【摘要】：背景作為人體中軸骨的主要承重結(jié)構(gòu),腰椎在脊柱的各項活動中常需處于壓縮、拉伸、側(cè)彎或扭曲等多種復雜的運動狀態(tài)中,其損傷及退變是人群常見的疾病之一,約60%成人及30%青少年有下腰痛(LBP)病史。既往研究顯示下腰痛與椎間盤退行性疾病密切相關(guān),而近年來的研究則提出腰椎終板Modic改變是LBP的危險因素。Modic改變根據(jù)MRI信號變化將退變終板分為3種亞型:Ⅰ型Modic改變表現(xiàn)為終板在T1像上信號降低,T2像信號增高,提示急性炎癥,骨組織輕微損傷及微骨折;Ⅱ型Modic改變表現(xiàn)為T1及T2像信號均增高,提示終板損傷、炎性改變及組織脂肪變;Ⅲ型Modic改變則表現(xiàn)為終板區(qū)域信號在T1及T2像均減低,提示區(qū)域骨化。Modic改變的人群整體發(fā)病率約為5.8%,其發(fā)生率及影響面積隨年齡增大而升高,以單純Ⅱ型Modic改變最為多見(64.2%),其次為Ⅰ/Ⅱ型混合(18.1%)及單純Ⅰ型(16%)。絕大部分Modic改變均發(fā)生于下腰椎位置(L4/L5及L5/S1,83%),其在椎間盤的上/下終板之間分布并無顯著差異,且77.9%的Modic改變在同一椎間盤的上/下方成對出現(xiàn)。有關(guān)Modic改變的成因現(xiàn)存有多種理論,其中最為經(jīng)典的是力學損傷機制。椎體終板是腰椎解剖結(jié)構(gòu)的薄弱部位,因此傳統(tǒng)理論認為椎間盤內(nèi)應(yīng)力可集中于終板,致使終板發(fā)生退變。終板的部分區(qū)域應(yīng)力集中的發(fā)生可引發(fā)終板裂隙,終板下部分區(qū)域骨質(zhì)逐漸鈣化,而Modic改變則反映了相應(yīng)的急性出血水腫(Ⅰ型)-慢性炎癥修復(Ⅱ型)-區(qū)域骨化(Ⅲ型)的進程。Modic改變的另一種病變機制可能涉及營養(yǎng)方面因素,研究顯示維生素D攝入量與Modic改變發(fā)生率之間存在正相關(guān),其可能與維生素D對鈣離子代謝的影響相關(guān)。而吸煙及高血脂引起的血管功能障礙亦被認為與Modic改變發(fā)生率呈正相關(guān)。此外,近年來痤瘡丙酸桿菌(P.acnes)感染與Modic改變引起了廣泛的爭議及研究。P.acnes的出現(xiàn)與Modic改變的發(fā)生高度相關(guān),而雙盲病例對照研究顯示抗生素治療對伴有Ⅰ型Modic改變的腰背痛患者具有較好的治療效果。本研究的興趣在于進一步研究Modic改變的發(fā)生機制,在細胞培養(yǎng),動物模型兩個層面分別觀察低毒性細菌感染及組織缺血對腰椎間盤及其相鄰終板結(jié)構(gòu)的影響,通過影像學,分子生物學及病理學手段進行評估。目的通過體外細胞培養(yǎng)及動物模型研究,了解骨性終板區(qū)感染、缺血等損傷對腰椎間盤及終板的影響,探究其損傷機制,進而探索相應(yīng)病因下治療或預防椎間盤疾病的新策略。方法1.獲取大鼠椎間盤髓核細胞及終板細胞進行體外培養(yǎng),給予不同的刺激(P.acnes細菌上清液,缺氧生長環(huán)境或血管生長抑制劑)觀察細胞生長情況,并收集細胞進行基因表達和細胞活性檢測。2.構(gòu)建經(jīng)肌間隙開放手術(shù)入路的兔腰椎間盤髓核穿刺通道,并以此通道注射痤瘡丙酸桿菌建立相應(yīng)動物模型。收集標本進行影像學檢查,基因表達,分子生物學測試,病理學檢測等。3.構(gòu)建兔腰椎間盤終板下穿刺通道,并以此通道注射痤瘡丙酸桿菌或博來霉素(血管抑制劑)建立相應(yīng)動物模型。收集標本進行影像學檢查,基因表達,分子生物學測試,病理學檢測等評估模型。結(jié)果1.低毒性細菌感染或缺氧對椎間盤細胞的刺激作用1.1低毒性細菌感染抑制髓核及終板細胞的生長細胞培養(yǎng)結(jié)果顯示,當共培養(yǎng)的細菌上清液濃度大于4*106CFU/mL時,髓核細胞發(fā)生大量死亡;當濃度小于2.5*105CFU/mL時,髓核細胞生長受到明顯抑制。當共培養(yǎng)的細菌上清液濃度大于2*106CFU/mL時,終板軟骨細胞發(fā)生大量死亡;當濃度小于5*105 CFU/mL時,終板軟骨細胞生長受到明顯抑制。1.2低毒性細菌感染刺激髓核及終板細胞炎癥反應(yīng)基因的表達基因表達結(jié)果顯示一定濃度的P.acnes上清液刺激可顯著提高髓核及終板細胞的細胞外基質(zhì)降解相關(guān)基因如MMP-9、ADAMTSs等的表達,同時下調(diào)細胞外基質(zhì),如蛋白聚糖、膠原蛋白等的表達水平。1.3血管生長抑制劑博來霉素對健康終板細胞無顯著損傷作用使用62.5μg/ml血管生長抑制劑博來霉素培養(yǎng)正常終板細胞48小時后,終板細胞存活率及活性與對照組相比沒有統(tǒng)計學差異;細胞產(chǎn)物表達(Ⅰ型膠原、Ⅱ型膠原、蛋白聚糖等)與對照組相比差異無統(tǒng)計學意義。1.4缺氧環(huán)境對髓核及終板細胞的刺激作用細胞培養(yǎng)結(jié)果顯示,終板細胞在缺氧(5%二氧化碳,3%氧分壓)環(huán)境下增殖速度減慢約40%,細胞代謝水平降低,無氧代謝產(chǎn)物增加,細胞活性下降;膠原及蛋白聚糖等細胞外加基質(zhì)合成減少,同時降解基質(zhì)的MMPs及ADAMTSs表達增加。髓核細胞在缺氧環(huán)境中活性未見明顯變化,僅有HIF-1α及ADAMTS-5表達上調(diào)。2.腰椎間盤內(nèi)低毒性細菌感染對在體腰椎終板及髓核的影響2.1腰椎間盤內(nèi)低毒性細菌感染引發(fā)顯著的的終板區(qū)域及椎間盤MRI退變表現(xiàn)通過開放手術(shù)經(jīng)側(cè)方肌間隙入路暴露椎間盤并進行穿刺,椎間盤內(nèi)注射P.acnes后,可發(fā)現(xiàn)在術(shù)后2周時即有椎間盤信號降低表現(xiàn)。至術(shù)后9月時,幾乎所有實驗椎間盤均發(fā)生不同程度退變。術(shù)后3月有部分節(jié)段終板區(qū)域發(fā)生Ⅰ型Modic改變,并在術(shù)后6月開始部分轉(zhuǎn)變?yōu)棰蛐蚆odic改變。2.2腰椎間盤內(nèi)低毒性細菌感染引發(fā)的椎間盤基質(zhì)降解及終板區(qū)域炎癥反應(yīng)P.acnes椎間盤內(nèi)感染可顯著增加椎間盤組織中細胞外基質(zhì)(如蛋白聚糖等)的分解,同時引發(fā)的炎癥反應(yīng)破壞了終板軟骨結(jié)構(gòu),進一步促進了椎間盤的退變。qPCR顯示髓核內(nèi)前炎癥因子如IL-1β、TNF-α、IFN-γ及基質(zhì)降解酶MMP-9、ADAMTS-5表達顯著上調(diào),同時終板區(qū)域IL-1β、TNF-α及ADAMTS-5表達量顯著升高。提示P.acnes感染引起了較為劇烈的椎間盤及終板區(qū)域炎性反應(yīng)。2.3腰椎間盤內(nèi)低毒性細菌感染與終板下骨質(zhì)的吸收與重構(gòu)P.acnes感染椎間盤內(nèi)后9月,終板骨結(jié)構(gòu)發(fā)生明顯骨吸收樣改變,骨小梁較為稀疏,骨密度顯著降低,骨相對體積顯著降低而BS/BV顯著升高,骨小梁厚度明顯降低。病理切片亦顯示在部分區(qū)域可見終板區(qū)域異位骨化增生情況。3.