从巴戟天多糖对银屑病AMSCs免疫调控的干预探讨“补肾培元”法治疗银屑病的作用机制
发布时间：2018-09-01 06:27
【摘要】：研究背景 银屑病(Psoriasis),是一种常见而顽固难治的慢性、炎症性皮肤病。其累及全球约2%的人口,以红斑鳞屑为主要表现,主要病理学改变是角质形成细胞异常增生、角化过度及角化不全,并有多种炎性细胞如单核细胞和淋巴细胞浸润。由于病因不清,尚无根治的有效方法,银屑病给患者和家属带来身心双重伤害。其病机错综复杂,以T淋巴细胞亚群介导的免疫功能紊乱在银屑病的发生和发展过程中起关键作用,研究证实Th1/Th2细胞平衡失调是银屑病发病的重要原因,但近年来研究发现Th17和Treg两种不同的T细胞亚群在银屑病发生过程中也起着重要作用。干细胞(Stem Cell.SC),是人体的起源细胞,具有自我复制和多向分化的潜能。依据细胞来源与发育潜能性的不同可以将干细胞分为成体干细胞(adult sterm cells,ASC)和胚胎干细胞(embryonic stem cells,ESC),成体干细胞是未分化细胞,存在于一种已经分化组织中,具有自我更新而且能够特化形成组成该类型组织的细胞。其中,间充质干细胞(mesenchymal stem cell,)是成体干细胞中的一员,是一种具有多分化潜能的非造血系祖细胞,起源于中胚层,广泛存在于骨髓、脂肪、胎盘、脐带、表皮等组织中,可以分化为多种中胚层来源细胞谱系,在体外特定诱导条件下.不仅能分化成同胚层的骨骼、脂肪和软骨细胞,还能够跨胚层分化为内胚层的神经或心肌细胞等。除具有干细胞多向分化的潜能外,间充质干细胞还具有特殊的低免疫原性和强有力的免疫调节能力,能够分泌众多细胞因子对T淋巴细胞产生免疫调节作用,可以抑制促炎性T淋巴细胞的活化、增殖和分化成熟以及改变免疫细胞分泌的细胞因子,使其倾向于抗炎和免疫耐受功能,也可以促进抑制炎症反应的T淋巴细胞的优势分化,以帮助机体恢复免疫平衡。间充质细胞对T淋巴细胞的免疫抑制作用在古代中医学中并没有对此类细胞的论述,但现在许多文献证明,间充质干细胞在来源、分布和功能上与中医上的“肾精”有许多相似相同之处,干细胞具有肾精的属性。另外,目前也有相关研究证实,补肾培元类中药具有调节MSCs生物学特性的功能,中药复方、单味中药以及中药有效组分等多种形式都能够影响干细胞的生物学特性及功能。“玄府闭郁,肾精亏损”是宋坪主任医师研究团队在继承朱仁康等名老专家学术思想,论治银屑病取得较好临床疗效的基础上,结合银屑病的发病特点,提出的银屑病的核心病机,并对应以开通玄府、补肾培元方法治疗,临床上主要是在麻黄、桂枝等开通玄府药物基础上,加仙灵脾、仙茅、威灵仙、补骨脂、桑寄生、杜仲、续断、肉苁蓉等补肾培元类药物,取得了良好疗效,但是其具体的作用机制目前还不清楚。因此,本课题在深入学习传承导师“开通玄府、补肾培元”治疗银屑病的理论和临床实践经验,总结前期工作的基础上,首次对“开通玄府、补肾培元”治疗银屑病的可能分子机制提出研究假说,认为银屑病的发病与T细胞免疫紊乱密切相关,作为T细胞免疫网络失衡的上游环节,银屑病患者MSC免疫调节功能存在异常,是导致银屑病发病的关键,开通玄府、补肾培元法可通过干预MSC的功能,调节外周血Th17和Treg细胞及分泌的细胞因子,发挥治疗银屑病的作用。本研究采用流式细胞术、PCR、ELISA等生物信息学技术,对银屑病患者和健康人AMSCs在生物学特性和免疫调控功能进行比较,并进一步研究银屑病患者AMSCs对患者外周血淋巴细胞的免疫调控作用,在此基础上以中药单体淫羊藿多糖和巴戟天多糖为干预手段,围绕单体对AMSCs的生物学特性和免疫调控作用探讨运用“开通玄府、补肾培元”理论指导治疗银屑病临床疗效的分子机制,验证假说的客观性,阐释其理论内涵,为今后“开通玄府、补肾培元”法治疗银屑病提供工作基础。研究目的 1.对比银屑病患者和健康人来源的脂肪间充质干细胞(adipose mesenchymal stem cells,AMSCs)的一般生物学特性和免疫调控功能差异。2.研究两组细胞对银屑病患者外周血淋巴细胞(peripheral blood lymphocyte,PBL)的免疫调控作用。3.研究巴戟天多糖和淫羊藿多糖干预的银屑病患者AMSCs对患者PBL的免疫调控的作用,以从侧面揭示“开通玄府、补肾培元”的理论内涵。研究方法 1.体外分离培养AMSCs,培养至第3代备用,采用流式细胞术对银屑病患者和健康人AMSCs的细胞表型和周期进行鉴定;采用油红0和碱性磷酶染色法鉴定两种AMSCs的成脂、成骨分化能力;用PCR和ELISA的方法检测银屑病患者和健康人AMSCs表达或分泌抑炎或促炎因子情况。2.将银屑病患者和健康人AMSCs分别加入患者PBL培养体系中,并设外周血淋巴细胞单独培养对照组,采用流式细胞术检测银屑病患者PBL中Treg和Th17比例,用PCR和ELISA检测银屑病患者PBL表达或分泌抑炎及促炎因子情况。3.用中药单体巴戟天多糖和淫羊藿多糖干预银屑病患者AMSCs,采用CCK-8实验筛选药物干预浓度,采用流式细胞术和成脂、成骨染色检测中药干预后银屑病患者AMSCs的生物学特性变化,采用PCR方法检测中药干预后的银屑病患者AMSCs表达抑炎和促炎因子情况。进一步将药物干预后的银屑病患者AMSCs与银屑病患者外周血淋巴细胞共培养,并设健康对照组。采用流式细胞术检测患者PBL中Treg和Th17比例,用PCR和ELISA检测患者外周血淋巴细胞表达或分泌抑炎和促炎因子情况。研究结果 论文的第一部分,我们发现健康人和银屑病患者来源的两组AMSCs细胞生长形态无显著变化,具有相同的表型,都表达CD29+,CD44+,CD73+,CD45-,CD31-及HLADR-。而且,都可分化成骨细胞和成脂细胞,但银屑病组AMSCs增殖趋缓(p0.05),与健康对照组相比成脂分化能力较强。另外,银屑病患者较健康人AMSCs表达或分泌的抑炎因子IL-10、IDO、TGF-β等下降(IL-10 p0.05,TGF-β p0.01,IDOp0.001),TNF-α,IFN-Y等促炎细胞因子增强(p0.05)。论文的第二部分,研究发现与健康人AMSCs+PBL相比,银屑病患者AMSCs+PBL组中Treg淋巴细胞数量减少(p0.01),同时,降低了 Treg淋巴细胞的核转录因子Foxp3及PBL中IL-10和TGF-β水平的表达及分泌(p0.05),但是,银屑病患者AMSCs对外周血Th17淋巴细胞比例的改变与健康人AMSCs相比并无明显影响,对Th17淋巴细胞的转录因子RORγt及外周血淋巴细胞分泌促炎因子的抑制作用具有减弱的趋势,但结果无统计学差异。