CD40作为脊柱结核靶向治疗靶标分子的实验研究

Experimental study of CD40 as a target molecule for targeted treatment of spinal tuberculosis

中文关键词:  成骨细胞  CD40  靶标分子  结核分枝杆菌

英文关键词:Osteoblasts  CD40  Target molecule  Mycobacterium tuberculosis

中文摘要:

  【摘要】 目的：通过对不同感染复数结核分枝杆菌侵染的正常人成骨细胞表面CD40分子差异表达的实验研究，探讨CD40作为脊柱结核分子靶向治疗的靶标分子的可行性。方法：将冷冻保存的正常人成骨细胞按照美国模式菌种收集中心（American Type Culture Collection，ATCC）的说明进行复苏和培养，传代至3代冻存备用；之后选择Middlebrooke7H9液体培养基对荧光结核分枝杆菌（H37Ra-GFP）进行培养，培养至对数中期备用。把传代后的人成骨细胞随机分为A、B、C、D四组，A组为对照组，是未被结核分枝杆菌侵染的人成骨细胞组；B、C、D三组均为实验组，分别是被感染复数（multiplicity of infection，MOI）为1∶1000、1∶100、1∶10浓度的结核分枝杆菌侵染的人成骨细胞组。然后采用实时定量PCR与蛋白免疫印迹（Western Blot，WB）的方法，分别检测不同感染复数结核分枝杆菌诱导人成骨细胞中CD40 mRNA的表达变化及成骨细胞表面CD40分子的表达变化。结果：传代后的人成骨细胞经荧光结核分枝杆菌侵染后，观察发现各组细胞感染程度良好。A组实时定量PCR反应结果表现为人成骨细胞中存在CD40 mRNA的表达；A（1.0337±0.0715）、B（1.4083±0.1145）、C（1.7172±0.1294）、D（2.0378±0.1573）四组之间的表达存在差异（F=74.005，P<0.05）；B、C、D三组分别与A组相比时，均存在差异（P<0.05），表现为结核分枝杆菌侵染组B、C、D三组成骨细胞中CD40 mRNA的表达均高于未侵染组A组；B、C、D三组，两两比较时，均存在差异（P<0.05），表现为从B组到D组成骨细胞中CD40 mRNA的表达依次上调。而通过蛋白免疫印迹法分析成骨细胞表面CD40分子的表达结果发现与上述mRNA表达上调的结果完全吻合。结论：结核分枝杆菌侵染人成骨细胞后可诱导成骨细胞中CD40 mRNA以及成骨细胞表面CD40分子的表达增多，并且感染复数越高，细胞表面CD40分子的表达越多。而CD40分子可与其所对应的核酸适体特异结合，这就为使用CD40分子作为靶标分子，对脊柱结核进行靶向治疗提供了理论基础。

英文摘要:

  【Abstract】 Objectives: To explore the possibility of CD40 as a target molecule for targeted therapy of spinal tuberculosis, and to investigate the differential expression of CD40 molecules on the surface of normal human osteoblasts infected by different multiplicity of mycobacterium tuberculosis. Methods: Normal human osteoblasts from American Type Culture Collection(ATCC) were used in this study and logarithmic phase culture of mycobacterium tuberculosis(H37Ra-GFP) was used for infection experiment. The human osteoblasts were randomly divided into four groups: A, B, C and D. Group A was the control group, which was not infected by H37Ra-GFP. The other groups were all treatment groups, these groups were infected by Mycobacterium tuberculosis with the different multiplicity of infection(MOI) 1∶1000, 1∶100, 1∶10. The expressions of CD40 mRNA in the human osteoblasts induced by Mycobacterium tuberculosis with different infections were detected by real-time quantitative PCR. Western blot was used to analyze the changes of CD40 protein levels on the surface of osteoblasts. Results: The analysis of mycobacterium tuberculosis uptake by osteoblast showed different MOI of bacterial infection caused dose-dependently increasing the uptake. Real-time quantitative PCR results showed that the up-regulated expression levels of CD40 mRNA in human osteoblasts had been observed. There were significant differences(F=74.005, P<0.05) in the expressions of CD40 mRNA in group A(1.0337±0.0715), B(1.4083±0.1145), C(1.7172±0.1294) and D(2.0378±0.1573). Compared with group A, the expression of CD40 mRNA in mycobacterium tuberculosis infection group B, C, D was higher than that in non-infection group A, and there were significant differences(P<0.05). There were differences in group B, C and D when comparing with each other(P<0.05), and the expression of CD40 mRNA from group B to group D was up-regulated. The Western Blot results showed that the expression of CD40 on osteoblast surface was correlated well with the CD40 mRNA expression. Conclusions: The CD40 mRNA levels were dose-dependently up-regulated by different dose of mycobacterium tuberculosis infection. And the CD40 protein levels were correlated well with the mRNA levels, that showed the higher the multiplicity of infection, the more expression of CD40 molecules. Considering CD40 molecules can specifically bind to their corresponding aptamers, this study provides a theoretical basis for targeted treatment of spinal tuberculosis using CD40 molecules as targeting molecules.