异基因骨髓源间充质干细胞对BXSB小鼠T、B细胞的影响

目的：探讨异基因骨髓源间充质干细胞（Bonemarrow derived mesenchymal stem cells，BMSCs）对BXSB小鼠T、B细胞增殖、活化及功能成熟等方面的影响。方法：采用。H．TdR掺入法、FACS法、EIJSA等方法检测BMSCs对BXSB小鼠T、B细胞的影响。结果：BALB／c小鼠的BMSCs在不影响BXSB小鼠T细胞诱导活化的基础上抑制其诱导增殖；可降低由ConA诱导的CD4^＋IL^＋细胞的数量，而提高由ConA诱导的CD4^＋IFN-γ^＋细胞数量。对于B细胞，BALB／c小鼠的BMSCs可以抑制其增殖、活化及IgG的分泌。另外，BALB／c小鼠的BMSCs可以抑制BXSB小鼠B细胞上CIMOL的异位表达。结论：异基因BMSCs对自身免疫病小鼠的T、B细胞有一定的调节作用。     

骨髓源间充质干细胞在异基因小鼠免疫器官内的分布及其免疫功能调节作用

目的：探讨骨髓源间充质干细胞(Bone marrow mesenchymal stem cells，BMSC)在异基因小鼠免疫器官内的分布及其免疫调节作用。方法：以CM-Dil荧光染料示踪BMSC的体内分布情况，并辅以PCR检测Y染色体的方法进一步鉴定；体外实验采用MTT法、ELISA和FACS等方法检测BMSC的免疫调节作用。结果：BMSC可进入并较长期(30天)存在于异基因小鼠免疫器官内；在体外，BALB／C小鼠的BMSC对由ConA诱导的BALB／C和C57BL／6(B6)和BXSB小鼠的T细胞增殖均有抑制作用；而对前两种小鼠由12S诱导的B细胞增殖和分泌k方面表现为促进作用，对BXSB小鼠由IPS诱导的B细胞增殖和k分泌有抑制作用。BALB／C小鼠的BMSC对BALB／C和B6小鼠由ConA诱导的IL-4生成细胞的数量无明显影响，却可降低由ConA诱导的两种品系小鼠的IFN-γ生成细胞的数量；但对于BXSB小鼠却不同，BALB／C的BMSC可降低由ConA诱导的BXSB小鼠的IL-4生成细胞的数量，而提高由ConA诱导的IFN-γ生成细胞的数量。结论：异基因BMSC不但可进入受体的免疫器官，且可较长期(30天)存在；另外，BMSC对同基因正常、异基因正常和异基因自身免疫病的个体均有一定程度的免疫调节作用。     

Cyclophosphamide inhibits the development of diabetes in the diabetes-prone BB rat

Abstract Aims/hypothesis. Cyclophosphamide has been shown to augment the diabetic process in NOD mouse and BB rat models of Type I (insulin-dependent) diabetes mellitus. Because cyclophosphamide has, however, been shown to increase immunoregulatory cell activity, we examined if cyclophosphamide treatment increases immunoregulatory cell activity and inhibits the diabetic process in BB rats. Methods. The development of insulitis and diabetes was explored in BB rats treated with saline and cyclophosphamide (60 to 175 mg/kg body weight). Subsets of spleen cells were assessed by flow cytometry and cytokine gene expression by RT-PCR. To determine if cyclophosphamide induces immunoregulatory cell activity, the development of diabetes was assessed in BB rats injected with spleen cells from rats treated with saline and cyclophosphamide. Results. All dosages of cyclophosphamide decreased the development of diabetes. The degree of insulitis was lower in pancreata from 55-day-old rats treated with cyclophosphamide than those from controls. Cyclophosphamide caused no alterations in the numbers of NK cells, T-cell subsets, or RT6.1⁺ T cells. The adoptive transfer of spleen cells from cyclophosphamide-treated rats to BB rats inhibited the development of diabetes. Cyclophosphamide treatment decreased IL-12, IL-1β, IL-2, IFN-γ and TNF-α gene expressions in mononuclear spleen cells but IL-4 gene expression increased. Conclusion/interpretation. These findings show that cyclophosphamide treatment decreases the development of diabetes by inhibiting the development of insulitis. This inhibitory action of cyclophosphamide on the diabetic process seems to be mediated by the induction of immunoregulatory cell activity. The suppression of cytokines that promote Th1 cell differentiation by cyclophosphamide treatment could also play a part in the diabetes sparing effect of cyclophosphamide. [Diabetologia (2000) 43: 986–994] Received: 10 December 1999 and in revised form: 13 April 2000     

