本文選題：鞘氨醇-1磷酸1　切入點(diǎn)：FTY720　出處：《中國人民解放軍醫(yī)學(xué)院》2013年博士論文　論文類型：學(xué)位論文

【摘要】：目的：本研究通過建立小鼠同種異基因角膜移植模型，予以全身應(yīng)用鞘氨醇-1-磷酸受體調(diào)節(jié)劑1（S1P1），觀察S1P1對小鼠角膜移植植片免疫排斥的影響；探索S1P1聯(lián)合用藥模式對小鼠角膜移植植片免疫排斥的影響；通過檢測體內(nèi)免疫因子水平和蛋白的表達(dá)，探索S1P1抑制角膜植片免疫排斥的作用機(jī)制。 方法：1、建立C57BL/6小鼠為供體，BALB/C小鼠為受體的同種異基因角膜移植實(shí)驗(yàn)?zāi)Ｐ�，隨機(jī)分為五組，每組7只，術(shù)后采用腹腔注射給藥，自手術(shù)當(dāng)日開始，A組為不含藥物的安慰劑對照組；B組S1P1(sphingosine1-phosphate receptor-1)5mg/kg/d；C組地塞米松（dexamethasone）1mg/kg/d；D組雷帕霉素（rapamycin）2mg/kg/d；E組環(huán)孢霉素A（cyclosporine A，CsA）5mg/kg/d，每日1次，連續(xù)給藥14d。術(shù)后10天拆除小鼠角膜移植植片縫線，術(shù)后2周內(nèi)每日于手術(shù)顯微鏡下觀察小鼠角膜植片變化情況并記錄植片生存率指數(shù)，超出2周時間后隔日觀察1次，至發(fā)生排斥后處死實(shí)驗(yàn)動物。 2、BALB/C小鼠隨機(jī)分為A、B、C、D、E、F、G七組，每組7只，建立C57BL/6-BALB/C小鼠角膜移植模型。A組為不含藥物的安慰劑對照組，B組為S1P1（5mg/kg）單獨(dú)用藥組；C組為地塞米松（1mg/kg）單獨(dú)用藥組；D組為S1P1（5mg/kg）聯(lián)合雷帕霉素（2mg/kg）用藥組，E組為S1P1（5mg/kg）聯(lián)合環(huán)孢霉素（5mg/kg）用藥組，F(xiàn)組為雷帕霉素（2mg/kg）單獨(dú)用藥組，G組為環(huán)孢霉素（5mg/kg）單獨(dú)用藥組。術(shù)后采用腹腔注射給藥，自手術(shù)當(dāng)日開始，每日1次，連續(xù)給藥14d。術(shù)后10天拆除角膜縫線。對各組小鼠移植后角膜排斥時間進(jìn)行觀察，術(shù)后2周內(nèi)每日1次，超出2周者隔日1次，記錄植片生存曲線，至發(fā)生排斥后處死實(shí)驗(yàn)動物。 3、BALB/C小鼠隨機(jī)分為七組，每組5只，建立C57BL/6-BALB/C小鼠角膜移植模型。術(shù)后分別給予上述分組方案注藥，連續(xù)腹腔注射14d。術(shù)后14d時處死小鼠，取頸部引流淋巴結(jié)、腹腔腸系膜淋巴結(jié)、外周血清、脾臟等行流式細(xì)胞學(xué)檢測，探討CD4+T淋巴細(xì)胞和CD4+CD25+Foxp3+調(diào)節(jié)性T細(xì)胞的分布和聚集狀態(tài)。 4、BALB/C小鼠同上隨機(jī)分為七組，每組10只，建立C57BL/6-BALB/C小鼠角膜移植模型。術(shù)后分別給予上述分組方案注藥，連續(xù)腹腔注射14d。術(shù)后14d時處死小鼠，所有處死小鼠取外周血清行Elisa法檢測IL-2、IL-10、IFN-及TGF-1細(xì)胞因子含量。每組取5只小鼠角膜標(biāo)本行RealtimePCR法檢測小鼠角膜植片中IL-2、IL-10、IFN-、TGF-1及Foxp3mRNA表達(dá)情況，另對5只小鼠移植后角膜植片進(jìn)行常規(guī)HE染色，并對CD4+T細(xì)胞以及細(xì)胞因子IL-2、IL-10、IFN-、TGF-1進(jìn)行免疫熒光染色。 結(jié)果：1、同種異體角膜移植安慰劑對照組角膜植片平均存活時間僅為（15.14±2.5d）；S1P1聯(lián)合雷帕霉素組移植角膜植片出現(xiàn)排斥時間明顯延長為（38.71±9.2d）；S1P1聯(lián)合環(huán)孢霉素組角膜植片存活時間為（32.71±7.2d）；S1P1單獨(dú)用藥組角膜植片存活時間為（37.85±0.8d），三組用藥組與對照組比較均具有統(tǒng)計學(xué)差異（p＜0.01)。 2、流式細(xì)胞儀結(jié)果顯示S1P1單獨(dú)用藥組能顯著增加頸部淋巴結(jié)和腸系膜淋巴結(jié)中CD4+T細(xì)胞及Treg細(xì)胞的比例（p＜0.01)，而在S1P1聯(lián)合雷帕霉素組檢出的頸部淋巴結(jié)和腸系膜淋巴結(jié)中CD4+T細(xì)胞及Treg細(xì)胞的比例，與對照組相比，也有統(tǒng)計學(xué)意義（p＜0.05)。 3、S1P1聯(lián)合雷帕霉素組小鼠角膜植片中TGF-β1和IL-10mRNA表達(dá)較對照組明顯增高(p0.01),體現(xiàn)出協(xié)同抑制作用；而在CsA單獨(dú)用藥組中IL-2和IFN-γmRNA表達(dá)降低(p0.05)。