腰椎終板區(qū)低毒性細菌感染及血管抑制對在體腰椎終板及髓核的損傷作用3.1腰椎終板區(qū)低毒性細菌感染引發(fā)的終板區(qū)域及髓核MRI影像學變化通過X線透視下經(jīng)皮穿刺椎間盤終板下骨,并經(jīng)該通道注射P.acnes后,可發(fā)現(xiàn)在術(shù)后6月均無顯著椎間盤退變表現(xiàn)。在術(shù)后6月時,約1/3椎體鄰近終板區(qū)域發(fā)生T1及T2像MRI信號升高表現(xiàn),提示發(fā)生Ⅱ型Modic改變。3.2腰椎終板區(qū)低毒性細菌感染誘發(fā)炎癥損傷P.acnes終板下感染時,終板下骨內(nèi)前炎癥因子如IL-1β、TNF-α、IFN-γ等表達顯著升高,提示P.acnes感染引起的溫和而慢性的終板區(qū)域炎癥樣反應(yīng)。同一時間內(nèi),椎間盤內(nèi)部并無顯著炎癥因子上升或炎性反應(yīng)表現(xiàn)。3.3腰椎終板區(qū)低毒性細菌感染作用下的骨結(jié)構(gòu)改變病理切片及Micro-CT檢查結(jié)果顯示,相比直接感染椎間盤內(nèi)而言,終板下骨區(qū)域P.acnes感染并無明顯終板骨性結(jié)構(gòu)重塑反應(yīng),終板下骨僅可見輕度骨吸收反應(yīng)。同時,病理檢查顯示部分樣本(3例)可見終板裂隙。3.4抑制血管生成在終板區(qū)域引發(fā)的終板區(qū)域MRI影像學變化通過X線透視下經(jīng)皮穿刺椎間盤終板下骨,并經(jīng)該通道注射博來霉素抑制血管生成后,可發(fā)現(xiàn)在術(shù)后6月均無顯著椎間盤退變表現(xiàn)。在術(shù)后2月、3月及6月時,約1/3椎體鄰近終板區(qū)域發(fā)生T1及T2像MRI信號降低表現(xiàn),提示發(fā)生Ⅲ型Modic改變,即終板區(qū)域骨化表現(xiàn)。3.5抑制血管生成在終板區(qū)域誘發(fā)的骨結(jié)構(gòu)改變對實驗動物終板下骨Micro-CT掃描結(jié)果顯示骨小梁結(jié)構(gòu)稀疏,但厚度增加,BMD升高,骨相對體積增加,提示終板下骨區(qū)域發(fā)生區(qū)域部分吸收,同時在大范圍內(nèi)發(fā)生骨化反應(yīng)。終板下骨內(nèi)炎癥因子如IL-1β、TNF-α表達升高。結(jié)論1.低毒性細菌上清液在髓核及軟骨終板細胞體外培養(yǎng)時可顯著抑制細胞生長,下調(diào)細胞外基質(zhì)蛋白表達水平,同時上調(diào)基質(zhì)降解酶,如MMPs、ADAMTSs的表達。同時,髓核細胞在缺氧環(huán)境下增殖速度減緩,代謝水平降低,細胞外基質(zhì)合成減少而降解增加。血管抑制劑博來霉素在體外培養(yǎng)時并沒有顯著細胞毒性,不會損害髓核細胞及終板細胞的活性及代謝水平。2.低毒性細菌直接感染腰椎間盤可在術(shù)后3-9月引發(fā)時間依賴性的終板Modic改變及椎間盤退變,激活髓核及終板內(nèi)IL-1β、TNF-α、IFN-y等多條炎癥通路,并促使終板下骨結(jié)構(gòu)發(fā)生骨吸收及骨重構(gòu)改變。3.低毒性細菌直接感染終板下骨可引發(fā)相應(yīng)區(qū)域輕度慢性炎癥反應(yīng),并在6月后表現(xiàn)為Modic Ⅱ型改變及偶發(fā)終板裂隙,但椎間盤本身并無顯著退變表現(xiàn)。抑制終板區(qū)域血管生成可引發(fā)終板區(qū)域骨化表現(xiàn)及Ⅲ型Modic改變,并伴有炎性反應(yīng)。
[Abstract]:BACKGROUND As the main load-bearing structure of the axial bone in the human body, the lumbar spine often needs to be compressed, stretched, curled or twisted in various complex motion states. Its injury and degeneration is one of the common diseases in the population. About 60% of adults and 30% of adolescents have a history of low back pain (LBP). Modic changes in the lumbar endplate were classified into three subtypes according to the changes in MRI signals. Type I Modic changes were characterized by decreased signal intensity on T1 images and increased signal intensity on T2 images, suggesting acute inflammation, slight injury to bone tissue and minimal fracture. The change of DIC showed that the signal of T1 and T2 increased, suggesting endplate injury, inflammation and fatty degeneration; the change of type III Modic showed that the signal of endplate area decreased on T1 and T2 images, suggesting regional ossification. The overall incidence of Modic change was about 5.8%, and its incidence and affected area increased with age, while the change of type II Modic was simple. Most of the Modic changes occurred in the lower lumbar spine (L4/L5 and L5/S1, 83%). There was no significant difference in the distribution of Modic between the upper and lower endplates, and 77.9% of Modic changes occurred in pairs above and below the same intervertebral disc. There are many theories about the cause of formation, among which the most classic one is the mechanism of mechanical damage. The vertebral endplate is the weak part of the lumbar vertebral anatomy, so the traditional theory holds that the stress in the intervertebral disc can be concentrated in the endplate, resulting in