白细胞介素-10抑制脊髓损伤后早期炎症的实验研究

甲基强的松龙能有效的抑制脊髓损伤（SCI）后炎症反应已得到世人的公认。并被成功的运用于临床。近年来，有学者在研究脊髓伤后炎症递质时发现，白介素-10（IL-10）对脊髓损伤后早期炎性反应亦具有明显的抑制作用，但对IL-10的作用机理知之甚少。本研究旨在通过观察IL-10对脊髓损伤后脊髓组织中核转录因子（NF）-kB的表达的影响探讨IL-10的抗炎作用，为临床应用提供实验依据。     

钛颗粒在刺激人单核/巨噬细胞引起人工关节松动中的作用

目的分析不同直径、不同浓度的钛颗粒对人单核／巨噬细胞的作用。方法应用梯度离心法分离人单核／巨噬细胞，去除钛颗粒表面的内毒素，将直径（15．47±5．18）μm和（2．54±0．86）μm的钛颗粒，分别以颗粒体积（μm^3）：细胞数比为10：1及100：1的浓度刺激人单核／巨噬细胞。在钛颗粒刺激细胞24h后，运用ELISA检测培养上清液中的骨溶解介质白介素-1β（IL-1β）、白介素-6（IL-6）和细胞坏死因子-α（TNF-α）的浓度。结果钛颗粒刺激单核／巨噬细胞可分泌IL-6、TNF-α和IL-1β；颗粒体积：细胞数比为100：1时，平均直径2．54μm的钛颗粒刺激单核／巨噬细胞产生IL-6、TNF-α和IL-1β的作用均大于直径15.47μm的钛颗粒，差异有统计学意义（P〈0.05）。结论不含内毒素的钛颗粒本身具有刺激单核／巨噬细胞产生溶骨介质的作用；高浓度、直径小的钛颗粒可刺激人单核／巨噬细胞分泌更多的溶骨介质，具有较强的生物活性。     

唑来膦酸钠在抑制钛颗粒诱导骨溶解中作用的体外实验研究

目的：研究唑来膦酸钠（ ZOL）对钛颗粒诱导的骨溶解的影响。方法分离6～8周C57BL/6J小鼠长骨中的前体破骨细胞（ OCP）并分为6组,A组：OCP＋细胞培养液,B组：OCP＋巨噬细胞集落刺激因子（M-CSF）＋NF-κB 受体活化因子配体（RANKL）＋细胞培养液,C组：OCP＋钛颗粒＋细胞培养液,D组：OCP＋上清液（钛颗粒刺激巨噬细胞24 h后上清液）＋细胞培养液,E组：OCP＋M-CSF＋RANKL＋ZOL＋细胞培养液,F组：OCP＋上清液＋ZOL＋细胞培养液。每组细胞分别接种在玻璃盖玻片、皮质骨磨片和含骨检测表面的96孔板上,10 d后检测玻璃盖玻片上细胞抗酒石酸磷酸酶（ TRAP）的表达及皮质骨磨片上骨吸收陷窝的形成,并以骨检测表面的骨吸收面积为指标比较各组破骨细胞的骨吸收活性。结果 B组、D组、E组和F组的OCP均能分化为能被TRAP染色成阳性的破骨细胞并形成骨吸收陷窝,其余组均未发现TRAP染色阳性的破骨细胞和骨陷窝。加入ZOL的F组骨吸收面积（5.54％±1.25％）较D组（10.34％±1.69％）明显减少,差异具有统计学意义（t＝5.61,P＜0.01）。结论在体外实验中钛颗粒并不能直接刺激前体破骨细胞向破骨细胞转化;唑来膦酸钠可以抑制钛颗粒诱导的骨溶解作用。     

巨噬细胞对钛和钴铬钼颗粒吞噬差异性的研究

目的:探究RAW 264.7巨噬细胞分别与钛(Ti)颗粒和钴铬钼合金(CoCrMo)颗粒共培养时的吞噬差异性及其机制。方法:将RAW 264.7(中国科学院上海细胞库)分空白组、Ti组(100 μg/ml)、CoCrMo组(100 μg/ml),分别在干预1、24、48、72 h后进行观察,计算各组RAW 264.7细...     

