银屑病患者骨髓造血细胞体外增殖活性研究

目的进一步了解银屑病患者骨髓造血细胞是否有异常。方法采用密度梯度离心法分离银屑病患者及正常对照组骨髓单一核细胞,然后在含有甲基纤维素的半固体细胞因子培养体系中进行体外高增殖潜能集落形成细胞(HPP-CFC)、粒-巨噬细胞系集落形成单位(CFU-GM)及红系集落形成单位(CFU-E)集落形成实验,培养一定时期后计数集落。结果与正常对照比较,银屑病患者的HPP-CFC及CFU-GM的集落形成数显著减少,而CFU-E形成的集落数无统计学意义。结论银屑病患者骨髓造血细胞体外增殖能力明显降低,这种异常的造血细胞可能在银屑病的免疫发病机制中发挥一定作用。     

银屑病患者骨髓高增殖潜能集落形成细胞P16基因mRNA表达的研究

目的：研究银屑病患者骨髓高增殖潜能集落形成细胞（HPP-CFC）的集落形成能力及P16基因mRNA表达,探讨银屑病患者骨髓造血干细胞体外增殖活性及原因。方法：密度梯度离心法分离银屑病患者及正常对照骨髓单个核细胞,培养于含人干细胞因子（SCF）、人粒-巨噬细胞系集落剌激因子（GM-CSF）、白介素（IL）-3、IL-6细胞因子组合的甲基纤维素半固体培养基,培养14d时计数HPP-CFC集落,然后收集集落。提取纯化集落细胞的总RNA,应用反转录（RT）-PCR方法检测HPP-CFC集落细胞的P16基因mRNA的表达。结果：①在甲基纤维素半固体培养基中,银屑病患者骨髓HPP-CFC集落数显著低于正常对照组,且集落形态较小;②银屑病患者骨髓HPP-CFC集落细胞的P16基因mRNA表达高于正常对照组,差异有统计学意义。结论：银屑病患者骨髓HPP-CFC集落形成能力降低;银屑病患者骨髓HPP-CFC的P16基因mRNA表达阳性率增高,推测P16基因可能参与了银屑病患者骨髓造血干细胞体外增殖活性异常的发生。     

单一核细胞条件培养液与细胞因子组合培养体系对骨髓HPP-CFC集落形成影响的研究

目的：观察两种培养体系对骨髓高增殖潜能集落形成细胞（high proliferative potential colony forming cell,HPP-CFC）集落形成的影响。方法：密度梯度离心法分离骨髓单一核细胞,分别在植物血凝素（PHA）刺激的单一核细胞条件培养液（phytohemagglutinin monocyte medium,PHA-MCM）及细胞因子组合支持下进行甲基纤维素半固体培养,14d时倒置显微镜下观察并计数两种培养体系中形成的集落数目及直径。结果：PHA-MCM与细胞因子组合均可支持HPP-CFC的集落形成,但细胞因子组合培养体系的集落数目明显高于PHA-MCM体系（P〈0.05）,而且集落直径明显大于后者。结论：在HPP-CFC的体外培养中,细胞因子组合培养体系明显优于PHA-MCM体系,是骨髓HPP-CFC较理想的体外培养体系,为银屑病及其它皮肤病的骨髓造血细胞研究奠定了一定的实验基础。     

斑块状银屑病外周血单一核细胞p16基因甲基化状态的研究

目的探讨银屑病患者外周血单一核细胞p16基因甲基化状态。方法采用甲基化特异的聚合酶链反应对34例斑块状银屑病患者和35例正常人外周血单一核细胞p16基因甲基化状态进行分析,银屑病的严重程度按PASI评分评估。结果斑块状银屑病外周血单一核细胞p16基因甲基化率高于正常人,差异有统计学意义(P<0.05),但单一核细胞p16基因甲基化状态对疾病的严重程度和病程无明显影响(P>0.05)。结论斑块状银屑病外周血单一核细胞p16基因甲基化率明显增高,在银屑病的发病机制中可能起某些作用。     

SLE患者外周血CD4~+ T淋巴细胞DNA中p16基因启动子区甲基化研究

目的 检测SIJE患者外周血CD4~+T淋巴细胞DNA中p16基因的甲基化状态及其在SLE发病机制中的作用.方法 以Taqman探针为基础的实时定量PCR(Methylight)方法检测28例SLE患者和20例正常人CD4~+T淋巴细胞中p16基因启动子区甲基化状态.结果 SLE患者CD4~+T淋巴细胞的p16基因启动子区甲基化率(35.7%,10/28)高于对照组(10.0%,2/20).两者比较筹异有统计学意义(x~2=4.11,P<0.05).SLE患者疾病严重程度评分与对应的标准甲基化指数无统计学相父性(r_s=-0.29,P>0.05).结论 SLE患者CD4~+T淋巴细胞p16基凶甲基化异常,提示p16基因的高甲基化在SLE的发病中起一定作用.     