免疫熒光染色證實(shí)角膜植片中上述細(xì)胞因子含量同mRNA表達(dá)是基本一致的。S1P1單獨(dú)用藥組和S1P1聯(lián)合雷帕霉素組均能明顯減少CD4+T細(xì)胞在角膜植片中的浸潤。 4、Elisa血清學(xué)檢測結(jié)果發(fā)現(xiàn)，，在S1P1單獨(dú)用藥組，IL-10和TGF-β1含量較對照組有明顯升高（p＜0.01)。在S1P1聯(lián)合雷帕霉素組，未發(fā)現(xiàn)有統(tǒng)計學(xué)差異（p＞0.05)，在S1P1單獨(dú)用藥組，IFN-γ含量升高有統(tǒng)計學(xué)意義（p＜0.05)，IL-2的表達(dá)則各組之間未見顯著性差異（p＞0.05)。 結(jié)論：1、S1P1聯(lián)合雷帕霉素、S1P1聯(lián)合環(huán)孢霉素均能夠顯著延長小鼠角膜移植術(shù)后植片的存活時間，體現(xiàn)出S1P1聯(lián)合用藥對小鼠角膜移植植片發(fā)生免疫排斥具有協(xié)同抑制作用。 2、S1P1能夠顯著影響淋巴細(xì)胞的分布，增加頸部引流淋巴結(jié)和腹腔腸系膜淋巴結(jié)中CD4+T細(xì)胞和Treg細(xì)胞的比率。S1P1聯(lián)合雷帕霉素亦能夠增加引流至局部淋巴結(jié)之淋巴細(xì)胞，但程度不及前者。 3、S1P1聯(lián)合雷帕霉素能夠顯著增加角膜植片中細(xì)胞因子TGF-β1和IL-10mRNA和蛋白的表達(dá)，體現(xiàn)出協(xié)同抑制作用。S1P1單獨(dú)用藥或S1P1聯(lián)合雷帕霉素均能夠減少CD4+T細(xì)胞在角膜植片中的浸潤。 4、S1P1能夠顯著升高血清中細(xì)胞因子IL-10和TGF-β1的表達(dá)水平，IFN-γ的表達(dá)也有升高。
[Abstract]:Objective: This study through the establishment of murine allogeneic corneal transplantation model, to systemic application of sphingosine -1- phosphate receptor modulators (S1P1), 1 S1P1 observation of immune rejection of graft effect of mouse corneal allograft; explore the S1P1 combination model plant effect on mice immune rejection of corneal transplantation; by detecting the expression level and immune factor protein, explore the S1P1 inhibition mechanism of corneal sheet immune rejection.
Methods: 1 C57BL/6 mice as donors and BALB/C mice as allogeneic corneal transplantation receptor in experimental model, randomly divided into five groups, 7 rats in each group, were treated with intraperitoneal injection, starting from the day of surgery, group A placebo control group; group B (S1P1 sphingosine1-phosphate receptor-1) 5mg/kg/d; group C dexamethasone (dexamethasone) 1mg/kg/d; D (rapamycin) 2mg/kg/d group, rapamycin group E; cyclosporine A (cyclosporine A, CsA 5mg/kg/d), 1 times a day, for removal of mouse corneal allograft drug 10 days after 14D. graft suture, 2 weeks after operation in the surgical microscope daily the mouse corneal changes and graft survival rate index records, beyond 2 weeks to the day after the observation of 1 times, to the rejection were the experimental animal.
