pRNA纳米微粒对脊柱结核分枝杆菌生长影响的实验研究

Experimental study of the effects of pRNA nanoparticles on the growth of mycobacterium tuberculosis in the spine

中文关键词:  脊柱结核  纳米微粒  成骨细胞  结核分枝杆菌  菌落形成单位

英文关键词:Spinal tuberculosis  Nanoparticles  Osteoblasts  Mycobacterium tuberculosis  Colony forming units

中文摘要:

  【摘要】 目的：观察pRNA-3WJ-siLNA gapmer（Mce4）-aptamer（CD40）纳米微粒（简称pRNA纳米微粒）在脊柱结核细胞模型中对结核分枝杆菌生长的影响。方法：利用荧光结核分枝杆菌感染人成骨细胞建立脊柱结核细胞模型。先培养及准备人成骨细胞，人成骨细胞培养至第三代；然后构建培养荧光结核分枝杆菌；将培养良好的人成骨细胞用1×Trypsin-EDTA胰蛋白酶消化之后以70%密度在100mm细胞培养皿种植细胞，经24h培养使细胞稳定之后，将1麦氏比浊管的对数生长中期荧光结核分枝杆菌以感染复数（multiplicity of infection，MOI）1∶10感染培养的人成骨细胞6h，用不加血清培养液清洗3次，在培养箱中培养24h；然后将感染成功的人成骨细胞随机分为A组（空白对照组）及B、C、D组（三个实验组），三个实验组分别置入本研究团队构建的0.1μM、1μM、10μM浓度的脊柱结核靶向治疗pRNA纳米微粒溶液，共培养36h之后，加入细胞裂解液裂解细胞，裂解产物用Middlebrook 7H9培养液稀释20倍，之后均匀涂抹到琼脂平板，放入到37℃，5% CO2细胞培养箱中培养21d，使用Gel Doc XR+system凝胶成像系统观察菌落影像，使用Bio-Rad Discovery Quantity One?誖1-D analysis软件对四组的所有菌落进行了测定，统计分析各组人成骨细胞中结核分枝杆菌菌落形成单位（colony forming unit，CFU）。结果：人成骨细胞培养良好；成功构建荧光结核分枝杆菌（H37Ra-GFP）；荧光结核分枝杆菌结合并进入人成骨细胞。经统计分析A、B、C、D四组菌落形成单位（272.67±67.06、183.33±8.74、154.33±25.72、76.67±11.02）的差异存在统计学意义（F=14.68，P<0.05）；且B、C、D三组的菌落形成单位分别与A组相比时，均明显低于A组（P<0.05），B、C、D三组菌落形成单位依次下调，三组菌落形成单位两两比较存在差异，并且有统计学意义（P<0.05）。结论：pRNA纳米微粒以浓度梯度的方式抑制结核分枝杆菌在人成骨细胞中的生长存活，甚至低浓度也具备抑制结核分枝杆菌生长的能力。

英文摘要:

  【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.