白细胞介素-6、白细胞介素-10及异丙酚对中性粒细胞内caspase-3活性的影响

白细胞介素(IL)-6可抑制中性粒细胞的凋亡,延长中性粒细胞的寿命,而IL-10和异丙酚则可以对抗IL-6的这种作用。但这些细胞因子和药物影响中性粒细胞凋亡过程的机制尚不清楚。caspase家族在细胞凋亡过程中起着重要作用,激活的caspase作用于另外caspase的前体,可使其激活,形成级联反应。在细胞内caspase作用于DNA修复蛋白和细胞骨架蛋白,从而导致细胞凋亡。caspase-3是caspase家族     

PMMA骨水泥颗粒对大鼠巨噬细胞RANK表达的影响

目的 观察PMMA骨水泥颗粒对体外培养的大鼠巨噬细胞RANK表达的影响.方法 大鼠腹腔灌洗液筛选培养巨噬细胞,CD68单抗免疫细胞化学法鉴定细胞纯度,分别观察不同浓度PMMA骨水泥颗粒及用骨水泥颗粒作用不同时间对巨噬细胞RANK mRNA表达的影响,用半定量逆转录-聚合酶链反应(RT-PCR)方法测定RANK mRNA表达量.结果 PMMA骨水泥颗粒对体外培养的巨噬细胞RANK mRNA表达的影响表现出浓度相关性和时间相关性,当浓度在0.1‰～5‰(v/v)时RANK mRNA表达明显升高 (P<0.05),当作用时间在24 h左右时RANK mRNA表达升高最显著(P<0.05).结论 PMMA骨水泥颗粒能够提高大鼠腹腔巨噬细胞RANK mRNA的表达,为巨噬细胞向具有骨质吸收功能的细胞类型的转化提供必要的前提条件.     

白细胞介素-10基因转染对活化巨噬细胞炎症介质产生的抑制作用

目的 探讨炎症诱导启动子指导人白细胞介素-10(hIL-10)基因的表达及其对炎症介质产生的效应.方法 诱导性真核表达载体pSAA3 hIL-10脂质体转染体外培养巨噬细胞,LPS(10 mg/L)活化后观察巨噬细胞hIL-10的表达(RT-PCR,ELISA法),测定肿瘤坏死因子-α(TNF-α)、IL-6的产生(ELISA法).结果 RT-PCR证明hIL-10可在巨噬细胞表达;巨噬细胞LPS(10 mg/L)活化12 h有hIL-10的表达(2.67 μg/L),活化24 h及48 h的巨噬细胞上清hIL-10显著增加(1.87～5.71倍);转基因预处理显著下调活化细胞TNF-α(25.0%～61.4%,P＜0.05)的产生,显著减少IL-6产生(54.8%～63.9%,P＜0.05).结论 LPS可活化pSAA3 hIL-10表达体系在巨噬细胞诱导表达,白细胞介素-10基因转染显著下调活化MΦ炎症介质的产生.     

白细胞介素10基因转染抑制小鼠心脏移植排斥反应的实验研究

目的　探讨白细胞介素 1 0 (IL 1 0 )基因转染对小鼠心脏移植排斥反应的抑制作用。方法　采用小鼠颈部心脏移植模型。随机将心脏移植后的小鼠分为 4组 :(1 )对照组 :心脏移植后每天用生理盐水 1ml灌胃 ;(2 )环孢素A(CsA)组 :心脏移植后每天用CsA 5mg/kg 灌胃 ;(3)IL 1 0组 :心脏移植时用IL 1 0重组腺病毒 30 0 μl(腺病毒滴度为 5× 1 0 1 1 pfu/ml)经主动脉根部灌注小鼠供心。(4)IL 1 0 CsA半剂量组 :在IL 1 0组的基础上 ,每天用CsA 2 .5mg/kg灌胃。观察移植心脏的存活时间及心脏跳动情况。结果　IL 1 0组、IL 1 0 CsA半剂量组和CsA组移植心脏存活时间均较对照组显著延长 (P <0 .0 1 ) ;IL 1 0组移植心脏存活时间较CsA组明显延长 (P <0 .0 5 ) ;IL 1 0 CsA半剂量组移植心脏存活时间最长 ,优于IL 1 0组 (P <0 .0 5 )和CsA组 (P <0 .0 1 )。结论　IL 1 0基因转染对心脏移植排斥反应有较强的免疫抑制作用 ,可明显延长移植心脏的存活时间 ,并且与CsA有协同作用 ,共同应用时 ,可减少CsA的用量。     

