体外扩增并重建NOD/SCID小鼠造血及免疫功能

背景:细胞因子介导的脐血造血细胞体外扩增有望能解决脐血移植数量不足的问题。目的:实验拟确立在无基质培养条件下体外扩增脐血单个核细胞最合适的细胞因子组合及干细胞因子、FLT3配基协同促聚核细胞生成因子对造血及免疫重建功能的影响。方法:将新鲜脐血标本分离的单个核细胞接种于含有不同细胞因子组合的无血清无基质培养体系中培养7d,根据不同细胞因子组合分组,将3因子干细胞因子+FLT3配基+促聚核细胞生成因子组合在无血清无基质条件下扩增培养7d前后的脐血单个核细胞移植给经亚致死量照射的NOD/SCID小鼠。在扩增培养0,7d检测脐血CD34+细胞数及CD34+CXCR4+,CD34+CD62L+,CD34+CD49d+的细胞数。脐血移植6周后通过流式细胞仪,PCR法检测存活小鼠体内的人源性细胞。结果与结论:移植6周后,存活小鼠体内均可检测到人源性CD45+细胞,扩增脐血移植组的NOD/SCID小鼠存活率和人特异性基因捡出率均高于新鲜脐血移植组和高于生理盐水移植组(P0.05)。扩增脐血组存活NOD/SCID小鼠骨髓中可检测到人髓系细胞(CD33+),T淋巴细胞(CD4+),B淋巴细胞(CD19+)和人造血干细胞成分(CD34+)细胞的表达。结果提示干细胞因子+FLT3配基+促聚核细胞生成因子3因子组合脐血造血细胞体外扩增是最合适的细胞因子组合,其扩增的脐血单个核细胞能够植入并重建NOD/SCID小鼠的造血及免疫功能。     

异基因骨髓间充质干细胞移植对EAE小鼠的治疗作用

目的 探讨异基因骨髓间充质干细胞(BMSC)对实验性自身免疫性脑脊髓膜炎(EAE)的治疗作用及相关免疫调节机制.方法 用髓鞘少突胶质细胞糖蛋白(MOG)多肽片段诱导建立C57BL/6J小鼠EAE模型;分离纯化培养BALB/c小鼠BM-SC,对EAE模型小鼠进行移植,移植前后对EAE小鼠进行神经功能评分;流式细胞术检测小鼠淋巴器官CD4+ CD25+ Foxp3+T细胞(Treg)的数量;荧光定量RT-PCR检测小鼠脾脏IL-2、IL-4、IL-17和IL-23 mRNA水平的表达.结果 异基因BMSC移植后小鼠神经功能评分明显改善;BMSC移植组小鼠胸腺、脾脏、淋巴结Treg数量显著增加;脾脏中IL-2、IL-17表达显著下降,同时IL-4、IL-23表达显著增加.结论 异基因BMSC移植通过调节免疫系统的Treg数量和CD4+T细胞分泌细胞因子的水平对EAE起治疗作用.     

骨髓间充质干细胞对肾病综合征大鼠CD4+CD25+调节性T细胞的影响

背景：CD4⁺CD25⁺调节性T细胞功能降低、数量下降已被公认为是肾病综合征患儿免疫失调的重要表现。骨髓间充质干细胞具有免疫调节功能,可上调CD4⁺CD25⁺调节性T细胞,抑制淋巴细胞增殖,并已在许多免疫性疾病方面成功应用。 目的：观察骨髓间充质干细胞移植对原发性肾病综合征大鼠外周血CD4⁺CD25⁺调节性T细胞的影响。 方法：自SD大鼠分离骨髓间充质干细胞,经体外传代培养及鉴定后制备细胞悬液。30只SD大鼠随机均分为3组,生理盐水组和干细胞移植组尾静脉注射阿霉素建立阿霉素肾病大鼠模型,干细胞移植组注射阿霉素当日尾静脉射干细胞1×10⁷;正常组不做处理。 结果与结论：①与正常组比较,生理盐水组大鼠均出现肾病综合征表现,以腹水、大量蛋白尿、低蛋白血症、高胆固醇血症为特征;干细胞移植组较生理盐水移植组则有明显改善(P < 0.05)。②造模第28天,干细胞移植组和生理盐水组大鼠造模后外周血CD4⁺CD25⁺Treg/ CD4⁺ Treg均明显高于正常组(P < 0.05);干细胞移植组与生理盐水组比较差异无显著性意义(P > 0.05)。③造模第28天,干细胞移植组大鼠外周血单个核细胞FoxP3mRNA的表达显著高于生理盐水组和正常组(P < 0.05)。提示骨髓间充质干细胞移植对阿霉素肾病综合征模型有一定的治疗作用,其机制可能与上调FoxP3在组织局部的表达而诱导CD4⁺CD25⁺Treg产生有关。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