说明银屑病患者AMSCs对抑制炎性细胞的免疫调控减弱,而对促炎性细胞的免疫调控减弱不明显。论文的第三部分,通过检测两种多糖对AMSCs抑制外周血淋巴细胞增殖的作用,结果发现淫羊藿多糖没有促进银屑病患者AMSCs抑制外周血淋巴增殖的作用,而巴戟天多糖浓度为28ug/ml、56ug/ml、112ug/ml时,外周血淋巴细胞受到明显抑制,其中巴戟天多糖在28ug/ml时对银屑病患者AMSCs抑制外周血淋巴细胞增殖的作用最明显(P0.001)。选择MOP在28ug/ml的浓度进一步实验研究。通过研究补肾药巴戟天多糖在银屑病患者AMSCs对患者外周血PBMC免疫调控中的作用,发现巴戟天多糖对银屑病患者AMSCs的细胞表型、周期、成脂及成骨分化能力没有影响,但是巴戟天多糖能够增强银屑病患者AMSCs的免疫抑炎功能,增加抑炎因子TGF-β、IL-10、IDO及抑炎相关配体PD-L1及受体TLR3的mRNA表达(IL-10p0.05),同时,能够减弱银屑病患者AMSCs的免疫促炎功能,减少促炎因子TNF-α及抑炎相关受体TLR4 mRNA表达。将巴戟天多糖干预后的银屑病患者AMSCs与PBL共培养,与健康人AMSCs+PBL相比,结果发现,巴戟天多糖能够促进银屑病AMSCs增加Treg的比例,同时增加PBL抑炎因子TGF-β和IL-10的表达或分泌(TGF-β P0.05)。而巴戟天多糖对促进银屑病患者AMSCs下调Th17淋巴细胞的比例没有影响,对减少外周血淋巴细胞促炎因子IL-17和IL-23的表达或分泌作用较弱。研究结论 1.银屑病患者AMSCs较健康人的增殖和成脂分化能力下降,表达及分泌抑炎因子的能力明显减弱。2.银屑病患者AMSCs促进Treg淋巴细胞增殖的能力较健康人减弱,并且促使外周血淋巴细胞中FoxP3、IL-10和TGF-β的表达的能力减弱。3.巴戟天多糖能够增强银屑病患者AMSCs的分泌抑炎因子的能力。同时,巴戟天多糖处理后的银屑病AMSCs对Treg细胞的促增殖能力增强,并且患者AMSCs对外周血淋巴细胞分泌抑炎因子的能力增强,因此,巴戟天多糖具有增强AMSC的抑炎功能的作用。4.研究采用分子生物学前沿技术探讨了补肾培元中药单体促进银屑病患者免疫抑炎功能作用,增强AMSCs对外周血免疫调控的细胞分子机制,从一个侧面阐释了“开玄补肾”的理论内涵。
[Abstract]:Background Psoriasis is a common and refractory chronic, inflammatory skin disease that affects about 2% of the world's population. It is characterized by erythematous scales. The main pathological changes are abnormal proliferation of keratinocytes, hyperkeratosis and incomplete keratosis, and infiltration of many inflammatory cells such as monocytes and lymphocytes. The pathogenesis of psoriasis is complicated. Immune dysfunction mediated by T lymphocyte subsets plays a key role in the occurrence and development of psoriasis. Studies have confirmed that the imbalance of Th1/Th2 cells is an important cause of psoriasis. In recent years, it has been found that two different T cell subsets, Th17 and Treg, play an important role in the pathogenesis of psoriasis. Stem cells (Stem Cell. SC) are human origin cells with the potential of self-replication and multi-differentiation. Ells, ASC, and embryonic stem cells (ESC), adult stem cells are undifferentiated cells, which exist in a differentiated tissue, have self-renewal and can specialize to form the cells that make up this type of tissue. Non-hematopoietic progenitor cells, derived from the mesoderm and widely distributed in bone marrow, fat, placenta, umbilical cord, epidermis and other tissues, can differentiate into a variety of mesoderm-derived cell lineages under specific induction conditions in vitro. They can not only differentiate into homozygotic skeleton, fat and chondrocytes, but also differentiate into endodermal nerves across the embryonic layer. Mesenchymal stem cells (MSCs) have special immunogenicity and strong immunomodulatory ability. They can secrete many cytokines to produce immunomodulatory effects on T lymphocytes. They