口服Ⅱ型胶原蛋白诱导免疫耐受治疗佐剂关节炎的研究

目的： 观察口服Ⅱ型胶原蛋白（ｔｙｐｅＩＩｃｏｌｌａｇｅｎ, ＣＩＩ） 对大鼠佐剂关节炎（ａｄｊｕｖａｎｔａｒｔｈｒｉｔｉｓ, ＡＡ） 的治疗作用。方法： 采用酶消化法, 提取牛软骨ＣＩＩ, 经口插管灌注将ＣＩＩ给予佐剂关节炎ＳＤ大鼠。结果： 口服ＣＩＩ可推迟ＡＡ的发病, 降低发病率, 明显减轻病变关节的炎症反应和使病程缩短。结论： 口服ＣＩＩ可诱导抗原特异性免疫耐受并对淋巴细胞的增殖反应有抑制作用, 而且此种免疫耐受作用可以剂量依赖的方式通过淋巴细胞转移。     

口服CⅡ对类风湿性关节炎局部细胞因子的影响

类风湿性关节炎 (ＲＡ)是一种常见的自身免疫性疾病 ,细胞因子的分泌异常在发病过程中起重要作用[1] 。治疗此类疾病的一种新方法是通过口服蛋白质抗原诱导机体产生对自身抗原的免疫耐受。在本实验中 ,我们观察了可溶型Ⅱ型胶原蛋白 (ＣⅡ)对大鼠佐剂型关节炎 (ＡＡ)的近期疗效 ,并测定了治疗后大鼠关节局部ＩＦＮ γ、ＴＮＦ α水平的变化 ,对Ⅱ型胶原蛋白治疗类风湿关节炎的可能作用机制进行了初步探讨。1　材料与方法1.1　动物模型建立　实验所用雄性 8周龄ＳＤ大鼠由首都医科大学动物中心提供。大鼠左后足垫皮下注射弗氏完全佐剂 (ＣＦ…     

重组人Ⅱ型胶原蛋白免疫原性多肽表达的研究

目的 构建编码人Ⅱ型胶原蛋白250-270多肽片段（HuCⅡ250-270)的多聚基因，以获得高效表达，高度可溶和便于纯化的重组多肽基因。方法 选用大肠杆菌（E.coli)偏爱密码子优化HuCⅡ250-270的基因组成，通过化学合成和PCR法，获得HuCⅡ250-270的基因，并利用BanHI和BglⅡ同裂酶进行酶切位点串联获得6聚体，对影响重组HuCⅡ250-270表达的各种因素进行系统研究，以获得优化表达的条件，对表达产物进行分离纯化。结果 酶切分析和DNA测序证实，6聚目的基因的构建正确，融合表达的量明显高于非融合表达，在优化条件下，可达30%，表达产物的可溶性明显提高（在90%以上），其相对分子质量（Mr)与预期的相符，纯化后的纯度达90%以上。结论 实现了HuCⅡ250-270的高效表达，为研究类风湿性关节炎的发病机制和治疗提供了实验依据。为在原核细胞中高效表达的胶原蛋白的功能性小片段，提供了可供参考的借鉴。     

口服鸡Ⅱ型胶原治疗大鼠实验性类风湿性关节炎的研究

本文研究口服鸡Ⅱ型胶原（CCⅡ）治疗大鼠类风湿性关节炎的作用。以CCⅡ和完全弗氏佐剂免疫Wistar大鼠,建立大鼠的类风湿性关节炎（RA）模型,用CCⅡ进行口服,观察实验组和对照组动物关节炎的发病程度。并以ELISA法对发病大鼠血清中的抗CCⅡ抗体的水平进行检测。结果表明,口服CCⅡ可以减轻大鼠关节炎的发病程度。其中口服6μg、60μgCCⅡ的实验组大鼠关节炎指数均对照组明显减低,且差异有显著性意义（P＜0．01;P＜0．05）。而口服600μgCCⅡ的实验组大鼠关节炎指数虽较对照组大鼠降低,胆二者相比,无显著性差异（P＞0．05）。ELISA检测结果显示,口服低剂量（6μg和60μg）可溶性CCⅡ对大鼠体抗CCⅡ抗体的产生有一定的抑制作用。口服CCⅡ对关节炎大鼠的发病程度有较明显的抑制作用。为应用口服耐受治疗RA等自身免疫性疾病提供了实验基础。     

