淫羊藿苷对钛颗粒诱导假体周围骨溶解抑制作用的研究

发布时间：2018-01-15 13:10

  本文关键词：淫羊藿苷对钛颗粒诱导假体周围骨溶解抑制作用的研究　出处：《苏州大学》2014年硕士论文　论文类型：学位论文

  更多相关文章： 钛颗粒 骨溶解 骨膜 炎症因子 淫羊藿苷 磨损颗粒 骨溶解 炎症抑制 RANKL/RANK 下调

【摘要】：第一部分钛颗粒诱导小鼠颅骨骨溶解模型的建立和评价 目的：建立钛（Ti）颗粒诱导的小鼠颅骨骨溶解模型，模拟磨损颗粒诱导假体无菌性松动的病理过程。 方法：7-8周龄健康雄性C57BL/6小鼠20只，随机分为两组：（A）对照组；（B）Ti组。Ti组小鼠接受手术将无菌去内毒素的Ti颗粒5毫克植入小鼠颅骨骨面上诱导骨溶解；对照组小鼠接受相同的手术步骤，但将无菌生理盐水置于颅骨骨面作为对照。两周后所有小鼠过量麻醉处死，取颅骨标本做研究使用。以颅骨骨面冠、矢状缝交点为中心选中直径为5mm的圆形“感兴趣区域（ROI）”作为研究对象。苏木精伊红（HE）染色法检测两组颅骨标本中骨溶解及炎性细胞浸润情况，Paint.NET软件测量骨膜厚度，酶联免疫吸附实验（ELISA）检测小鼠颅骨ROI区域体外培养上清液中肿瘤坏死因子α（TNF-α）和白细胞介素1β（IL-1β）的含量，实时定量逆转录-聚合酶链反应(QRT-PCR)法检测TNF-α、IL-1β基因mRNA转录量。 结果：实验过程中无动物死亡。颅骨标本大体观察见局部皮肤组织无红肿、渗液、皮疹及流脓等；Ti覆盖颅骨骨面粗糙，凹凸不平。HE染色结果发现，Ti组小鼠颅骨骨面有明显的虫蚀样改变，骨膜内可见大量细胞浸润，炎性细胞居多，成纤维细胞较少。Paint.NET软件测量结果表明Ti组小鼠颅骨骨膜增厚为(0.30±0.03)mm，与对照组(0.06±0.01)mm比较，差异有统计学意义(p0.05)。ELISA结果显示Ti组颅骨培养上清中TNF-α, IL-1β含量分别为(1440.12±82.33)ng/l和(872.13±42.39)ng/l，与对照组(235.01±35.41)ng/l和(140.02±21.07)ng/l比较，差异有统计学意义(p0.05)。RT-PCR结果显示，经Ti刺激后，Ti组颅骨标本中TNF-α, IL-1β基因mRNA含量增多至7.31±0.40和9.62±0.85，与对照组（标准化为1）比较，差异有显著性意义(p0.05)。 结论：Ti颗粒诱导的小鼠颅骨骨溶解模型简单、可重复强，能较为准确的模拟关节假体无菌性松动的病理过程。 第二部分淫羊藿苷对钛颗粒诱导骨溶解的影响 目的：探讨淫羊藿苷（IC）对Ti颗粒诱导小鼠颅骨骨溶解的治疗作用及其机制。 方法：7-8周龄健康雄性C57BL/6小鼠80只，随机分为四组：（A）对照组，（B）IC组，（C）Ti+安慰剂组，（D）Ti+IC组。C和D组Ti颗粒植入小鼠颅骨上建立骨溶解模型，A及B组小鼠仅接受相同的手术步骤，无Ti颗粒植入。建模当天B组和D组小鼠以IC（200毫克/千克/天）灌胃给药，A和C组以相同剂量安慰剂（PBS）治疗，两周后所有小鼠过量麻醉处死，取颅骨备用。利用micro-ct扫描不同处理组ROI区域，根据三维重建图像观察各组小鼠颅骨表面溶骨馅窝数量，CT自带软件定量计算颅骨标本ROI区域骨量（BV）,骨矿化密度（BMD）,骨表面积（BS）,骨表面积/骨量比率（BS/BV）；HE染色法检测骨溶解及炎性细胞浸润情况，Paint.NET软件测量骨膜厚度；TRAP染色检测成熟破骨细胞；免疫组织化学染色检测RANKL和RANK的表达变化；ELISA检测颅骨培养上清中TNF-α，IL-1β的含量；QRT-PCR检测RANKL、 RANK、TNF-α和IL-1β基因mRNA表达变化。 结果： 1. Micro-CT三维重建图像显示对照组未放置Ti颗粒的小鼠颅骨表面光滑，未见明显的骨溶解迹象，而Ti+安慰剂组小鼠颅骨骨面蚀骨现象明显，骨溶解陷窝密布；相较于Ti+安慰剂组，Ti+IC组小鼠经IC口服治疗后溶骨现象得到遏制，蚀骨陷窝数量明显减少。 Micro-CT3D分析定量检测：对照组中BV,BMD，BS，BS/BV值分别为(3.79±0.23)mm3,(0.71±0.05)mg/mm2，(35.71±1.98)mm2,(9.41±1.06)1/mm。Ti+安慰剂组中BV,BMD值降至(2.03±0.12)mm3,(0.58±0.03)mg/mm2,BS，BS/BV值增高至(45.73±3.84)mm2,(22.50±2.31)1/mm,与对照组相比，差异有统计学意义（P0.05）；经IC口服干预后,Ti+IC组中BV,BMD值增至(3.51±0.23)mm3,(0.69±0.04)mg/mm2,BS，BS/BV值降至(37.21±1.98)mm2,(10.57±1.29)1/mm，与Ti+安慰剂组相比，差异有统计学意义（P0.05）。 2.HE染色：对照组骨膜内细胞总量较少，成纤维细胞居多，炎性细胞较少，骨膜较薄；Ti+安慰剂组骨膜内可见大量细胞浸润，炎性细胞居多，成纤维细胞较少。