多種影像學(xué)聯(lián)合組織病理學(xué)檢查對(duì)脊柱結(jié)核早期的評(píng)價(jià)
發(fā)布時(shí)間：2018-09-03 15:03
【摘要】：目的：應(yīng)用ATCC25177結(jié)核分枝桿菌菌種(凍干粉)在兔腰椎建立脊柱結(jié)核病模型,探討X線、CT (Computed tomography)、核磁共振(Magnetic resonance imaging,MRI)常規(guī)掃描、MRI彌散成像(Diffusion weighted imaging, DWI)、磁共振灌注成像(Perfusion weighted imaging, PWI)及組織病理學(xué)檢查在兔脊柱結(jié)核發(fā)病早期的影像診斷中的應(yīng)用價(jià)值。方法：首先選擇實(shí)驗(yàn)兔60只依據(jù)手術(shù)方法不同及注入試劑不同分為4組：A組25只：兔腰椎椎體內(nèi)經(jīng)手術(shù)鉆孔注入5.0mg/ml的結(jié)核菌液0.2ml；B組25只：椎間盤(pán)內(nèi)直接注入5.0mg/ml的結(jié)核菌液0.2ml；C組5只：兔腰椎椎體內(nèi)注入生理鹽水0.2ml；D組5只：兔腰椎椎間盤(pán)內(nèi)注入生理鹽水0.2ml。(C組與D組為陰性對(duì)照)。選定的每只新西蘭大白兔腰6椎體作為實(shí)驗(yàn)的觀察椎體,腰4椎體可作為自身對(duì)照椎體。術(shù)后1-8周時(shí)隨機(jī)處死A、B兩組實(shí)驗(yàn)兔行熒光定量PCR檢查、HE染色、膿液結(jié)核菌培養(yǎng)等組織病理學(xué)檢查與解剖學(xué)大體觀察；C、D兩組實(shí)驗(yàn)兔在同樣時(shí)間段處死實(shí)驗(yàn)兔行上述檢查。主要觀察指標(biāo)：比較分析兩種手術(shù)方法造模的成功率、抗酸染色的陽(yáng)性率；比較分析PCR熒光定量法在脊柱結(jié)核早期檢測(cè)的優(yōu)勢(shì)、與抗酸染色的陽(yáng)性率比較；對(duì)結(jié)核病灶邊緣骨組織成骨細(xì)胞和破骨細(xì)胞計(jì)數(shù)及骨組織計(jì)量學(xué)分析。再根據(jù)實(shí)驗(yàn)兔存活率最高且結(jié)核種植區(qū)椎體感染最優(yōu)的手術(shù)方法,建立影像學(xué)動(dòng)物模型：選擇實(shí)驗(yàn)兔40只依據(jù)術(shù)中注入試劑不同分為2組：A組30只：兔腰椎椎體內(nèi)經(jīng)手術(shù)鉆孔注入5.0mg/ml的結(jié)核菌液0.2ml；B組10只：兔腰椎椎體內(nèi)注入生理鹽水0.2ml；(B組為陰性對(duì)照)。選定的每只新西蘭大白兔腰6椎體作為本實(shí)驗(yàn)的干預(yù)椎體,腰4椎體可作為空白對(duì)照椎體。術(shù)后4周、6周、8周時(shí)行X線檢查、CT檢查、MRI常規(guī)檢查及MRI-DWI彌散加權(quán)成像檢查及MRI-PWI灌注成像掃描。分析各影像學(xué)掃描方法在脊柱結(jié)核早期影像圖像中的意義及各掃描方法中各個(gè)參數(shù)的意義,并將MRI-DWI彌散加權(quán)成像中的參數(shù)與MRI-PWI灌注成像的參數(shù)做相關(guān)性分析。比較各影像學(xué)檢查方法對(duì)脊柱結(jié)核早期評(píng)價(jià)中的優(yōu)缺點(diǎn)。結(jié)果：通過(guò)兩組不同手術(shù)方法造模成功率的比較,A組腰椎側(cè)方入路于腰6椎體處鉆孔注入結(jié)核菌液的實(shí)驗(yàn)兔手術(shù)成功率高于B組腰椎側(cè)方入路于腰5-6椎間盤(pán)處直接注入結(jié)核菌液的實(shí)驗(yàn)兔；通過(guò)熒光定量PCR方法可以檢測(cè)出脊柱結(jié)核膿液中結(jié)核的特異性TB-DNA含量,并可檢出最低TB-DNA含量,且優(yōu)于抗酸染色法；建立的實(shí)時(shí)熒光定量PCR的線性關(guān)系很好,Ct值與結(jié)核桿菌的DNA模板含量呈線性關(guān)系線性范圍為4×102～107Copies/mL, r=0.99503;通過(guò)組織病理學(xué)染色觀察到脊柱結(jié)核破壞早期,主要是以結(jié)核菌滲出期間椎體骨炎及增生期間的骨質(zhì)破壞為主的組織病理學(xué)表現(xiàn)。通過(guò)影像學(xué)的實(shí)驗(yàn)研究證明：A組術(shù)后8周時(shí),三種影像學(xué)檢查方法對(duì)骨質(zhì)破壞、椎旁膿腫、硬模、與脊髓受壓的診斷敏感性都有意義。術(shù)后8周時(shí),MRI對(duì)脊柱結(jié)核造成的骨質(zhì)破壞與CT檢查的診斷敏感性比較,P值0.05,說(shuō)明兩者在術(shù)后8周這個(gè)時(shí)間段時(shí)對(duì)診斷骨質(zhì)破壞的敏感性上無(wú)明顯差異性,對(duì)于診斷椎旁膿腫與硬模、脊髓是受壓的敏感性MRICTX線；手術(shù)造模后第4周時(shí)MRI及MRI-DWI就出現(xiàn)異常信號(hào),且兔脊柱成像時(shí)DWI序列中研究b值在600s/mm2左右時(shí)可獲得較為清晰的圖像；第4、6、8周感染組病變椎體的ADC值高于正常椎體(P0.05)；通過(guò)對(duì)4類ADC圖像及ADC值的比較分析,得出：用8個(gè)b值的DWI擬合出的ADC8b圖其信噪比最高,圖像質(zhì)量最好；ADChigh值可以反映出病變椎體內(nèi)部結(jié)核桿菌的活性；ADClow值隨著時(shí)間延長(zhǎng)呈現(xiàn)逐漸增加的趨勢(shì),說(shuō)明在低b值的DWI上,病變椎體內(nèi)部血流灌注的增加比水分子運(yùn)動(dòng)受限對(duì)該值的貢獻(xiàn)要明顯；ADCperf值隨著結(jié)核桿菌對(duì)脊柱椎體造成的破壞而呈現(xiàn)逐漸增大的趨勢(shì)；脊柱結(jié)核A組的實(shí)驗(yàn)兔的CT值和破骨細(xì)胞數(shù)間存在負(fù)相關(guān)、骨小梁體積分?jǐn)?shù)和破骨細(xì)胞數(shù)之間存在負(fù)相關(guān)。通過(guò)對(duì)MRI灌注成像的參數(shù)分析得出：A組實(shí)驗(yàn)組術(shù)后4、6、8周除達(dá)峰時(shí)間(TTP)隨著觀察時(shí)間的延長(zhǎng)而降低以外,其他灌注參數(shù)都隨時(shí)間的延長(zhǎng)而增加,增加幅度高于B組對(duì)照組,且除達(dá)峰時(shí)間(TTP)與DWI中的ADCperf呈負(fù)相關(guān)外,首過(guò)強(qiáng)化率(Efirst)、首過(guò)強(qiáng)化速率(Vfirst)、早期強(qiáng)化率(Ee)、早期強(qiáng)化速率(Ve)、信號(hào)最大強(qiáng)化率(Emax)、信號(hào)增強(qiáng)率(SER)、增強(qiáng)峰值(PH)、信號(hào)最大上升斜率(MSI)、排泄率(washout)都與ADCperf呈正相關(guān)。結(jié)論：通過(guò)在新西蘭大白兔腰椎局部種植結(jié)核分枝桿菌與腰椎間盤(pán)直接注入結(jié)核桿菌兩種手術(shù)方法的實(shí)驗(yàn)研究,可以構(gòu)建出兔脊柱結(jié)核模型,通過(guò)大體、解剖學(xué)、熒光定量PCR法及組織病理學(xué)檢查,證實(shí)建造模型成功。其中在新西蘭大白兔腰椎局部種植結(jié)核分枝桿菌脊柱結(jié)核模型的構(gòu)建方法簡(jiǎn)便且成功率較高,對(duì)脊柱結(jié)核早期的組織病理學(xué)研究等的動(dòng)物實(shí)驗(yàn)研究奠定了一定基礎(chǔ)。通過(guò)X線、CT與常規(guī)MRI及MRI-DWI、 MRI-PWI成像的影像學(xué)檢查方法在診斷早期脊椎結(jié)核中的優(yōu)勢(shì)互補(bǔ)可以提高檢查敏感度。通過(guò)對(duì)MRI-DWI中多b值的分析及MRI-PWI中各參數(shù)的分析,可以對(duì)脊柱結(jié)核早期血流動(dòng)力學(xué)做出定量指標(biāo)的評(píng)價(jià)。為臨床骨科對(duì)脊柱結(jié)核的早期診療提供有價(jià)值的影像依據(jù)。
