TIM-3分子在器官移植免疫中的作用基础和临床研究初探

发布时间：2018-05-12 00:27

本文选题：TIM-3 + 肾移植　；参考：《中国人民解放军军医进修学院》2012年博士论文

【摘要】：肾移植患者术后外周血TIM-3mRNA水平的检测对急性排斥反应的诊断及预后判断的价值目的：考察TIM-3mRNA表达水平的监测在肾移植术后急性排斥反应的诊断和预后评估方面的作用，为肾移植后急性排斥反应的无创性诊断寻求潜在的新的预警分子。方法：本研究中包含AR组(急性排斥反应组，n=24)，NO-AR组(非急性排斥组，n=20)。连续留取手术后血液样本（术后1，4，7，14，21天）和急性排斥反应时血液样本（冲击治疗前、治疗后1，4，7天）。另肾移植术后满1年，肾功能良好患者作为Stable组（肾功能稳定组，n=18）以进一步对照。RT-PCR的方法检测TIM-3mRNA的表达水平。ROC曲线检验TIM-3mRNA表达水平在诊断急性排斥反应时的灵敏度和特异度。相关性分析考察TIM-3mRNA表达与肾移植术后血肌酐水平的关系。结果：TIM-3mRNA在急性排斥反应发生时表达水平明显升高，与NO-AR组和Stable组相比均有显著性差异（286.72±86.28vs126.10±28.31，286.72±86.28vs96.91±17.88, p=0.00）。ROC曲线分析TIM-3mRNA表达水平在诊断急性排斥反应时的灵敏度和特异度分别可达到90.91%和90.00%。抗排斥治疗后TIM-3mRNA表达水平下降。TIM-3mRNA表达水平与肾移植术后血肌酐水平有较好的相关性(r~2=0.716, p=0.00)。结论：肾移植术后患者外周血中TIM-3mRNA表达水平的检测可以作为无创诊断急性排斥反应的指标。但是TIM-3mRNA在急排冲击治疗后的表达趋势并不能判定急性排斥反应的预后，这主要是因为TIM-3mRNA在急性排斥患者不同个体间表达差异很大所致。 Galectin-9腹腔注射对同种异体小鼠皮肤移植物存活的影响及可能的分子机制目的：研究TIM-3分子配体Galectin-9腹腔注射对同种异基因小鼠移植皮片存活的影响；并初步研究其对体内Teff和Treg免疫平衡的调控作用。方法：60只C57BL/6小鼠（受者），30只BALB/c小鼠（供者），建立同种异基因小鼠皮肤移植模型。对照组C57BL/6小鼠术后连续7天给予生理盐水腹腔注射；实验组C57BL/6小鼠术后连续7天给予Galectin-9100ug腹腔注射。术后连续记录并留取影像资料以观察移植皮片的存活时间，累积生存函数计算皮片MST（Median Survival Time，中位存活时间）；术后7天移植皮片HE染色观察皮片内的淋巴细胞浸润情况；FCM（流式细胞术）检测小鼠脾脏、外周血CD4~+TIM-3~+T、CD4~+TIM-1~+T、Th17、CD4~+CD25~+Foxp3~+Treg所占T淋巴细胞亚群的比例；ELISA法检测外周血细胞因子IL-2、IL-4、IL-17及IFN-γ的表达水平。结果： 1. Galectin-9腹腔注射可以延长同种异体小鼠皮肤移植物的存活时间，对照组和实验组移植皮片的MST分别为12.5天和16天； 2. Galectin-9腹腔注射减轻同种异基因皮肤移植术后第7天移植皮片内的淋巴细胞浸润； 3. Galectin-9腹腔注射对体内Teff细胞比例有下调的作用，但对Treg细胞比例无影响。不同淋巴细胞亚群变化如下： ①CD4~+TIM-3~+T实验组术后7天脾脏组织和外周血的比例均下降（脾4.82±1.01%vs2.84±0.89%，血7.13±1.82%vs4.94±1.56%，p0.05）；术后14天无差异（脾3.34±1.10%vs3.44±0.98%，血2.79±0.87%vs2.67±0.84%，p0.05）。 ②Th17实验组术后7天，外周血的比例下降（12.42±3.67%vs5.79±1.68%，p0.05），脾脏组织Th17无变化（5.54±1.38%vs5.78±1.64%，p0.05）；术后14天，外周血中比例无差异（5.21±1.96%vs5.91±2.03%，p0.05），实验组脾Th17比例下降（4.61±1.78%vs7.91±2.34%，p0.05）。 ③CD4~+TIM-1~+T脾脏组织中的比例术后7天，14天均无差异(7天3.79±1.35%vs4.27±1.52%；14天3.19±1.26%vs3.92±1.34%，p0.05)；外周血中7天无差异（34.74±4.80%vs34.50±5.00%，p0.05），14天时实验组比例有上升（52.80±8.79%vs34.86±5.67%，p0.05）。 ④CD4~+CD25~+Foxp3~+Treg术后7天、14天其在脾脏组织和外周血淋巴细胞中的比例均无差异。脾（7天10.57±2.56%vs10.02±3.04%，14天11.49±2.87%vs12.93±3.12%，p0.05）；血（7天8.00±1.78%vs8.20±2.01%，14天9.76±2.45%vs10.90±2.67%，p0.05）。 4. Galectin-9腹腔注射可以显著降低术后7天外周血IFN-γ（51.33±8.16pg/ml vs98.41±15.01pg/ml，p=0.00）和IL-17的表达水平（48.50±8.48pg/mlvs15.17±5.46pg/ml，p=0.00）；但对IL-2（23.17±4.22pg/ml vs26.50±3.78pg/ml，p=0.180）和IL-4(86.83±13.33pg/ml vs99.67±11.64pg/ml，p=0.107)表达水平无影响。结论：Galectin-9腹腔注射可以延长同种异体小鼠皮肤移植的存活时间，并明显减轻同种异体小鼠皮肤移植物中的淋巴细胞浸润；该作用与其特异性下调Th17和CD4~+TIM-3~+T有关；同时Galectin-9不改变CD4~+CD25~+Foxp3~+Treg细胞在CD4~+T淋巴细胞群中的比例
[Abstract]:The value of the detection of TIM - 3 mRNA in peripheral blood after renal transplantation in the diagnosis and prognosis of acute rejection