斑块状银屑病外周血中性粒细胞P16基因甲基化状态的研究

目的：探讨银屑病外周血中性粒细胞P16基因甲基化状态及其在疾病发生机制中的作用。方法：采用甲基化特异的PCR对34例斑块状银屑病患者外周血中性粒细胞的P16基因Ep基化状态进行分析，并与35例健康人做对比。结果：斑块状银屑病外周血中性粒细胞P16基因甲基化率高于正常对照组，差异有显著性（P〈0．05），但对病程、PASI积分无明显影响（P〉0．05）。结论：本研究表明斑块状银屑病外周血中性粒细胞P16基因甲基化明显异常，可能参与斑块状银屑病的发病。     

银屑病患者外周血单一核细胞的培养上清液对骨髓造血干细胞和祖细胞集落形成的影响

目的 研究银屑病患者外周血单一核细胞(PBMC)的培养上清液对骨髓造血干细胞、祖细胞集落形成的影响.方法 密度梯度离心法分离骨髓单一核细胞,采用甲基纤维素半固体集落培养法研究银屑病患者及正常人PBMC培养上清液对骨髓高增殖潜能集落形成细胞、红系集落形成单位及粒细胞-巨噬细胞系集落形成单位集落形成的影响.结果 银屑病患者PBMC培养上清液作用后,骨髓高增殖潜能集落形成细胞、红系集落形成单位及巨噬细胞系集落形成单位集落形成数较自然增殖组及正常人PBMC培养上清液组显著减少(P<0.05,P<0.01);而正常人PBMC培养上清液作用组与自然增殖组差异无统计学意义(P>0.05).结论 银屑病患者PBMC培养上清液对骨髓造血干细胞、祖细胞集落形成可能有影响.     

寻常性银屑病患者表皮p16INK4a基因启动子高甲基化与临床资料的相关性研究

目的：探讨斑块状银屑病患者表皮p16^INK4a基因启动子甲基化状态,并分析与其临床资料的相关性。方法：按银屑病皮损面积和严重度指数（PASI）评分评估患者病情严重程度。采用甲基化特异PCR（MAP）方法检测p16^INK4a甲基化状态。结果：①银屑病患者皮损和非皮损表皮p16^INK4a基因的甲基化率分别为32．14％（9／28）和7．14％（2／28）,皮损处明显高于非皮损处（P〈0．05）,正常对照组中无表皮p16^INK4a基因甲基化（0/38）;②进行期皮损表皮p16^INK4a基因的甲基化率明显高于稳定期皮损（P〈0．05）;③皮损表皮p16^INK4a基因甲基化阳性患者的PASI评分明显高于甲基化阴性患者（P〈0．05）。结论：斑块状银屑病患者皮损表皮p16^INK4a基因启动子甲基化率明显增高,并与皮损严重程度和活动性有关,提示p16^INK4a基因启动子高甲基化在银屑病的发病中可能起作用。     

Functional characterization of CD4+CD25+ regulatory T cells differentiated in vitro from bone marrow-derived haematopoietic cells of psoriasis patients with a family history of the disorder