Paint.NET软件测量结果表明对照组、IC组、Ti+安慰剂组、Ti+IC组颅骨骨膜厚度分别为(0.07±0.01)mm，(0.06±0.01)mm，(0.28±0.04)mm，(0.12±0.02) mm。Ti+安慰剂组与对照组相比差异有显著性意义(P 0.05)；Ti+IC组与Ti+安慰剂组相比差异有显著性意义(P 0.05)。 3. TRAP染色结果显示，Ti+安慰剂组可见多个细胞胞浆呈紫红色、细胞核3个以上的TRAP阳性细胞，对照组和IC组阳性深染多核细胞较少；各组多核细胞计数显示：对照组（5.0±1.3）个，IC组未检出，Ti+安慰剂组（36.0±4.8）个，Ti+IC组（11.0±2.3）个，Ti+安慰剂组与对照组，Ti+IC组与Ti+安慰剂组相比较，差异有显著性意义（P0.05）。IC显著减少Ti颗粒诱导的TRAP阳性细胞形成。 4. ELISA检测结果显示：对照组上清中TNF-α和IL-1β蛋白浓度分别为（237.13±37.36）ng/l和（133.09±19.64）ng/l；Ti+安慰剂组中，二者浓度增长至（1447.52±78.23）ng/l和（889.14±45.94）ng/l，与对照组相比，，差异有显著性意义（P0.05）；Ti+IC组TNF-α和IL-1β浓度下降至（568.86±42.32）ng/l和（279.39±19.44）ng/l，与Ti+安慰剂组相比，差异有统计学意义（P0.05）。 5.免疫组化染色显示：相较于对照组，Ti+安慰剂组中RANKL、RANK阳性深染位点分布密集，区域广泛，Ti+IC组中阳性深染位点分布稀疏，区域狭窄。这表明钛颗粒诱导RANKL、RANK蛋白表达上调，IC通过减少RANKL、RANK表达量抑制骨溶解。 6.实时定量逆转录-聚合酶链反应(QRT-PCR)分析显示：设定对照组TNF-α和IL-1β mRNA表达量为1，IC组中二者表达量分别为0.81±0.13，0.83±0.12； Ti+安慰剂组为7.60±0.60；9.80±0.94；Ti+IC组为2.40±0.18；2.10±0.23；与对照组相比，Ti+安慰剂组中TNF-α和IL-1β mRNA表达量上调明显，差异有统计学意义（P0.05）；经IC治疗后，Ti+IC组TNF-α和IL-1β基因转录量下调显著，差异有统计学意义（P0.05）。这表明，淫羊藿苷对钛颗粒诱导骨溶解的抑制作用涉及TNF-α和IL-1β基因下调。 Ti+安慰剂组中RANKL和RANK转录量分别为9.02±1.05，11.01±0.97,与对照组相比，差异有统计学意义（P0.05）。RANKL和RANK mRNA转录量在Ti颗粒刺激下明显上调。相反的，Ti+IC组中IC干预治疗后RANKL和RANK基因表达量显著减少为2.50±0.36，3.03±0.31，与Ti+安慰剂组相比，差异有统计学意义（P0.05）。 结论：IC显著抑制钛颗粒诱导的炎症反应，下调RANKL/RANK分子表达，抑制钛颗粒诱导的小鼠颅骨骨溶解，口服IC有可能成为预防和治疗假体周围骨溶解的有效方法。
[Abstract]:The establishment and evaluation of mouse calvarial osteolysis induced by titanium particles of the first part
Objective: to establish a titanium (Ti) particles induced mouse calvarial osteolysis model, simulate the wear particle induced aseptic loosening of the prosthesis in the pathological process.
Methods: 7-8 weeks old 20 healthy male C57BL/6 mice were randomly divided into two groups: control group (A); (B) Ti group.Ti group mice received Ti surgery will be sterile endotoxin to particle induced osteolysis 5 mg were implanted into mouse calvarial bone surface; the control mice received the same surgical procedures, but will be sterile the saline in the skull bone surface as control. After two weeks, all mice were killed by overdose anesthesia, the skulls do research. Using the skull bone surface coronal, sagittal suture point as the center selected 5mm diameter