[Abstract]:Objective: To establish a rabbit model of spinal tuberculosis by using ATCC 25177 Mycobacterium tuberculosis strain (lyophilized powder) in lumbar spine. To investigate the effects of X-ray, CT, magnetic resonance imaging (MRI), diffusion weighted imaging (DWI), magnetic resonance perfusion imaging (PWI) and magnetic resonance imaging (MRI) on spinal tuberculosis. Methods: Sixty rabbits were divided into four groups according to different operation methods and different injection reagents: 25 rabbits in group A were injected with 5.0mg/ml tuberculous fluid 0.2ml into the lumbar vertebra by surgical drilling; 25 rabbits in group B were injected with 5.0 ml tuberculous fluid directly into the intervertebral disc. 5 rabbits in group C: 0.2 ml normal saline was injected into the lumbar vertebra of rabbits; 5 rabbits in group D: 0.2 ml normal saline was injected into the lumbar intervertebral disc of rabbits (group C and group D were negative control). Each rabbit in group C was selected as the experimental observation vertebra, and the lumbar vertebra in group 4 was used as the self-control vertebra. The rabbits in group A and group B were executed by fluorescence quantitative PCR, HE staining, culture of pus tuberculosis and other histopathological examination and general anatomical observation. Comparing with the positive rate of acid-fast staining, the advantages of PCR fluorescence quantitative method in the early detection of spinal tuberculosis were analyzed, and the counts of osteoblasts and osteoclasts and bone histomorphometry were analyzed. Model: 40 rabbits were divided into two groups according to different intraoperative injection reagents: group A: 30 rabbits were injected with 5.0mg/ml tuberculosis fluid into the lumbar vertebra by surgical drilling; group B: 10 rabbits were injected with 0.2ml normal saline into the lumbar vertebra; (group B was negative control). Each New Zealand white rabbit was selected to intervene in this experiment. The vertebral body and the lumbar 4 vertebral body can be used as the blank control vertebral body.X-ray examination, CT examination, MRI routine examination, MRI-DWI diffusion-weighted imaging and MRI-PWI perfusion imaging were performed at 4 weeks, 6 weeks and 8 weeks after operation.The significance of each imaging scanning method in the early imaging of spinal tuberculosis and the significance of each parameter in each scanning method were analyzed. Correlation analysis was made between the parameters of diffusion weighted imaging and those of MRI-PWI perfusion imaging. The advantages and disadvantages of each imaging method in early evaluation of spinal tuberculosis were compared. Results: By comparing the success rate of two different surgical methods, the lateral approach of lumbar spine in group A was used to drill into the lumbar 6 vertebra and inject tuberculosis fluid into the lumbar 6 vertebra. The success rate was higher than that of group B, which was injected tuberculosis fluid directly into the lumbar 5-6 intervertebral disc via the lateral approach of lumbar spine. Well, the linear relationship between CT value and DNA template content of Mycobacterium tuberculosis ranged from 4 *102 to 107 Copies/mL, r = 0.99503. Histopathological staining showed that