【作者】 高飞；

【导师】 杨述华；

【作者基本信息】 华中科技大学 ， 外科学， 2013， 博士

【摘要】 第一部分细菌脂多糖诱导的小鼠全身系统性炎症对骨再生的影响目的：骨折修复有赖于骨骼系统和免疫系统的密切配合。在诸如某些病理情况如多发性创伤,糖尿病,风湿性关节炎,肥胖,衰老等某些基础炎症水平高于正常的情况均能导致骨再生的速度和质量下降。本实验的目的在于应用细菌脂多糖构建一种高基础炎症状态模型,以探讨单纯炎症状态下骨折愈合的细胞生物学机制。方法：对10周大C57BL/6老鼠用三点加压模型造成非稳定性骨折后,连续7天经腹腔注射LPS(3μg/天/老鼠),对照组老鼠注射同等剂量的LPS。分别在骨折的第1天,第3天,第7天,第10天,第14天,第21天心脏采血法收集血浆,并收集骨折侧肢体。用双抗体夹心ELISA方法检测血浆中IL-6水平。骨折侧肢体经过固定,脱钙,透明,石蜡包埋后进行石蜡切片。间隔10张切片分别行Hall Bryant染色。显微镜计算每张切片上骨痂大小及成分。选取Cab2T3和ADTC5两种细胞系分别在不同浓度LPS存在下进行成骨化和成软骨化诱导,应用茜素红和阿尔新蓝染色判断成骨化和成软骨化程度。结果：LPS连续7天注射后,LPS组老鼠未出现明显发热,体重降低,活动减少等症状。血清学检测发现,在骨折后24小时LPS与PBS对照组血浆内IL-6水平达到峰值,但是LPS组IL-6峰值水平远高于PBS对照组。骨折后3天,对照组血浆中炎性因子即恢复至基础水平,但是LPS组在7天时才缓慢恢复至基础水平。体内实验结果表明,在骨折后第7天,第10天和第14天,LPS组骨痂大小较对照比明显减小。新生软骨和新生骨体积LPS组也显著小于对照组,但软骨减少更为明显。细胞实验结果提示,LPS能明显抑制成骨作用和成软骨化作用。结论：通过腹腔注射LPS可以系统性基础炎症水平增高,系统性炎症水平升高能导致局部骨再生减缓,同时影响骨再生质量。第二部分骨折修复过程中巨噬细胞的作用及其表型演变目的：骨折发生后,正常机体能有效募集各种炎性细胞对组织损伤相关刺激进行应答,清除炎症并启动组织修复程序。巨噬细胞作为先天性免疫系统的重要的效应细胞,能发挥清除坏死组织,吞噬细胞碎片,提呈抗原等作用。此外,巨噬细胞能根据细胞外微环境尤其是周围组织炎症水平呈现不同的极化状态,表现不同的生物学功能。在上皮组织伤口愈合中,巨噬细胞的极化和功能变化已经大量见诸报道,本研究旨在应用Ccr2-/-基因缺陷型老鼠研究巨噬细胞在骨折愈合过程中的作用以及在骨折过程中的表型的动态改变。方法：实验采用10周大小的Ccr2-/-老鼠作为干预组,年龄与性别相匹配的正常C57BL/6老鼠作为对照组,判断Ccr2基因在骨折愈合过程中的作用。运用Yet40和Yarg两种YFP荧光标记老鼠来判断骨折愈合不同阶段M1和M2的数量。三点加压法制造非稳定性骨折后,不同时间点收集骨折侧肢体,经过固定,脱钙,透明,石蜡包埋后进行石蜡切片,间隔10张切片分别行Hall Bryant染色,显微镜下计算每张切片上骨痂大小及成分。另选取每10张组织切片行免疫组织化学染色,计数巨噬细胞和中性粒细胞。骨折后分别于第1,3,5,7,10,14天在显微镜下分离骨痂,提取总RNA,逆转录后用qPCR方法检测M1和M2相关基因。在骨折愈合的第1天和第7天,选取Yet40和Yarg荧光标记鼠组织切片做抗YFP染色探讨骨折修复过程中巨噬细胞的表型转化。结果：骨折后第3天,与正常组相比,Ccr2-/-老鼠骨折局部巨噬细胞明显减少,但是中性粒细胞数量并未出现显著下降。骨折第7天,Ccr2-/-基因缺陷鼠较野生型老鼠相比,骨折后骨痂大小显著性减小(P=0.27),新生骨痂中骨含量和软骨含量两组之间并无明显差异。骨折第14天,骨痂大小无明显差异,对照组新生骨含量明显多于Ccr2-/-缺陷组。骨折后第21天,Ccr2-/-组骨痂中新生骨体积和软骨体积均高于对照组。Yet40和Yarg组实验组提示,骨折后第1天,M1数量明显高于M2,至骨折第7天,结果显示M1巨噬细胞数量减少而M2数量增多。结论：Ccr2基因缺陷老鼠能阻止巨噬细胞向机体损伤部位募集从而导致骨折愈合延缓,新生骨数量和质量明显降低,进一步说明巨噬细胞在骨修复过程中发挥重要作用。骨折愈合过程中,巨噬细胞表型由Ml逐渐向M2表型转变,从促炎症表型向抗炎症表型转变,这一转变对清除创伤后局部炎症,促进组织修复发挥积极作用。第三部分年龄相关性巨噬细胞极化对成骨细胞成骨化作用的影响目的：巨噬细胞作为一种主要的先天性免疫应答细胞和抗原提呈细胞在骨折愈合中发挥重要作用,年龄能影响巨噬细胞的表型和功能,同时巨噬细胞也能根据细胞外微环境的改变而呈现不同的细胞表型。在组织损伤发生后,一般认为巨噬细胞首先呈现促炎表型M1,引发炎症和机体免疫应答,随着炎症的消退,巨噬细胞逐渐转变为一种抗炎表型M2,分泌细胞外基质合成胶原等促进组织修复。本部分研究旨在探讨年龄对巨噬细胞极化状态的影响,以及在这种年龄相关的极化状态下,巨噬细胞与成骨细胞的相互作用。方法：不同年龄组老鼠处死后,收集骨髓中单核细胞。用含有5%CMG12-14细胞系上清液的a-MEM培养基培养单核细胞诱导生成小鼠巨噬细胞。10ng/ml IFN-γ与lOng/ml LPS加入培养基过夜,诱导生成M1型巨噬细胞,1ng/ml IL-4培养12小时诱导生成M2型巨噬细胞。用ELISA方法分别测量细胞培养上清液中有关抗炎因子和促炎因子的浓度。用Giess试剂盒测量M1巨噬细胞上清中的NO含量,用Q-PCR方法检测M2巨噬细胞中的YM1和FIZZ1基因的含量,证实巨噬细胞已活化为目的表型。分别用三种共培养的方式培养巨噬细胞和成骨前体细胞1)标准法混合培养2)条件培养基培养3)Trans-well小室培养。用Q-PCR的方法分别在诱导分化的第3天,第7天,第10天,第14天分别检查成骨分化的相关基因ALP, Colla和Osterix表达情况。在共培养14天后用茜素红染色和碱性磷酸酶活性检测的方法来判断成骨化作用。结果：18月组小鼠巨噬细胞IL-6水平较10周组明显升高。M1标志物NO和M2标志物YM1和FIZZ1,18月组均高于10周组。Q-PCR结果显示,10周和18周未活化骨髓巨噬细胞对成骨细胞成骨化并未表现出统计学差异性。对于M1巨噬细胞条件培养基培养条件下分化的成骨细胞,早期时间点成骨化相关基因表达水平高于对照,但是晚期时间点表达水平低于同年龄组对照。M2条件培养基下,10周组各个时间点成骨化基因均高于正常,而18周M2组与同年龄组对照并无统计学差异。标准法混合培养体系下,各年龄组茜素红染色显示钙结节并无显著差异。在条件培养基培养和Transwell培养体系下,10周M2组较同年龄M1组相比显著促进钙结节生成。18月组M1和M2组钙结节数量均低于同一年龄正常对照组。结论：体外实验证实衰老组巨噬细胞较正常成年组呈现出一种更高的基础炎性状态,衰老状况下巨噬细胞本身仍然具有向M1和M2极化的能力,并分泌M1,M2特异性标记物的能力。这一研究同样也说明在长期的炎症环境下,成骨细胞成骨作用受损,同时M2巨噬细胞表型能促进成骨细胞分化及成骨作用。因此,调控巨噬细胞的极化可能是促进如衰老,肥胖,糖尿病等某些长期慢性炎症情况下骨折修复再生的关键。更多还原