BACKGROUND: In psoriasis CD4+CD25+ regulatory T cells are functionally deficient. The imbalance between regulatory and effector T-cell functions is important for inducing psoriasis. It is reasonable to speculate that the dysfunctional activity of CD4+CD25+ regulatory cells may originate partly from the abnormal haematopoietic cells determined mainly by genetic background. OBJECTIVES: To test the hypothesis that haematopoietic stem cells are responsible for dysfunctional CD4+CD25+ regulatory cells in psoriasis. METHODS: Bone marrow-derived CD34+ haematopoietic cells from patients with psoriasis (with a family history of psoriasis) and from normal controls were differentiated into T cells in vitro. CD4+CD25+ T cells were isolated by an immunomagnetic bead method, and proliferation activity and capacity for cytokine secretion were determined. Furthermore, the ability of CD4+CD25+ T cells to suppress the proliferative responses of allogeneous peripheral blood CD4+CD25- effector T cells was assessed in vitro. RESULTS: The differentiated CD4+CD25+ T cells of psoriatic origin showed similar characteristics to those of normal volunteers, including proliferation activity and secretion profile of the cytokines interleukin (IL)-2, IL-4, IL-8, IL-10 and interferon (IFN)-gamma. However, proliferation and secretion levels of the cytokines IL-2 and IL-10 for CD4+CD25+ cells of psoriatic CD34+ cell origin were significantly lower than those of normal controls in response to streptococcal superantigen (Strep-A). In particular, CD4+CD25+ T cells differentiated from psoriatic CD34+ cells were functionally insufficient to restrain effector T-cell proliferation. CONCLUSIONS: CD4+CD25+ T cells differentiated in vitro from haematopoietic cells of patients with psoriasis are impaired in regulatory function. The dysfunction of psoriatic CD4+CD25+ T cells may be due to inherent genetic programming passed down from bone marrow-derived haematopoietic cells.     

银屑病患者骨髓基质细胞体外培养生长状况的研究

目的研究银屑病患者骨髓基质细胞(bone marrow stromal cells,BMSCs)体外培养的情况,包括细胞的形态改变、贴壁情况和增殖状态。方法密度梯度离心法分离BMSCs,接种2d后首次换液时去除未贴壁细胞,通过倒置显微镜观察BMSCs的形态改变、生长和贴壁情况,MTT法检测BMSCs增殖活性。结果与对照组相比较,银屑病组BMSCs形态发生改变、贴壁晚,增殖活性低(8d时P<0.05)。结论银屑病患者骨髓基质细胞活性异常,可能对银屑病患者骨髓造血微环境产生一定影响。     

寻常性银屑病患者HLAⅠ类分子A、B、C位点甲基化的研究

目的探讨寻常性银屑病患者表皮中HLA Ⅰ类分子重链A、B、C位点启动子区甲基化与疾病的关系。方法 甲基化特异性PCR技术检测46例寻常性银屑病患者皮损和非皮损表皮中的HLAⅠ类分子重链A、B、C位点启动子区域CpG岛的甲基化率,与28例正常人皮肤组织进行对照研究。PASI评分评价银屑病患者皮损的严重程度。结果 在银屑病患者皮损、非皮损和正常人皮肤组织中,重链A位点均未检测到发生甲基化,B位点的甲基化率分别是4.35％( 2/46)、4.35％( 2/46)和0,C位点的甲基化率分别是4.35％ (2/46)、21.74％( 10/46)和0。3组样本比较,C位点启动子区甲基化率非皮损明显高于皮损和正常人皮肤组织,但与病情严重程度无关;HLA Ⅰ类分子重链A和B位点差异均无统计学意义。结论 寻常性银屑病患者HLA Ⅰ类分子C位点启动子区甲基化异常。     

Abnormal hematopoiesis in psoriasis--in vitro studies

Using mononuclear cells (MNC) of patients suffering from psoriasis for production of colony-stimulating factors (CSF) an abnormal colony formation in bone marrow of healthy volunteers was detected (colony-forming assay): Number of colonies per 10(5) bone marrow cells after cultivation with 10(6) MNC of patients suffering from psoriasis vulgaris: 65 +/- 14 (n = 8); psoriasis arthropathica: 63 +/- 11 (n = 8). Control values (10(5) bone marrow cells plus 10(6) MNC of healthy donors): 116 +/- 23 (n = 10). Statistical differences: psoriasis vulgaris: p less than 0.001; psoriasis arthropathica: p less than 0.001. The mechanisms responsible for these abnormalities in hematopoiesis are still unknown. They may be of pathogenic significance in psoriasis.     