人类Ⅱ型胶原蛋白活性肽段多聚基因的构建和表达

类风湿性关节炎(rheumatoid arthritis,RA)的发展机理尚未完全阐明,目前认为与自身免疫密切相关,Ⅱ型胶愿蛋白(type Ⅱ collagen,CⅡ)是最可能的自身抗原(1).国外的研究提示,CⅡ的这种免疫活性主要一现在其250-270片段(hCⅡ250-270)上[1,2].本文用基因工程的方法,选用大肠杆菌(E.coli)偏爱密码子,化学合成优化该片段的基因组成,利用酶切位点将其串联成六聚体,并在E.coli中进行表达,获得大量重组hCⅡ250-270的类似物,为进一步研究该段活性肽的功能提供条件.     

组织工程支架的隔离层对新生软骨Ⅱ型胶原蛋白表达的影响

目的研究隔离层在组织工程骨软骨复合支架中,对新生软骨Ⅱ型胶原蛋白表达的影响。方法将向软骨方向诱导后的兔骨髓间充质干细胞接种到骨软骨复合支架的软骨支架上,然后将具有隔离层和没有隔离层的骨软骨复合支架分别植入兔膝关节骨软骨全层缺损处。术后3月和6月分别取材,进行新生软骨Ⅱ型胶原蛋白蛋白的免疫组化染色、Western blot法检测Ⅱ型胶原蛋白的表达、实时荧光定量PCR(qRT-PCR)测定Ⅱ型胶原蛋白相关基因的表达。结果与无隔离层的对照组相比,具有隔离层的骨软骨复合支架所形成的新生软骨的Ⅱ型胶原蛋白和基因的表达均明显增强(P<0.05)。结论隔离层加入骨软骨复合支架后,能显著提高新生组织工程软骨Ⅱ型胶原蛋白表达的能力。     

Ⅰ、Ⅱ型胶原蛋白对人软骨细胞生物学特性的影响CSCD

背景:实验证明胶原蛋白底物具有刺激成软骨的作用,但关于不同类型胶原蛋白刺激成软骨作用的能力仍存在争议。目的:观察Ⅰ、Ⅱ型胶原蛋白对体外培养人软骨细胞生物学特性的影响。方法:将P3代人软骨细胞分别加入普通培养板、Ⅰ型胶原蛋白包被培养板、Ⅱ型胶原蛋白包被培养板继续培养。培养10 d内,MTT法绘制细胞生长曲线;培养28 d后,采用ELISA法、聚合酶链反应、二甲基亚甲基蓝比色等方法检测3种培养板中软骨细胞分泌Ⅰ胶原蛋白、Ⅱ型胶原蛋白及糖胺多糖的量。结果与结论:Ⅱ型胶原蛋白包被培养板中软骨细胞数量最多,增殖速度为Ⅰ型胶原蛋白包被培养板的2倍、普通培养板的5倍。Ⅱ型胶原蛋白包被培养板中软骨细胞分泌Ⅰ型胶原蛋白最少,与普通培养板板检测结果差异有显著性意义(P<0.01),与Ⅰ型胶原蛋白包被培养板检测结果差异无显著性意义;Ⅱ型胶原蛋白包被培养板中软骨细胞分泌Ⅱ型胶原蛋白、糖胺多糖最多,与其他两种培养板检测结果差异有显著性意义(P<0.01)。表明胶原蛋白包被培养板培养软骨细胞优于普通培养板,其中Ⅱ型胶原蛋白包被培养板在培养软骨细胞时更能维持细胞形态,延长去分化现象出现的时间,更利于细胞再分化。     