【Abstract】 Part ⅠLipopolysaccharide-induced systemic inflammation affects bone healing in a murine tibia fracture modelObjective:The early stages of fracture repair require close coordination between the immune system and skeletal system. Conditions such as diabetes, rheumatoid arthritis, smoking, obesity and aging adversely affect bone healing. Common to all these conditions is a sustained inflammation. It has been shown that lipopolysaccharide-induced systemic inflammation results in fracture callus of inferior mechanical characteristics in a rat femur fracture model. The aim of this study was.to compare fracture healing in a murine tibia fracture model in the setting of LPS-induced systemic inflammation to that of control animalsMethods:Sustained inflammation was created by the injection of lipopolysaccharide intra-peritoneally (3μg/mouse/day).Control animals received equivalent volume of phosphate-buffered saline vehicle.Concurrently, diaphyseal tibial fractures were created and stabilized with intramedullary pins. Plasma and tibiae were collected on D1, D3, D7, D10, D14and D21.Plasma was isolated from whole blood by cadiac puncture and centrifuge at2,000xg for20minutes. All plasma samples were immediately stored at-20℃after isolation. IL-6levels were detected by ELISA. Prepared ELISA plates were read as suggested by the manufacturer. Concentrations were expressed in pg/mL. All samples were run in triplicate. Harvested limbs were fixed in4%paraformaldehyde and decalcified in EDTA for14days after which they were paraffin embedded and sectioned at5pm. Every tenth section was stained using a Trichrome stain. Adjacent sections were stained using the method of Hall and Bryant. Sections were then examined and tissue types were quantified by stereology. Calculated volumes were expressed in mm3. Mean volumes were compared using the student’s T-test with a significance level of p<0.05employed.Cab2T3and ADTC5cell lines were plated in standard12well plates.100μg/ml ascobic acid2-phosphate and5mM β-glycerophosphate were added into2%FBS a-MEM to induce osteogenesis.25μg/ml ascobic acid2-phosphate and1%ITS mixture were added into5%FBS a-MEM to induce chondrogenesis. Alizarin red and Alician blue were applied for access ostegenesis and chondrogenesis.Results:During the seven day injection period no behavioral differences were noted between treated and control animals. No symptoms of systemic illness were noted in either group. For the LPS group, the IL-6level rose shapely to concentration of209.9pg/mL24hours after fracture whereas the maximum IL-6level reached by controls was23.5pg/mL IL-6levels decreased in both groups to near-basal levels by post-fracture day3in controls and day7in the LPS-treated animals. At Day7the treatment group trended towards a smaller mean callus volume (Mean difference:15.5mm3; p=0.21). By Day10and14there was no difference in callus size between groups. The difference in callus size was primarily related to an increased volume of cartilage and undifferentiated tissue in control animals. Hall-Bryant quadruple stain confirmed the larger callus in control animals (Mean difference:17.4; p<0.076). The size difference noted was present in all tissue types suggesting an overall smaller callus, rather than a difference in one cell type. LPS inhibit both osteogenesis and chondrogenesis in vitro and related to LPS concentration.Conclusion:Sustained inflammation delayed the bone healing process, both quantity and quality of new bone was impaired. Part ⅡMacrophage plasticity and polarization plays an important role in bone healing processObjective:After a fracture occurs, the architecture and