Breg、PD1、PDL1通过耐受性树突状细胞在实验性自身免疫性脑脊髓炎中的治疗作用及机制
发布时间：2018-07-18 14:33
【摘要】：研究背景及目的意义:多发性硬化(multiple sclerosis,MS)是中枢神经系统慢性炎症性自身免疫疾病,以脑和脊髓的轴突破坏及髓鞘脱失为主要病理特点。MS患者临床表现的严重程度不一,重者可致身体残疾,极大程度地降低患者的生活质量。实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)是MS基础实验研究常用的动物模型。树突状细胞(dendritic cell,DC)作为专职的抗原提呈细胞,在MS的发病过程中起重要作用。耐受性树突状细胞(tolerogenic dendritic cell,t DC)是一种特殊类型的DC,通过诱导调节性T细胞(regulatory T cell,Treg)的形成和促进抑炎因子的分泌,对MS具有潜在的治疗价值。程序性死亡受体(programmed death 1,PD1)和程序性死亡配体1(programmed death ligand 1,PDL1)属于B7超家族,通过发挥负性调节的作用在MS的发病过程中起一定的治疗作用。调节性B细胞(regulatory B cell,Breg)是B细胞的一种,可通过分泌抑炎因子在MS的发病过程中起抑制疾病发展的作用。1,25(OH)2D3是一种免疫调节剂,能够有效诱导t DC(VD3-DC)的形成,并将所诱导VD3-DC回输给发病的EAE小鼠后可发挥一定的治疗作用。本实验课题拟在体外先诱导骨髓来源的单核细胞成为DC,后经过具有免疫调节作用的1,25(OH)2D3诱导形成VD3-DC,检测正常DC和VD3-DC表面PDL1的表达,后将正常DC和VD3-DC回输给已发病的EAE小鼠:(1)观察回输治疗后EAE小鼠临床症状的变化;(2)检测脾脏和淋巴结T细胞表面PD1的表达;(3)检测脾脏和淋巴结B细胞中Breg的比例;(4)采用HE和LFB染色观察小鼠脊髓内炎性细胞浸润以及脱髓鞘病变情况。进而探究Breg、PD1、PDL1通过1,25(OH)2D3诱导的VD3-DC在实验性自身免疫性脑脊髓炎中的治疗作用及机制。研究方法:(1)用MOG35-55加完全弗氏佐剂(complete freund's adjuvant,CFA)的混合乳液,经背部皮下注入C57BL/6小鼠中,于免疫当天和2天后分别经腹腔注射百日咳毒素(pertussis toxin,PTX)建立EAE动物模型;(2)培养基内分别加入白介素-4(interleukin-4,IL-4)和粒细胞巨噬细胞刺激因子(granulocyte-macrophage colony-stimulating factor,GM-CSF)(各10ng/ml)来刺激小鼠股骨和胫骨骨髓来源的单个核细胞诱导DC。诱导DC的同时在其培养基内加入1,25(OH)2D3(10-8M)来诱导形成VD3-DC。然后分别向正常DC、VD3-DC中加入脂多糖(lipopolysaccharide,LPS)刺激24h,促进其成熟,通过流式细胞术检测DC和VD3-DC表面PDL1的表达;(3)将EAE小鼠分为三组,在DC/VD3-DC诱导的第8天,用MOG抗原孵育4小时后,将MOG抗原洗尽,分别将PBS、DC和VD3-DC经尾静脉回输给EAE小鼠(EAE小鼠诱导第10、13、16天),观察各组小鼠临床症状变化;(4)在回输治疗发病高峰期,分离经过回输治疗后的EAE小鼠脾脏和淋巴结细胞,通过流式细胞术检测细胞表面PD1的表达及Breg的比例;(5)在回输治疗发病高峰期,分离小鼠脊髓,通过苏木精-伊红染色(hematoxylin-eosin staining,HE)和卢克斯快蓝染色(luxol fast blue,LFB),观察对比治疗前后EAE小鼠脊髓炎性细胞的浸润和髓鞘脱失情况。研究结果:(1)根据我们前期实验可知,通过相同的实验方法可以成功诱导出EAE小鼠动物模型和VD3-DC。本实验通过流式细胞术检测可知与正常DC相比,VD3-DC表面PDL1的表达增多,且差异具有统计学意义;(2)与PBS对照组相比,DC和VD3-DC回输给EAE小鼠,可有效减轻其临床症状,且VD3-DC的效果更显著;(3)与PBS对照组相比,VD3-DC组和DC组脾脏和淋巴结T细胞表面PD1的表达均增加,且有统计学意义;但VD3-DC组和正常DC组之间PD1的表达差异无统计学意义;(4)与PBS对照组相比,VD3-DC组脾脏B细胞表面Breg(CD19+CD5+CD1d+)的比例显著增加,而正常DC组脾脏B细胞中Breg的比例减少,差异无统计学意义;同时VD3-DC组和正常DC组淋巴结B细胞中Breg的比例均有所增加,但差异无统计学意义;(5)根据HE和LFB染色可知,与PBS对照组相比,DC和VD3-DC回输EAE小鼠后,可降低小鼠脊髓炎性细胞浸润和髓鞘脱失程度,且以VD3-DC作用更显著。结论:(1)VD3-DC细胞表面PDL1的表达增加;(2)VD3-DC回输EAE后可有效减轻其临床症状;(3)VD3-DC回输可增加EAE小鼠脾脏和淋巴结B细胞中Breg的比例以及脾脏和淋巴结T细胞表面PD1的表达;(4)VD3-DC回输EAE后可显著降低脊髓内炎性细胞浸润和髓鞘脱失程度。
[Abstract]:Research background and purpose: multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system. The main pathological features of the brain and spinal cord destruction and myelin loss are the different severity of the clinical manifestations of.MS patients. The heavy persons can cause physical disability and greatly reduce the quality of life of the patients. Experimental autoimmune encephalomyelitis (experimental autoimmune encephalomyelitis, EAE) is a common animal model in MS basic experimental research. Dendritic cell (DC), as a full-time antigen presenting cell, plays an important role in the pathogenesis of MS. The specific type of DC is of potential therapeutic value for MS by inducing the formation of regulatory T cells (regulatory T cell, Treg) and promoting the secretion of anti inflammatory factors. The programmed death receptor (programmed death 1, PD1) and programmed death ligand 1 (programmed death 1) belong to the superfamily, by playing a negative regulatory role. Regulatory B cell (Breg) is a kind of B cell (regulatory B cell, Breg), which can inhibit the development of the disease by secreting anti inflammatory factors in the pathogenesis of MS, and.1,25 (OH) 2D3 is an immune regulator. The EAE mice of the disease can play a certain