the early stage of spinal tuberculosis destruction was mainly due to osteitis during exudation and bone destruction during proliferation of Mycobacterium tuberculosis. The results showed that: 8 weeks after operation, the three imaging methods had significance in the diagnosis of bone destruction, paravertebral abscess, hard model, and spinal cord compression. There was no significant difference in bad sensitivity. For the diagnosis of paravertebral abscess and hard model, the spinal cord was compression-sensitive MRICT X-ray; abnormal signal appeared on MRI and MRI-DWI at the 4th week after operation, and the B value in DWI sequence at about 600 s/mm2 in rabbit spinal imaging could obtain a clearer image; at the 4th, 6th and 8th week after infection, the vertebral body of the lesion could be seen clearly. ADC value was higher than that of normal vertebra (P 0.05). By comparing and analyzing four kinds of ADC images and ADC values, it was concluded that the ADC 8b image fitted with 8 b values had the highest signal-to-noise ratio and the best image quality; ADChigh value could reflect the activity of tuberculosis bacillus in the lesion vertebra; ADClow value showed a gradual increasing trend with time, indicating that the ADC 8b image fitted with 8 b values had the highest signal-to-noise ratio and the best image On DWI with low B value, the increase of blood perfusion in the lesion vertebra was more obvious than that of water molecule movement limitation; ADCperf value increased gradually with the destruction of spinal vertebra caused by Mycobacterium tuberculosis; there was a negative correlation between CT value and the number of osteoclasts in experimental rabbits with spinal tuberculosis group A, and the volume fraction of trabecular bone was increased. By analyzing the parameters of MRI perfusion imaging, it was found that except the peak time (TTP) decreased with the prolongation of the observation time, the other perfusion parameters in group A increased with the prolongation of the observation time, and the increase amplitude was higher than that in group B, and the peak time (TTP) and the ADCpe in DWI were higher than that in group B. In addition, the first pass enhancement rate (Efirst), first pass enhancement rate (Vfirst), early enhancement rate (Ee), early enhancement rate (Ve), signal maximum enhancement rate (Emax), signal enhancement rate (SER), peak enhancement rate (PH), signal maximum slope of increase (MSI), excretion rate (washout) were positively correlated with ADCperf. The rabbit spinal tuberculosis model was established by implanting Mycobacterium tuberculosis in the lumbar spine and injecting Mycobacterium tuberculosis directly into the lumbar intervertebral disc. The model was successfully established by gross, anatomical, fluorescence quantitative PCR and histopathological examination. The method of constructing tuberculosis model is simple and the success rate is high. It has laid a foundation for the animal experimental study of early spinal tuberculosis, such as histopathology. The complementary advantages of X-ray, CT, conventional MRI and MRI-DWI, MRI-PWI imaging in the diagnosis of early spinal tuberculosis can improve the sensitivity of examination. The Multi-b value analysis in MRI-DWI and the analysis of parameters in MRI-PWI can make quantitative evaluation on the early hemodynamics of spinal tuberculosis and provide valuable imaging basis for clinical orthopedics in the early diagnosis and treatment of spinal tuberculosis.