vascular supply of the bone are disrupted. This results in a loss of mechanical stability, a decrease in tissue oxygenation and nutrient supply, and the release of biological factors into the site of injury. During the inflammatory stage of repair, cells, including macrophages, neutrophils, and degranulating platelets, infiltrate to the fracture site and release cytokines. Macrophages exhibit varying phenotypes depending on the inflammatory environment in which they are located. In this study we use the CCR2-/-mice which could block macrophages immigrate to fracture site and Yet40and Yarg mice which is to understand the polarization during bone healing process according to the inflammation macroenvironment changes.Methods:Male,10week old Ccr2-/-mice were used in this study. Age-and gender-matched, congenic C57BL/6L mice were used as wild-type controls. Two lines of mice YET40and YARG is given as a gift by Dr.Richard Locksley.Tibial fractures were created by three-point bending. Harvested limbs were fixed in4%paraformaldehyde and decalcified in EDTA for14days after which they were paraffin embedded and sectioned at10μm. Every tenth section was stained using the method of Hall and Bryant. Sections were then examined and tissue types were quantified by stereology. Calculated volumes were expressed in mm3. Mean volumes were compared using the student’s T-test with a significance level of p<0.05employed.Total RNA were extracted from the fractured tibiae by D1,D3,D5,D7,D10and D14. cDNA synthesis and real-time PCR were proformed. Macrophages and neutrophils in the callus at D3after fracture were viasulized by immunostaining using macrophage-specific (F4/80or Mac-3) and neutrophile specific (MCA771G) antibodies.Ml and M2macrophage identified by anti-YFP antibody.Results:Results from this study prove that a lack of Ccr2affects early fracture healing. At3days after injury, Ccr2-/-mice had significantly fewer macrophages at the fracture site while a similar number of neutrophils were observed in both wild-type and mutant mice. At day7, the Callus in the Ccr2-/-mice were significantly smaller than the wild type mice (P=0.27),but there is no difference in total volume of new bone or cartilage in the callus.At day14,there was no difference in callus size but less new bone in Ccr2-/-mice.At day21,both callus size and volume of new bone remained large in the Ccr2-/-mice,but both were decrease in the wild type mice.For the Ml and M2profile study, we observed a large number of IL12p40-YFP positive cells and a lower number of Arg-1-YFP cells at the fracture site at day1.At Day7we observed a decrease in "M1"cells and an increase in "M2" cells.By using the real time PCR, we found that M2gene expression "disolves" rapidly and return back to normal level during the time course of bone fracture healing while the M1gene expression last relatively longer during the whole bone healing process.Conclusion:These results indicate that a deficiency of Ccr2reduces the infiltration of macrophages and impairs the bone fracture healing. The phenotype of macrophage shift from a pro-inflammation "M1" to an anti-inflammation "M2" during the bone healing process. Part ⅢAge-related changes in macrophage polarization affect osteogenesisObjective:Aging affects many cellular functions of macrophages, and macrophages exhibit various phenotypes depending