can inhibit the activation, proliferation, differentiation and maturation of proinflammatory T lymphocytes and change immunity. The cytokines secreted by epidemic cells tend to resist inflammation and immune tolerance, and also promote the dominant differentiation of T lymphocytes that inhibit inflammation in order to help restore the immune balance of the body. It has been proved that mesenchymal stem cells have many similarities with kidney essence in origin, distribution and function, and stem cells have the properties of kidney essence. "Xuanfu depression, kidney essence deficiency" is the core pathogenesis of psoriasis proposed by Song Ping's research team on the basis of inheriting Zhu Renkang's academic ideas and achieving better clinical efficacy in the treatment of psoriasis. To open Xuanfu, invigorate the kidney and peel the yuan method of treatment, mainly in ephedra, Guizhi and other open Xuanfu drugs on the basis of adding Xianling spleen, Xianmao, Wellingxian, psoralea, mulberry parasitic, Eucommia ulmoides, Dipsacus, Cistanche deserticola and other invigorate the kidney and peel the yuan drugs, and achieved good results, but the specific mechanism is still unclear. Based on the theory and clinical practice of "opening Xuanfu and invigorating the kidney and peiyuan" in the treatment of psoriasis, and on the basis of summing up the previous work, a hypothesis was put forward for the first time to study the possible molecular mechanism of "opening Xuanfu and invigorating the kidney and peiyuan" in the treatment of psoriasis. In the upstream link of the disequilibrium of epidemic network, the abnormal immune regulation function of MSC in psoriasis patients is the key to the pathogenesis of psoriasis. Opening Xuanfu and invigorating kidney and Peiyuan can regulate the peripheral blood Th17 and Treg cells and secreted cytokines by interfering with the function of MSC. Flow cytometry, PCR, ELISA were used in this study. The biological characteristics and immune regulation function of AMSCs in psoriatic patients and healthy people were compared by bioinformatics techniques. The immune regulation effect of AMSCs on peripheral blood lymphocytes of patients with psoriasis was further studied. On this basis, the monomer of Herba Epimedii polysaccharides and Morinda officinalis polysaccharides were used as intervention means to surround the monomer on AMS. To explore the biological characteristics and immune regulation function of Cs in the treatment of psoriasis by using the theory of "opening Xuanfu, invigorating kidney and peiyuan" to guide the molecular mechanism of the clinical efficacy of psoriasis, verify the objectivity of the hypothesis, and explain its theoretical connotation, so as to provide a basis for the future treatment of psoriasis by "opening Xuanfu, invigorating kidney and peiyuan". The general biological characteristics and immunoregulatory function of adipose mesenchymal stem cells (AMSCs) from healthy volunteers and healthy volunteers were compared. 