Objective : To investigate the role of TIM - 3 mRNA expression in the diagnosis and prognosis of acute rejection after renal transplantation , and to find potential new early - warning molecules for non - invasive diagnosis of acute rejection after renal transplantation .

Methods : The study included AR group ( acute rejection group , n = 24 ) , NO - AR group ( non - acute rejection group , n = 20 ) . Blood samples ( 1 , 4 , 7 , 14 , 21 days after operation ) and acute rejection were continuously retained after operation ( 1 , 4 , 7 , 14 , 21 days after treatment ) . The expression level of TIM - 3 mRNA was detected by RT - PCR . The expression level of TIM - 3 mRNA was determined by RT - PCR . The relationship between TIM - 3 mRNA expression and serum creatinine level in renal transplantation was investigated by ROC curve .

Results : The expression level of TIM - 3 mRNA in acute rejection was significantly higher than that in NO - AR group and stable group ( 286 . 72 卤 86.28 vs 126 . 10 卤 28.31 , 286 . 72 卤 86.28 vs 96.91 卤 17.88 , p = 0.00 ) . The sensitivity and specificity of TIM - 3 mRNA in diagnosis of acute rejection were 90.91 % and 90.00 % , respectively . TIM - 3 mRNA expression level of TIM - 3 mRNA decreased after anti - rejection treatment ( r ~ 2 = 0.716 , p = 0.00 ) .

Conclusion : The expression level of TIM - 3 mRNA in peripheral blood of patients after renal transplantation can be used as an index for non - invasive diagnosis of acute rejection . However , the expression of TIM - 3 mRNA in acute rejection after acute rejection can not determine the prognosis of acute rejection .

Effect of Galectin - 9 Intraperitoneal Injection on the Survival of Skin Graft in Mice and Possible Molecular Mechanism

Objective : To study the effect of TIM - 3 ligand Galectin - 9 intraperitoneal injection on survival of allogeneic mice .
The regulation of Teff and Treg in vivo was studied .