白细胞介素-10与器官移植

白细胞介素 1 0 (IL 1 0 )是近年发现的细胞因子网络中为数不多的抑制性细胞因子 ,主要由TH2细胞产生 ,能够抑制IL 2、IL 1、IFN γ、TNF α等多种细胞因子的合成及活性。越来越多的证据表明器官移植后产生免疫耐受时出现IL 1 0的高表达 ,而移植后发生排异反应时则表现为IL 1 0的低表达。移植后应用IL 1 0或将IL 1 0基因转染移植物能够延长移植物的存活。     

白细胞介素—10基因转染对活化巨噬细胞炎症介质产生的抑？…

目的 探讨炎症诱导启动子指导人白细胞介素－１０（ｈＩＬ－１０）基因的表达及其对炎症介质产生的效应。方法 诱导性真核表达勒体ｐＳＡＡ３ｈＩＬ－１０脂质体转染体外培养巨噬细胞，ＬＰＳ活化后观察巨噬细胞ｈＩＬ－１０的表达（ＲＴ－ＰＣＲ，ＥＬＩＳＡ法），测定肿瘤坏死因子－α（ＴＮＦ－α）、ＩＬ－６的产生（ＥＬＩＳＡ法）。结果 ＲＴ－ＰＣＲ证明ｈＩＬ－１０可在巨噬细胞表达；巨噬细胞ＬＰＳ活化１２ｈ有ｈＩＬ＿     

辛伐他汀对钛颗粒刺激人单核细胞形成破骨细胞的影响

目的研究体外辛伐他汀对钛颗粒刺激单核细胞形成破骨细胞的影响,探讨辛伐他汀防治人工关节无菌性松动的可能性。方法体外分离培养人外周血单个核细胞并分成5组,A组为钛颗粒刺激组（单核细胞和磨屑混合培养）,B组为10^-5mol／L辛伐他汀组（单核细胞、磨屑混合培养＋10^-5mol／L辛伐他汀）,C组为10^-6mol／L辛伐他汀组（单核细胞、磨屑混合培养＋10^-6mol／L辛伐他汀）,D组为10^-7mol／L辛伐他汀组（单核细胞、磨屑混合培养＋10^-7mol／L辛伐他汀）,E组为单核细胞组。各组细胞培养24h后取上清液,用ELISA法检测上清液中肿瘤坏死因子（TNF-α）、单核细胞趋化蛋白-1（MCP-1）的含量。分别培养10d、18d后进行TRAP染色阳性细胞计数,采用扫描电镜检测骨磨片的吸收陷窝,观察钛颗粒对破骨细胞形成的影响。结果磨屑刺激单个核细胞分泌溶骨因子,辛伐他汀抑制磨损颗粒刺激单核／巨噬细胞分泌TNF-α及MCP-1;且破骨细胞数明显减少,骨吸收陷窝数减少,与钛颗粒组刺激组比较,差异均有统计学意义（P〈0．05）。结论辛伐他汀通过抑制TNF-α、MCP-1的释放而有效防止磨屑诱导的骨溶解,有望成为防治人工关节无菌性松动的一种有潜力的药物。     

Polyethylene and titanium particles induce osteolysis by similar, lymphocyte-independent, mechanisms.

Periprosthetic osteolysis is a major clinical problem that limits the long-term survival of total joint arthroplasties. Osteolysis is induced by implant-derived wear particles, primarily from the polyethylene bearing surfaces. This study examined two hypotheses. First, that similar mechanisms are responsible for osteolysis induced by polyethylene and titanium particles. Second, that lymphocytes do not play a major role in particle-induced osteolysis. To test these hypotheses, we used the murine calvarial model that we have previously used to examine titanium-induced osteolysis. Polyethylene particles rapidly induced osteolysis in the murine calvaria 5-7 days after implantation. The polyethylene-induced osteolysis was associated with large numbers of osteoclasts as well as the formation of a thick periosteal fibrous tissue layer with numerous macrophages containing phagocytosed polyethylene particles. Polyethylene-induced osteolysis was rapidly repaired and was undetectable by day 21 after implantation. Lymphocytes were noted in the fibrous layer of wild-type mice. However, the amount of osteolysis and cytokine production induced by polyethylene particles was not substantially affected by the lack of lymphocytes in Pfp/Rag2 double knock out mice. All of these findings are similar to our observations of osteolysis induced by titanium particles. These results provide strong support for both of our hypotheses: that similar mechanisms are responsible for osteolysis induced by polyethylene and titanium particles and that lymphocytes do not play a major role in particle-induced osteolysis.     