2. To study the immunoregulatory effect of AMSCs on peripheral blood lymphocyte (PBL) in patients with psoriasis. 3. To study the intervention of Morinda officinalis polysaccharide and Epimedium polysaccharide. The effect of AMSCs on the immune regulation of PBL in patients with psoriasis was studied in order to reveal the theoretical connotation of "opening Xuanfu, invigorating kidney and nourishing kidney and nourishing kidney and nourishing kidney and nourishing kidney and nourishing kidney and nourishing kidney and nourishing kidney and nourishing kidney". Phosphatase staining was used to identify the adipogenesis and osteogenic differentiation ability of AMSCs. PCR and ELISA were used to detect the expression or secretion of anti-inflammatory or pro-inflammatory factors in AMSCs from psoriatic patients and healthy people. 2. AMSCs from psoriatic patients and healthy people were added into PBL culture system respectively, and peripheral blood lymphocytes were cultured separately as control group. Treg and Th17 in PBL of psoriasis patients were detected by cytometry, and the expression or secretion of anti-inflammatory and pro-inflammatory factors in PBL of psoriasis patients were detected by PCR and ELISA. The biological characteristics of AMSCs in patients with psoriasis were detected by colorimetric assay, and the expression of anti-inflammatory and pro-inflammatory factors in patients with psoriasis were detected by PCR. The ratio of Treg and Th17 in PBL was detected by cytometry, and the expression or secretion of anti-inflammatory and pro-inflammatory factors in peripheral blood lymphocytes were detected by PCR and ELISA. 44 +, CD73 +, CD45 -, CD31 - and HLADR -. Moreover, all of them can differentiate into osteoblasts and adipocytes, but the proliferation of AMSCs in psoriasis group slowed down (p0.05), and the adipogenic differentiation ability was stronger than that in healthy control group. In addition, the expression or secretion of anti-inflammatory factors IL-10, IDO, TGF-beta in psoriasis patients were lower than that in healthy people (IL-10 p0.05, TGF-beta p0.01, IDOp 0.001), T-10. In the second part of the paper, the number of Treg lymphocytes in the AMSCs + PBL group was decreased (p0.01) and the expression and secretion of the nuclear transcription factors Foxp3 and IL-10 and TGF-beta in the Treg lymphocytes were decreased (p0.05) compared with those in the healthy subjects (p0.05). AMSCs in patients with psoriasis had no