血浆外泌体样小体免疫调节作用的初步研究

Objective To identify the exosomes-like vesicles from the plasma and study their biologic characteristics and regulatory effect.Methods The exosomes-like vesicles were purified from healthy donors plasma with a series of high-speed centrifugations and ultrafiltration.Morphology was identified by transmission electron microscopy and biologic characteristics by Western blot and flow cytometry.CIM+T ceils and CD4+ CD25+CD127 low Treg cells were purified from peripheral blood mononuclear cells(PBMCs)by Magnetic cell sorting.After exosomes-like vesicles cultured with CD4+ T cells or CD4+ CD25+CD127low Treg cells,cell proliferation and apoptosis were assayed.Phosphorylated β-catenin level in Wnt signaling by phosflow.Results Exosomes-like vesicles from plasma were similar to previously described exosomes in shapes and size and expressed exosome marker proteins CD63 and CD81 as well as the MHC-Ⅱ molecule.costimulatory molecules CD86 etc.Mter co-cultured with CD4+ T cells,exosomes- like vesicles inhibited the proliferation of the T cells in a dose-dependent manner.After Treg cells cultured with exosomes-like vesicles for 14days,the survival rate of the Treg cells was 57.07%,while that of the control Treg was 30.91%.Frizzled receptors 2,3,4and LRP6 gene mRNA expressed(the relative gray value was 48.50、34.84、23.85、49.73)in the Treg cells by RT-PCR,and Wnt molecular expressed in exosomes-like vesicles.After Treg ceils cocultured with exosomes-like vesicles,the MFI of phosphorylated β-catenin decreased(from 20.06±2.99 to 12.41±2.08),and the expression of Bcl-2 mRNA was upregulated significantly(the relative gray value from 0.45 to 84.97).Conclusions Exosomes-like vesicles existed in human plasma and express immune regulatory molecules.They can suppress the proliferation of activated CD4+ T cels induce their apoptosis and prolong the survival of natural Treg cells via Wnt signaling pathway.     

血浆外泌体样小体对巨噬细胞Wnt5A-Ca2+通路影响的初步研究

目的 研究血浆中外泌体(exosomes)样小体对正常人巨噬细胞免疫调节作用的影响.方法 采用超速离心结合膜过滤的方法,从健康献血者血浆中分离纯化外泌体样小体.通过分离健康献血者外周血单个核细胞,贴壁培养并分离巨噬细胞.外泌体样小体与巨噬细胞共孵育后,采用流式细胞术、RT-PCR、Western blot法分别检测巨噬细胞胞质Ca2+、相关基因和蛋白的变化.结果 外泌体样小体与巨噬细胞孵育一段时间后,巨噬细胞胞质内Ca2+平均荧光强度上调;外泌体样小体对巨噬细胞的促炎因子如IL-1β、IL-6、TNF-α的表达有促进作用,在共培养2 h时,IL-1β、TNF-α基因表达达到高峰,分别比对照组表达上调0.85倍和1.69倍,而IL-6基因表达上调水平在共培养8 h时达到高峰,是对照组的3.7倍,而对抑炎因子IL-10的表达具有抑制作用;同时发现巨噬细胞表达Wnt5A受体Frizzled5,但是外泌体样小体对其基因表达没有明显影响;外泌体样小体能使钙调蛋白依赖性激酶Ⅱ(CaMKⅡ)发生磷酸化.结论 血浆外泌体样小体能上调巨噬细胞炎症因子基因的表达,并且可能通过活化巨噬细胞的Wnt5A-Ca2+信号通路促进炎性细胞因子的分泌.