【學(xué)位授予單位】：新疆醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R529.2
[Abstract]:Objective: To establish a rabbit model of spinal tuberculosis by using ATCC 25177 Mycobacterium tuberculosis strain (lyophilized powder) in lumbar spine. To investigate the effects of X-ray, CT, magnetic resonance imaging (MRI), diffusion weighted imaging (DWI), magnetic resonance perfusion imaging (PWI) and magnetic resonance imaging (MRI) on spinal tuberculosis. Methods: Sixty rabbits were divided into four groups according to different operation methods and different injection reagents: 25 rabbits in group A were injected with 5.0mg/ml tuberculous fluid 0.2ml into the lumbar vertebra by surgical drilling; 25 rabbits in group B were injected with 5.0 ml tuberculous fluid directly into the intervertebral disc. 5 rabbits in group C: 0.2 ml normal saline was injected into the lumbar vertebra of rabbits; 5 rabbits in group D: 0.2 ml normal saline was injected into the lumbar intervertebral disc of rabbits (group C and group D were negative control). Each rabbit in group C was selected as the experimental observation vertebra, and the lumbar vertebra in group 4 was used as the self-control vertebra. The rabbits in group A and group B were executed by fluorescence quantitative PCR, HE staining, culture of pus tuberculosis and other histopathological examination and general anatomical observation. Comparing with the positive rate of acid-fast staining, the advantages of PCR fluorescence quantitative method in the early detection of spinal tuberculosis were analyzed, and the counts of osteoblasts and osteoclasts and bone histomorphometry were analyzed. Model: 40 rabbits were divided into two groups according to different intraoperative injection reagents: group A: 30 rabbits were injected with 5.0mg/ml tuberculosis fluid into the lumbar vertebra by surgical drilling; group B: 10 rabbits were injected with 0.2ml normal saline into the lumbar vertebra; (group B was negative control). Each New Zealand white rabbit was selected to intervene in this experiment. The vertebral body and the lumbar 4 vertebral body can be used as the blank control vertebral body.X-ray examination, CT examination, MRI routine examination, MRI-DWI diffusion-weighted imaging and MRI-PWI perfusion imaging were performed at 4 weeks, 6 weeks and 8 weeks after operation.The significance of each imaging scanning method in the early imaging of spinal tuberculosis and the significance of each parameter in each scanning method were analyzed. Correlation analysis was made between the parameters of diffusion weighted imaging and those of MRI-PWI perfusion imaging. The advantages and disadvantages of each imaging method in early evaluation of spinal tuberculosis were compared. Results: By comparing the success rate of two different surgical methods, the lateral approach of lumbar spine in group A was used to drill into the lumbar 6 vertebra and inject tuberculosis fluid into the lumbar 6 vertebra. The success rate was higher than that of group B, which was injected tuberculosis fluid directly into the lumbar 5-6 intervertebral disc via the lateral approach of lumbar spine. Well, the linear relationship between CT value and DNA template content of Mycobacterium tuberculosis ranged from 4 *102 to 107 Copies/mL, r = 0.99503. Histopathological staining showed that the early stage of spinal tuberculosis destruction was mainly due to osteitis during exudation and bone destruction during proliferation of