the degeneration of the endplate. Another mechanism of Modic changes may involve nutritional factors. Studies have shown that there is a positive correlation between vitamin D intake and the incidence of Modic changes, which may be related to vitamin D pairs. In addition, the prevalence of Modic changes has been widely debated and studied in recent years. The presence of P. acnes is highly correlated with the occurrence of Modic changes, while the double-blind case-control study shows that the presence of P. acnes is highly correlated with the occurrence of Modic changes. Antibiotics have been shown to be effective in the treatment of low back pain with type I Modic alterations. The interest of this study is to further investigate the mechanism of Modic alterations. The effects of low toxic bacterial infection and tissue ischemia on the structure of lumbar intervertebral disc and its adjacent endplates were observed at cell culture and animal model levels, respectively. Objective To investigate the effects of bone endplate infection and ischemia on the lumbar intervertebral disc and endplate, explore the mechanism of injury, and then explore new strategies for the treatment or prevention of intervertebral disc diseases under the corresponding etiology. Methods 1. Rat intervertebral disc nucleus pulposus cells and endplate cells were cultured in vitro. Different stimuli (P. acnes bacterial supernatant, hypoxic growth environment or angiogenesis inhibitors) were given to observe the growth of cells, and the cells were collected for gene expression and cell activity detection. 2. The rabbit lumbar intervertebral disc nucleus pulposus pulposus pulposus perforation was constructed by open muscle space operation. Samples were collected for imaging examination, gene expression, molecular biology test, pathological examination and so on. 3. The rabbit lumbar intervertebral disc endplate puncture passage was constructed, and the corresponding animal model was established by injecting propionibacter acnes or bleomycin (vascular inhibitor) into the passage. Results 1. Stimulation of intervertebral disc cells by hypotoxic bacterial infection or hypoxia 1. Low toxic bacterial infection inhibited the growth of nucleus pulposus and endplate cells. The results showed that the concentration of co-cultured bacterial supernatant was high. At 4*106 CFU/mL, a large number of nucleus pulposus cells died; when the concentration was less than 2.5*105 CFU/mL, the growth of nucleus pulposus cells was significantly inhibited. When the concentration of co-cultured bacterial supernatant was more than 2*106 CFU/mL, a large number of endplate chondrocytes died; when the concentration was less than 5*105 CFU/mL, the growth of endplate chondrocytes was significantly inhibited.1.2 with low toxicity. The expression of inflammatory response genes in nucleus pulposus and endplate cells was stimulated by bacterial infection. The results showed that the expression of extracellular matrix degradation related genes such as MMP-9 and ADAMTSs was significantly increased and the expression of extracellular matrix, such as proteoglycan and collagen, was down-regulated by stimulation with certain