significant effect on the percentage of Th17 lymphocytes in peripheral blood compared with AMSCs in healthy volunteers. The inhibitory effect of AMSCs on the transcription factor RORgamt of Th17 lymphocytes and the secretion of pro-inflammatory factors from peripheral blood lymphocytes was weakened, but the results showed no statistical difference. Epimedium polysaccharide did not promote the proliferation of peripheral blood lymphocytes in psoriasis patients, but Morinda officinalis polysaccharide concentration was 28ug/ml, 56u. The effect of Morinda officinalis polysaccharide on the proliferation of peripheral blood lymphocytes in psoriasis patients was the most obvious (P 0.001) when the concentration of Morinda officinalis polysaccharide was 28 ug/ml. The concentration of MOP at 28 ug/ml was selected for further experimental study. It was found that Morinda officinalis polysaccharides had no effect on the phenotype, cycle, adipogenesis and osteogenic differentiation of AMSCs in psoriasis patients, but Morinda officinalis polysaccharides could enhance the immunosuppressive function of AMSCs in psoriasis patients, increase the mRNA expression of anti-inflammatory factors TGF-beta, IL-10, IDO and anti-inflammatory ligand PD-L1 and receptor TLR3. The results showed that Morinda officinalis polysaccharide could promote the increase of Treg in AMSCs of psoriasis patients compared with AMSCs+PBL in co-culture with PBL. At the same time, the expression or secretion of PBL anti-inflammatory factors TGF-beta and IL-10 were increased (TGF-beta P 0.05). Morinda officinalis polysaccharides had no effect on promoting the down-regulation of Th17 lymphocyte proportion in patients with psoriasis, but had weak effect on reducing the expression or secretion of IL-17 and IL-23 in peripheral blood lymphocyte. The ability of AMSCs to promote the proliferation of Treg lymphocytes in psoriatic patients was weaker than that in healthy people, and the ability of FoxP3, IL-10 and TGF-beta expression in peripheral blood lymphocytes was weaker. 3. Morinda officinalis polysaccharide could enhance the ability of AMSC in psoriatic patients. Meanwhile, AMSC s from Morinda officinalis after polysaccharide treatment can enhance the proliferation of Treg cells and the ability of AMSC s to secrete anti-inflammatory factors from peripheral blood lymphocytes. Therefore, Morinda officinalis polysaccharides can enhance the anti-inflammatory function of AMSC. 4. Molecular biology frontier technology was used in this study. This paper discusses the effect of Kidney-Tonifying and yuan-tonifying Chinese herbal monomer on the immunosuppressive function of psoriasis patients and the cellular and molecular mechanism of AMSCs on peripheral blood immune regulation.
