| Objective:To investigate effects and the mechanism of interleukin-10-treated immature dendritic cells unite CTLA4-Ig on lupus-prone B6.MRL-Faslpr/J mice.To explore new methods of immuneization to treat SLE.Methods:1.To culture interleukin-10-treated immature dendritic cells of upus-prone mice:With erythrocyte lysis buffer of mice,we extracted precursors of dendritic cells from mice bone marrow. We started cell primary culture by induction of cytokines. At the fifth day of the culture, the experiment was divided into four groups:(1)the sixth day-DC:Continue to culture to the sixth day;(2)the nith day-DC:Continue to culture to the ninth day;(3)the sixth day IL-10-treated-DC:medium was changed on5th day of IL-10,and cultuerd to sixth day;(4)the ninth day IL-10-treated-DC:medium was changed on5th day of IL-10, and later still the next day the medium was changed and each of IL-10, cultured to ninth day.We compared differences in morphology and phenotype among the four groups.2. Effects of interleukin-10-treated immature dendritic cells unite CTLA4-Ig on lupus-prone B6. MRL-Faslpr/J mice:select30four mouths old female lupus-prone B6.MRL-Faslpr/J mice, trials were divided into four groups, V1～V3group:seven per group,V4was nine.V1group:injected6-IL-10-imDC though tail vein;V2group:injected6-IL-10-imDC+CTLA-4Ig though tail vein;V3group:injected CTLA-4Ig though tail vein;V4group:intravenous injection of equal volume of PBS naming placebo control group.V5group: select six four moths old female C57BL/6J mice only reared under the same conditions has not been any interventionas called normal control group. Detecting each group’s concentration of IL-17A, ANA and anti-ds-DNA antibody in the mice’s serum and the mice’s24h urine protein and the percentage of Th17,Treg cells in homogenate of spleens and compare the differences of each group.Results:1. In cultured cells, detected by flow cytometry of DCs surface markers CD11c positive rate of89.2%, the purity of the cultured DCs was89.2%.6-IL-10-to-DC expressed MCHⅡ, CD40, CD86, CD80were lowest,and the expression of MCHⅡ, CD86, CD80, CD40in9-CD were highest. Difference was all statistically significant (all P<0.05).2.Before the intervention, the mice’s24h urine protein and the concentration of IL-17A,ANA and anti-ds-DNA antibody in the mice’s serum were all not significantly statistic difference between V1-V4group(all P>0.05),and they were all hight than V5group,the difference was statistically significant(all P<0.05). 3. After two weeks of the last intervention,V1and V2,V3group,the24-hour urine protein and the concentration of serum’s ANA and anti-ds-DNA antibody compared with before intervention all decreased, the difference were all statistically significant(all P<0.05).The difference of the above indexs of V5group before and after the observation were all not statistically significant(all P>0.05).After intervention,V4group’s24-hour urine protein increased,the difference was statistically significant(P<0.05), serum’s ANA and anti-ds-DNA antibody before and after the intervention were all not significant difference(all P>0.05).4. After two weeks of the last intervention,each groups24h urinary protein from low to high followed by:V5group which was(758.67±357.20)μg,V2group which was(788.29±114.44)μg,the V3group was (1036.86±155.14) μg,the V1group was(1059.43±141.57)μg and theV4group which was (1692±252.60) μg, each group24h urinary urine protein were statistically significant different (F=23.738,P<0.001),where V1group V3group differences were not statistically significant(P>0.05),among other groups were all statistically significant(all P<0.05). The ANA antibody of the V1group was (10.36±1.68) pg/ml and V3group was (10.10±1.42) pg/ml and V2group was (9.95±0.98) pg/ml, they were all lower than V4group which was (13.37±2.95) pg/ml and higher than V5group (7.65±1.82) pg/ml.The difference of the ANA antibodies of the above groups were statistically significant.(F=8.090,P=0.000), of which the V1and V2, V3, V2group and the V3group were all not statistically significant differences(all P>0.05),and among other groups were all statistically significant(all P<0.05).The anti-ds-DNA antibody of the V1group was (17.716±1.89) IU/ml and the V3group was (17.34±2.37) IU/ml, higher than the V2group which was (13.11±2.10) IU/ml,V1-V3groups’s were all lower than V4 group which was (23.61±4.71) IU/ml and all higher than and V5group which was (11.24±1.53) IU/ml.The difference of the anti-ds-DNA antibodies of the above groups were statistically significant (X2=23.994,P<0.001),which use SNK two two comparison show that V1group and the V3group, V2and V5were all not statistically significant differences (all P>0.05), and among other groups were all statistically significant differences (all P<0.05).5. After two weeks of the last intervention,the IL-17A of the V3group was(176.61±27.61)pg/ml and the V1group was(174.57±25.39)pg/ml, higher than the V2group which was(139±0.98)pg/ml, V1-V3groups’s were all lower than V4group which was (300.53±84.09)pg/ml and all higher than and V5group which was (118.80±30.35) pg/ml.The difference of IL-17A of the above groups were statistically significant differences (X2=26.415,P<0.001), which used SNK two two comparison showed that V1group and the V3group, V2and V5were all not statistically significant differences(all P>0.05), and among other groups were all statistically significant differences (all P<0.05), including the V4group were significantly higher than V5group. The percentage of Th17cells in homogenate of spleens of the V1group was(2.20±0.47)%and V3group was(2.12±0.47)%,higher than the V2group (1.64±0.30)%,and V1-V3groups‘s were all lower than V4group which was (3.483±1.01)%and higher than V5group which was(1.01±0.49)%.The difference of the percentage of Th17cells in homogenate of spleens of the above groups were statistically significant differences(X2=23.177,P<0.001),which used SNK two two comparison showed that V1group and the V3group, V2and V5were all not statistically significant differences(all P>0.05), and among other groups were all statistically significant differences(all P<0.05), including the V4group were significantly higher than V5group. 6. The percentage of Th17cells in homogenate of spleens of V4group’s lupus-prone B6.Fas mice was positively correlated with IL-17A and anti-ds-DNA antibody in serum and the24h urine protein,however it was not correlated with ANA in serum, And IL-17A in serum which is the main secrete factor of Th17cells was also positively correlated with anti-ds-DNA antibody in serum and the24h urine protein, and it was also not correlated with ANA in serum.7. After two weeks of the last intervention,the percentage of Treg cells in homogenate of spleens of the V2group was(6.39±0.87)%, higher than the VI group which was(3.70±1.02)%and V3group which was(3.55±1.12)%and each of the V1～V3group’s was higher than V4group which was(1.88±0.69)%and was lower than V5group which was(10.95±1.85)%.The percentage of Treg cells in homogenate of spleens were statistically significant different (F=67.065, P<0.001), where V1group V3group’s difference was not statistically significant(P>0.05),and among other groups were all statistically significant(all P<O.05).Conclusions:interleukin-10-treated imDC, CTLA4-Ig or interleukin-10-treated imDC unite CTLA4-Ig injiected into the same genetic backgroup lupus mice can all induce immune tolerance and make the lupus mouse serum ANA antibody, ds-DNA antibodies and24-hour urinary protein decreased significantly. And the intervention effect of the unite application on the lupus mice was better than the two alone.The decline of Th17cells and IL-17A in serum which is the main secrete factor of Th17and increase Treg cells may be one of the important immunological mechanisms for the role of the intervention effect of interleukin-10-treated imDC, CTLA-4Ig or the two united to B6.MRL-Faslpr/J lupus mice.IL-17A levels in serum may become a new indicator to determine the severity and efficacy of SLE disease. |
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