concentration of P.acnes supernatant. Ping.1.3 Bleomycin, an angiogenesis inhibitor, did not cause significant damage to healthy endplate cells. After culturing normal endplate cells with Bleomycin, a 62.5 ug/ml angiogenesis inhibitor, for 48 hours, there was no significant difference in the survival rate and activity of endplate cells compared with the control group; the expression of cell products (type I collagen, type II collagen, proteoglycan, etc.) Compared with the control group, there was no significant difference. 1.4 The stimulation effect of hypoxia on nucleus pulposus and endplate cells showed that under hypoxia (5% carbon dioxide, 3% oxygen partial pressure), the proliferation rate of endplate cells slowed down by about 40%, the level of cell metabolism decreased, anaerobic metabolites increased, and cell activity decreased. The activity of nucleus pulposus cells did not change significantly in hypoxic environment, but the expression of HIF-1a and ADAMTS-5 was up-regulated. 2. The effect of low toxic bacterial infection in lumbar intervertebral disc on lumbar endplate and nucleus pulposus in vivo 2.1 Low toxic bacterial infection in lumbar intervertebral disc caused significant changes. The degeneration of endplate area and intervertebral disc on MRI was found at 2 weeks after injection of P. acnes. By 9 months after operation, almost all experimental discs had degenerated to varying degrees. Type I Modic changes occurred in the region of the endplate and partially converted to type II Modic changes at 6 months postoperatively. 2.2 Decomposition of the intervertebral disc matrix caused by low toxic bacterial infections in the lumbar intervertebral disc and inflammatory reaction in the endplate region P. acnes intradiscal infection significantly increased the decomposition of extracellular matrix (such as proteoglycan) in the intervertebral disc tissue, as well. QPCR showed that the expression of pro-inflammatory factors such as IL-1 beta, TNF-a, IFN-gamma, matrix degrading enzymes MMP-9 and ADAMTS-5 in nucleus pulposus was significantly up-regulated, while the expression of IL-1 beta, TNF-a and ADAMTS-5 in the endplate region was significantly increased. Intense inflammatory reaction in the intervertebral disc and endplate. 2.3 Low toxic bacterial infection in the lumbar intervertebral disc and absorption and remodeling of bone under the endplate in the intervertebral disc after P. acnes infection in September, the bone structure of the endplate showed obvious bone resorption-like changes, trabeculae were sparse, bone mineral density was significantly reduced, relative bone volume was significantly reduced, BS/BV was significantly increased, and bone trabeculae were significantly increased. Pathological sections also showed heterotopic ossification and hyperplasia of the endplate region in some areas. 3. Damage of low toxic bacterial infection and vascular inhibition in the lumbar endplate and nucleus pulposus in vivo. 3. MRI imaging changes of the endplate region and nucleus pulposus caused by low toxic bacterial infection in the lumbar endplate region were examined by X-ray fluoroscopy. No significant disc degeneration was found at 6 months after the operation. At 6 months after the operation, T1 and T2 MRI signals were elevated in about 1/3 of the adjacent endplate areas, suggesting the occurrence of type II Modic changes. 