本文選題：TIM-3 + 腎移植　； 參考：《中國人民解放軍軍醫(yī)進(jìn)修學(xué)院》2012年博士論文

【摘要】：腎移植患者術(shù)后外周血TIM-3mRNA水平的檢測對急性排斥反應(yīng)的診斷及預(yù)后判斷的價(jià)值 目的：考察TIM-3mRNA表達(dá)水平的監(jiān)測在腎移植術(shù)后急性排斥反應(yīng)的診斷和預(yù)后評估方面的作用，為腎移植后急性排斥反應(yīng)的無創(chuàng)性診斷尋求潛在的新的預(yù)警分子。 方法：本研究中包含AR組(急性排斥反應(yīng)組，n=24)，NO-AR組(非急性排斥組，n=20)。連續(xù)留取手術(shù)后血液樣本（術(shù)后1，4，7，14，21天）和急性排斥反應(yīng)時(shí)血液樣本（沖擊治療前、治療后1，4，7天）。另腎移植術(shù)后滿1年，腎功能良好患者作為Stable組（腎功能穩(wěn)定組，n=18）以進(jìn)一步對照。RT-PCR的方法檢測TIM-3mRNA的表達(dá)水平。ROC曲線檢驗(yàn)TIM-3mRNA表達(dá)水平在診斷急性排斥反應(yīng)時(shí)的靈敏度和特異度。相關(guān)性分析考察TIM-3mRNA表達(dá)與腎移植術(shù)后血肌酐水平的關(guān)系。 結(jié)果：TIM-3mRNA在急性排斥反應(yīng)發(fā)生時(shí)表達(dá)水平明顯升高，與NO-AR組和Stable組相比均有顯著性差異（286.72±86.28vs126.10±28.31，286.72±86.28vs96.91±17.88, p=0.00）。ROC曲線分析TIM-3mRNA表達(dá)水平在診斷急性排斥反應(yīng)時(shí)的靈敏度和特異度分別可達(dá)到90.91%和90.00%�？古懦庵委熀骉IM-3mRNA表達(dá)水平下降。TIM-3mRNA表達(dá)水平與腎移植術(shù)后血肌酐水平有較好的相關(guān)性(r~2=0.716, p=0.00)。 結(jié)論：腎移植術(shù)后患者外周血中TIM-3mRNA表達(dá)水平的檢測可以作為無創(chuàng)診斷急性排斥反應(yīng)的指標(biāo)。但是TIM-3mRNA在急排沖擊治療后的表達(dá)趨勢并不能判定急性排斥反應(yīng)的預(yù)后，這主要是因?yàn)門IM-3mRNA在急性排斥患者不同個(gè)體間表達(dá)差異很大所致。 Galectin-9腹腔注射對同種異體小鼠皮膚移植物存活的影響及可能的分子機(jī)制 目的：研究TIM-3分子配體Galectin-9腹腔注射對同種異基因小鼠移植皮片存活的影響；并初步研究其對體內(nèi)Teff和Treg免疫平衡的調(diào)控作用。 方法：60只C57BL/6小鼠（受者），30只BALB/c小鼠（供者），建立同種異基因小鼠皮膚移植模型。對照組C57BL/6小鼠術(shù)后連續(xù)7天給予生理鹽水腹腔注射；實(shí)驗(yàn)組C57BL/6小鼠術(shù)后連續(xù)7天給予Galectin-9100ug腹腔注射。術(shù)后連續(xù)記錄并留取影像資料以觀察移植皮片的存活時(shí)間，累積生存函數(shù)計(jì)算皮片MST（Median Survival Time，中位存活時(shí)間）；術(shù)后7天移植皮片HE染色觀察皮片內(nèi)的淋巴細(xì)胞浸潤情況；FCM（流式細(xì)胞術(shù)）檢測小鼠脾臟、外周血CD4~+TIM-3~+T、CD4~+TIM-1~+T、Th17、CD4~+CD25~+Foxp3~+Treg所占T淋巴細(xì)胞亞群的比例；ELISA法檢測外周血細(xì)胞因子IL-2、IL-4、IL-17及IFN-γ的表達(dá)水平。 結(jié)果： 1. Galectin-9腹腔注射可以延長同種異體小鼠皮膚移植物的存活時(shí)間，對照組和實(shí)驗(yàn)組移植皮片的MST分別為12.5天和16天； 2. Galectin-9腹腔注射減輕同種異基因皮膚移植術(shù)后第7天移植皮片內(nèi)的淋巴細(xì)胞浸潤； 3. Galectin-9腹腔注射對體內(nèi)Teff細(xì)胞比例有下調(diào)的作用，但對Treg細(xì)胞比例無影響。不同淋巴細(xì)胞亞群變化如下： ①CD4~+TIM-3~+T實(shí)驗(yàn)組術(shù)后7天脾臟組織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）；術(shù)后14天無差異（脾3.34±1.10%vs3.44±0.98%，血2.79±0.87%vs2.67±0.84%，p0.05）。 ②Th17實(shí)驗(yàn)組術(shù)后7天，外周血的比例下降（12.42±3.67%vs5.79±1.68%，p0.05），脾臟組織Th17無變化（5.54±1.38%vs5.78±1.64%，p0.05）；術(shù)后14天，外周血中比例無差異（5.21±1.96%vs5.91±2.03%，p0.05），實(shí)驗(yàn)組脾Th17比例下降（4.61±1.78%vs7.91±2.34%，p0.05）。 ③CD4~+TIM-1~+T脾臟組織中的比例術(shù)后7天，14天均無差異(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天無差異（34.74±4.80%vs34.50±5.00%，p0.05），14天時(shí)實(shí)驗(yàn)組比例有上升（52.80±8.79%vs34.86±5.67%，p0.05）。 ④CD4~+CD25~+Foxp3~+Treg術(shù)后7天、14天其在脾臟組織和外周血淋巴細(xì)胞中的比例均無差異。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腹腔注射可以顯著降低術(shù)后7天外周血IFN-γ（51.33±8.16pg/ml vs98.41±15.01pg/ml，p=0.00）和IL-17的表達(dá)水平（48.50±8.48pg/mlvs15.17±5.46pg/ml，p=0.00）；但對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)表達(dá)水平無影響。 結(jié)論：Galectin-9腹腔注射可以延長同種異體小鼠皮膚移植的存活時(shí)間，并明顯減輕同種異體小鼠皮膚移植物中的淋巴細(xì)胞浸潤；該作用與其特異性下調(diào)Th17和CD4~+TIM-3~+T有關(guān)；同時(shí)Galectin-9不改變CD4~+CD25~+Foxp3~+Treg細(xì)胞在CD4~+T淋巴細(xì)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 鹵 86.28 vs 126 . 10 鹵 28.31 , 286 . 72 鹵 86.28 vs 96.91 鹵 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 - 緯 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 鹵 1.01 % vs 2.84 鹵 0.89 % , blood 7.13 鹵 1 . 82 % vs 4.94 鹵 1 . 56 % , p . 05 ) ;
There was no difference 14 days after operation ( spleen 3.34 鹵 1.10 % v3.44 鹵 0.98 % , blood 2.79 鹵 0.87 % v2.67 鹵 0.84 % , p0.01 ) .