The effectiveness of polyethylene versus titanium particles in inducing osteolysis in vivo.

Bearing surface wear and periprosthetic osteolysis due to wear particles are among the most common reasons for joint replacement failure. A murine calvarial model of wear particle-induced osteolysis has been used to identify different biologic factors associated with this problem and to test nonsurgical methods of modulating the host response to particulate debris. This model has utilized titanium particles, however, in clinical practice the most common source of particulate debris is polyethylene particles from bearing surface wear. We now report a calvarial model of wear particle-induced osteolysis based on commercially available polyethylene particles. We found that compared to sham surgery osteoclast recruitment and bone resorption can be induced by introduction of the titanium particles or polyethylene particles. However, bone resorption was significantly higher with polyethylene particles compared to titanium particles (p=0.02). We consider the polyethylene based murine calvarial model of wear particle-induced osteolysis a reliable and clinically relevant tool to understand the host factors and potential pharmacologic interventions that can influence wear debris generated osteolysis. This model might serve as an extension of the well-established titanium based bone resorption model.     

清炎颗粒对肾盂肾炎小鼠外周血IL-8与IL-10的影响

目的：探讨清炎颗粒治疗肾盂肾炎的免疫学机制。方法：采用孙氏方法改进造模,治疗组分别按剂量3.12、6.24、12.48g·kg^-1·d^-1予清炎颗粒,模型组予生理盐水,对照组予至灵胶囊灌胃,喂养至3d、7d、15d、30d、60d取血清,用ELISA法检测其IL-8、IL-10的水平。结果：清炎颗粒能显著降低不同时段血清IL-8的水平、提高不同时段血清IL-10的水平。结论：免疫调控可能是清炎颗粒治疗肾盂肾炎的作用机制之一。     

Adherent endotoxin mediates biological responses of titanium particles without stimulating their phagocytosis.

Aseptic loosening of orthopaedic implants is thought to be primarily due to stimulation of cytokine production by wear particles from the implants. The cytokines increase osteoclast differentiation, leading to osteolysis and implant loosening. Accumulating evidence indicates that adherent endotoxin mediates the biological responses induced by the wear particles. One mechanism by which adherent endotoxin may act is by increasing phagocytosis of the wear particles. To test this hypothesis, the effect of adherent endotoxin on phagocytosis of titanium particles was determined. First, we developed reliable confocal and fluorescence microscopy methods to examine both the attachment and internalization steps of phagocytosis. Use of these methods showed that adherent endotoxin does not detectably alter the rate or the extent of phagocytosis of titanium particles by RAW 264.7 cells. Despite this lack of an effect on phagocytosis, adherent endotoxin dramatically increases the ability of RAW 264.7 cells to produce TNF-alpha and induce osteoclast differentiation. Thus, adherent endotoxin mediates these biological responses by a mechanism that does not rely on increased phagocytosis. These results also demonstrate that phagocytosis is not sufficient to induce cytokine production and osteoclast differentiation but do not rule out the possibility that phagocytosis is required for induction of these responses by titanium particles with adherent endotoxin.     

Effect of pamidronate on the stimulation of macrophage TNF-alpha release by ultra-high-molecular-weight polyethylene particles: a role for apoptosis.