【学位授予单位】：中国中医科学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R275
本文编号：2216385
[Abstract]:Background Psoriasis is a common and refractory chronic, inflammatory skin disease that affects about 2% of the world's population. It is characterized by erythematous scales. The main pathological changes are abnormal proliferation of keratinocytes, hyperkeratosis and incomplete keratosis, and infiltration of many inflammatory cells such as monocytes and lymphocytes. The pathogenesis of psoriasis is complicated. Immune dysfunction mediated by T lymphocyte subsets plays a key role in the occurrence and development of psoriasis. Studies have confirmed that the imbalance of Th1/Th2 cells is an important cause of psoriasis. In recent years, it has been found that two different T cell subsets, Th17 and Treg, play an important role in the pathogenesis of psoriasis. Stem cells (Stem Cell. SC) are human origin cells with the potential of self-replication and multi-differentiation. Ells, ASC, and embryonic stem cells (ESC), adult stem cells are undifferentiated cells, which exist in a differentiated tissue, have self-renewal and can specialize to form the cells that make up this type of tissue. Non-hematopoietic progenitor cells, derived from the mesoderm and widely distributed in bone marrow, fat, placenta, umbilical cord, epidermis and other tissues, can differentiate into a variety of mesoderm-derived cell lineages under specific induction conditions in vitro. They can not only differentiate into homozygotic skeleton, fat and chondrocytes, but also differentiate into endodermal nerves across the embryonic layer. Mesenchymal stem cells (MSCs) have special immunogenicity and strong immunomodulatory ability. They can secrete many cytokines to produce immunomodulatory effects on T lymphocytes. They can inhibit the activation, proliferation, differentiation and maturation of proinflammatory T lymphocytes and change immunity. The cytokines secreted by epidemic cells tend to resist inflammation and immune tolerance, and also promote the dominant differentiation of T lymphocytes that inhibit inflammation in order to help restore the immune balance of the body. It has been proved that mesenchymal stem cells have many similarities with kidney essence in origin, distribution and function, and stem cells have the properties of kidney essence. "Xuanfu depression, kidney essence deficiency" is the core pathogenesis of psoriasis proposed by Song Ping's research team on the basis of inheriting Zhu Renkang's academic ideas and achieving better clinical efficacy in the treatment of psoriasis. To open Xuanfu, invigorate the kidney and peel the yuan method of treatment, mainly in ephedra, Guizhi and other open Xuanfu drugs on the basis of adding Xianling spleen, Xianmao, Wellingxian, psoralea, mulberry parasitic, Eucommia ulmoides, Dipsacus, Cistanche deserticola and other invigorate the kidney and peel the yuan drugs, and achieved good results, but the specific mechanism is still unclear. Based on the theory and clinical practice of "opening Xuanfu and invigorating the kidney and peiyuan" in the treatment of psoriasis, and on the basis of summing up the previous work, a hypothesis was put forward for the first time to study the possible molecular mechanism of "opening Xuanfu and invigorating the kidney and peiyuan" in the treatment of psoriasis. In the upstream link of the disequilibrium of epidemic network, the abnormal immune regulation function of MSC in psoriasis patients is the key to the pathogenesis of psoriasis. Opening Xuanfu and invigorating kidney and Peiyuan can regulate the peripheral blood Th17 and Treg cells and secreted cytokines by interfering with the function of MSC. Flow cytometry, PCR, ELISA were used in this study. The biological characteristics and immune regulation function of AMSCs in psoriatic patients and healthy people were compared by bioinformatics techniques. The immune regulation effect of AMSCs on peripheral blood lymphocytes of patients with psoriasis was further studied. On this basis, the monomer of Herba Epimedii polysaccharides and Morinda officinalis polysaccharides were used as intervention means to surround the monomer on AMS. To explore the biological characteristics and immune regulation function of Cs in the treatment of psoriasis by using the theory of "opening Xuanfu, invigorating kidney and peiyuan" to guide the molecular mechanism of the clinical efficacy of psoriasis, verify the objectivity of the hypothesis, and explain its theoretical connotation, so as to provide a basis for the future treatment of psoriasis by "opening Xuanfu, invigorating kidney and peiyuan". The general biological characteristics and immunoregulatory function of adipose mesenchymal stem cells (AMSCs) from healthy volunteers and healthy volunteers were compared. 2. To study the immunoregulatory effect of AMSCs on peripheral blood lymphocyte (PBL) in patients with psoriasis. 