结核杆菌裂解物刺激后的成骨细胞来源外泌体对破骨细胞的影响

Experimental study on the effect of osteoblast-derived exosomes stimulated by mycobacterium tuberculosis lysate on osteoclasts

中文关键词:  外泌体  成骨细胞  破骨细胞  结核杆菌裂解物

英文关键词:Exosomes  Osteoblasts  Osteoclasts  Mycobacterium tuberculosis lysate

中文摘要:

  【摘要】 目的：研究结核分枝杆菌裂解物（MTL）刺激后的成骨细胞来源外泌体（MTL-OB-Exo）对破骨细胞形成的影响。方法：采用细胞增殖与毒性实验（Cell Counting Kit-8，CCK-8）确定MTL的最佳剂量为35.9ng/ml，用最佳剂量的MTL刺激小鼠成骨细胞，采用差速离心法分离、提取MTL-OB-Exo和正常小鼠成骨细胞来源外泌体（OB-Exo），经透射电镜观察其形态及大小，采用蛋白质印迹法（Western Blot，WB）检测外泌体跨膜蛋白CD9和成骨细胞特异性碱性磷酸酶（AKP）的表达。将小鼠破骨细胞的前体细胞单核巨噬细胞（RAW264.7），按不同的干预方式分为三组：实验组，MTL-OB-Exo（10ng/ml）；对照组，OB-Exo（10ng/ml）；空白组，不予任何处理。培养4d后，在光学显微镜下观察RAW264.7的分化情况，并采用抗酒石酸酸性磷酸酶染色法（tartrate resistant acid-phosphatase，TRAP），观察各组破骨细胞的形态并计数，比较破骨细胞形成的数量。同样的分组条件下，将干预后的细胞在骨磨片上培养10d，采用亚甲蓝染色，观察各组骨吸收陷窝的形态并计数，比较骨吸收陷窝形成的数量。结果：MTL-OB-Exo为直径30～100nm的圆形或椭圆形结构，高表达CD9和AKP蛋白。采用不同的方式对RAW264.7进行干预后，实验组破骨细胞形成的数量为39.60±1.95个，显著高于对照组（30.20±1.64个）和空白组（28.80±1.58个），差异有统计学意义（P<0.05）；实验组骨吸收陷窝数量为21.40±1.52个，显著高于对照组（16.20±1.30个）和空白组（14.40±1.52个），差异有统计学意义（P<0.05）。结论：MTL-OB-Exo可以诱导并增强破骨细胞的形成，造成骨质破坏。

英文摘要:

  【Abstract】 Objectives: To study the effect of osteoblast-derived exosomes(MTL-OB-Exo) stimulated by mycobacterium tuberculosis lysate(MTL) on the formation of osteoclasts. Methods: Cell Counting Kit-8 (CCK-8) was used to determine that 35.9ng/ml was the optimal dose of MTL, which was used to stimulate mouse osteoblasts. Differential centrifugation was used to separate and extract MTL-OB-Exo and normal mouse osteoblast-derived exosomes(OB-Exo). The morphology and size were observed by transmission electron microscopy, and Western blot(WB) was used to detect the expression of exosomal transmembrane protein CD9 and osteoblast-specific alkaline phosphatase(AKP). Mouse mononuclear macrophages(RAW264.7), the precursor cells of osteoclasts, were divided into three groups according to different intervention methods: experimental group, MTL-OB-Exo (10ng/ml); control group, OB-Exo (10ng/ml); blank group, no treatment. After 4 days of culture, RAW264.7 was observed under light microscope and tartrate resistant acid phosphatase(TRAP) staining was used. The morphology of osteoclasts in each group was observed and counted, and the difference in the number of osteoclasts was compared. In the same intervention group, the cells after intervention were cultured on the bone grinding plate for 10 days. The morphology of bone resorption lacunae in each group was observed and counted by methylene blue staining, and the difference in the number of bone resorption lacunae was compared. Results: MMTL-OB-Exo was of a round or round-like structure with a diameter of 30-100nm, and it highly expressed CD9 and AKP proteins. After the intervention of RAW264.7 in different ways, the number of osteoclasts formed in the experimental group was 39.60±1.95, which was significantly higher than that in the control group(30.20±1.64) and the blank group(28.80±1.58), and the difference was statistically significant(P<0.05). The number of bone resorption lacunae in the experimental group was 21.40±1.52, which was significantly higher than that in the control group(16.20±1.30) and the blank group(14.40±1.52), and the difference was statistically significant(P<0.05). Conclusions: MTL-OB-Exo can induce and enhance the formation of osteoclasts, result in bone destruction.