Myeloid-derived suppressor cells and regulatory T cells share common immunoregulatory pathways-related microRNAs that are dysregulated by acute lymphoblastic leukemia and chemotherapy

BackgroundRelapse remains a critical challenge in children with acute lymphoblastic leukemia (ALL). The emergence of immunoregulatory cells, including myeloid-derived suppressor cells (MDSCs), and T regulatory (T_reg) cells, has been considered one potential mechanism of relapse in children with ALL.AimThis study aimed to address the microRNAs (miRNAs) related to MDSCs and T_reg cells and to explore their targeted immunoregulatory pathways.MethodsAffymetrix microarray was used for global miRNA profiling in B-ALL pediatric patients before, during, and after induction of chemotherapy. Bioinformatics analysis was performed on MDSCs and T_reg cells-related dysregulated miRNAs, and miR-Pathway analysis was performed to explore their targeted immunoregulatory pathways.Results516 miRNAs were dysregulated in ALL patients as compared to the healthy donor. Among them, 13 miRNAs and 8 miRNAs related to MDSCs and T_reg cells, respectively, were common in all patients. Besides, 12 miRNAs were shared between MDSCs and T_reg cells; 4 of them were common in all patients. Four immune-related pathways; TNF, TGF-β, FoxO, and Hippo were found implicated.ConclusionOur pilot study concluded certain miRNAs related to MDSCs and T_reg cells, these miRNAs were linked to immunoregulatory pathways. Our results open avenues for testing those miRNA as molecular biomarkers for the immunosuppressive tumor microenvironment.     

Protective effect of nitric oxide on hepatopulmonary syndrome from ischemia-reperfusion injury

AIM: To evaluate immunological protection of nitric oxide (NO) in hepatopulmonary syndrome and probable mechanisms of ischemia-reperfusion (IR) injury in rat liver transplantation.METHODS: Sixty-six healthy male Wistar rats were randomly divided into three groups (11 donor/recipient pairs). In group II, organ preservation solution was lactated Ringer’s solution with heparin 10  000/μL at 4 °C. In groups I and III, the preservation solution added, respectively, L-arginine or N^G-L-arginine methyl ester (L-NAME) (1 mmol/L) based on group II, and recipients were injected with L-arginine or L-NAME (50 mg/kg) in the anhepatic phase. Grafted livers in each group were stored for 6 h and implanted into recipients. Five rats were used for observation of postoperative survival in each group. The other six rats in each group were used to obtain tissue samples, and executed at 3 h and 24 h after transplantation. The levels of alanine aminotransferase (ALT), tumor necrosis factor (TNF)-α and NO metabolites (NOx) were detected, and expression of NO synthase, TNF-α and intercellular adhesion molecule 1 (ICAM-1) was examined by triphosphopyridine nucleotide diaphorase histochemical and immunohistochemical staining.RESULTS: By supplementing L-arginine to strengthen the NO pathway, a high survival rate was achieved and hepatic function was improved. One-week survival rate of grafted liver recipients in group I was significantly increased (28.8 ± 36.6 d vs 4 ± 1.7 d, P < 0.01) as compared with groups II and III. Serum levels of ALT in group I were 2-7 times less than those in groups II and III (P < 0.01). The cyclic guanosine monophosphate (cGMP) levels in liver tissue and NOx in group I were 3-4 times higher than those of group II after 3 h and 24 h reperfusion, while in group III, they were significantly reduced as compared with those in group II (P < 0.01). The levels of TNF-α in group I were significantly lower than in group II after 3 h and 24 h reperfusion (P < 0.01), while being significantly higher in group III than group II (P < 0.01). Histopathology revealed more severe tissue damage in graft liver and lung tissues, and a more severe inflammatory response of the recipient after using NO synthase inhibitor, while the pathological damage to grafted liver and the recipient’s lung tissues was significantly reduced in group I after 3 h and 24 h reperfusion. A small amount of constitutive NO synthase (cNOS) was expressed in liver endothelial cells after 6 h cold storage, but there was no expression of inducible NO synthase (iNOS). Expression of cNOS was particularly significant in vascular endothelial cells and liver cells at 3 h and 24 h after reperfusion in group II, but expression of iNOS and ICAM-1 was low in group I. There was diffuse strong expression of ICAM-1 and TNF-α in group III at 3 h after reperfusion.CONCLUSION: The NO/cGMP pathway may be critical in successful organ transplantation, especially in treating hepatopulmonary syndrome during cold IR injury in rat orthotopic liver transplantation.     

