布鲁氏菌及结核分枝杆菌侵染大鼠成骨细胞对肿瘤坏死因子-α及白细胞介素-1β表达的影响

The expression levels and effects of TNF-α and IL-1β after Brucella.ovis and Mycobacterium tuberculosis infecting osteoblasts

中文关键词:  绵羊布鲁氏菌  结核分枝杆菌  成骨细胞  肿瘤坏死因子-α  白细胞介素-1β

英文关键词:Brucella.ovis  Mycobacterium tuberculosis  Osteoblasts  Tumor necrosis factor-α  Interleukin-1β

中文摘要:

  【摘要】 目的：测定绵羊布鲁氏菌与结核分枝杆菌分别侵染成骨细胞后肿瘤坏死因子-α（tumor necrosis factor，TNF-α）及白细胞介素-1β（interleukin，IL-1β）的表达水平，初步探讨布鲁氏菌性脊柱炎相较于脊柱结核骨损伤程度不同的原因。方法：取新生2～5d的SD大鼠颅骨提取原代成骨细胞并培养至第3代，用碱性磷酸酶（ALP）染色法对成骨细胞进行鉴定。用制备好的细菌培养基分别培养绵羊布鲁氏菌及结核分枝杆菌并计数以配制转染液；荧光显微镜观察并记录荧光标记结核分枝杆菌在转染复数（MOI）为1∶10、1∶20时的成骨细胞，提取绵羊布鲁氏菌以MOI为1∶100侵染的成骨细胞在不同时间的裂解液，使用平板接种法进行计数，确定结核分枝杆菌及布鲁氏菌能否侵染成骨细胞。分别用绵羊布鲁氏菌及结核分枝杆菌以不同MOI及时间侵染成骨细胞，采用CCK-8检测两种细菌侵染后细胞的存活率，选择最佳MOI及时间。将成骨细胞分为三组，结核分枝杆菌侵染组（用最佳MOI结核分枝杆菌侵染）、绵羊布鲁氏菌组侵染组（用最佳MOI绵羊布鲁氏菌侵染）和阴性对照组，采用细胞免疫组化染色观察三组细胞表面TNF-α及IL-1β表达情况；采用酶联免疫吸附实验（ELISA）测量三组细胞培养上清液中TNF-α及IL-1β表达水平；采用蛋白免疫印迹（Western Blot）检测三组细胞中TNF-α及IL-1β蛋白表达水平。采用单因素方差分析和t检验比较三组间的差异，P<0.05为差异有统计学意义。结果：分离培养的成骨细胞ALP染色后细胞质呈蓝色，细胞核呈绿色，符合成骨细胞特点。绵羊布鲁氏菌及结核分枝杆菌均可以侵染成骨细胞；结核分枝杆菌组在MOI为1∶20、24h，绵羊布鲁氏菌组在MOI为1∶100、24h细胞存活率均为50%，以此作为最佳对比MOI及时间。细胞免疫组化、ELISA及Western Blot结果显示结核分枝杆菌及绵羊布鲁氏菌侵染成骨细胞后均能导致TNF-α及IL-1β的表达高于阴性对照组（P<0.05），且结核分枝杆菌组的表达水平明显高于绵羊布鲁氏菌组（P<0.05）。结论：结核分枝杆菌侵染成骨细胞后TNF-α及IL-1β的表达水平高于绵羊布鲁氏菌侵染的成骨细胞，可能导致脊柱结核与布鲁氏菌性脊柱炎骨质损伤程度不同。

英文摘要:

  【Abstract】 Objectives: To measure the expression levels of tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β) in osteoblasts infected by Brucella.ovis and Mycobacterium tuberculosis and to explore the reasons of different degrees of bone injury in Brucella spondylitis and spinal tuberculosis. Methods: Primary osteoblasts were extracted from the skulls of SD rats at 2-5 days after birth and cultured to the third generation, and alkaline phosphatase(ALP) staining was applied to identify osteoblasts. Brucella.ovis and Mycobacterium tuberculosis were cultured with prepared bacterial culture medium and counted to prepare the transfection medium. The osteoblasts of fluorescent Mycobacterium tuberculosis were observed and recorded under fluorescence microscope when the multiplicity of infection(MOI) was 1∶10 and 1∶20. The lysates of osteoblasts infected with Brucella.ovis with MOI of 1:100 were extracted at different time, and the plate streak method was used for counting to determine whether Mycobacterium tuberculosis and Brucella.ovis can infect osteoblasts. The osteoblasts were infected with Brucella ovis and Mycobacterium tuberculosis at different MOI and time, and CCK-8 was used to detect the cell survival rate after infection by the two kinds of bacteria to choose the best MOI and time. The osteoblasts were divided into three groups: Mycobacterium tuberculosis infection group(infected with the best MOI Mycobacterium tuberculosis), Brucella.ovis infection group (infected with the best MOI Brucella.ovis) and negative control group. Cell immunohistochemistry staining was used to observe the TNF-α and IL-1β expression levels on the cells′ surface of the three groups; Enzyme-linked immunosorbent assay(ELISA) was used to measure TNF-α and IL-1β expression levels on the cell supernatant of the three groups; Western Blot was used to detect the TNF-α and IL-1β protein expression levels in the three groups of cells. One-way ANOVA and T test were used to compare the differences among the three groups, P<0.05, the difference was statistically significant. Results: After ALP staining, the cytoplasm of osteoblasts was blue and the nucleus was green, which conformed to the characteristics of osteoblasts. Both Brucella.ovis and Mycobacterium tuberculosis can infect osteoblasts. The MOI of Mycobacterium tuberculosis group was 1∶20 and 24h, and that of Brucella ovis group was 1∶100 and 24h, respectively, and the cell survival rate was 50%, thus the MOI and time were the best comparison. Cellular immunohistochemistry, Western Blot and ELISA showed that after Mycobacterium tuberculosis and Brucella.ovis infected osteoblasts, the expression of TNF-α and IL-1β increased, and the expression levels of TNF-α and IL-1β in the Mycobacterium tuberculosis group were significantly higher than those in the Brucella.ovis group(P<0.05). Conclusions: The expression levels of TNF-α and IL-1β in osteoblasts infected with Mycobacterium tuberculosis were higher than those of Brucella.ovis, which may be one of the reasons for the different degrees of bone damage between spinal tuberculosis and Brucella spondylitis.