Abstract：

Objective To study the influence of human plasma exosomes-like vesicles on the regulatory function of macrophages. Methods The exosomes-like vesicles were purified from healthy donors plasma with a series of high-speed centrifugation and ultrafiltration. Macrophages were derived from cultured human blood monocytes. The molecular markers of macrophages were assayed by FACS. After cultured with exosomes-like vesicles, the changes of macrophages cytoplasma Ca2 + , and related genes and proteins were assayed by FACS, RT-PCR and Western Blot, respectively. Results After cultured with exosomes-like vesicles, mean fluoresecent intensity (MFI) of macrophages cytoplasma Ca2+ was increased. The vesicles enhanced macrophages to express cytokines genes, the expression of IL-1 β and TNF-α genes being increased by 0.85 and 1.69 times respectively at 2 h, and that of IL-6 gene 3.7 times compared with the control at 8 h. However, the vesicles inhibited the expression of macrophages IL-10 gene, had no influence on the Frizzled5 receptor expression and could induce CaMKⅡ phosphorylation. Conclusions Exosomes-like vesicles can up-regulat macrophages expression of inflammatory cytokines genes, and increase the secretion of inflammatory cytokines by activating the Wnt5A- Ca2+ signaling pathway.     

多发性硬化患者外周血CD8+CD28-调节性T细胞的变化

Objective To investigate the levels of peripheral blood CD8+ CD28- regulatory lymphocytes and their clinical values in the patients with multiple sclerosis (MS). Method From October 2005 to August 2008, 51 patients with active rehpsing-remitting MS were enrrolled from Department of Neurology of the First Affil-iated Hospital of Wenzliou Medical College. The diagnostic criteria for MS were the 2005 revisions to the "McDon-ald criteria". All the admitted patients received 1 g of methylprednisoione per day intravenously for 5 days, fol-lowed by 60 mg prednisone per day orally for 12 days,and tapered in 6 weeks. Fourteen patients were reevaluated after corticosteroid therapy. Twenty healthy individuals ,as normal controls,matched for age and sex with the MS patients were also enrolled in this study. The percentages of peripheral blood T cells (CD8+ CD28-, CD8+CD28+, CD8+, CD4+ CD8-) were measured by flow cytometric analysis. Parametric statistical analysis were per-formed using standard methods, and linear regression analysis was conducted using Pearson correlation test. Re-sults (1)Compared with controls,the patients with active MS had significantly lower percentage of CD8+ CD28-T cells [(18.48±9.89)% vs. (24.48±4.86)%, P <0.01], and higher percentage of CD8+ CD28+ T cells [(12.23±4.31) % vs. (8.55±3.49) %, P <0.01]. (2)The percentage of CD8+ CD28- T cells was negative-ly correlated with that of CD4+ CDS- T cells (r = -0.488, P < 0.01). (3) After corticosteroid therapy, the per-eentage of peripheral blood CD8+ CD28- / CD8+ CD28+ T cells didn' t significantly decrease or increase in 14 ac-tive MS patients (P > 0.05). Conclusions The decrease of peripheral blood CD8+ CD28- regulatory T cells might be associated with the pathogenesis of MS, and CD8+ CD28- regulatory T cells perhaps played their roles through CD4+ T cells. Corticosteroid therapy could not reverse the levels of CD8+ CD28- T cells.     

多发性硬化患者外周血CD8+CD28-调节性T细胞的变化

Objective To investigate the levels of peripheral blood CD8+ CD28- regulatory lymphocytes and their clinical values in the patients with multiple sclerosis (MS). Method From October 2005 to August 2008, 51 patients with active rehpsing-remitting MS were enrrolled from Department of Neurology of the First Affil-iated Hospital of Wenzliou Medical College. The diagnostic criteria for MS were the 2005 revisions to the "McDon-ald criteria". All the admitted patients received 1 g of methylprednisoione per day intravenously for 5 days, fol-lowed by 60 mg prednisone per day orally for 12 days,and tapered in 6 weeks. Fourteen patients were reevaluated after corticosteroid therapy. Twenty healthy individuals ,as normal controls,matched for age and sex with the MS patients were also enrolled in this study. The percentages of peripheral blood T cells (CD8+ CD28-, CD8+CD28+, CD8+, CD4+ CD8-) were measured by flow cytometric analysis. Parametric statistical analysis were per-formed using standard methods, and linear regression analysis was conducted using Pearson correlation test. Re-sults (1)Compared with controls,the patients with active MS had significantly lower percentage of CD8+ CD28-T cells [(18.48±9.89)% vs. (24.48±4.86)%, P <0.01], and higher percentage of CD8+ CD28+ T cells [(12.23±4.31) % vs. (8.55±3.49) %, P <0.01]. (2)The percentage of CD8+ CD28- T cells was negative-ly correlated with that of CD4+ CDS- T cells (r = -0.488, P < 0.01). (3) After corticosteroid therapy, the per-eentage of peripheral blood CD8+ CD28- / CD8+ CD28+ T cells didn' t significantly decrease or increase in 14 ac-tive MS patients (P > 0.05). Conclusions The decrease of peripheral blood CD8+ CD28- regulatory T cells might be associated with the pathogenesis of MS, and CD8+ CD28- regulatory T cells perhaps played their roles through CD4+ T cells. Corticosteroid therapy could not reverse the levels of CD8+ CD28- T cells.     