克氏针经皮撬拨治疗JudetⅡ型桡骨颈骨折的临床效果观察

目的探讨克氏针经皮撬拨治疗JudetⅡ型桡骨颈骨折的疗效。方法 2012年7月~2014年11月,通过应用克氏针经皮撬拨复位治疗9例JudetⅡ型桡骨颈骨折,根据Mayo肘关节功能评分(Mayo elbow performance score,MEPS)和疼痛视觉模拟(VAS)评分进行疗效评判。结果 9例患者均获得随访,末次随访时MEPS评分及VAS评分与术前比较,差异均有统计学意义(P0.05),其中按MEPS评分优8例,良1例,中、差0例,整体优良率100%。结论克氏针经皮撬拨治疗JudetⅡ型桡骨颈骨折具有良好的临床疗效,是有效的治疗方法之一。     

骨质疏松大鼠膝关节组织总胶原、Ⅰ型及Ⅱ型胶原蛋白的含量变化

背景：目前研究认为胶原蛋白在骨细胞的增殖与分化、骨质的形成和吸收、骨基质矿化等过程中起着非常重要的作用。 目的：观察骨质疏松模型大鼠膝关节组织总胶原、Ⅰ型及Ⅱ型胶原蛋白含量变化。 方法：SD雌性大鼠40只随机分为实验组和对照组,实验组大鼠行双侧卵巢切除16周后,取膝关节软骨和交叉韧带,采用微量羟脯氨酸测定法及酶联免疫吸附法测定大鼠膝关节软骨及前交叉韧带组织总胶原、Ⅰ型及Ⅱ型胶原蛋白含量。 结果与结论：骨质疏松膝关节模型大鼠膝关节软骨、前交叉韧带总胶原、Ⅰ型及Ⅱ型胶原蛋白含量明显下降(P < 0.05),Ⅰ型/Ⅱ型胶原比值也显著降低(P < 0.05),提示骨质疏松可引起膝关节内组织总胶原、Ⅰ型及Ⅱ型胶原含量及其比值改变,与膝关节骨性关节炎的发生密切相关。     

类风湿性关节炎患者血清抗Ⅱ型胶原抗体的检测及意义

目的探讨类风湿性关节炎(RA)患者血清抗Ⅱ型胶原(CⅡ)抗体阳性的意义. 方法分别以人Ⅱ型胶原蛋白(HCⅡ)及牛Ⅱ型胶原蛋白CⅡ(BCⅡ)作为包被抗原进行间接 ELISA法,检测30例RA患者,对照组(30例非RA的风湿病患者和34例健康人)血清抗CⅡ抗体. 结果以HCⅡ及BCⅡ作为包被抗原检测RA患者血清抗CⅡ抗体的阳性率分别约为30.0%和33.3%;对照组血清中抗体阳性率均约为1.6%;二者差异极其显著(P＜0.01).抗CⅡ抗体阳性的RA患者RF的阳性率高于抗CⅡ抗体阴性的RA患者.与HCⅡ和BCⅡ均发生阳性反应的血清8例,均为早期RA患者. 结论抗CⅡ抗体可作为判断RA患者病情的辅助手段之一,牛CⅡ可替代人CⅡ进行有关RA发病机制中B细胞免疫的实验研究.     

Large mononuclear Ia-positive veiled cells in Peyer's patches. II. Localization in rat Peyer's patches.

Dendritic Ia-positive veiled cells were seen in frozen sections of rat Peyer's patches within the lymphoepithelium, in the reticular area just underneath and in the T-dependent interfollicular area. From immunohistochemical stainings with anti-IgA, IgE, IgG, IgM and monoclonal anti T lymphocyte sera it is concluded that these Ia-positive cells do not belong to lymphocyte series. The ultrastructure of these Ia-positive veiled cells resembled the morphology of the veiled cells found in Peyer's patch cell suspensions and in skin lymph. It is suggested that these cells form an antigen presenting cell system in mucosa-associated lymphoid tissue, similar to the antigen presenting cell system in the skin.     

Induction of oral tolerance in rats without Peyer's patches.

The induction of oral tolerance after ingestion of antigen has been reported in several animal models. The precise mechanisms responsible for this unresponsiveness are not well understood. As some investigations have suggested a key role of Peyer's patches suppressor T cells, an animal model was developed in which the PP were surgically removed. Using this model, the influence of the PP on the induction of oral tolerance against SRBC was investigated. In order to induce tolerance, the rats were fed SRBC on four consecutive days. On Day 5 they were i.p. challenged by injection of SRBC, and 5 days later the number of immunoglobulin-secreting cells against SRBC was determined within the spleen. Using this protocol, the oral tolerance induction could be shown very clearly in control animals as well as in rats without PP. Therefore, tolerance induction is possible in the absence of PP-T cells. Other mechanisms must be responsible for the tolerance induction in this model.     