In Th17 experimental group , the proportion of peripheral blood decreased ( 12.42 鹵 3.67 % vs 5.79 鹵 1 . 68 % , p < 0.05 ) . Th17 in spleen tissue was not changed ( 5.54 鹵 1.38 % vs 5.78 鹵 1.64 % , p < 0.05 ) .
The proportion of spleen Th17 in the experimental group was decreased ( 4.61 鹵 1.78 % vs 7.91 鹵 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 鹵 1.35 % vs 4.27 鹵 1.52 % ) .
3.19 鹵 1.26 % vs3 . 92 鹵 1 . 34 % , p < 0 . 05 ) .
There was no difference in the peripheral blood for 7 days ( 34.74 鹵 4.80 % vs 34.50 鹵 5.00 % , P < 0.05 ) . The proportion of the experimental group increased ( 52.80 鹵 8.79 % vs 34.86 鹵 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 鹵 2.56 % vs 10.02 鹵 3.04 % , 14 days 11.49 鹵 2.87 % vs 12.93 鹵 3.12 % , p0.01 ) ;
Blood ( 8 . 00 鹵 1 . 78 % vs 8.20 鹵 2 . 01 % , 14 - day 9.76 鹵 2.45 % vs 10.90 鹵 2.67 % , p . 05 ) .

4 . Intraperitoneal injection of Galectin - 9 significantly reduced the expression of IFN - 緯 ( 51.33 鹵 8.16pg / ml vs 98.41 鹵 15.01pg / ml , p = 0.00 ) and IL - 17 ( 48.50 鹵 8.48pg / ml vs 15.17 鹵 5.46pg / ml , p = 0.00 ) .
But the level of IL - 2 ( 23.17 鹵 4.22pg / ml vs 26.50 鹵 3.78pg / ml , p = 0.180 ) and IL - 4 ( 86.83 鹵 13.33pg / ml vs 99.67 鹵 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

【學(xué)位授予單位】：中國人民解放軍軍醫(yī)進(jìn)修學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R392

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