Experimental study on substance P in the regulation of degranulation of cultured murine mast cells
10.3760/cma.j.issn.1673-0860.2016.09.008
- VernacularTitle:P物质调控肥大细胞脱颗粒的实验研究
- Author:
Fengli CHENG
1
;
Qiuting LI
;
Changqing ZHAO
;
Yunfang AN
;
Xueping QI
Author Information
1. 山西医科大学第二医院耳鼻咽喉头颈外科
- Keywords:
Mast cell;
Substance P;
Receptor;
Interleukin-4
- From:
Chinese Journal of Otorhinolaryngology Head and Neck Surgery
2016;51(9):675-680
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the effect of interleukin-4 (IL-4) stimulation on the expression of FcεR Ⅰ αt and NK-1R on mature mast cells(MC) cultured and differentiated from mouse bone marrow stem cells,and then to study if these MC also respond to substance P (SP) both in FcεR Ⅰ α and NK-1R dependent manners.Methods Bone marrow cells were aseptically flushed from BALB/c mouse femurs into complete RPMI 1640,followed by culture with stem cell factor (SCF 100 μg/L),IL-3 (15 μg/ L) and IL-4 (0,10,15,20 and 25 μg/L,respectively).The culture medium was changed once a week.The morphological changes of culture cells were observed under inverted microscope.After 4 weeks culture,the cells were collected and appraised by toluidine blue staining and flow cytometry.The expressions of surface CD117,FceR Ⅰ α and NK-1R on these cells were detected by flow cytometry and Western blot.Bone marrow MC were activated with SP (0,0.01,0.1,1.0 and 10 mg/L,respectively) for 30 min.The histamine released into the supernatant and stored in the protoplasm was quantified by enzyme linked immunosorbent assay (ELISA).The percentage of histamine release was calculated as a percent of total histamine content.Results When different concentrations of IL-4 (0,10,15,20,25 μg/L)were added into RPMI 1640,the positive rates of CD117 on MC surface were expressed as (94.8 ± 1.3)％,(95.7 ±2.5)％,(94.1 ± 1.3)％,(96.6 ± 1.0)％,and (96.6 ± 1.1)％,respectively,and there was no significant difference among these groups (F =8.51,P ＞ 0.05).The positive rates of FcεR Ⅰ α were expressed as (81.5 ±2.6)％,(84.2 ± 1.8)％,(91.8 ±2.0)％,(91.6 ± 1.6)％,and (93.0 ± 2.6) ％,respectively,and there was statistically increasing among these groups (F =15.76,P ＜ 0.05).Then MC were activated by SP (0,0.0l,0.1,1.0,10 mg/L),histamine from 20 μg/L IL-4 group were released (20.08±1.50)％,(32.76 ±2.99)％,(42.90 ±3.36)％ 、(50.21 ±1.29)％,(56.10± 3.60)％,as similar as from 0 μg/L IL-4 were (19.37 ±2.02),(19.50 ±1.50),(21.77 ±1.91),(32.00 ±2.50),(33.56 ± 1.25),there was significantly different when compared with each other (all P ＜0.05).Bone marrow MC were shown to have the highest expression of FcεR Ⅰ α and NK-1R in culture of 20 μg/L IL-4 by the detection of Western blot,meanwhile these MC could be activated to degranulate by a lower concentration of SP (0.01 mg/L),with the release rate of histamine from MC showing a positive correlation with SP concentrations.On the other hand,MC with high expression of FcεR Ⅰα and little expression of NK-1 R cultured with 0 μg/L IL-4,could also be activated by a much higher concentration of SP (1.0 mg/L).Conclusions Bone marrow mast cells were shown to be successfully differentiated and to express NK-1R and FceR Ⅰ α upon co-culture with SCF and IL-3 or SCF,IL-3 and IL-4.When IL-4 was added into RPMI 1640,bone marrow MC could highly produce FcεR Ⅰ α and NK-1 R,thus building a better model of MC degranulation regulated by SP.And SP-controlled MC degranulation may be mediated through both FcεR Ⅰ α (immunologically) and NK-1R (non-IgE mediated or non-immunologically) pathway.
