Breg、PD1、PDL1通過耐受性樹突狀細(xì)胞在實(shí)驗(yàn)性自身免疫性腦脊髓炎中的治療作用及機(jī)制
發(fā)布時(shí)間：2018-07-18 14:33
【摘要】：研究背景及目的意義:多發(fā)性硬化(multiple sclerosis,MS)是中樞神經(jīng)系統(tǒng)慢性炎癥性自身免疫疾病,以腦和脊髓的軸突破壞及髓鞘脫失為主要病理特點(diǎn)。MS患者臨床表現(xiàn)的嚴(yán)重程度不一,重者可致身體殘疾,極大程度地降低患者的生活質(zhì)量。實(shí)驗(yàn)性自身免疫性腦脊髓炎(experimental autoimmune encephalomyelitis,EAE)是MS基礎(chǔ)實(shí)驗(yàn)研究常用的動(dòng)物模型。樹突狀細(xì)胞(dendritic cell,DC)作為專職的抗原提呈細(xì)胞,在MS的發(fā)病過程中起重要作用。耐受性樹突狀細(xì)胞(tolerogenic dendritic cell,t DC)是一種特殊類型的DC,通過誘導(dǎo)調(diào)節(jié)性T細(xì)胞(regulatory T cell,Treg)的形成和促進(jìn)抑炎因子的分泌,對(duì)MS具有潛在的治療價(jià)值。程序性死亡受體(programmed death 1,PD1)和程序性死亡配體1(programmed death ligand 1,PDL1)屬于B7超家族,通過發(fā)揮負(fù)性調(diào)節(jié)的作用在MS的發(fā)病過程中起一定的治療作用。調(diào)節(jié)性B細(xì)胞(regulatory B cell,Breg)是B細(xì)胞的一種,可通過分泌抑炎因子在MS的發(fā)病過程中起抑制疾病發(fā)展的作用。1,25(OH)2D3是一種免疫調(diào)節(jié)劑,能夠有效誘導(dǎo)t DC(VD3-DC)的形成,并將所誘導(dǎo)VD3-DC回輸給發(fā)病的EAE小鼠后可發(fā)揮一定的治療作用。本實(shí)驗(yàn)課題擬在體外先誘導(dǎo)骨髓來源的單核細(xì)胞成為DC,后經(jīng)過具有免疫調(diào)節(jié)作用的1,25(OH)2D3誘導(dǎo)形成VD3-DC,檢測(cè)正常DC和VD3-DC表面PDL1的表達(dá),后將正常DC和VD3-DC回輸給已發(fā)病的EAE小鼠:(1)觀察回輸治療后EAE小鼠臨床癥狀的變化;(2)檢測(cè)脾臟和淋巴結(jié)T細(xì)胞表面PD1的表達(dá);(3)檢測(cè)脾臟和淋巴結(jié)B細(xì)胞中Breg的比例;(4)采用HE和LFB染色觀察小鼠脊髓內(nèi)炎性細(xì)胞浸潤(rùn)以及脫髓鞘病變情況。進(jìn)而探究Breg、PD1、PDL1通過1,25(OH)2D3誘導(dǎo)的VD3-DC在實(shí)驗(yàn)性自身免疫性腦脊髓炎中的治療作用及機(jī)制。研究方法:(1)用MOG35-55加完全弗氏佐劑(complete freund's adjuvant,CFA)的混合乳液,經(jīng)背部皮下注入C57BL/6小鼠中,于免疫當(dāng)天和2天后分別經(jīng)腹腔注射百日咳毒素(pertussis toxin,PTX)建立EAE動(dòng)物模型;(2)培養(yǎng)基內(nèi)分別加入白介素-4(interleukin-4,IL-4)和粒細(xì)胞巨噬細(xì)胞刺激因子(granulocyte-macrophage colony-stimulating factor,GM-CSF)(各10ng/ml)來刺激小鼠股骨和脛骨骨髓來源的單個(gè)核細(xì)胞誘導(dǎo)DC。