骨髓源性间充质干细胞联合来氟米特对小鼠T淋巴细胞的免疫调节作用

目的 探讨骨髓源性间充质干细胞(BMSCs)在体外联合来氟米特(LEF)对小鼠T淋巴细胞的免疫调节作用.方法 直接贴壁筛选法分离培养小鼠BMSCs,流式细胞分析(FCM)鉴定BMSCs的纯度.用EZ-SepTMm Mouse 1X分离异体BALB/c小鼠的脾淋巴细胞,在刀豆球蛋白A(ConA)诱导的同时,先经LEF处理,洗去LEF后,脾淋巴细胞再和BMSCs共培养.分组:A组:脾淋巴细胞;B组:脾淋巴细胞+BMSCs;C组:LEF处理的脾淋巴细胞;D组:LEF处理的脾淋巴细胞+BMSCs.用四甲基噻唑蓝(MTT)比色法检测各组T淋巴细胞的增殖情况,流式细胞术分析各组T淋巴细胞的活化及凋亡情况,定量反转录-聚合酶链反应(RT-PCR)检测各组T淋巴细胞的白细胞介素(IL)-2、IL-10基因水平.结果 在体外,BM-SCs处理组(B组)T淋巴细胞的A570 nm值为0.578±0.042,LEF处理组(C组)A570 nm值为0.5024±0.040,两组A570 nm值均显著低于对照组(A组)A570 nm值为0.778±0.035(P<0.01).BMSCs联合LEF组(D组)A570 nm值为0.218±0.033,显著低于BMSCs处理组及LEF处理组(P<0.01). BMSCs联合LEF对CD3+CD69+、CD3+CD28+表达、IL-2基因表达均无明显影响.BMSCs单独或联合LEF组T淋巴细胞凋亡率分别为(2.29±0.32)%、(4.22±0.98)%,较A组T淋巴细胞凋亡率(8.08±1.20)%均明显下降.BMSCs处理组和LEF处理组T淋巴细胞IL-10基因相对表达分别为:0.098±0.039、0.054±0.022明显低于A组(IL-10基因相对表达为1.000)(P<0.01),MSCs联合LEF组IL-10基因相对表达为:0.023±0.015,显著低于BMSCs处理组及LEF处理组(P<0.01).结论 BMSCs联合LEF对小鼠T淋巴细胞有一定程度的协同免疫调节作用.     

沙利度胺对人T淋巴细胞的免疫调节作用

目的 探讨沙利度胺(Thd)对健康人外周血T淋巴细胞的免疫调节作用.方法 健康人外周血T淋巴细胞经不同浓度Thd作用后,用MTT比色法检测其增殖情况,用流式细胞术检测其早期凋亡及CD28、CD152表达,用RT PCR检测其白细胞介素6(IL-6)、IL-10、肿瘤坏死因子α(TNF-α)mRNA表达水平.结果 在体外,与阴性对照组相比,500μg/ml Thd组可促进T淋巴细胞的增殖、早期凋亡,抑制T淋巴细胞CD28的表达.100μg/ml Thd组可促进T淋巴细胞CD152表达.各剂量组均能抑制IL-6、TNF-α mRNA表达,100μg/ml和500μg/ml Thd可增强IL-10 mRNA的表达.结论 Thd可以通过影响T淋巴细胞的增殖、早期凋亡以及CD152、CD28,IL-6、TNF-α、IL-10 mRNA的表达,对健康人T淋巴细胞具有免疫调节作用.     