therapeutic role. This experimental subject intends to induce mononuclear cells derived from bone marrow in vitro to become DC, and then 1,25 (OH) 2D3 induced by immunoregulation to form VD3-DC, to detect the expression of PDL1 on the surface of normal DC and VD3-DC, and then return normal DC and VD3-DC to the infected EAE mice: (1) observation and transfusion. Changes in clinical symptoms of EAE mice after treatment; (2) detection of the expression of PD1 on the surface of T cells in the spleen and lymph nodes; (3) to detect the proportion of Breg in the spleen and lymph node B cells; (4) the infiltration of inflammatory cells and demyelinating lesions in the spinal cord of the mice were observed by HE and LFB staining. Breg, PD1, PDL1 were investigated by 1,25 (OH). The therapeutic effect and mechanism of the experimental autoimmune encephalomyelitis. Methods: (1) a mixed emulsion of complete freund's adjuvant (CFA) and MOG35-55 was injected into the C57BL/6 mice subcutaneously on the back, and the EAE animal model was established by intraperitoneal injection of pertussis toxin (pertussis toxin, PTX) on the day of immunization and 2 days after the immunization. (2) -4 (interleukin-4, IL-4) and granulocyte macrophage stimulating factor (granulocyte-macrophage colony-stimulating factor, GM-CSF) (10ng/ml) were added to the culture base to induce mononuclear cells from the bone marrow of the femur and tibia to induce DC. induced DC, while adding 1,25 (OH) into the culture base to induce the DC. Lead to VD3-DC. and then add lipopolysaccharide (lipopolysaccharide, LPS) to normal DC and VD3-DC to stimulate 24h, promote its maturation and detect the expression of PDL1 on the surface of DC and VD3-DC by flow cytometry; (3) the EAE mice are divided into three groups. After eighth days of DC/VD3-DC induction, they are incubated with MOG antigen for 4 hours. DC through the tail vein to EAE mice (EAE mice induced day 10,13,16) and observe the changes of clinical symptoms in each group. (4) the spleen and lymph node cells of EAE mice were separated after the return transfusion treatment, and the expression of PD1 and the proportion of Breg on the cell surface were detected by flow cytometry; (5) the peak of the treatment was at the peak. During the period, the spinal cord of mice was separated and the infiltration of inflammatory cells and the loss of myelin sheath in EAE mice were observed by hematoxylin-eosin staining (HE) and lux fast blue staining (Luxol fast blue, LFB). The results of the study were: (1) according to our previous experiments, the same experimental method could be successfully induced. The EAE mouse model and VD3-DC. test showed that the expression of PDL1 on the surface of VD3-DC increased in comparison with the normal DC, and the difference was statistically significant. (2) compared with the PBS control group, DC and VD3-DC could effectively reduce the clinical symptoms of EAE mice, and the effect of VD3-DC was more significant; (3) compared with the PBS control group, The expression of PD1 on the surface of T cells of spleen and lymph node in group VD3-DC and DC group increased and had statistical significance, but there was no significant difference in the expression of PD1 between the VD3-DC group and the normal DC group. (4) the proportion of Breg (CD19+CD5+CD1d+) in the spleen B cells in the VD3-DC group was significantly increased compared with that of the PBS control group, and the proportion of the spleen cells in the normal group was proportional to the proportion of