多发性硬化患者外周血CD8+CD28-调节性T细胞的变化

Objective To investigate the levels of peripheral blood CD8+ CD28- regulatory lymphocytes and their clinical values in the patients with multiple sclerosis (MS). Method From October 2005 to August 2008, 51 patients with active rehpsing-remitting MS were enrrolled from Department of Neurology of the First Affil-iated Hospital of Wenzliou Medical College. The diagnostic criteria for MS were the 2005 revisions to the "McDon-ald criteria". All the admitted patients received 1 g of methylprednisoione per day intravenously for 5 days, fol-lowed by 60 mg prednisone per day orally for 12 days,and tapered in 6 weeks. Fourteen patients were reevaluated after corticosteroid therapy. Twenty healthy individuals ,as normal controls,matched for age and sex with the MS patients were also enrolled in this study. The percentages of peripheral blood T cells (CD8+ CD28-, CD8+CD28+, CD8+, CD4+ CD8-) were measured by flow cytometric analysis. Parametric statistical analysis were per-formed using standard methods, and linear regression analysis was conducted using Pearson correlation test. Re-sults (1)Compared with controls,the patients with active MS had significantly lower percentage of CD8+ CD28-T cells [(18.48±9.89)% vs. (24.48±4.86)%, P <0.01], and higher percentage of CD8+ CD28+ T cells [(12.23±4.31) % vs. (8.55±3.49) %, P <0.01]. (2)The percentage of CD8+ CD28- T cells was negative-ly correlated with that of CD4+ CDS- T cells (r = -0.488, P < 0.01). (3) After corticosteroid therapy, the per-eentage of peripheral blood CD8+ CD28- / CD8+ CD28+ T cells didn' t significantly decrease or increase in 14 ac-tive MS patients (P > 0.05). Conclusions The decrease of peripheral blood CD8+ CD28- regulatory T cells might be associated with the pathogenesis of MS, and CD8+ CD28- regulatory T cells perhaps played their roles through CD4+ T cells. Corticosteroid therapy could not reverse the levels of CD8+ CD28- T cells.     

抗CD44单抗IM7对慢性粒细胞白血病CD34+细胞黏附和迁移能力影响的体外研究

Objective To explore the effects of anti-CD44 monoclonal antibody-IM7 on the in vitro adhesion and migration of chronic myeloid leukemia stem cell(CML-LSC) and its mechanism. Methods CD34+ CD38- CD123+ leukemic stem cells (LSC) from 20 newly-diagnosed chronic myeloid leukemia ( CML) patients BM cells and CD34+ CD38- hematopoietic stem cells (HSC) from 20 full-term newborn cord blood cells were isolated with EasySepTM magnet beads. The CD44 expression of the LSC and HSC was detected by flow cytometry (FCM), and the adhesion and migration ability of the LSC and HSC pre- and post-incubated with IM7 in vitro by MTT assay and transendothelial migration assay, respectively. Results ①After incubated with IM7, the LSC and HSC CD44 expression rates were (86.60±2.10)% vs. (25.40±1.70)% (P<0.05), respectively. ②The adhesive ability of the LSC to endothelial cells was decreased markedly after incubated with IM7, the OD value (A570) changing from pre- incubation of (0.62±0.11) to post-incubation of (0.34±0.07), while there was little change of A570 in the HSC group. ③The migration abilityof the LSC group was inhibited evidently after incubated with IM7, the inhibition rate being 46%～63%, while little change of that in HSC group was detected. ④The adhesive ability of the LSC group to marrow stromal cells was decreased markedly after incubated with IM7, while little change was found in that of HSC group. Conclusion The anti-CD44 monoclonal antibody-IM7 can effectively inhibit the adhesion and migration abilities of the LSC in vitro, which might provide a theoretical evidence for targeting therapy.     