MSCs联合雷帕霉素对异基因鼠脾淋巴细胞的免疫调节作用

目的探讨体外骨髓间充质干细胞（MSCs）联合雷帕霉素对BALB/c鼠T、B淋巴细胞的免疫调节作用及可能机制。方法从5～6周龄BALB/c鼠骨髓中分离培养MSCs并鉴定其纯度。用EZ SepTM Mouse 1X分离异体BALB/c鼠脾脏淋巴细胞,分别在刀豆蛋白A（ConA）、脂多糖（LPS）刺激下,用MSCs和/或雷帕霉素处理,MTT法检测淋巴细胞的增殖,流式细胞术检测T、B淋巴细胞CD69、CD28和CD86的表达和T淋巴细胞凋亡情况,Real time PCR测定T淋巴细胞IL 10 mRNA、IFN γ mRNA的表达水平。结果MSCs明显抑制T、B淋巴细胞增殖,联合雷帕霉素组抑制作用更加显著（P＜0.01）。MSCs可抑制T淋巴细胞的凋亡,联合雷帕霉素组较单独MSCs组抑制作用更加明显(P＜0.01)。MSCs联合雷帕霉素具有协同促进IFN γ mRNA表达和协同抑制IL 10 mRNA表达的作用。单独MSCs或MSCs联合雷帕霉素对T、B淋巴细胞CD69、CD28和CD86的表达无显著影响。结论MSCs联合雷帕霉素对BALB/c鼠T、B淋巴细胞有免疫负调节作用,可能与协同抑制淋巴细胞增殖、T淋巴细胞凋亡和干预IFN γ mRNA、IL 10 mRNA表达有关。     

人参皂甙Rg1对老年大鼠免疫功能的调节作用

已知老年机体免疫功能的降低与淋巴细胞增殖能力的减弱和白细胞介素-2(IL-2)产生减少有密切关系。以老年大鼠免疫功能为主要研究对象,首次发现人参皂甙Rg1无论体内给药还是体外实验均能选择性增强老年大鼠脾淋巴细胞增殖能力和IL-2的产生与释放,采用Northern和Western印迹分析法证明,Rg₁可明显促进IL-2基因和蛋白的表达,表现在IL-2mRNA和IL-2蛋白含量的显著增加。值得注意的是,在同样的条件下,Rg₁对青年大鼠免疫功能的影响并不显著,由此可以认为Rg1一种“免疫调节剂”,而并非单纯的“免疫增强剂”。     

Immunoregulatory Cells for Transplantation Tolerance and Graft-versus-Leukemia Effect

Various immunoregulatory cells that inhibit graft-versus-host disease (GVHD) and induce the graft-versus-leukemia (GVL) effect are found after allogeneic hematopoietic stem cell transplantation. These cells comprise CD4+CD25+ regulatory T-cells, regulatory dendritic cells (rDCs), gamma(delta) T-cells, natural killer (NK) T-cells, and NK cells and T-cells with inhibitory NK receptors. Although the first 4 types of cells effectively inhibit GVHD in animal models, with rDCs showing an inhibitory effect on GVHD in humans as well, the GVL effect was observed only in rDCs. Additional analyses are required to determine whether these cells can inhibit GVHD and exert the GVL effect in humans. In contrast, NK cells and T-cells with inhibitory NK receptors have been shown in humans to possess a suppressive activity against GVHD while preserving the GVL effect. These results indicate that immunoregulatory cells may be used to modulate GVHD and the GVL effect in clinical settings.