Research On Expression Of Innade Cholinergic System And LTB4-BLT2 In DC And It's Role In JIA Inflammatory Reaction

Part I Induction and Cultivation of Bone Marrow(MB)-derived Dendritic cell from MiceObjective:To establish the method of isolation bone marrow (BM)-derived DC by optimizing condition, study the morphological structure and bio-characteristics during the induction and identification maturation. Methods:1. One marrow (BM)-derived DC from healthy BALB/c mice were incubated and differentiated with rmGM-CSF and rmIL-4, and stimulated to mature with LPS in vitro.2. The change of DC form was observed by invert light microscope.3. The marker molecule was identified by flow cytometry.4. ELISA method was used to measure IL-12 concentration in DC cultivation supernatan.5. Cells were digested with 0.4% fresh trypsin at 37°c. Results:1. Adherent cells were observed 4h later. On the second day, a few cell processes were observed. 2d later, cells became irregular, bigger and the processes increased gradually, showing the morphology of immature DC. Then, 6d later, the cell changed obviously with LPS, showing a typical dendritic morphology of mature DC. Meanwhie, the percentage of dendritic cell population was observed to 85-90%.2. Flow cytometic analysis showed that expression percentage (%) of relative specific markers CD_11c , MHC class II, costimulatory molecules (ie, CD80, CD86) before and after maturity were 41.783 ± 6.106, 32.300 ± 4.586, 21.683 ± 2.875, 27.523 ± 3.708 and 75.687 ± 11.671, 71.767 ± 9.199, 63.293 ± 8.119,71.970 ± 10.485, respectively. Mature DC was increasing more significantly ascompared with immature DC (p<0.01). 3. IL-12 concentration of DC culture supernates in immature DC and mature DC was37.7±7.6 pg/ml and 712±134.425 pg/ml, respectively. The IL-12 concentration inmature DC culture supernates was higher than immature DC (p<0.01). Conclusion:Bone marrow cells can be cultivated into immature and mature DC successfully by induction and differentiation with rmGM-CSFand rmIL-4, and stimulation with LPS by optimizing condition. The method was good to induction and differentiation, cell growth and further research. Part II Expression of innate Cholinergic System and LTB4-BLT signalling pathway in DC(1). Expression of cholinergic system (nAChR α 7, ChAT and AChE) in mature DC and effects of AChR α 7 by MECObjective:To probe whether there was expression of nicotinic acetylcholine receptor subunit α 7(nAChR α 7), choline acetyltransferase (ChAT), acetylcholinesterase (AChE) and its regulation in mature dendritic cell(DC). Method:1. Fixing expression on protein of cholinergic system (nAChR α 7, ChAT and AChE) were measured with immuocytochemistry and immunofluorescence methods.2. Fixed quantity expression on protein of cholinergic system and nAChR α 7 expression with mecamylamine (MEC) in 12h was measured by flow cytometry.3. mRNA expression of nAChR α 7, ChAT and AChE was measured by RT-PCR method.Results:1. nAChR α 7 distributed principally in DC membrane, while ChAT and AChE in DC cytoplasm. 2.Protein expression percentage (%) of cholinergic system (nAChR α 7 , ChAT and AChE) was 41.267 ± 3.470, 37.467 ± 4.441 and49.867 ± 5.620, respectively. Expression of AChE was stronger as compared with ChAT (p<0.05), and there was a trend toward increasing as compared with nAChR α 7.2. mRNA expression of cholinergic system nAChR α 7, ChAT and AChE in DC was observed.3. The expression of nAChR α 7 was down regulated by MEC as compared with the group without MEC (p<0.05).Conclusion:1. An innate cholinergic system existed in mature DC.2. The innate cholinergic system in DC may be relate to immunomodulation.3. The functional status of innate cholinergic system in DC was affected by extrinsic factor (i. e, MEC).(2) Expression of LTB4—BLT signalling pathway in DCObjective:To investigate LTB4-BLT signalling pathway (LTB4-BLT1 and LTB4-BLT2) in DC Methods:1. ELISA method was used to measure LTB4 concentration in culture supernates of immature DC and mature DC.2. Both immunocytochemistry method and immunofluorescent antibody technique were used to measure protein-fixing expression of BLT2 in mature DC.3. RT-PCR method was used to measure expression of BLT (including BLT1, BLT2) in immature and mature DC.Results:1. LTB4 concentration was 10.667 ± 2.394pg/ml, 7.089 ± 1.810pg/ml, 3.222 ± 0.995pg/ml and 14.217 ±3.396pg/ml respectively, in 2d, 4d, 6d (immaturation) and 8d (maturation) during the course of DC induction, differentiation and maturation, respectively. LTB4 level showed a trend toward decreasing progressively during DC differentiation. While, LTB4 concentration in mature DC cultivante supernatant was higher than immature DC (p<0.05).2. BLT2 distributed in DC cytoplasm. Especially, expression was more obviously in cytoplasmic organoids.3. The expression level of BLT1 mRNA and BLT2 mRNA was stronger in mature DC than immature DC (p<0.05) respectively, which were measured by semiquantitative RT-PCR assay.Conclusions:1. LTB4-BLT signalling pathway in DC was possiblly established by its LTB4 secretion and BLTexpression.2. Both signalling pathway of LTB4-LTBland LTB4-LTB2 may be one cause, which resulted in differentiation and maturation of DC.3. Both signalling pathway may be play an important role in inflammatory reaction and immunoregulation.Part