on the inflammatory environment in which they are located. After injury macrophages exhibit a pro-inflammatory phenotype, termed M1, and then the macrophages polarize to an anti-inflammatory phenotype (M2). The M2phenotype is associated with stimulation of healing. We hypothesized that the phenotype and function of macrophages changes with age, and these changes would affect osteogenesis. Therefore, we developed a macrophage/pre-osteoblast co-culture system to assess the inter-reaction between these two cell types as a function of age.Methods:Bone marrow monocytes were isolated from mice of different age(4weeks,10weeks,6months,18months). Cells were differentiated into macrophages (BMM) in vitro. lOng/ml IFN-Y combined with lOng/ml LPS were introduced to the media to activate BMM to a pro-inflammatory phenotype (M1) and lng/ml IL-4was used to drive macrophages towards an anti-inflammatory phenotype (M2). Pro-inflammatory cytokines and anti-inflammatory cytokines were tested by ELISA from R&D Systems. M1marker Nitric oxide (NO) was assayed using the Giess reagent system and M2marker levels (YM1and FIZZ1) were quantified by Q-PCR.Macrophages of different phenotypes were then co-cultured with pre-osteoblast cell lines (Cab2t3) in three configurations.1) Standard co-culture2) macrophage-conditioned media and Cab2t3cells3) trans-well co-culture. We used qPCR to quantify ALP, Colla and osterix expression at different time points (D3, D7, D10) during osteoblast differentiation. Alizarin red staining was used to quantify mineralization and alkaline phosphatase was quantified to assess osteogenesisResults:1.BMM from aged mice secret higher levels of pro-inflammatory cytokines:IL-6levels in10weeks BMM and M2group were significance lower than18months BMM and M2group, while IL-10does not appear to be different. BMM from10weeks old mice secrete lower levels of NO and down-regulated gene expression of YM1and FIZZ1compared with BMM generated from18months old mice.2.Effect of different phenotype of macrophages on the bone cells differentiation:In10weeks and18months group, ALP, Coll a and osterix gene expression were up-regualted in M1groups by early time point but reduced in later time point. In M2group ALP, Colla and osterix gene expression were increased in10weeks group in all the time points but not significantly different in18groups.3.Effect of different phenotype of macrophages on the bone cells osteogenesis:In standard co-culture system:osteogenesis of Cab2T3cells did not show any difference between different age group. In condition media and Trans-well co-culture system, Cab2t3cells have more osteogenic nodules in M2group than Ml group from10weeks mice. However macrophages from18months mice, less nodules were detected in both M1and M2groups compare with BMM group.Conclusion:We demonstrate that macrophages from aged mice exhibit a more pro-inflammatory phenotype compare with macrophages from young adult animals. Our results suggest that osteogenesis may be impaired in inflammatory environments that do not resolve, while M2macrophages are beneficial for osteogenesis in adult animals. This is consistent with other conclusions that M2cells are good for tissue repair and regeneration. These data support our hypothesis and suggest that regulating macrophage polarity may be important for bone repair in aged patients.更多还原