抗CD44单抗IM7对慢性粒细胞白血病CD34+细胞黏附和迁移能力影响的体外研究

Objective To explore the effects of anti-CD44 monoclonal antibody-IM7 on the in vitro adhesion and migration of chronic myeloid leukemia stem cell(CML-LSC) and its mechanism. Methods CD34+ CD38- CD123+ leukemic stem cells (LSC) from 20 newly-diagnosed chronic myeloid leukemia ( CML) patients BM cells and CD34+ CD38- hematopoietic stem cells (HSC) from 20 full-term newborn cord blood cells were isolated with EasySepTM magnet beads. The CD44 expression of the LSC and HSC was detected by flow cytometry (FCM), and the adhesion and migration ability of the LSC and HSC pre- and post-incubated with IM7 in vitro by MTT assay and transendothelial migration assay, respectively. Results ①After incubated with IM7, the LSC and HSC CD44 expression rates were (86.60±2.10)% vs. (25.40±1.70)% (P<0.05), respectively. ②The adhesive ability of the LSC to endothelial cells was decreased markedly after incubated with IM7, the OD value (A570) changing from pre- incubation of (0.62±0.11) to post-incubation of (0.34±0.07), while there was little change of A570 in the HSC group. ③The migration abilityof the LSC group was inhibited evidently after incubated with IM7, the inhibition rate being 46%～63%, while little change of that in HSC group was detected. ④The adhesive ability of the LSC group to marrow stromal cells was decreased markedly after incubated with IM7, while little change was found in that of HSC group. Conclusion The anti-CD44 monoclonal antibody-IM7 can effectively inhibit the adhesion and migration abilities of the LSC in vitro, which might provide a theoretical evidence for targeting therapy.     

多发性硬化患者外周血CD8+CD28-调节性T细胞的变化

Objective To investigate the levels of peripheral blood CD8+ CD28- regulatory lymphocytes and their clinical values in the patients with multiple sclerosis (MS). Method From October 2005 to August 2008, 51 patients with active rehpsing-remitting MS were enrrolled from Department of Neurology of the First Affil-iated Hospital of Wenzliou Medical College. The diagnostic criteria for MS were the 2005 revisions to the "McDon-ald criteria". All the admitted patients received 1 g of methylprednisoione per day intravenously for 5 days, fol-lowed by 60 mg prednisone per day orally for 12 days,and tapered in 6 weeks. Fourteen patients were reevaluated after corticosteroid therapy. Twenty healthy individuals ,as normal controls,matched for age and sex with the MS patients were also enrolled in this study. The percentages of peripheral blood T cells (CD8+ CD28-, CD8+CD28+, CD8+, CD4+ CD8-) were measured by flow cytometric analysis. Parametric statistical analysis were per-formed using standard methods, and linear regression analysis was conducted using Pearson correlation test. Re-sults (1)Compared with controls,the patients with active MS had significantly lower percentage of CD8+ CD28-T cells [(18.48±9.89)% vs. (24.48±4.86)%, P <0.01], and higher percentage of CD8+ CD28+ T cells [(12.23±4.31) % vs. (8.55±3.49) %, P <0.01]. (2)The percentage of CD8+ CD28- T cells was negative-ly correlated with that of CD4+ CDS- T cells (r = -0.488, P < 0.01). (3) After corticosteroid therapy, the per-eentage of peripheral blood CD8+ CD28- / CD8+ CD28+ T cells didn' t significantly decrease or increase in 14 ac-tive MS patients (P > 0.05). Conclusions The decrease of peripheral blood CD8+ CD28- regulatory T cells might be associated with the pathogenesis of MS, and CD8+ CD28- regulatory T cells perhaps played their roles through CD4+ T cells. Corticosteroid therapy could not reverse the levels of CD8+ CD28- T cells.     

