多种影像学联合组织病理学检查对脊柱结核早期的评价

发布时间：2018-09-03 15:03

【摘要】：目的：应用ATCC25177结核分枝杆菌菌种(冻干粉)在兔腰椎建立脊柱结核病模型,探讨X线、CT (Computed tomography)、核磁共振(Magnetic resonance imaging,MRI)常规扫描、MRI弥散成像(Diffusion weighted imaging, DWI)、磁共振灌注成像(Perfusion weighted imaging, PWI)及组织病理学检查在兔脊柱结核发病早期的影像诊断中的应用价值。方法：首先选择实验兔60只依据手术方法不同及注入试剂不同分为4组：A组25只：兔腰椎椎体内经手术钻孔注入5.0mg/ml的结核菌液0.2ml；B组25只：椎间盘内直接注入5.0mg/ml的结核菌液0.2ml；C组5只：兔腰椎椎体内注入生理盐水0.2ml；D组5只：兔腰椎椎间盘内注入生理盐水0.2ml。(C组与D组为阴性对照)。选定的每只新西兰大白兔腰6椎体作为实验的观察椎体,腰4椎体可作为自身对照椎体。术后1-8周时随机处死A、B两组实验兔行荧光定量PCR检查、HE染色、脓液结核菌培养等组织病理学检查与解剖学大体观察；C、D两组实验兔在同样时间段处死实验兔行上述检查。主要观察指标：比较分析两种手术方法造模的成功率、抗酸染色的阳性率；比较分析PCR荧光定量法在脊柱结核早期检测的优势、与抗酸染色的阳性率比较；对结核病灶边缘骨组织成骨细胞和破骨细胞计数及骨组织计量学分析。再根据实验兔存活率最高且结核种植区椎体感染最优的手术方法,建立影像学动物模型：选择实验兔40只依据术中注入试剂不同分为2组：A组30只：兔腰椎椎体内经手术钻孔注入5.0mg/ml的结核菌液0.2ml；B组10只：兔腰椎椎体内注入生理盐水0.2ml；(B组为阴性对照)。选定的每只新西兰大白兔腰6椎体作为本实验的干预椎体,腰4椎体可作为空白对照椎体。术后4周、6周、8周时行X线检查、CT检查、MRI常规检查及MRI-DWI弥散加权成像检查及MRI-PWI灌注成像扫描。分析各影像学扫描方法在脊柱结核早期影像图像中的意义及各扫描方法中各个参数的意义,并将MRI-DWI弥散加权成像中的参数与MRI-PWI灌注成像的参数做相关性分析。比较各影像学检查方法对脊柱结核早期评价中的优缺点。结果：通过两组不同手术方法造模成功率的比较,A组腰椎侧方入路于腰6椎体处钻孔注入结核菌液的实验兔手术成功率高于B组腰椎侧方入路于腰5-6椎间盘处直接注入结核菌液的实验兔；通过荧光定量PCR方法可以检测出脊柱结核脓液中结核的特异性TB-DNA含量,并可检出最低TB-DNA含量,且优于抗酸染色法；建立的实时荧光定量PCR的线性关系很好,Ct值与结核杆菌的DNA模板含量呈线性关系线性范围为4×102～107Copies/mL, r=0.99503;通过组织病理学染色观察到脊柱结核破坏早期,主要是以结核菌渗出期间椎体骨炎及增生期间的骨质破坏为主的组织病理学表现。通过影像学的实验研究证明：A组术后8周时,三种影像学检查方法对骨质破坏、椎旁脓肿、硬模、与脊髓受压的诊断敏感性都有意义。术后8周时,MRI对脊柱结核造成的骨质破坏与CT检查的诊断敏感性比较,P值0.05,说明两者在术后8周这个时间段时对诊断骨质破坏的敏感性上无明显差异性,对于诊断椎旁脓肿与硬模、脊髓是受压的敏感性MRICTX线；手术造模后第4周时MRI及MRI-DWI就出现异常信号,且兔脊柱成像时DWI序列中研究b值在600s/mm2左右时可获得较为清晰的图像；第4、6、8周感染组病变椎体的ADC值高于正常椎体(P0.05)；通过对4类ADC图像及ADC值的比较分析,得出：用8个b值的DWI拟合出的ADC8b图其信噪比最高,图像质量最好；ADChigh值可以反映出病变椎体内部结核杆菌的活性；ADClow值随着时间延长呈现逐渐增加的趋势,说明在低b值的DWI上,病变椎体内部血流灌注的增加比水分子运动受限对该值的贡献要明显；ADCperf值随着结核杆菌对脊柱椎体造成的破坏而呈现逐渐增大的趋势；脊柱结核A组的实验兔的CT值和破骨细胞数间存在负相关、骨小梁体积分数和破骨细胞数之间存在负相关。通过对MRI灌注成像的参数分析得出：A组实验组术后4、6、8周除达峰时间(TTP)随着观察时间的延长而降低以外,其他灌注参数都随时间的延长而增加,增加幅度高于B组对照组,且除达峰时间(TTP)与DWI中的ADCperf呈负相关外,首过强化率(Efirst)、首过强化速率(Vfirst)、早期强化率(Ee)、早期强化速率(Ve)、信号最大强化率(Emax)、信号增强率(SER)、增强峰值(PH)、信号最大上升斜率(MSI)、排泄率(washout)都与ADCperf呈正相关。结论：通过在新西兰大白兔腰椎局部种植结核分枝杆菌与腰椎间盘直接注入结核杆菌两种手术方法的实验研究,可以构建出兔脊柱结核模型,通过大体、解剖学、荧光定量PCR法及组织病理学检查,证实建造模型成功。其中在新西兰大白兔腰椎局部种植结核分枝杆菌脊柱结核模型的构建方法简便且成功率较高,对脊柱结核早期的组织病理学研究等的动物实验研究奠定了一定基础。通过X线、CT与常规MRI及MRI-DWI、 MRI-PWI成像的影像学检查方法在诊断早期脊椎结核中的优势互补可以提高检查敏感度。通过对MRI-DWI中多b值的分析及MRI-PWI中各参数的分析,可以对脊柱结核早期血流动力学做出定量指标的评价。为临床骨科对脊柱结核的早期诊疗提供有价值的影像依据。
[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.
【学位授予单位】：新疆医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R529.2

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