2 BALB/C mice were randomly divided into A, B, C, D, E, F, G seven groups, 7 rats in each group, the establishment of C57BL/6-BALB/C mouse corneal transplantation model.A group as placebo control group, B group S1P1 (5mg/kg) monotherapy group; group C, dexamethasone (1mg/kg) monotherapy group D; group S1P1 (5mg/kg) combined with rapamycin (2mg/kg) treatment group, E group, S1P1 (5mg/kg) combined with cyclosporine (5mg/kg) treatment group, F group of rapamycin (2mg/kg) monotherapy group, G group of cyclosporine (5mg/kg) monotherapy group. Postoperative intraperitoneal injection, since the beginning of the day of operation, 1 times a day, 10 days for corneal suture removal after operation. Drug 14D. of mice after corneal transplantation rejection were observed within 2 weeks after the operation, 1 times a day, more than 2 weeks every other day for 1 times, recorded the graft survival curve to rejection after death experimental animals.
3 BALB/C mice were randomly divided into seven groups, 5 rats in each group, the establishment of C57BL/6-BALB/C mouse corneal transplantation model. After the grouping scheme injection were given by intraperitoneal injection after 14D. mice were sacrificed at 14d, the cervical lymph nodes, abdominal mesenteric lymph node, peripheral blood and spleen by flow cytometry to investigate the distribution of CD4+T lymphocyte, and CD4+CD25+Foxp3+ regulatory T cells and aggregation.
4, BALB/C mice were randomly divided into seven groups, 10 rats in each group, the establishment of C57BL/6-BALB/C mouse corneal transplantation model. After the grouping scheme injection were given by intraperitoneal injection after 14D. mice were sacrificed at 14d, all mice were sacrificed from peripheral serum Elisa assay for detection of IL-2, IL-10, IFN- and TGF-1 cytokines 5 mice in each group. The corneal specimens for detection of mouse corneal graft in RealtimePCR IL-2, IL-10, IFN-, TGF-1 and the expression of Foxp3mRNA, another of the 5 mice after transplantation of corneal graft were stained by HE, and the CD4+T cells and cell factor IL-2, IL-10, IFN-, TGF-1 immunofluorescence staining.
Results: 1, placebo control group corneal allograft corneal graft average survival time was (15.14 + 2.5D); combined with rapamycin group S1P1 transplantation corneal graft rejection was prolonged for (38.71 + 9.2d); S1P1 combined with cyclosporine group corneal graft survival time was (32.71 + 7.2d) S1P1; single drug group corneal graft survival time (37.85 + 0.8d), three groups of treatment group and control group were statistically significant (P < 0.01).
2, flow cytometry results showed that S1P1 monotherapy group could significantly increase the cervical and mesenteric lymph nodes in CD4+T cells and the percentage of Treg cells (P < 0.01), while the detection in S1P1 combined with rapamycin group of cervical lymph node and mesenteric lymph nodes in CD4+T cells and Treg cell ratio, compared with the control group also had statistical significance (P < 0.05).
3, S1P1 combined with rapamycin group mice corneal graft in TGF- beta 1 and IL-10mRNA expression was significantly higher than the control group (P0.01), reflecting the synergistic inhibitory effect of mRNA and IL-2; in CsA alone group and IFN- expression decreased (P0.05). Immunofluorescence staining showed that the corneal grafts in the same cytokine content the expression of mRNA is.S1P1 alone group and S1P1 combined with rapamycin group consistent could significantly decrease CD4+T cells in corneal infiltration in the film.
4, the detection results of Elisa serological found in S1P1 alone group, IL-10 and TGF- beta 1 group was increased significantly compared with the control (P < 0.01). In S1P1 combined with rapamycin group, there was no statistic difference (P > 0.05), in the S1P1 alone group, IFN- increased the content of statistical significance (gamma P < 0.05), there was no significant difference between the expression of IL-2 in each group (P > 0.05).
Conclusion: 1, S1P1 combined with rapamycin, S1P1 combined with cyclosporine can significantly prolong the survival time of mice after keratoplasty, showing that S1P1 combination has synergistic inhibition effect on corneal allograft rejection.
2, S1P1 can significantly affect the distribution of lymphocytes, increase the ratio of CD4+T cells and Treg cells in cervical draining lymph nodes and celiac mesenteric lymph nodes..S1P1 combined with rapamycin can also increase the number of lymphocytes draining into local lymph nodes, but the degree is not as good as the former.
3, S1P1 combined with rapamycin can significantly increase the expression of cytokines TGF- beta 1 and IL-10mRNA and protein in corneal graft, showing synergistic inhibition..S1P1 alone or S1P1 combined with rapamycin can reduce CD4+T cell infiltration in corneal graft.
4, S1P1 could significantly increase the expression level of cytokine IL-10 and TGF- beta 1 in serum, and the expression of IFN- gamma also increased.

【學(xué)位授予單位】：中國人民解放軍醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2013
【分類號】：R779.65

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