【作者】 黄翠丽；

【导师】 林有坤；

【作者基本信息】 广西医科大学 ， 皮肤性病学， 2012， 硕士

【摘要】 目的：初步探讨IL-10修饰的未成熟树突状细胞与CTLA-4Ig联合应用对狼疮鼠的影响及其机制,为免疫疫苗治疗SLE的进一步研究打下基础。方法：1、IL-10修饰的狼疮小鼠骨髓源性imDC培养：无菌提取小鼠股骨及胫骨骨髓细胞进行体外原代培养。在培养至第5d时将实验分为4组：(1)6-DC组：继续培养至第6d；(2)9-DC组：继续培养至第9d；(3)6-IL-10-DC组：于第5d换液时加入IL-10继续培养至第6d；(4)9-IL-10-DC组：于第5d换液时加入IL-10,以后仍隔天换液并每次均加入IL-10,培养至第9d。倒置显微镜下观察细胞形态和数量的变化。流式细胞仪鉴定细胞的纯度和成熟度(CD11c为小鼠DC表面的特异性标志,因此用其鉴定细胞及其纯度,CD80、CD86及MCHⅡ则随着其成熟而增多。)2、IL-10修饰的imDC与CTLA-4Ig联合应用对同系狼疮鼠的作用：选取4个月大小雌性B6.MRL-Faslpr/J狼疮小鼠30只,随机分为4组,V1-V3组每组7只,V4组9只。V1组：静脉注射6-IL-10-imDC;V2组：静脉注射6-IL-10-imDC+CTLA-4Ig；V3组：静脉注射CTLA-4Ig;V4组：静脉注射等体积PBS即安慰剂对照组；V5组：选取6只4个月大小的雌性C57BL/6J小鼠：仅在相同条件下饲养而未予任何干预,即为正常对照组。检测各组小鼠24h尿蛋白、血清中ANA和抗dsDNA抗体的含量、血清中IL-17A的含量及脾脏中Th17细胞和Treg细胞的比例,比较各组的差异。结果：1、在培养的细胞中,应用流式细胞仪检测显示DCs的表面标志CD11c的阳性率达89.2%,即所培养的DCs的纯度达89.2%。而6-IL-10-DC表达MCHⅡ、CD40、CD86、CD80最低,9-CD表达MCHⅡ、CD86、CD80、CD40最高。其中6-DC与9-IL-10-DC差异无统计学意义(P>0.05),其余各组差异均具有统计学意义(均P<0.05)。2、干预前V1、V2、V3、V4组24h尿蛋白、血清中的ANA、抗ds-DNA抗体比较差异无统计学意义(均P>0.05),均高于V5组,差异有统计学意义(均P<0.05)。3、末次干预2W后V1、V2、V3组的24小时尿蛋白、血清ANA及抗ds-DNA均较干预前下降,差异均有统计学意义(均P<0.05),而V5组观察开始与结束时以上指标差异均无统计学意义(均P>0.05),V4组干预后24h尿蛋白较前升高,差异有统计学意义(P<0.05),而其血清ANA及血清抗ds-DNA抗体干预前后差异均无统计学意义(均P>0.05)。4、末次干预2W后,各组24h尿蛋白定量由低到高依次为V5组(758.67±357.20)μg、V2组(788.29±114.44)μg、V3组(1036.86±155.14)μg、V1组(1059.43±141.57)μg、V4组的(1692±252.60)μg。以上各组24h尿蛋白比较差异有统计学意义(F=23.738,P=0.000),两两比较发现V1组与V3组、V2组与V5组差异无统计学意义(均P>0.05),其余组间差异均有统计学意义(均P<0.05)。血清ANA抗体水平V1组为(10.36±1.68)pg/ml、V3组为(10.10±1.42)pg/ml、V2组为(9.95±0.98)pg/ml,V1-V3组均较V4组的(13.37±2.95)pg/ml低,但高于V5组的(7.65±1.82)pg/ml,以上各组ANA抗体比较差异有统计学意义(F=8.090,P<0.001),但其中V1组与V2、V3组,V2组与V3组差异均无统计学意义(均P>0.05),其余组间差异有统计学意义(均P<0.05)。血清抗ds-DNA抗体水平V1组为(17.716±1.89)IU/ml、V3组为(17.34±2.37)IU/ml,高于V2组的(13.11±2.10)IU/ml,V1-V3组均较V4组的(23.61±4.71)IU/ml低,但高于V5组的(11.24±1.53)IU/ml,以上各组抗ds-DNA抗体水平比较差异有统计学意义(X2=23.994,P<0.001),SNK两两比较发现V1组与V3,V2与V5差异均无统计学意义(均P>0.05),而其余组间差异均有统计学意义(均P<0.05)。5、末次干预2W后血清中IL-17A的浓度V3组为(176.61±27.61)pg/ml,V1组的为(174.57±25.39)pg/ml,均高于V2组的(139±0.98)pg/ml,但V1-V3组的均较V4组的(300.53±84.09)pg/ml低,而较V5组的(118.80±30.35)pg/ml高,以上各组血清中IL-17A的浓度比较差异有统计学意义(X2=26.415,P<0.001),其中SNK两两比较示V1组与V3,V2与V5差异均无统计学意义(均P>0.05),而其余组间差异均有统计学意义(均P<0.05),包括V4组较V5组明显升高。Vl组的脾脏淋巴细胞Th17细胞的比例为(2.20±0.47)%、V3组的为(2.12±0.47)%,均高于V2组的(1.64±0.30)%,V1-V3组均低于V4组的(3.483±1.01)%,较V5组的(1.01±0.49)%的高,以上各组脾脏淋巴细胞Th17细胞的比例的浓度比较差异有统计学意义(X2=23.177,P<0.001),其中SNK两两比较示V1组与V3,V2与V5差异均无统计学意义(均P>0.05),而其余组间差异均有统计学意义(均P<0.05),包括V4组较V5组明显升高。6、V4组狼疮小鼠的脾脏Th17细胞的比例与血清中的IL-17A、ds-DNA抗体及24h尿蛋白呈正相关,与血清中的ANA无相关；血清中的IL-17A含量与抗ds-DNA及24h尿蛋白呈正相关,与血清中的ANA无相关。7、末次干预2W后脾脏淋巴细胞Treg细胞的比例V2组为(6.39±0.87)%,高于V1组的(3.70±1.02)%及V3组的(3.55±1.12)%,V1-V3组均高于V4组的(1.88±0.69)%,均低于V5组的(10.95±1.85)%。以上各组脾脏淋巴细胞Treg细胞的比例比较差异有统计学意义(F=67.065,P<0.001),其中V1组与V3组差异无统计学意义(P>0.05),其余组间差异均有统计学意义(均P<0.05)。结论：IL-10修饰的imDC、CTLA4-Ig或IL-10修饰的imDC与CTLA4-Ig联合应用输入同基因狼疮鼠体内,均可以诱导其产生免疫耐受,能使狼疮鼠血清ANA抗体、ds-DNA抗体及24h尿蛋白明显下降。且联合应用对于狼疮鼠的干预效果优于两者单独应用。下调Th17细胞及其主要细胞因子IL-17A的水平、上调Treg细胞的表达可能是IL-10修饰的imDC.CTLA4-Ig或两者联合应用对B6.MRL-Faslpr/J狼疮小鼠干预作用的重要免疫学机制之一。血清中的IL-17A水平有可能会成为判断SLE病情严重程度和疗效的新指标。更多还原

【Abstract】 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.更多还原