誘導(dǎo)DC的同時(shí)在其培養(yǎng)基內(nèi)加入1,25(OH)2D3(10-8M)來誘導(dǎo)形成VD3-DC。然后分別向正常DC、VD3-DC中加入脂多糖(lipopolysaccharide,LPS)刺激24h,促進(jìn)其成熟,通過流式細(xì)胞術(shù)檢測(cè)DC和VD3-DC表面PDL1的表達(dá);(3)將EAE小鼠分為三組,在DC/VD3-DC誘導(dǎo)的第8天,用MOG抗原孵育4小時(shí)后,將MOG抗原洗盡,分別將PBS、DC和VD3-DC經(jīng)尾靜脈回輸給EAE小鼠(EAE小鼠誘導(dǎo)第10、13、16天),觀察各組小鼠臨床癥狀變化;(4)在回輸治療發(fā)病高峰期,分離經(jīng)過回輸治療后的EAE小鼠脾臟和淋巴結(jié)細(xì)胞,通過流式細(xì)胞術(shù)檢測(cè)細(xì)胞表面PD1的表達(dá)及Breg的比例;(5)在回輸治療發(fā)病高峰期,分離小鼠脊髓,通過蘇木精-伊紅染色(hematoxylin-eosin staining,HE)和盧克斯快藍(lán)染色(luxol fast blue,LFB),觀察對(duì)比治療前后EAE小鼠脊髓炎性細(xì)胞的浸潤(rùn)和髓鞘脫失情況。研究結(jié)果:(1)根據(jù)我們前期實(shí)驗(yàn)可知,通過相同的實(shí)驗(yàn)方法可以成功誘導(dǎo)出EAE小鼠動(dòng)物模型和VD3-DC。本實(shí)驗(yàn)通過流式細(xì)胞術(shù)檢測(cè)可知與正常DC相比,VD3-DC表面PDL1的表達(dá)增多,且差異具有統(tǒng)計(jì)學(xué)意義;(2)與PBS對(duì)照組相比,DC和VD3-DC回輸給EAE小鼠,可有效減輕其臨床癥狀,且VD3-DC的效果更顯著;(3)與PBS對(duì)照組相比,VD3-DC組和DC組脾臟和淋巴結(jié)T細(xì)胞表面PD1的表達(dá)均增加,且有統(tǒng)計(jì)學(xué)意義;但VD3-DC組和正常DC組之間PD1的表達(dá)差異無統(tǒng)計(jì)學(xué)意義;(4)與PBS對(duì)照組相比,VD3-DC組脾臟B細(xì)胞表面Breg(CD19+CD5+CD1d+)的比例顯著增加,而正常DC組脾臟B細(xì)胞中Breg的比例減少,差異無統(tǒng)計(jì)學(xué)意義;同時(shí)VD3-DC組和正常DC組淋巴結(jié)B細(xì)胞中Breg的比例均有所增加,但差異無統(tǒng)計(jì)學(xué)意義;(5)根據(jù)HE和LFB染色可知,與PBS對(duì)照組相比,DC和VD3-DC回輸EAE小鼠后,可降低小鼠脊髓炎性細(xì)胞浸潤(rùn)和髓鞘脫失程度,且以VD3-DC作用更顯著。結(jié)論:(1)VD3-DC細(xì)胞表面PDL1的表達(dá)增加;(2)VD3-DC回輸EAE后可有效減輕其臨床癥狀;(3)VD3-DC回輸可增加EAE小鼠脾臟和淋巴結(jié)B細(xì)胞中Breg的比例以及脾臟和淋巴結(jié)T細(xì)胞表面PD1的表達(dá);(4)VD3-DC回輸EAE后可顯著降低脊髓內(nèi)炎性細(xì)胞浸潤(rùn)和髓鞘脫失程度。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R744.51
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