Abnormal histone modifications in PBMCs from patients with psoriasis vulgaris

Excessive keratinocyte proliferation is thought to be responsible for the formation and development of psoriasis vulgaris. Evidence indicates that epigenetic modifications are associated with aberrant gene expression, however, nothing is known about the status of histone modifications in psoriasis vulgaris. We investigated alterations in histone modifications in patients with psoriasis vulgaris. Global histone H3/H4 acetylation and H3K4/H3K27 methylation in peripheral blood mononuclear cells from 30 psoriatic patients and 20 healthy control subjects were quanti?ed by the EpiQuik(TM) global histone H3/H4 acetylation and H3K4/H3K27 methylation assay kit. The mRNA levels of 12 members of 3 classes of chromatin modifier genes were measured by real-time quantitative polymerase chain reaction. Compared with normal controls, global histone H4 hypoacetylation was observed in PBMCs from psoriasis vulgaris patients. There was a negative correlation between the degree of histone H4 acetylation and disease activity in patients as measured by PASI. Global levels of H3 acetylation, H3K4/H3K27 methylation did not significantly differ between psoriatic patients and controls. mRNA levels of P300, CBP and SIRT1 were significantly reduced in PBMCs from patients with psoriasis vulgaris compared with healthy controls, while mRNA expression levels of HDAC1, SUV39H1 and EZH2 was significantly increased in psoriatic patients.We conclude that histone modifications are aberrant in the PBMCs of psoriasis vulgaris patients.     

银屑病角质形成细胞p16启动子甲基化的研究

目的 探讨p16启动子甲基化与银屑病发生发展的关系.方法 采用甲基化特异性PCR及基因测序技术检测p16启动子.结果 26例银屑病患者皮损和非皮损甲基化率分别为23.08%(6/26)和19.23%(5/26),明显高于正常对照(P<0.05);12例进行期皮损和非皮损p16启动子甲基化率均为41.67%(5/12),14例静止期分别为7.14%(1/14)和0,进行期甲基化率明显高于静止期(P<0.05).结论 p16启动子甲基化与银屑病的发生发展相关,且与病期相关.     

系统性红斑狼疮患者血浆DNA p16基因启动子的甲基化状态

目的 探讨系统性红斑狼疮(SLF)患者中p16基因的甲基化状态及其在发病机制中的作用。方法 检测45例SLE患者及20例健康对照者血浆DNA甲基化状态。应用甲基化特异PcR(MSP)方法检测p16基因启动子区甲基化状态,并分析与临床表现及常规实验室检查结果之间的关联。结果 在SLE患者血浆DNA中p16基因呈现高甲基化状态,占64.44%(29/45),而健康对照组中只有1例检测到p16基因高甲基化(1/20,5%),两组间差异有统计学意义(x²=19.69,P<0.01)。活动期SLE组p16基因呈高甲基化状态者所占比率83.33%(20/24),比非活动期SLE组42.85%,(9/21)高,差异也有统计学意义(x²=8.01;P<0.01)。但初发SLE组p16基因呈高甲基化状态者所占比例(71.43%,15/21)与复发SLE组(58.33%,14/24)相比差异无统计学意义(P=0.37)。具有下列临床表现的SLE患者其血浆p16基因高甲基化状态者所占比率相对较高:关节炎(57.5%,15/26),白细胞减少(42.3%,11/26),血沉增快(56%,14/25)。结论 活动期SLE患者血浆DNAp16基因启动子区呈现明显高甲基化状态,并与关节炎、白细胞减少及血沉增快等临床表现相关;提示p16基因启动子区高甲基化可能在SLE的发病机制中起作用。     

系统性红斑狼疮患者外周血单一核细胞及中性粒细胞p16基因甲基化状态研究

目的：研究系统性红斑狼疮（SLE）患者外周血单一核细胞（PBMC）及中性粒细胞（PMNL）的P16基因甲基化状态,探讨其在SLE发病中的作用。方法：采用甲基化特异的PCR方法对25例活动期SLE患者PBMC及PMNL的P16基因甲基化状态进行分析,并与21名健康者进行对照？结果：SLE患者PBMCP16基因甲基化阳性率为76．0％（19／25）,对照组为47．6％（10／21）。两者比较差异有统计学意义（P〈0．05）;两组PMNL．P16基因甲基化率分别为54．3％（12／25）和45．7％（11／21）,差异无统计学意义（P〉0．05）。在PBMCP16基因高甲基化的SLE患者中,IgG升高者占78．9％（15／19）,与该细胞系P16基因甲基化阴性的SLE患者IgG平均水平比较,差异有统计学意义（P〈0．05）。结论：SLE患者PBMCP16基因甲基化明显异常,IgG升高与之相关,p16基因高甲基化可能在SLE的发病中起一定的作用。