III The Role of Inflammatory Reaction in JIA of InherentCholinergic System and LTB4-BLT2 Signalling Pathway in DCObjective:To investigate a role of inflammatory reaction in JIA of innade cholinergic system and LTB4-BLT2 signalling pathway in DC Methods:1. Flow cytometry was used to measure the expression of nAChR α 7 and BLT2 in DC with normal serum, JIA active phase serum and JIA active phase serum+MEC, respectively.2. ELISA method was used to measure IL-12 and LTB4 concentration of DC cultivante supernatant in normal serum group, JIA active phase serum group and JIA active phase serum+ MEC group, respectively.3. MTT method was used to measure lymphocyte proliferation from mice splenic in DC cultivation supernatan + normal serum group, DC culture supernates + JIA active phase serum group, DC culture supernates + JIA active phase serum +MEC group, respectively. 4. Flow cytometry was used to mensurate CD69 expression of micespleniclymphocyte in DC culture supernates in normal serum group, DC culturesupernates +JIA active phase serum group, DC culture supernates + JIA activephase serum + MEC group, respectively. Results1. Expression percentage (%) of DC nAChR α 7 was 39.80±4.12, 52.66±7.99 and 45.49 ± 7.73 at 18h in normal serum group, JIA active phase serum group and JIA active phase serum+ MEC group, respectively. The expression was stronger in JIA active phase serum than normal serum group (p<0.05). However, the expression was inhibited in JIA active phase serum+ MEC group.2. Expression percentage of BLT2 in DC was 27.57 ± 2.989, 45.947 ± 8.068 and 67.343 ± 11.394 in normal serum group, JIA active phase serum group and JIA active phase serum + MEC group, respectively at 18h. The expression was stronger in JIA active phase serum group than normal serum group (p<0.05). JIA active phase serum+ MEC group was further stronger than normal serum group (p<0.01).3. Before intervention, IL-12 concentration was actual cytokine expression in serum. The concentration of IL-12 in JIA active phase serum group and normal serum group was 975.525 ± 206.638pg/ml and 23.823 ± 3.555pg/ml, respectively. The expression level of IL-12 was higher in JIA active phase serum group than normal serum group (p<0.01). Then, at 18h, the concentration of IL-12 was 48.893 ± 9.562pg/ml, 1281.175 ± 228.586pg/ml and 1618.075 ± 292.81pg/ml in normal serum group, JIA active phase serum group and JIA active phase serum+ MEC group, respectively. Accordingly, the expression level of IL-12 was highest in JIA active phase serum with MEC group of three groups, and it was second in JIA active phase serum group, compared with JIA active phase serum with MEC group (p<0.05).4. Before intervention, LTB4 concentration was actual cytokine in serum. The expression level of LTB4 in JIA active phase serum group and normal serum group was 82.375±19.9pg/ml and 16.9±2.953pg/ml, respectively. The expression was higher in JIA active phase serum group than normal serum group (p<0.01). Then, at 18h, the concentration of LTB4 was 24.225±6.264pg/ml, 114.575±20.375pg/ml and142.05±24.574pg/ml in normal serum group, JIA active phase serum group and JIA active phase serum+ MEC group, respectively. The expression level was obviously higher in JIA active phase serum group and JIA active phase serum with MEC group than normal serum group (p<0,01). Meantime, the expression in JIA active phase serum with MEC group was also stronger than JIA serum group (p<0.05).5. Stimulation index ( SI ) to lymphocyte proliferation from mouse spleenwasl.234±0.276, 3.053±0.432 and .3.672±0.817 in DC culture supernates with normal serum group, DC culture supernates with JIA active phase serum group, DC culture supernates with JIA active phase serum and MEC group, respectively. The role of lymphocyte proliferation was the most manifest in DC culture supernates with JIA active phase serum and MEC group of three. Then, the role was the second in JIA active phase serum group, comoared with JIA active phase serum and MEC group (p<0.05).6. Expression percentage of CD69 with low dose ConA in 8h in lymphocyte was 11.075±2.812, 34.35 ± 5.17 and 43.35±7.12 in DC culture supernates with normal serum group, DC culture supernates with JIA active phase serum group, DC culture supernates with JIA active phase serum and MEC group, respectively. The expression was higher in DC culture supernates with JIA active phase serum group, DC culture supernates with JIA active phase serum and MEC group, compared with DC culture supernates with normal serum group (p<0.01). Meanwhile, The CD69 expression was also higher in DC culture supernates with JIA active phase serum and MEC group than JIA active phase serum without MEC group (p<0.05).Conclusions:1. JIA active phase serum could stimulate expression of nAChR α 7 and BLT2 in DC. However, MEC may inhibit nAChR α 7 expression to inhibit the anti-inflammatory effect of cholinergic system, which may be aggratate inflammatory reaction by stimulating BLT2.2. DC cholinergic system may be play anti-inflammatory effect by inhibiting the secretion of IL-12 and LTB4 in DC.3. Excessive activation and proliferation of immunological cell and augmentation of inflammatory reaction may be resulted in by disorder of cholinergic system andactivation of LTB4-BLT2 signalling pathway during the pathological course inJIA.