Differential cytokine mRNA expression in single lymphatic follicles of the calf ileal and jejunal Peyer's patches

The ruminant gut-associated lymphoid tissues are broadly classified into ileal and jejunal Peyer's patches (PP). We isolated single lymphatic follicles from ileal and jejunal PP and examined mRNA expression of 13 cytokines using RT-PCR. Four patterns of differential expression were identified. In Pattern 1, the cytokines IL-7, IL-10, IL-12, and IL-18 were detected in all follicles of both ileal and jejunal PP. In Pattern 2, the cytokines IL-2, IL-4, and IL-13 were expressed in most jejunal PP follicles, but were undetectable in the ileal PP follicles. The cytokines characterizing Pattern 3 (IL-1beta, IFN-gamma, and IL-6) were detected in all follicles of the jejunal PP, but were differentially expressed in each follicle of ileal PP. In Pattern 4, the cytokines IL-8, TNF-alpha, and GM-CSF were variably expressed in follicles of both ileal and jejunal PP. More detailed knowledge about differential expression of cytokines in ileal and jejunal PP will facilitate a better understanding of the immune responses of primary and secondary lymphoid organs in the bovine small intestine.     

Induction of oral tolerance to cellular immune responses in the absence of Peyer's patches

Systemic hyporesponsiveness occurs following oral administration of antigen (oral tolerance) and involves the uptake and processing of antigen by the gut-associated lymphoid tissue (GALT), which includes Peyer's patches (PP) lamina propria lymphocytes and mesenteric lymph nodes (MLN). Animals with targeted mutations of genes in the tumor necrosis factor (TNF) family have differential defects in the development of peripheral lymphoid organs including PP and MLN, and provide a unique opportunity to investigate the role of GALT structures in the induction of oral tolerance. Oral tolerance could not be induced in TNF/lymphotoxin (LT) alpha-/- mice, which are devoid of both PP and MLN, although these animals could be tolerized by intraperitoneal administration of antigen, demonstrating the requirement for GALT for oral tolerance induction. LTbeta-/- mice and LTalpha/LTbeta+/- animals do not have PP but could be orally tolerized, as measured by IFN-gamma production and delayed-type hypersensitivity responses by administration of both low or high doses of ovalbumin. To further investigate the requirement for PP, we tested the progeny of LTbeta-receptor-IgG-fusion-protein (LTbetaRigG)-treated mice, which do not form PP but have an otherwise intact immune system. Although these animals had decreased fecal IgA production, they could be orally tolerized. Our results demonstrate that PP are not an absolute requirement for the induction of either high- or low-dose oral tolerance, although oral tolerance could not be induced in animals devoid of both PP and MLN.     

Generation of gamma interferon responses in murine Peyer's patches following oral immunization.

To date, oral immunizations have been shown to generate only Th2 responses in murine Peyer's patches (PP), raising the possibility that T cells present in PP may be capable of mounting only Th2 responses or that the microenvironment of PP does not favor the generation of Th1 cells. However, it is also possible that antigens that can generate Th1 responses have not yet been used for oral immunizations. This study shows that T cells in PP of mice immunized orally with live Salmonella typhimurium secrete large amounts of gamma interferon (IFN-gamma) when they are stimulated with bacterial sonicate in vitro. Moreover, oral challenge of mice with live bacteria 4 months after immunization elicits a secondary IFN-gamma response in PP and mesenteric lymph nodes. Parenteral immunization does not generate an IFN-gamma T-cell response in PP, and parenteral challenge of orally immunized mice does not elicit a secondary response in PP. However, oral challenge of intraperitoneally immunized mice elicits a secondary IFN-gamma response in PP and mesenteric lymph nodes, and intraperitoneal challenge of orally immunized mice elicits a secondary response in the spleen. The data suggest that memory T cells recirculate between mucosal and nonmucosal compartments and that they may be recruited to the site of antigenic challenge.     

