本文關(guān)鍵詞：淫羊藿苷對(duì)鈦顆粒誘導(dǎo)假體周圍骨溶解抑制作用的研究　出處：《蘇州大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 鈦顆粒 骨溶解 骨膜 炎癥因子 淫羊藿苷 磨損顆粒 骨溶解 炎癥抑制 RANKL/RANK 下調(diào)

【摘要】：第一部分鈦顆粒誘導(dǎo)小鼠顱骨骨溶解模型的建立和評(píng)價(jià) 目的：建立鈦（Ti）顆粒誘導(dǎo)的小鼠顱骨骨溶解模型，模擬磨損顆粒誘導(dǎo)假體無(wú)菌性松動(dòng)的病理過(guò)程。 方法：7-8周齡健康雄性C57BL/6小鼠20只，隨機(jī)分為兩組：（A）對(duì)照組；（B）Ti組。Ti組小鼠接受手術(shù)將無(wú)菌去內(nèi)毒素的Ti顆粒5毫克植入小鼠顱骨骨面上誘導(dǎo)骨溶解；對(duì)照組小鼠接受相同的手術(shù)步驟，但將無(wú)菌生理鹽水置于顱骨骨面作為對(duì)照。兩周后所有小鼠過(guò)量麻醉處死，取顱骨標(biāo)本做研究使用。以顱骨骨面冠、矢狀縫交點(diǎn)為中心選中直徑為5mm的圓形“感興趣區(qū)域（ROI）”作為研究對(duì)象。蘇木精伊紅（HE）染色法檢測(cè)兩組顱骨標(biāo)本中骨溶解及炎性細(xì)胞浸潤(rùn)情況，Paint.NET軟件測(cè)量骨膜厚度，酶聯(lián)免疫吸附實(shí)驗(yàn)（ELISA）檢測(cè)小鼠顱骨ROI區(qū)域體外培養(yǎng)上清液中腫瘤壞死因子α（TNF-α）和白細(xì)胞介素1β（IL-1β）的含量，實(shí)時(shí)定量逆轉(zhuǎn)錄-聚合酶鏈反應(yīng)(QRT-PCR)法檢測(cè)TNF-α、IL-1β基因mRNA轉(zhuǎn)錄量。 結(jié)果：實(shí)驗(yàn)過(guò)程中無(wú)動(dòng)物死亡。顱骨標(biāo)本大體觀察見局部皮膚組織無(wú)紅腫、滲液、皮疹及流膿等；Ti覆蓋顱骨骨面粗糙，凹凸不平。HE染色結(jié)果發(fā)現(xiàn)，Ti組小鼠顱骨骨面有明顯的蟲蝕樣改變，骨膜內(nèi)可見大量細(xì)胞浸潤(rùn)，炎性細(xì)胞居多，成纖維細(xì)胞較少。Paint.NET軟件測(cè)量結(jié)果表明Ti組小鼠顱骨骨膜增厚為(0.30±0.03)mm，與對(duì)照組(0.06±0.01)mm比較，差異有統(tǒng)計(jì)學(xué)意義(p0.05)。ELISA結(jié)果顯示Ti組顱骨培養(yǎng)上清中TNF-α, IL-1β含量分別為(1440.12±82.33)ng/l和(872.13±42.39)ng/l，與對(duì)照組(235.01±35.41)ng/l和(140.02±21.07)ng/l比較，差異有統(tǒng)計(jì)學(xué)意義(p0.05)。RT-PCR結(jié)果顯示，經(jīng)Ti刺激后，Ti組顱骨標(biāo)本中TNF-α, IL-1β基因mRNA含量增多至7.31±0.40和9.62±0.85，與對(duì)照組（標(biāo)準(zhǔn)化為1）比較，差異有顯著性意義(p0.05)。 結(jié)論：Ti顆粒誘導(dǎo)的小鼠顱骨骨溶解模型簡(jiǎn)單、可重復(fù)強(qiáng)，能較為準(zhǔn)確的模擬關(guān)節(jié)假體無(wú)菌性松動(dòng)的病理過(guò)程。 