Methods : 60 C57BL / 6 mice ( recipients ) and 30 BALB / c mice ( donors ) were established to establish a model of allogeneic mouse skin transplantation .
Galectin - 9100ug was injected intraperitoneally for 7 days after operation in experimental group C57BL / 6 mice . Image data was recorded continuously and the survival time of graft skin was observed . The cumulative survival function was used to calculate MST ( Median Survival Time , median survival time ) .
After 7 days post - operation , HE staining was used to observe the infiltration of lymphocytes in the skin .
FCM ( flow cytometry ) was used to detect the percentage of T lymphocyte subsets in spleen , peripheral blood CD4 ~ + TIM - 3 ~ + T , CD4 ~ + TIM - 1 ~ + T , Th17 , CD4 ~ + CD25 ~ + Foxp3 ~ + Treg .
The levels of IL - 2 , IL - 4 , IL - 17 and IFN - 纬 in peripheral blood were detected by ELISA .

Results :

1 . Intraperitoneal injection of Galectin - 9 could prolong the survival time of allograft skin grafts , and MST in control group and experimental group were 12.5 days and 16 days , respectively .

2 . Intraperitoneal injection of Galectin - 9 alleviated the infiltration of lymphocytes in skin grafts on the 7th day after allogeneic skin transplantation .

3 . The percentage of Teff cells in vivo was downregulated by intraperitoneal injection of Galectin - 9 , but there was no effect on the proportion of Treg cells . The changes of lymphocyte subsets were as follows :

( 1 ) The proportion of spleen and peripheral blood was decreased 7 days after operation of CD4 ~ + TIM - 3 ~ + T test group ( spleen 4.82 卤 1.01 % vs 2.84 卤 0.89 % , blood 7.13 卤 1 . 82 % vs 4.94 卤 1 . 56 % , p . 05 ) ;
There was no difference 14 days after operation ( spleen 3.34 卤 1.10 % v3.44 卤 0.98 % , blood 2.79 卤 0.87 % v2.67 卤 0.84 % , p0.01 ) .

In Th17 experimental group , the proportion of peripheral blood decreased ( 12.42 卤 3.67 % vs 5.79 卤 1 . 68 % , p < 0.05 ) . Th17 in spleen tissue was not changed ( 5.54 卤 1.38 % vs 5.78 卤 1.64 % , p < 0.05 ) .
The proportion of spleen Th17 in the experimental group was decreased ( 4.61 卤 1.78 % vs 7.91 卤 2.34 % , p < 0.05 ) .

( 3 ) There was no difference in the proportion of CD4 ~ + TIM - 1 ~ + T spleen in 7 days and 14 days ( 3.79 卤 1.35 % vs 4.27 卤 1.52 % ) .
3.19 卤 1.26 % vs3 . 92 卤 1 . 34 % , p < 0 . 05 ) .
There was no difference in the peripheral blood for 7 days ( 34.74 卤 4.80 % vs 34.50 卤 5.00 % , P < 0.05 ) . The proportion of the experimental group increased ( 52.80 卤 8.79 % vs 34.86 卤 5.67 % , p0.01 ) at 14 days .

( 4 ) The ratio of CD4 ~ + CD25 ~ + Foxp3 ~ + Treg in spleen and peripheral blood lymphocytes was no difference between 7 days and 14 days . The spleen ( 10.57 卤 2.56 % vs 10.02 卤 3.04 % , 14 days 11.49 卤 2.87 % vs 12.93 卤 3.12 % , p0.01 ) ;
Blood ( 8 . 00 卤 1 . 78 % vs 8.20 卤 2 . 01 % , 14 - day 9.76 卤 2.45 % vs 10.90 卤 2.67 % , p . 05 ) .

4 . Intraperitoneal injection of Galectin - 9 significantly reduced the expression of IFN - 纬 ( 51.33 卤 8.16pg / ml vs 98.41 卤 15.01pg / ml , p = 0.00 ) and IL - 17 ( 48.50 卤 8.48pg / ml vs 15.17 卤 5.46pg / ml , p = 0.00 ) .
But the level of IL - 2 ( 23.17 卤 4.22pg / ml vs 26.50 卤 3.78pg / ml , p = 0.180 ) and IL - 4 ( 86.83 卤 13.33pg / ml vs 99.67 卤 11.64pg / ml , p = 0.107 ) had no effect .

Conclusion : Intraperitoneal injection of Galectin - 9 can prolong the survival time of allogeneic mouse skin graft and significantly reduce the infiltration of lymphocytes in skin grafts of allogeneic mice .
Th17 and CD4 ~ + TIM - 3 ~ + T were downregulated by this effect .
Galectin - 9 does not change the proportion of CD4 ~ + CD25 ~ + Foxp3 ~ + Treg cells in CD4 ~ + T lymphocyte population

【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R392

【参考文献】