The demonstration that one of the mechanisms of action of bisphosphonates (BPs) is the induction of osteoclast and macrophage apoptosis, suggests a potent therapeutic role for the BPs and other apoptosis-modulating agents in the management of periprosthetic osteolysis. The purpose of this study was to improve our understanding of the basic underlying molecular events leading to the inhibitory effect of pamidronate on the macrophage response to ultra-high-molecular-weight polyethylene (UHMWPE) particles. Murine J774 macrophages were incubated for 0-72 h in the presence of UHMWPE particles and/or pamidronate. TNF-alpha release was measured by ELISA while poly(ADP-ribose)polymerase (PARP) expression was measured by Western blot. DNA was analyzed on agarose. The appearance of PARP fragment and the fragmentation of DNA were used as markers of apoptosis. We observed a dose-dependent response to UHMWPE particles with TNF-alpha release reaching 4, 10, and 19 times control with 10, 25, and 125 particles/macrophage, respectively. UHMWPE particles (25 particles/macrophage) stimulate TNF-alpha release by a factor of 10, 7, and 6 after 24, 48, and 72 h, respectively, indicating a rapid stimulating effect of UHMWPE particles on TNF-alpha release. Our results also showed that at 10 particles/macrophage, pamidronate inhibits UHMWPE-induced TNF-alpha release by 12%, 14%, and 23% respectively after 24, 48, and 72 h (p<0.05 vs. 24 and 48 h). With 25 particles/macrophage, the inhibition of TNF-alpha reached 9%, 12%, and 15% after 24, 48, and 72 h (p<0.05 vs. 24 h), respectively. There is no significant difference between the inhibition by pamidronate of TNF-alpha release induced by 125 particles/macrophages at 24, 48, and 72 h. When cells are pre-incubated for 48 h with pamidronate prior to addition of UHMWPE particles for 24 h, we observed an increased inhibition of TNF-alpha compared to the co-incubation protocol. The inhibiting effect of pamidronate reaches 56% when pre-incubated with macrophages prior to incubation with 10 particles of UHMWPE/macrophage (p<0.05 vs. co-incubation).Co-incubation of pamidronate with UHMWPE particles also led to the appearance of the proteolytic PARP fragment after 24 h incubation. We also demonstrated the stimulation of DNA fragmentation (DNA laddering) after 48-72 h with pamidronate. The proteolytic cleavage of PARP, an early event in the induction of apoptosis, precedes the inhibition of UHMWPE particle-induced TNF-alpha release by pamidronate whereas the fragmentation of DNA, a late apoptotic event, parallels this inhibition. Our results suggest the induction of macrophage apoptosis is associated with the inhibitory effect of pamidronate on TNF-alpha release. There is a need for the development of medical management of periprosthetic osteolysis. The demonstration that drugs such as pamidronate induce specific apoptosis-related pathways in macrophages contributes data for a rational approach in the treatment and/or prevention of periprosthetic osteolysis.     

钛颗粒诱导破骨细胞分化过程中活化T细胞核因子c1的表达

目的探讨钙调磷酸酶（CN）／活化T细胞核因子（NFAT）途经在磨损颗粒诱导破骨细胞分化调节中的作用。方法MTT法检测11R—VIVIT肽和钛（Ti）颗粒对小鼠骨髓单核／巨噬细胞系细胞（BMMs）活力的影响;RT—PCR法分析Ti颗粒诱导破骨细胞分化过程中NFATcl mRNA表达,并观察1R—VIVIT肽对Ti颗粒诱导NFATcl表达的影响;TRAP染色进行破骨细胞分化鉴定。结果11R—VIVIT肽和Ti颗粒均不影响体外培养中的BMMs活力;Ti颗粒显著刺激破骨细胞分化（P〈0.01）和NFATcl mRNA表达;应用11R—VIVIT肽阻断CN／NFAT途径可显著抑制Ti颗粒诱导的NFATcl表达。结论Ti颗粒刺激BMMs向破骨细胞分化;其作用机制可能与激活CN／NFAT途径有关。     

Ⅲ型前列腺炎前列腺液中IFN-γ、IL-10、IL-4水平变化与临床症状的相关性

目的探讨Ⅲ型前列腺炎前列腺按摩液中细胞因子IFN-γ、IL-10、Il-4水平变化对临床症状的影响。方法按照美国国立卫生院（NIH）分类方法诊断Ⅲ型前列腺炎共76例。ⅢA型前列腺炎47例，ⅢB型前列腺炎29例。采用美国国立卫生研究院的慢性前列腺炎症状评分表（NIH—CPSI），评估疼痛症状评分、排尿症状评分；采用双抗体夹心酶联免疫法（ELISA）检测前列腺液（EPS）中IFN-γ、IL-10、IL-4水平，分析各细胞因子变化和症状评分的相关性。结果ⅢA、ⅢB型前列腺炎两组疼痛症状和排尿症状评分比较无显著性差异（P〉0．05）；IFN-γ、IL-10、IL-4均和疼痛症状评分呈正相关（P〈0．05），其中IL-10起主要相关；IFN-γ、IL-10、IL-4均和排尿症状评分无相关（P〉0．05）。结论Ⅲ型前列腺炎EPS中IFN-γ、IL-10、IL-4水平和疼痛症状有关，其中IL-10可能起主要作用；但IFN-γ、IL-10、IL-4和排尿症状无相关。