苏木水提物在同种皮肤移植中诱导免疫耐受的实验研究

目的:探讨苏木水提物在小鼠同种皮肤移植中诱导耐受的作用。方法:C57BL/6和BALB/c小鼠分别为供体和受体,建立皮肤移植模型。实验分为对照组、苏木组、苏木加半量环孢素A(CsA)组、CsA组。术前3d给药,术后逐日观察移植皮片存活情况;术后3、7d时检测脾组织中CD4+ CD25+T细胞数量变化及Foxp3 mRNA表达情况。结果:与对照组比,苏木组、苏木加半量CsA组、CsA组移植皮片存活时间明显延长(P<0.01),移植后3、7d时,CD4 +CD25+T细胞数量及Foxp3的相对表达量显著增多(P<0.05)。结论:苏木水提物可延长同种移植物的存活时间,上调CD4+ CD25+T细胞及Foxp3的活性,有较强的免疫抑制作用,有望用于诱导免疫耐受。     

表达SDF-1/HOXB4融合基因的间充质干细胞联合脐血CD34~+干细胞共移植对辐射损伤小鼠的影响

目的观察表达SDF-1/HOXB4融合基因的间充质干细胞(mesenchymal stem cells,MSCs)联合脐血CD34+造血干细胞(hematopoietic stem cells,HSCs)共移植对辐射损伤小鼠的影响。方法表达SDF-1、HOXB4和SDF-1/HOXB4基因的3个腺病毒载体分别转染正常人骨髓MSCs,将其联合脐血CD34+细胞经尾静脉移植到辐射损伤的NOD/SCID小鼠体内(MSCs 8×105细胞/只,CD34+1×105细胞/只),分别为SDF-1/MSCs+CD34+组(SDF-1组)、HOXB4/MSCs+CD34+组(HOXB4组)、SDF1-HOXB4/MSCs+CD34+组(S-H组),另外3组为未转染MSCs+CD34+组(MSC-HSC组)、单纯CD34+组(HSC组)、单纯辐照组(IR组)。检测移植后各组小鼠存活率、外周血象恢复、骨髓病理变化及人源CD45+细胞植入率。结果 S-H组小鼠存活率高,且外周血WBC、HGB、PLT和骨髓造血功能恢复快。在移植后6周骨髓CD45+细胞植入率(47.43%±8.89%)较其余各组高。结论表达SDF-1/HOXB4融合基因的间充质干细胞(MSCs)联合脐血CD34+造血干细胞(HSCs)共移植能促进造血重建及植入,提高移植成功率。     

静脉输注过氧化氢溶液上调小鼠脾脏和外周血CD4+Foxo3+调节性T细胞

背景：现已证实,天然生成的调节性T细胞在维护机体免疫稳态、抑制炎性反应、抗移植排斥、抑制抗肿瘤免疫应答以及防治自身免疫病中都有重要的功能和作用。 目的：探讨静脉输注过氧化氢对BALB/c小鼠CD4⁺CD25⁺Foxo3⁺ 调节性T细胞的影响。 方法：取BALB/c小鼠12只,随机分为两组。H₂O₂处理组小鼠尾静脉注射含有H₂O₂的PBS;PBS组小鼠静脉注射无菌PBS。分别在注射2周后处死小鼠,采集小鼠外周血、脾脏和胸腺,采用流式细胞仪检测其中CD4⁺CD25⁺ Foxp3⁺T细胞比例。 结果与结论：H₂O₂处理组小鼠外周血和脾脏CD4⁺Foxp3⁺ T细胞比例、CD4⁺Foxp3⁺/CD4⁺细胞比例均高于对照组小鼠(P < 0.05)。提示静脉注射H₂O₂能上调小鼠外周血和脾脏内CD4⁺Foxp3⁺调节性T细胞的比例。     

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。     

Immunoglobulin synthesis in nude (nu/nu), nu/+ and reconstituted nu/nu mice infected with a demyelinating strain of Semliki Forest virus

Infections with the avirulent (A7/74) strain of Semliki Forest virus which causes primary demyelination of the central nervous system in mice have been studied further in nude athymic (nu/nu) mice and their immunocompetent (nu/+) litter mates to measure the production of immunoglobulins. This has been done by radial immunodiffusion and enzyme-linked immunosorbent assays. Half the nude mice examined were able to synthesize specific IgG but at levels 1,000-fold lower than their nu/+ littermates. The majority of nude mice reconstituted with spleen cells from nu/+ mice 1 day before infection with virus were able to synthesize specific IgG nearly as well as the nu/+ animals.     