the spleen cells in the normal group. The difference was not statistically significant; at the same time, the proportion of Breg in the lymph node B cells of the VD3-DC group and the normal DC group increased, but the difference was not statistically significant. (5) according to HE and LFB staining, compared with the PBS control group, DC and VD3-DC retransfusion of EAE mice could reduce the infiltration of myeloamedullary cells and the degree of myelinating in the mice, and the VD3-DC action was used. Conclusion: (1) the expression of PDL1 on the surface of VD3-DC cells increased; (2) VD3-DC could effectively reduce its clinical symptoms after EAE transfusion; (3) VD3-DC transfusion could increase the proportion of Breg in the spleen and lymph node B cells in EAE mice and the expression of PD1 of the spleen and lymph node T cell surface; (4) the infiltration of inflammatory cells in the spinal cord could significantly decrease the infiltration of inflammatory cells in the spinal cord. And the degree of loss of myelin sheath.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R744.51
[Abstract]:Research background and purpose: multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system. The main pathological features of the brain and spinal cord destruction and myelin loss are the different severity of the clinical manifestations of.MS patients. The heavy persons can cause physical disability and greatly reduce the quality of life of the patients. Experimental autoimmune encephalomyelitis (experimental autoimmune encephalomyelitis, EAE) is a common animal model in MS basic experimental research. Dendritic cell (DC), as a full-time antigen presenting cell, plays an important role in the pathogenesis of MS. The specific type of DC is of potential therapeutic value for MS by inducing the formation of regulatory T cells (regulatory T cell, Treg) and promoting the secretion of anti inflammatory factors. The programmed death receptor (programmed death 1, PD1) and programmed death ligand 1 (programmed death 1) belong to the superfamily, by playing a negative regulatory role. Regulatory B cell (Breg) is a kind of B cell (regulatory B cell, Breg), which can inhibit the development of the disease by secreting anti inflammatory factors in the pathogenesis of MS, and.1,25 (OH) 2D3 is an immune regulator. The EAE mice of the disease can play a certain therapeutic role. This experimental subject intends to induce mononuclear cells derived from bone marrow in vitro to become DC, and then 1,25 (OH) 2D3 induced by immunoregulation to form VD3-DC, to detect the expression of PDL1 on the surface of normal DC and VD3-DC, and then return normal DC and VD3-DC to the infected EAE mice: (1) observation and transfusion. Changes in clinical symptoms of EAE mice after treatment; (2) detection of the expression of PD1 on the surface of T cells in the spleen and lymph nodes; (3) to detect the proportion of Breg in the spleen and lymph node B cells; (4) the infiltration of inflammatory cells and demyelinating lesions in the spinal cord of the mice were observed by HE and LFB staining. Breg, PD1, PDL1 were investigated by 1,25 (OH). The therapeutic effect and mechanism of the experimental autoimmune encephalomyelitis. Methods: (1) a mixed emulsion of complete freund's adjuvant (CFA) and MOG35-55 was injected into the C57BL/6 mice subcutaneously on the back, and the EAE animal model was established by intraperitoneal injection of pertussis toxin (pertussis toxin, PTX) on the day of immunization and 2 days