circular region of interest (ROI) "as the object of study. Hematoxylin eosin (HE). Staining group two skull specimens of bone dissolution and inflammatory cells, Paint.NET software to measure the thickness of periosteum, enzyme-linked immunosorbent assay (ELISA) and tumor necrosis factor alpha in the supernatant were detected in vitro ROI skull region (TNF- alpha) and interleukin 1 beta (IL-1 beta The content of TNF- alpha and mRNA transcription of IL-1 beta gene were detected by real-time quantitative reverse transcription polymerase chain reaction (QRT-PCR) method.
Results: no animal died during the experiment. The skull specimens generally observe local skin tissue without redness, oozing, rash and pus; Ti covering the skull bone surface rough, uneven.HE staining results showed that Ti mice bone surface has eroded significantly changes in the periosteum showed a large amount of inflammatory cell infiltration. The majority of cells, a measuring result of fiber cells less.Paint.NET software showed that Ti mice skull periosteum thickening (0.30 + 0.03) mm, and the control group (0.06 + 0.01) mm comparison, the difference was statistically significant (P0.05).ELISA showed that Ti group culture supernatant skull TNF- alpha, IL-1 beta content respectively (1440.12. 82.33) and ng/l (872.13 + 42.39) ng/l, and the control group (235.01 + 35.41) ng/l and (140.02 + 21.07) ng/l comparison, the difference was statistically significant (P0.05).RT-PCR results showed that after stimulation with Ti, TNF- alpha Ti group skull specimens, IL-1 beta gene mRNA content increased It was 7.31 + 0.40 and 9.62 + 0.85, compared with the control group (standardized 1), the difference was significant (P0.05).
Conclusion: mouse calvarial osteolysis model induced by Ti particles is simple and repeatable, can more accurately simulate aseptic loosening of the prosthesis in the pathological process.
The second part: the effect of Icariin on titanium particle induced osteolysis
Objective: To investigate the effects of icariin (IC) treatment effect and mechanism of Ti particles induced mouse calvarial osteolysis.
鏂规硶锛

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