3. To study the intervention of Morinda officinalis polysaccharide and Epimedium polysaccharide. The effect of AMSCs on the immune regulation of PBL in patients with psoriasis was studied in order to reveal the theoretical connotation of "opening Xuanfu, invigorating kidney and nourishing kidney and nourishing kidney and nourishing kidney and nourishing kidney and nourishing kidney and nourishing kidney and nourishing kidney and nourishing kidney". Phosphatase staining was used to identify the adipogenesis and osteogenic differentiation ability of AMSCs. PCR and ELISA were used to detect the expression or secretion of anti-inflammatory or pro-inflammatory factors in AMSCs from psoriatic patients and healthy people. 2. AMSCs from psoriatic patients and healthy people were added into PBL culture system respectively, and peripheral blood lymphocytes were cultured separately as control group. Treg and Th17 in PBL of psoriasis patients were detected by cytometry, and the expression or secretion of anti-inflammatory and pro-inflammatory factors in PBL of psoriasis patients were detected by PCR and ELISA. The biological characteristics of AMSCs in patients with psoriasis were detected by colorimetric assay, and the expression of anti-inflammatory and pro-inflammatory factors in patients with psoriasis were detected by PCR. The ratio of Treg and Th17 in PBL was detected by cytometry, and the expression or secretion of anti-inflammatory and pro-inflammatory factors in peripheral blood lymphocytes were detected by PCR and ELISA. 44 +, CD73 +, CD45 -, CD31 - and HLADR -. Moreover, all of them can differentiate into osteoblasts and adipocytes, but the proliferation of AMSCs in psoriasis group slowed down (p0.05), and the adipogenic differentiation ability was stronger than that in healthy control group. In addition, the expression or secretion of anti-inflammatory factors IL-10, IDO, TGF-beta in psoriasis patients were lower than that in healthy people (IL-10 p0.05, TGF-beta p0.01, IDOp 0.001), T-10. In the second part of the paper, the number of Treg lymphocytes in the AMSCs + PBL group was decreased (p0.01) and the expression and secretion of the nuclear transcription factors Foxp3 and IL-10 and TGF-beta in the Treg lymphocytes were decreased (p0.05) compared with those in the healthy subjects (p0.05). AMSCs in patients with psoriasis had no significant effect on the percentage of Th17 lymphocytes in peripheral blood compared with AMSCs in healthy volunteers. The inhibitory effect of AMSCs on the transcription factor RORgamt of Th17 lymphocytes and the secretion of pro-inflammatory factors from peripheral blood lymphocytes was weakened, but the results showed no statistical difference. Epimedium polysaccharide did not promote the proliferation of peripheral blood lymphocytes in psoriasis patients, but Morinda officinalis polysaccharide concentration was 28ug/ml, 56u. The effect of Morinda officinalis polysaccharide on the proliferation of peripheral blood lymphocytes in psoriasis patients was the most obvious (P 0.001) when the concentration of Morinda officinalis polysaccharide was 28 ug/ml. The concentration of MOP at 28 ug/ml was selected for further experimental study. It was found that Morinda officinalis polysaccharides had no effect on the phenotype, cycle, adipogenesis and osteogenic differentiation of AMSCs in psoriasis patients, but Morinda officinalis polysaccharides could enhance the immunosuppressive function of AMSCs in psoriasis patients, increase the mRNA expression of anti-inflammatory factors TGF-beta, IL-10, IDO and anti-inflammatory ligand PD-L1 and receptor TLR3. The results showed that Morinda officinalis polysaccharide could promote the increase of Treg in AMSCs of psoriasis patients compared with AMSCs+PBL in co-culture with PBL. At the same time, the expression or secretion of PBL anti-inflammatory factors TGF-beta and IL-10 were increased (TGF-beta P 0.05). Morinda officinalis polysaccharides had no effect on promoting the down-regulation of Th17 lymphocyte proportion in patients with psoriasis, but had weak effect on reducing the expression or secretion of IL-17 and IL-23 in peripheral blood lymphocyte. The ability of AMSCs to promote the proliferation of Treg lymphocytes in psoriatic patients was weaker than that in healthy people, and the ability of FoxP3, IL-10 and TGF-beta expression in peripheral blood lymphocytes was weaker. 3. Morinda officinalis polysaccharide could enhance the ability of AMSC in psoriatic patients. Meanwhile, AMSC s from Morinda officinalis after polysaccharide treatment can enhance the proliferation of Treg cells and the ability of AMSC s to secrete anti-inflammatory factors from peripheral blood lymphocytes. Therefore, Morinda officinalis polysaccharides can enhance the anti-inflammatory function of AMSC. 4. Molecular biology frontier technology was used in this study. This paper discusses the effect of Kidney-Tonifying and yuan-tonifying Chinese herbal monomer on the immunosuppressive function of psoriasis patients and the cellular and molecular mechanism of AMSCs on peripheral blood immune regulation.
【学位授予单位】：中国中医科学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R275
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