Antibody formation to double-stranded DNA in nu/nu and nu/+ mice.

Antibody formation to double-stranded (ds-) DNA in nude mice (nu/nu) and normal littermates (nu/+) was studied using a Millipore filter method. The mean antibody titre to ds-DNA in the sera of nu/nu mice was generally higher than that in nu/+ mice raised in the same environment. However, in both genotypes, anti-ds-DNA titres varied according to the environmental condition in which the animals were raised. Mice maintained germ-free showed no significant antibody formation to ds-DNA. Nu/+ thymus and spleen cells passively transferred to nu/nu mice reduced the antibody titre to ds-DNA in the recipients. Immunochemical studies indicated that the antibodies to ds-DNA were IgM. It is suggested that T cell-mediated regulatory mechanisms control antibody formation to ds-DNA and that some infections trigger antibody formation to ds-DNA.     

Coxsackievirus B3 persistence and myocarditis in NFR nu/nu and +/nu mice

NFR nude (nu/nu) and euthymic (+/nu) littermates were infected with coxsackievirus B3 (CBV-3) and assayed for virus persistence in the heart, pancreas and spleen, for development of myocarditis and for antibody production. The virus grew to higher titer and persisted longer in hearts of nu/nu mice. In both types of mice there was comparable myocarditis with a mononuclear cell inflammatory response, and there was some evidence of chronic lesions for up to 21 days in +/nu and 28 days in nu/nu mice. Antibody of the IgM class was present at 7 days in both strains of mice. Thereafter, +/nu mice produced high titers of virus-specific IgG antibody, while nu/nu mice produced little or no viral IgG antibody. The persistence of virus for up to 28 days in NFR nude mice is longer than has been reported previously, and offers an opportunity for further study of the role of T-cells in virus persistence and myocarditis.     

裸鼠实验性矽结节形成与免疫应答关系的探讨

先天性胸腺缺陷的裸鼠及正常ＢＡＬＢ／ｃ小鼠腹腔注射二氧化硅后，连续８周动态观察血清ＩｇＭ，ＩｇＧ，抗ｓｓＤＮＡ抗体的变化及其与矽结节形成的关系，结果表明：两组实验鼠在形成矽结节过程中，免疫反应不同，ＢＡＬＢ／ｃ鼠诱导形成的矽结节明显大于裸鼠组，ＢＡＬＢ／ｃ组鼠血清ＩｇＭ，ＩｇＧ，裸鼠组血清ＩｇＧ明显升高。     

Mycobacterium lepraemurium infection of nude athymic (nu/nu) mice.

Nude athymic (nu/nu) mice on a BALB/c background and their heterozygous euthymic litter mates (nu/+) were infected with either 10(8) or 10(6) Mycobacterium lepraemurium organisms intravenously or in the left hind footpad (LHF). After LHF infection with 10(8) M. lepraemurium organisms, nu/+ mice slowly developed a response that consisted of LHF swelling and local resistance to Listeria monocytogenes. The lower inoculum induced a proportionately lower response in nu/+ mice, but the nu/nu mice developed neither LHF swelling nor resistance to L. monocytogenes in response to either dose of M. lepraemurium. Counts of M. lepraemurium in the LHF revealed no difference between the nu/+ mice and nu/nu mice. After intravenous infection the nu/+ mice developed splenomegaly, but did not otherwise differ from nu/nu mice with respect to resistance to intravenous challenge with L. monocytogenes or growth of M. lepraemurium in the spleen. In light of the poor responsiveness of nu/+ mice in this experiment, they were then compared with CB6 and B6D2 mice, which are genetically susceptible and resistant to M. lepraemurium, respectively. These mice were infected with either 10(8) or 10(6) M. lepraemurium cells or 10(6) Mycobacterium bovis BCG cells in the LHF. Once again the nu/+ mice responded poorly to M. lepraemurium, the CB6 mice responded very strongly, and the B6D2 mice gave an intermediate response with respect to LHF swelling and resistance to L. monocytogenes. However, M. lepraemurium grew to higher numbers in the LHF of nu/+ and CB6 mice than in B6D2 mice, revealing, in CB6 mice, a dissociation between resistance to L. monocytogenes and M. lepraemurium. All three mouse strains responded strongly to M. bovis BCG, but there was a suggestion that nu/+ mice might be more susceptible to this agent than the other two strains. I concluded that the failure of nu/+ mice to restrict the growth of M. lepraemurium more than nu/nu mice was due to the intrinsic genetic susceptibility of both types of mice. In effect, the nu/+ mice behaved like nu/nu mice, as if they too were deficient in T lymphocytes that were responsive to M. lepraemurium.     