Ontogeny of Peyer's patches of the rat

The development of lymphoid and nonlymphoid cells in Peyer's patches (PP) of the rat was investigated using light microscopical methods (routine histological techniques, enzyme histochemistry and immunohistochemistry). In newborn rats PP were mainly populated by T lymphocytes and Ia-positive nonlymphoid cells, which most likely are interdigitating cells. At about 12 days after birth the B and T cells were localized in defined regions, the follicular (FA) and interfollicular area (IFA), respectively. Compartmentalization within the FA started about 14 days after birth. The first signs of the development of secondary follicles were seen from about 18 days onward. PP obtained their mature structure at about 4 weeks after birth. It is suggested that after PP had developed fully, cells having cytoplasmic IgA migrate via the high endothelial venules (HEV) to the lamina propria of the intestine; cIgM and IgG cells seem to develop locally within the FA.     

Modulation of IgE response and cytokine production in Peyer's patches and draining lymph nodes in sensitized mice made tolerant by oral dust mite administration.

Such allergic diseases as rhinitis and asthma are IgE-mediated type I reactions and are controlled primarily by Th2 cells. One of the major dust mites, Dermatophagoides pteronyssinus (Dp), is considered to cause allergic reactions. Oral tolerance, largely used to modulate immune response, opens the possibility of modulating Th2 allergic responses. We observed downmodulation of total and specific IgE antibody levels as well as the number of specific IgE-secreting cells with Dp feeding in previously sensitized mice. Analysis of the cytokine profile in mucosal lymphoid tissues in the protocol revealed altered patterns of interferon-gamma (IFN-gamma), interleukin-5 (IL-5), and transforming growth factor-beta (TGF-beta) secretion in Dp-fed animals. The results suggest that both the Th and B cell populations are modulated in mice made tolerant by oral Dp feeding. Understanding the mechanisms at the mucosal level that underlie oral tolerance can improve its use in allergy immunotherapy.     

Selective induction of Th2 cells in murine Peyer's patches by oral immunization.

We have used three different methods to determine the T helper (Th) cell response, including Th1 and Th2 types, in murine Peyer's patches (PP) following oral immunization with sheep red blood cells (SRBC). These include: (i) use of cytokine-specific (IFN-gamma and IL-5), single cell assays to estimate the frequencies of Th1 and Th2 cells respectively, (ii) cytokine-specific mRNA--cDNA dot blot and Northern gel hybridizations to detect levels of specific mRNA, and (iii) T cell cloning techniques to determine the frequency of Th1 and Th2 clones. Mice were immunized with SRBC by either the oral or i.p. route. The PP and splenic (SP) CD3+ and CD3+ CD4+ T cell subsets were isolated and cultured with antigen, feeder cells, and IL-2, and were assessed at various intervals (days 0, 1, 3, and 6) for numbers of T cells producing either IFN-gamma or IL-5 by use of an enzyme-linked immunospot (ELISPOT) procedure. Cultures of T cells from PP or SP of mice given SRBC by the oral route had a high frequency of IL-5 spot forming cells (SFC), with lower numbers of IFN-gamma SFC. However, cultures of CD3+ T cells and CD3+ CD4+ Th cells from spleens of i.p. immunized mice exhibited predominantly IFN-gamma SFC, with smaller but significant numbers of IL-5 SFC. This distinct pattern of cytokine production was supported by mRNA analysis where high IL-5 specific mRNA levels were noted in PP T cell cultures of orally primed mice, while IFN-gamma mRNA was predominant in the SP CD3+ T cell and CD3+ CD4+ Th cell cultures from i.p. immunized mice. When the frequencies of IFN-gamma or IL-5 SFC were assessed among cloned Th cells from orally- or systemically-immunized mice, 74% of Th cell clones from PP of mice orally immunized with SRBC were IL-5 producers (Th2 type), while 67% of Th cell clones from SP of mice immunized by the i.p. route were IFN-gamma producers (Th1 type). Our studies show that higher frequencies of IFN-gamma producing Th1-type cells occur in SP of mice given antigen by the systemic route, while oral immunization results in predominantly IL-5 producing, Th2-type cells in PP.