多发性硬化患者外周血CD8+CD28-调节性T细胞的变化

Objective To investigate the levels of peripheral blood CD8+ CD28- regulatory lymphocytes and their clinical values in the patients with multiple sclerosis (MS). Method From October 2005 to August 2008, 51 patients with active rehpsing-remitting MS were enrrolled from Department of Neurology of the First Affil-iated Hospital of Wenzliou Medical College. The diagnostic criteria for MS were the 2005 revisions to the "McDon-ald criteria". All the admitted patients received 1 g of methylprednisoione per day intravenously for 5 days, fol-lowed by 60 mg prednisone per day orally for 12 days,and tapered in 6 weeks. Fourteen patients were reevaluated after corticosteroid therapy. Twenty healthy individuals ,as normal controls,matched for age and sex with the MS patients were also enrolled in this study. The percentages of peripheral blood T cells (CD8+ CD28-, CD8+CD28+, CD8+, CD4+ CD8-) were measured by flow cytometric analysis. Parametric statistical analysis were per-formed using standard methods, and linear regression analysis was conducted using Pearson correlation test. Re-sults (1)Compared with controls,the patients with active MS had significantly lower percentage of CD8+ CD28-T cells [(18.48±9.89)% vs. (24.48±4.86)%, P <0.01], and higher percentage of CD8+ CD28+ T cells [(12.23±4.31) % vs. (8.55±3.49) %, P <0.01]. (2)The percentage of CD8+ CD28- T cells was negative-ly correlated with that of CD4+ CDS- T cells (r = -0.488, P < 0.01). (3) After corticosteroid therapy, the per-eentage of peripheral blood CD8+ CD28- / CD8+ CD28+ T cells didn' t significantly decrease or increase in 14 ac-tive MS patients (P > 0.05). Conclusions The decrease of peripheral blood CD8+ CD28- regulatory T cells might be associated with the pathogenesis of MS, and CD8+ CD28- regulatory T cells perhaps played their roles through CD4+ T cells. Corticosteroid therapy could not reverse the levels of CD8+ CD28- T cells.     

多发性硬化患者外周血CD8+CD28-调节性T细胞的变化

Objective To investigate the levels of peripheral blood CD8+ CD28- regulatory lymphocytes and their clinical values in the patients with multiple sclerosis (MS). Method From October 2005 to August 2008, 51 patients with active rehpsing-remitting MS were enrrolled from Department of Neurology of the First Affil-iated Hospital of Wenzliou Medical College. The diagnostic criteria for MS were the 2005 revisions to the "McDon-ald criteria". All the admitted patients received 1 g of methylprednisoione per day intravenously for 5 days, fol-lowed by 60 mg prednisone per day orally for 12 days,and tapered in 6 weeks. Fourteen patients were reevaluated after corticosteroid therapy. Twenty healthy individuals ,as normal controls,matched for age and sex with the MS patients were also enrolled in this study. The percentages of peripheral blood T cells (CD8+ CD28-, CD8+CD28+, CD8+, CD4+ CD8-) were measured by flow cytometric analysis. Parametric statistical analysis were per-formed using standard methods, and linear regression analysis was conducted using Pearson correlation test. Re-sults (1)Compared with controls,the patients with active MS had significantly lower percentage of CD8+ CD28-T cells [(18.48±9.89)% vs. (24.48±4.86)%, P <0.01], and higher percentage of CD8+ CD28+ T cells [(12.23±4.31) % vs. (8.55±3.49) %, P <0.01]. (2)The percentage of CD8+ CD28- T cells was negative-ly correlated with that of CD4+ CDS- T cells (r = -0.488, P < 0.01). (3) After corticosteroid therapy, the per-eentage of peripheral blood CD8+ CD28- / CD8+ CD28+ T cells didn' t significantly decrease or increase in 14 ac-tive MS patients (P > 0.05). Conclusions The decrease of peripheral blood CD8+ CD28- regulatory T cells might be associated with the pathogenesis of MS, and CD8+ CD28- regulatory T cells perhaps played their roles through CD4+ T cells. Corticosteroid therapy could not reverse the levels of CD8+ CD28- T cells.