3.2 low toxic bacterial infection in the lumbar endplate area induced inflammatory injury P. The expression of proinflammatory factors such as IL-1 beta, TNF-a and IFN-gamma increased significantly in sublaminar infection of acnes, suggesting mild and chronic inflammation-like reactions in the endplate region caused by P. acnes infection. There was no significant increase in inflammatory factors or inflammatory reaction in the intervertebral disc at the same time. 3.3 Low toxic bacterial infection in the lumbar endplate region was present. Pathological sections and micro-CT examination showed that there was no significant end-plate remodeling in P.acnes infection, and only slight bone resorption was observed in the subendplate bone. In addition, some samples (3 cases) showed end-plate fissures.3.4 inhibited blood vessels. No significant disc degeneration was observed at 6 months after surgery. T1 and T2 images were detected in about 1/3 of the adjacent endplate regions at 2 months, 3 months and 6 months after surgery. MRI signal reduction indicated type III Modic changes, i.e. ossification of the endplate area. 3.5 Inhibition of angiogenesis in the endplate area induced bone structural changes in experimental animals under the endplate bone micro-CT scan showed that the trabecular bone structure was sparse, but the thickness increased, BMD increased, relative bone volume increased, suggesting that the area of bone occurring under the endplate region. The expression of inflammatory cytokines such as IL-1beta and TNF-alpha was increased under the endplate. Conclusion 1. Low toxicity bacterial supernatant could inhibit the growth of nucleus pulposus and cartilage endplate cells, down-regulate the expression of extracellular matrix protein and up-regulate the expression of matrix degrading enzymes such as MMPs and ADAMTSs in vitro. At the same time, the proliferation rate of nucleus pulposus cells in hypoxic environment slowed down, the metabolic level decreased, the synthesis of extracellular matrix decreased and the degradation increased. Bleomycin, an inhibitor of blood vessel, had no significant cytotoxicity in vitro, and did not damage the activity and metabolic level of nucleus pulposus cells and endplate cells. 2. Low toxic bacteria directly infected the waist. The intervertebral disc can induce time-dependent changes of Modic and intervertebral disc degeneration from 3 to 9 months after operation, activate multiple inflammatory pathways such as IL-1 beta, TNF-a, IFN-y in the nucleus pulposus and endplate, and promote bone resorption and remodeling in the subendplate bone structure. 3. Low toxic bacteria directly infect the subendplate bone can cause mild chronic inflammatory reaction in the corresponding region. Inhibition of angiogenesis in the endplate region can induce ossification of the endplate region and change of Modic type III with inflammatory reaction.
【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R681.53

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