第二部分淫羊藿苷對(duì)鈦顆粒誘導(dǎo)骨溶解的影響 目的：探討淫羊藿苷（IC）對(duì)Ti顆粒誘導(dǎo)小鼠顱骨骨溶解的治療作用及其機(jī)制。 方法：7-8周齡健康雄性C57BL/6小鼠80只，隨機(jī)分為四組：（A）對(duì)照組，（B）IC組，（C）Ti+安慰劑組，（D）Ti+IC組。C和D組Ti顆粒植入小鼠顱骨上建立骨溶解模型，A及B組小鼠僅接受相同的手術(shù)步驟，無(wú)Ti顆粒植入。建模當(dāng)天B組和D組小鼠以IC（200毫克/千克/天）灌胃給藥，A和C組以相同劑量安慰劑（PBS）治療，兩周后所有小鼠過(guò)量麻醉處死，取顱骨備用。利用micro-ct掃描不同處理組ROI區(qū)域，根據(jù)三維重建圖像觀察各組小鼠顱骨表面溶骨餡窩數(shù)量，CT自帶軟件定量計(jì)算顱骨標(biāo)本ROI區(qū)域骨量（BV）,骨礦化密度（BMD）,骨表面積（BS）,骨表面積/骨量比率（BS/BV）；HE染色法檢測(cè)骨溶解及炎性細(xì)胞浸潤(rùn)情況，Paint.NET軟件測(cè)量骨膜厚度；TRAP染色檢測(cè)成熟破骨細(xì)胞；免疫組織化學(xué)染色檢測(cè)RANKL和RANK的表達(dá)變化；ELISA檢測(cè)顱骨培養(yǎng)上清中TNF-α，IL-1β的含量；QRT-PCR檢測(cè)RANKL、 RANK、TNF-α和IL-1β基因mRNA表達(dá)變化。 結(jié)果： 1. Micro-CT三維重建圖像顯示對(duì)照組未放置Ti顆粒的小鼠顱骨表面光滑，未見明顯的骨溶解跡象，而Ti+安慰劑組小鼠顱骨骨面蝕骨現(xiàn)象明顯，骨溶解陷窩密布；相較于Ti+安慰劑組，Ti+IC組小鼠經(jīng)IC口服治療后溶骨現(xiàn)象得到遏制，蝕骨陷窩數(shù)量明顯減少。 Micro-CT3D分析定量檢測(cè)：對(duì)照組中BV,BMD，BS，BS/BV值分別為(3.79±0.23)mm3,(0.71±0.05)mg/mm2，(35.71±1.98)mm2,(9.41±1.06)1/mm。Ti+安慰劑組中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,與對(duì)照組相比，差異有統(tǒng)計(jì)學(xué)意義（P0.05）；經(jīng)IC口服干預(yù)后,Ti+IC組中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，與Ti+安慰劑組相比，差異有統(tǒng)計(jì)學(xué)意義（P0.05）。 2.HE染色：對(duì)照組骨膜內(nèi)細(xì)胞總量較少，成纖維細(xì)胞居多，炎性細(xì)胞較少，骨膜較��；Ti+安慰劑組骨膜內(nèi)可見大量細(xì)胞浸潤(rùn)，炎性細(xì)胞居多，成纖維細(xì)胞較少。Paint.NET軟件測(cè)量結(jié)果表明對(duì)照組、IC組、Ti+安慰劑組、Ti+IC組顱骨骨膜厚度分別為(0.07±0.01)mm，(0.06±0.01)mm，(0.28±0.04)mm，(0.12±0.02) mm。Ti+安慰劑組與對(duì)照組相比差異有顯著性意義(P 0.05)；Ti+IC組與Ti+安慰劑組相比差異有顯著性意義(P 0.05)。 