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| FAN Hongjie,YIN Huquan,JIN Weidong.Experimental study of the effects of pRNA nanoparticles on the growth of mycobacterium tuberculosis in the spine[J].Chinese Journal of Spine and Spinal Cord,2021,(2):158-166. |
| Experimental study of the effects of pRNA nanoparticles on the growth of mycobacterium tuberculosis in the spine |
| Received:June 15, 2020 Revised:October 30, 2020 |
| English Keywords:Spinal tuberculosis Nanoparticles Osteoblasts Mycobacterium tuberculosis Colony forming units |
| Fund:国家自然科学基金资助项目（编号：81460336、81660370） |
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| English Abstract: |
| 【Abstract】 Objectives: To observe the effects of pRNA-3WJ-siLNA gapmer(Mce4) -aptamer(CD40) nanoparticles(pRNA nanoparticles) on the growth of mycobacterium tuberculosis growth in spinal tuberculosis cell model. Methods: Spinal tuberculosis cell model was established by infecting human osteoblasts with fluorescent mycobacterium tuberculosis. First human osteoblasts were cultivated and prepared, which were cultured to the third generation, then fluorescent mycobacterium tuberculosis was constructed and cultured, which was cultured to the mid-logarithmic growth phase to infect human osteoblasts. Good human osteoblasts cultured were digested with 1×Trypsin-EDTA trypsin at a density of 70% in a 100mm cell culture dish. After culturing the cells for 24h to stabilize the cells, the mycobacterium tuberculosis fluorescent medium in the logarithmic growth phase of a 1 McFarland Standard was infected with the cultured osteoblasts at a multiplicity of infection(MOI) 1∶10 for 6h, washed 3 times with serum-free culture medium, and cultured in an incubator for 24h. After that, human osteoblasts infected successfully were randomly divided into group A(the blank control group) and three experimental groups(group B, C and D), four groups were placed with pRNA nanoparticles at concentrations of 0μM, 0.1μM, 1μM, and 10μM constructed by the research team. After 36h of co-cultivation, cell lysate was added to lyse the cells. The lysate was diluted 20-fold with Middlebrook 7H9 culture solution, then evenly spread on agar plates, and placed in 37℃, 5% CO2 cell incubator for 21d. The gel Doc XR+system gel imaging was adopted to observe the colony images, and Bio-Rad Discovery Quantity One?誖 1-D analysis software was used to determine all the colonies in the four groups. The colony forming units of mycobacterium tuberculosis in each group of human osteoblasts were statistically analyzed. Results: Human osteoblasts were cultured well and fluorescent mycobacterium tuberculosis was successfully constructed. The fluorescent mycobacterium tuberculosis combined and entered human osteoblasts. After statistical analysis, the differences in colony forming units among the four groups A, B, C and D(272.67±67.06, 183.33±8.74, 154.33±25.72, 76.67±11.02) had statistical significance(F=14.68, P<0.05); and the colony forming units of groups B, C, and D were significantly lower than those of group A(P<0.05). The colony formation units of groups B, C, and D were lowered in turn, and there were differences between the three groups, and they were statistically significant(P<0.05). Conclusions: pRNA nanoparticles significantly inhibit the growth and survival of mycobacterium tuberculosis in osteoblasts in a concentration gradient manner, even low concentrations have the ability to inhibit the growth of mycobacterium tuberculosis. |
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