Mycobacterium tuberculosis. The results showed that: 8 weeks after operation, the three imaging methods had significance in the diagnosis of bone destruction, paravertebral abscess, hard model, and spinal cord compression. There was no significant difference in bad sensitivity. For the diagnosis of paravertebral abscess and hard model, the spinal cord was compression-sensitive MRICT X-ray; abnormal signal appeared on MRI and MRI-DWI at the 4th week after operation, and the B value in DWI sequence at about 600 s/mm2 in rabbit spinal imaging could obtain a clearer image; at the 4th, 6th and 8th week after infection, the vertebral body of the lesion could be seen clearly. ADC value was higher than that of normal vertebra (P 0.05). By comparing and analyzing four kinds of ADC images and ADC values, it was concluded that the ADC 8b image fitted with 8 b values had the highest signal-to-noise ratio and the best image quality; ADChigh value could reflect the activity of tuberculosis bacillus in the lesion vertebra; ADClow value showed a gradual increasing trend with time, indicating that the ADC 8b image fitted with 8 b values had the highest signal-to-noise ratio and the best image On DWI with low B value, the increase of blood perfusion in the lesion vertebra was more obvious than that of water molecule movement limitation; ADCperf value increased gradually with the destruction of spinal vertebra caused by Mycobacterium tuberculosis; there was a negative correlation between CT value and the number of osteoclasts in experimental rabbits with spinal tuberculosis group A, and the volume fraction of trabecular bone was increased. By analyzing the parameters of MRI perfusion imaging, it was found that except the peak time (TTP) decreased with the prolongation of the observation time, the other perfusion parameters in group A increased with the prolongation of the observation time, and the increase amplitude was higher than that in group B, and the peak time (TTP) and the ADCpe in DWI were higher than that in group B. In addition, the first pass enhancement rate (Efirst), first pass enhancement rate (Vfirst), early enhancement rate (Ee), early enhancement rate (Ve), signal maximum enhancement rate (Emax), signal enhancement rate (SER), peak enhancement rate (PH), signal maximum slope of increase (MSI), excretion rate (washout) were positively correlated with ADCperf. The rabbit spinal tuberculosis model was established by implanting Mycobacterium tuberculosis in the lumbar spine and injecting Mycobacterium tuberculosis directly into the lumbar intervertebral disc. The model was successfully established by gross, anatomical, fluorescence quantitative PCR and histopathological examination. The method of constructing tuberculosis model is simple and the success rate is high. It has laid a foundation for the animal experimental study of early spinal tuberculosis, such as histopathology. The complementary advantages of X-ray, CT, conventional MRI and MRI-DWI, MRI-PWI imaging in the diagnosis of early spinal tuberculosis can improve the sensitivity of examination. The Multi-b value analysis in MRI-DWI and the analysis of parameters in MRI-PWI can make quantitative evaluation on the early hemodynamics of spinal tuberculosis and provide valuable imaging basis for clinical orthopedics in the early diagnosis and treatment of spinal tuberculosis.
【學(xué)位授予單位】：新疆醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R529.2
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