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摘要:
目的 明确肝脏灌洗液中的单个核细胞（MC）的抗菌作用。 方法 体外实验：收集SD大鼠肝脏灌洗液中的MC细胞并分为两组，一组在培养液中添加白细胞介素（IL）-15，另一组不添加IL-15。将两组细胞分别与铜绿假单胞菌（P. aeruginosa）共培养4 h后收集上清并裂解细胞。以LB平板培养检测裂解液中细菌负荷，酶联免疫吸附试验（ELISA）检测培养上清中干扰素（IFN）-γ和肿瘤坏死因子（TNF）-α含量。体内实验：将40只SD大鼠自气管注入P. aeruginosa菌液（1×109 CFU/mL）并随机分为4组，每组10只：对照组，生理盐水灌胃；免疫抑制组，他克莫司（FK506）灌胃；MC细胞组，应用FK506后自尾静脉注射MC细胞1.0×108；IL-15预处理MC细胞组，应用FK506后自尾静脉注射IL-15预处理的MC细胞1.0×108。收集各组大鼠肺组织及肺泡灌洗液，以LB平板培养检测肺组织及肺泡灌洗液中的细菌负荷，ELISA、Western blot检测肺泡灌洗液、肺组织中IFN-γ和TNF-α含量。 结果 与单纯MC细胞比较，IL-15预处理的MC细胞体外抗菌能力显著提高，CFU仅为前者35%，且合成、释放IFN-γ、TNF-α的能力更强。与对照组大鼠比较，免疫抑制组大鼠肺脏内免疫细胞数量减少，肺组织和肺泡灌洗液中的细菌负荷量明显升高，而IFN-γ和TNF-α含量呈下降趋势。输入IL-15预处理MC细胞后可显著提高肺组织内免疫细胞数量，减轻细菌负荷量并增加IFN-γ和TNF-α分泌。 结论 肝脏灌洗液中的MC细胞具有良好的抗菌活性，在免疫抑制条件下可有效提升机体对机会性致病菌的免疫防御能力。 Abstract:Objective To evaluate the antibacterial effect of mononuclear cells (MCs) in the liver lavage solution. Methods For in vitro experiment, MCs were collected from the liver lavage solution of SD rats and divided into the supplement of interleukin (IL)-15 and non-supplement groups. The MCs were co-cultured with Pseudomonas aeruginosa (P. aeruginosa) for 4 h and then the supernatant was collected and MCs were lysed. The bacterial load in the lysate was detected after LB plate culture. The levels of interferon (IFN)-γ and tumor necrosis factor (TNF)-α in the supernatant were measured by enzyme-linked immune absorbent assay (ELISA). For in vivo experiment, 40 SD rats were administered via tracheal injection of P. aeruginosa solution at a dose of 1×109 CFU/mL and randomly divided into four groups (n=10). In the control group, physiological saline was given via gavage. In the immunosuppression group, tacrolimus (FK506) was delivered via gavage. In the MC group, MCs at a dose of 1.0×108 was given via intravenous injection after use of FK506. In the IL-15 pretreated-MC group, IL-15 pretreated-MCs at a dose of 1.0×108 were administered via intravenous injection after application of FK506. The lavage solution of pulmonary alveolus and the rat lung tissue were collected. The bacterial load was detected after LB plate culture. The expression of IFN-γ and TNF-α in the pulmonary alveolus and lung tissue were measured by ELISA and Western blot. Results Compared with MCs alone, IL-15 pretreated-MCs exhibited significantly higher antibacterial capability in vitro. The CFU was 35% of untreated MCs. The synthesis and release capabilities of IFN-γ and TNF-α were significantly enhanced. Compared with the control group, the quantity of immune cells in the lung tissue was decreased and the bacterial load in the lung tissue and the lavage solution of pulmonary alveolus was significantly increased, whereas the expression levels of IFN-γ and TNF-α tended to decline in the immunosuppression group. Administration of IL-15 pretreated-MCs significantly enhanced the quantity of immune cells in the lung tissue, decreased the bacterial load and increased the secretion of IFN-γ and TNF-α. Conclusions MCs in the liver lavage solution exhibit favorable antibacterial activity. Under immunosuppression condition, the defense capability of the host against the opportunistic pathogenic bacteria is significantly enhanced. -
Key words:
- Pseudomonas aeruginosa /
- Liver /
- Lavage solution /
- Mononuclear cell /
- Anti-infection
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图 1 肝脏灌洗液MC细胞对P. aeruginosa生长的影响
aP < 0.01
Figure 1. Effects of liver lavage fluid MCs on the growth of P. aeruginosa
图 2 肝脏灌注液中MC细胞分泌IFN-γ和TNF-α的能力
A图为IFN-γ，aP < 0.05；B图为TNF-α，bP < 0.01
Figure 2. The ability of MCs secreting IFN-and TNF-in liver perfusion fluid
图 3 各组大鼠肺脏内免疫细胞浸润情况
A图为生理盐水对照组；B图为免疫抑制组；C图为MC细胞组；D图为IL-15预处理MC细胞组
Figure 3. The infiltration of immune cells in the lungs of rats in each group (HE, ×200)
图 4 肺组织及肺泡灌洗液中细菌负荷量的检测
A图为肺组织；B图为肺泡灌洗液；aP < 0.05，bP < 0.01
Figure 4. Detection of bacterial load in lung tissue and alveolar lavage fluid
图 5 Western blot检测肺组织中IFN-γ和TNF-α含量
1为对照组；2为免疫抑制组；3为MC细胞组；4为IL-15预处理MC细胞组；aP < 0.01
Figure 5. The content of IFN-γ and TNF-α in lung tissue measured by Western blot
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