3. TRAP染色結(jié)果顯示，Ti+安慰劑組可見多個(gè)細(xì)胞胞漿呈紫紅色、細(xì)胞核3個(gè)以上的TRAP陽(yáng)性細(xì)胞，對(duì)照組和IC組陽(yáng)性深染多核細(xì)胞較少；各組多核細(xì)胞計(jì)數(shù)顯示：對(duì)照組（5.0±1.3）個(gè)，IC組未檢出，Ti+安慰劑組（36.0±4.8）個(gè)，Ti+IC組（11.0±2.3）個(gè)，Ti+安慰劑組與對(duì)照組，Ti+IC組與Ti+安慰劑組相比較，差異有顯著性意義（P0.05）。IC顯著減少Ti顆粒誘導(dǎo)的TRAP陽(yáng)性細(xì)胞形成。 4. ELISA檢測(cè)結(jié)果顯示：對(duì)照組上清中TNF-α和IL-1β蛋白濃度分別為（237.13±37.36）ng/l和（133.09±19.64）ng/l；Ti+安慰劑組中，二者濃度增長(zhǎng)至（1447.52±78.23）ng/l和（889.14±45.94）ng/l，與對(duì)照組相比，，差異有顯著性意義（P0.05）；Ti+IC組TNF-α和IL-1β濃度下降至（568.86±42.32）ng/l和（279.39±19.44）ng/l，與Ti+安慰劑組相比，差異有統(tǒng)計(jì)學(xué)意義（P0.05）。 5.免疫組化染色顯示：相較于對(duì)照組，Ti+安慰劑組中RANKL、RANK陽(yáng)性深染位點(diǎn)分布密集，區(qū)域廣泛，Ti+IC組中陽(yáng)性深染位點(diǎn)分布稀疏，區(qū)域狹窄。這表明鈦顆粒誘導(dǎo)RANKL、RANK蛋白表達(dá)上調(diào)，IC通過(guò)減少RANKL、RANK表達(dá)量抑制骨溶解。 6.實(shí)時(shí)定量逆轉(zhuǎn)錄-聚合酶鏈反應(yīng)(QRT-PCR)分析顯示：設(shè)定對(duì)照組TNF-α和IL-1β mRNA表達(dá)量為1，IC組中二者表達(dá)量分別為0.81±0.13，0.83±0.12； Ti+安慰劑組為7.60±0.60；9.80±0.94；Ti+IC組為2.40±0.18；2.10±0.23；與對(duì)照組相比，Ti+安慰劑組中TNF-α和IL-1β mRNA表達(dá)量上調(diào)明顯，差異有統(tǒng)計(jì)學(xué)意義（P0.05）；經(jīng)IC治療后，Ti+IC組TNF-α和IL-1β基因轉(zhuǎn)錄量下調(diào)顯著，差異有統(tǒng)計(jì)學(xué)意義（P0.05）。這表明，淫羊藿苷對(duì)鈦顆粒誘導(dǎo)骨溶解的抑制作用涉及TNF-α和IL-1β基因下調(diào)。 Ti+安慰劑組中RANKL和RANK轉(zhuǎn)錄量分別為9.02±1.05，11.01±0.97,與對(duì)照組相比，差異有統(tǒng)計(jì)學(xué)意義（P0.05）。RANKL和RANK mRNA轉(zhuǎn)錄量在Ti顆粒刺激下明顯上調(diào)。相反的，Ti+IC組中IC干預(yù)治療后RANKL和RANK基因表達(dá)量顯著減少為2.50±0.36，3.03±0.31，與Ti+安慰劑組相比，差異有統(tǒng)計(jì)學(xué)意義（P0.05）。 結(jié)論：IC顯著抑制鈦顆粒誘導(dǎo)的炎癥反應(yīng)，下調(diào)RANKL/RANK分子表達(dá)，抑制鈦顆粒誘導(dǎo)的小鼠顱骨骨溶解，口服IC有可能成為預(yù)防和治療假體周圍骨溶解的有效方法。
[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.
鏂規(guī)硶錛

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