【作者】 张宇；

【导师】 甄平；

【作者基本信息】 兰州大学 ， 外科学（专业学位）， 2014， 硕士

【摘要】 目的：探讨荧光定量逆转录聚合酶链反应(Real Time reverse transcriptase polymerase chain reaction, Real Time RT-PCR)在脊柱结核诊治中的检测意义及临床应用价值,分析经标准化疗后结核杆菌活菌在脊柱结核患者病椎中的分布规律,统计分析结核病灶内残存菌的相对数量,为脊柱结核病灶组织切除范围的界定提供科学的理论依据。方法：20例初治脊柱结核患者,根据CT显示脊柱结核病灶边缘是否有硬化确定全部病例为硬化型,患者均服用抗痨药物4周后行手术治疗。术中切取病灶组织,据结核杆菌侵袭椎体组织的不同区域分别进行取材并随机分为：A组(结核病灶中心的干酪样坏死组织区域)、B组(结核病灶边缘硬化带区域)、C组(B组区域以外的“亚健康骨”区域)三组,再将C组中病灶组织距硬化带边缘的不同范围将“亚健康骨”又分为C1组(硬化带边缘向外3mm结核病灶组织区域)、C2组(C1组区域边缘向外3mm结核病灶组织区域)、C3组(C2组区域边缘向外3mm结核病灶组织区域)、C4组(C3组区域边缘向外3mm结核病灶组织区域),以距离A组(结核病灶中心的干酪样坏死组织区域)最远的C4组检测结果作为标准化阳性对照。Trizol法提取各病灶组织的总RNA,运用荧光定量逆转录聚合酶链反应(Real Time RT-PCR)对结核分枝杆菌(mycobacterium tuberculosis, MTB)中目的基因MPT-64mRNA及内参基因GAPDH分别进行扩增。所得实验数据用SPSS17.0统计软件分析,以P<0.05表示差异有统计学意义。结果：荧光定量RT-PCR检测结果显示,各实验组术前服用异烟肼+利福平+链霉素、异烟肼+利福平+吡嗪酰胺、异烟肼+利福平+乙胺丁醇三组的结核杆菌MPT64mRNA表达量无明显差别,各服用抗痨药物不同组间C4组的表达水平最低,C2组、C3组各时期表达量不显著。6例患者术前服用INH+RFP+SM,A组、B组、Cl组、C2组间的mRNA表达量无明显差别(P>0.05),C2组与其余四组比较有显著差异(P<0.05),C4组的表达量远远低于A组、B组、C1组、C2组；7例患者术前服用INH+RFP+PZA,A组、B组、C1组、C2组与C4组相比均有显著差异(P<0.01)；7例患者术前服用INH+RFP+EMB,A组、B组、C1组、C2组的表达量明显高于其余C3、C4组(P<0.01)。平均报告时间：2天。结论：(1)运用荧光定量RT-PCR技术检测脊柱结核患者不同病灶组织中结核菌的相对含量及活性度,具有灵敏、迅速、特异性高等特点；(2)脊柱结核病灶硬化带区域中存留生物活性的结核菌,药物很难穿过硬化带,应手术彻底切除硬化带；(3)成功运用分子生物学方法研究脊柱结核患者不同病灶组织(尤其“亚健康骨”组织)中结核菌的存活与相对定量的分布规律；(4)“亚健康骨组织”中C2组(C1组区域边缘向外3mm结核病灶组织区域)以内结核菌数量相对较多,以外结核菌数锐减,因此术中应将包括硬化壁在内的病灶组织彻底清除,且硬化壁以外的病灶亦应清除,清除范围不小于5mm。并且术中应结合影像学资料判断具体病灶组织的切除范围,防止术后脊柱不稳。更多还原

【Abstract】 Objective:To investigate quantitative reverse transcriptase polymerase chain reaction (Real Time RT-PCR) in clinical diagnosis and treatment of spinal tuberculosis in the application of value analysis of Mycobacterium tuberculosis lesions in the spinal lesions of the existence of different circumstances, from the etiological point of view to explore surgical lesion of spinal tuberculosis the scope for the clinical diagnosis of spinal tuberculosis, treatment to help.Methods:20patients with previously untreated patients with spinal tuberculosis, spinal tuberculosis according to CT showed hardened edge to determine whether there is a hardening of all cases, the patients were taking anti-tuberculosis therapy for4weeks after surgery. Intraoperative take all tuberculosis patients with spinal tuberculosis organization, depending on the scope of the invasion of tuberculosis were drawn randomly divided into:group A(caseous necrotic tissue area tuberculosis center), group B (curing tuberculosis edge band area), group C (group B outside the region "sub bone " area) three groups, while group C in accordance with tuberculosis at different distances from the edge of the hardening with "sub-bone" into group C1(3mm hardened band edge out tuberculosis stove tissue area), group C2(C13mm outward edge of the region to organize regional tuberculosis), group C3(group C2area outside edge3mm tuberculosis tissue area), group C4(group C3area outside edge3mm tuberculosis organization area), the distance a (caseous necrotic tissue area tuberculosis center) group C4farthest detected as a result of standardization control. Extracted with Trizol various lesions of spinal tuberculosis Organization RNA, application of quantitative reverse transcriptase polymerase chain reaction (RT-PCR) for Mycobacterium tuberculosis (MTB) in the target gene MPT-64mRNA and reference gene GAPDH were amplified. Statistical analysis was performed using SPSS17.0software, P0.05was considered significant. Results:Real Time RT-PCR detected before each experimental group patients taking INH+RFP+SM, INH+RFP+PZA, INH+RFP+EMB expression of Mycobacterium tuberculosis MPT64mRNA no significant difference between the three groups, each taking a different anti-tuberculosis drugs Low expression levels between the groups group C4, group C2, group C3less the expression of each period. Table5shows that6patients before surgery taking INH+RFP+SM, group A,group B, group C1, mRNA expression levels between the group C2had no significant difference (P>0.05), there are differences between group C2and the other four groups (P<0.05), group A, group B, group C1, the expression of the group is much higher than group C2, C4;7patients before surgery taking INH+RFP+PZA, group A, group B, group C1, group C2no significant difference between them, but each compared with the group C4were significantly different (P<0.01);7patients with preoperative taking INH+RFP+EMB, group A, group B, group C1, group C2expression was significantly higher than the rest of the group C3, C4(P<0.01). Each taking different anti-tuberculosis drugs in the experimental group group C3and C4expression were significantly lower than the rest of the group expression of group (P<0.05). the average reporting time:2days.Conclusions:(1) using Real Time RT-PCR detection of the focus of spinal tuberculosis in Mycobacterium tuberculosis, with rapid,sensitive specificity high;(2) hardening the focus of spinal tuberculosis in the left wall of Mycobacterium tuberculosis with biological activity,complete surgical excision is conducive to the prevention of postoperative recurrence of tuberculosis.(3) from the bacteriological point of view,the successful use of methods of molecular biology research,analysis of the focus of spinal tuberculosis in different tissues(especially the "subhealth bone") the existence of Mycobacterium tuberculosis,survive the situation;(4)"sub-healthy bone tissue " in the group C2(C1area outside edge3mm tuberculosis tissue area) more than the sharp drop in the number of TB, surgery should be based on the specific circumstances of resection combined with image data to determine the scope,so as not to affect the postoperative spinal stability.更多还原