Regulation of chronic colitis in athymic nu/nu (nude) mice

The objective of this study was to assess the roles of NK cells,B cells and/or intraepithelial lymphocytes (IEL) in suppressingthe development of colitis in nude mice reconstituted with CD4⁺CD45RB^highT cells. BALB/c nude mice were lethally irradiated and reconstitutedwith bone marrow from different immunodeficient mice to generateathymic chimeras devoid of one or more lymphocyte populations.Transfer of CD4⁺C45RB^high T cells into chimeric recipients devoidof B cells, T cells and IEL produced severe colitis within 6–8weeks, whereas transfer of these same T cells into B cell- andT cell-deficient or T cell-deficient chimeras produced littleto no gut inflammation. In addition, we found that nude micedepleted of NK cells or RAG-1^–/– mice reconstitutedwith IEL failed to develop colitis following transfer of CD45RB^highT cells. Severe colitis could, however, be induced in nude miceby transfer of activated/T_h1 CD4⁺CD45RB^low T cells. Taken together,our data suggest that IEL, but not B cells or NK cells, playan important role in suppressing the development of chroniccolitis in this model. In addition, our data demonstrate thatsuppression of disease may be due to polarization of naive CD4⁺cells toward a non-pathogenic and/or regulatory phenotype.     

Lectin-binding proteins as potent mitogens for B lymphocytes from nu/nu mice

It was found that lectin-binding proteins (LB) from leguminosae seeds can serve as mitogens for B lymphocytes from athymic nu/nu mice. Most of the experiments were performed using a LB from Vicia faba as this was the most potent mitogen among all LB tested. When mixed B and T lymphocyte populations were stimulated by LB, a bell-shaped dose-response curve resulted which is also characteristic of the corresponding lectins; LB, however, act at much higher concentrations than the lectins. In control experiments, using lymphocytes from C3H/HeJ mice which have a genetic defect with respect to lipopolysaccharide responsiveness, the enhancement of DNA synthesis by LB was comparable to that observed with other strains. This indicates that stimulation by LB does not result from contamination by lipopolysaccharides. B lymphocytes from nu/nu mice were stimulated by LB as efficiently as mixed T and B lymphocyte populations whereas lectins per se had no effect on nu/nu lymphocytes. Cyclosporin A, a fungal metabolite, is known to specifically suppress T cell responses. Cyclosporin A did not influence the mitogenic activity of the LB from Vicia faba on unseparated spleen cells. Cell populations enriched for T cells (lymph node cells: nylon wool-passed or nylon wool-passed and anti-Ia plus complement-treated) responded poorly to LB, if at all, even in the presence of interleukin 2-containing supernatants.     

Functional Thy-1+ cells in cultures of spleen cells from nu/nu mice

Thy-1⁺ lymphocytes, detectable by quantitative serum absorption, arise in cultures of spleen cells from congenitally athymic (nu/nu) mice supplemented with supernatants from cultures of normal spleen cells stimulated with the T cell mitogen concanavalin A. Pretreatment of nu/nu spleen cells with appropriate anti-Thy-1 alloantibody and complement prior to culture reduces their capacity to generate Thy-1⁺ cells by about 90%. This shows that the majority of cells proliferating in these cultures are descendants of Thy-1⁺ cells which can be detected in the original nu/nu spleens. Experiments aimed at exploring the function of these Thy-1⁺ cells after culture in conditioned medium revealed that within one or two days after culture initiation, strong cooperative activity for a humoral response to xenogeneic erythrocytes can be detected. In mixtures of bone marrow-derived lymphocytes from various mouse strains with cultured nu/nu spleen cells, it was observed that T-B cooperation is not H-2-restricted. Attempts at inducing T cell-mediated cytotoxicity to alloantigens in such cultures of nu/nu spleen cells were unsuccessful. In contrast, nonspecific cytotoxicity which was attributed to natural killer cells was regularly observed and could be maintained in these cultures over extended periods.