after the immunization. (2) -4 (interleukin-4, IL-4) and granulocyte macrophage stimulating factor (granulocyte-macrophage colony-stimulating factor, GM-CSF) (10ng/ml) were added to the culture base to induce mononuclear cells from the bone marrow of the femur and tibia to induce DC. induced DC, while adding 1,25 (OH) into the culture base to induce the DC. Lead to VD3-DC. and then add lipopolysaccharide (lipopolysaccharide, LPS) to normal DC and VD3-DC to stimulate 24h, promote its maturation and detect the expression of PDL1 on the surface of DC and VD3-DC by flow cytometry; (3) the EAE mice are divided into three groups. After eighth days of DC/VD3-DC induction, they are incubated with MOG antigen for 4 hours. DC through the tail vein to EAE mice (EAE mice induced day 10,13,16) and observe the changes of clinical symptoms in each group. (4) the spleen and lymph node cells of EAE mice were separated after the return transfusion treatment, and the expression of PD1 and the proportion of Breg on the cell surface were detected by flow cytometry; (5) the peak of the treatment was at the peak. During the period, the spinal cord of mice was separated and the infiltration of inflammatory cells and the loss of myelin sheath in EAE mice were observed by hematoxylin-eosin staining (HE) and lux fast blue staining (Luxol fast blue, LFB). The results of the study were: (1) according to our previous experiments, the same experimental method could be successfully induced. The EAE mouse model and VD3-DC. test showed that the expression of PDL1 on the surface of VD3-DC increased in comparison with the normal DC, and the difference was statistically significant. (2) compared with the PBS control group, DC and VD3-DC could effectively reduce the clinical symptoms of EAE mice, and the effect of VD3-DC was more significant; (3) compared with the PBS control group, The expression of PD1 on the surface of T cells of spleen and lymph node in group VD3-DC and DC group increased and had statistical significance, but there was no significant difference in the expression of PD1 between the VD3-DC group and the normal DC group. (4) the proportion of Breg (CD19+CD5+CD1d+) in the spleen B cells in the VD3-DC group was significantly increased compared with that of the PBS control group, and the proportion of the spleen cells in the normal group was proportional to the proportion of the spleen cells in the normal group. The difference was not statistically significant; at the same time, the proportion of Breg in the lymph node B cells of the VD3-DC group and the normal DC group increased, but the difference was not statistically significant. (5) according to HE and LFB staining, compared with the PBS control group, DC and VD3-DC retransfusion of EAE mice could reduce the infiltration of myeloamedullary cells and the degree of myelinating in the mice, and the VD3-DC action was used. Conclusion: (1) the expression of PDL1 on the surface of VD3-DC cells increased; (2) VD3-DC could effectively reduce its clinical symptoms after EAE transfusion; (3) VD3-DC transfusion could increase the proportion of Breg in the spleen and lymph node B cells in EAE mice and the expression of PD1 of the spleen and lymph node T cell surface; (4) the infiltration of inflammatory cells in the spinal cord could significantly decrease the infiltration of inflammatory cells in the spinal cord. And the degree of loss of myelin sheath.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R744.51
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