鞘氨醇-1-磷酸受体调节剂1抑制小鼠异基因角膜移植排斥机制的实验研究

发布时间：2018-03-06 20:45

本文选题：鞘氨醇-1磷酸1　切入点：FTY720　出处：《中国人民解放军医学院》2013年博士论文　论文类型：学位论文

【摘要】：目的：本研究通过建立小鼠同种异基因角膜移植模型，予以全身应用鞘氨醇-1-磷酸受体调节剂1（S1P1），观察S1P1对小鼠角膜移植植片免疫排斥的影响；探索S1P1联合用药模式对小鼠角膜移植植片免疫排斥的影响；通过检测体内免疫因子水平和蛋白的表达，探索S1P1抑制角膜植片免疫排斥的作用机制。方法：1、建立C57BL/6小鼠为供体，BALB/C小鼠为受体的同种异基因角膜移植实验模型，随机分为五组，每组7只，术后采用腹腔注射给药，自手术当日开始，A组为不含药物的安慰剂对照组；B组S1P1(sphingosine1-phosphate receptor-1)5mg/kg/d；C组地塞米松（dexamethasone）1mg/kg/d；D组雷帕霉素（rapamycin）2mg/kg/d；E组环孢霉素A（cyclosporine A，CsA）5mg/kg/d，每日1次，连续给药14d。术后10天拆除小鼠角膜移植植片缝线，术后2周内每日于手术显微镜下观察小鼠角膜植片变化情况并记录植片生存率指数，超出2周时间后隔日观察1次，至发生排斥后处死实验动物。 2、BALB/C小鼠随机分为A、B、C、D、E、F、G七组，每组7只，建立C57BL/6-BALB/C小鼠角膜移植模型。A组为不含药物的安慰剂对照组，B组为S1P1（5mg/kg）单独用药组；C组为地塞米松（1mg/kg）单独用药组；D组为S1P1（5mg/kg）联合雷帕霉素（2mg/kg）用药组，E组为S1P1（5mg/kg）联合环孢霉素（5mg/kg）用药组，F组为雷帕霉素（2mg/kg）单独用药组，G组为环孢霉素（5mg/kg）单独用药组。术后采用腹腔注射给药，自手术当日开始，每日1次，连续给药14d。术后10天拆除角膜缝线。对各组小鼠移植后角膜排斥时间进行观察，术后2周内每日1次，超出2周者隔日1次，记录植片生存曲线，至发生排斥后处死实验动物。 3、BALB/C小鼠随机分为七组，每组5只，建立C57BL/6-BALB/C小鼠角膜移植模型。术后分别给予上述分组方案注药，连续腹腔注射14d。术后14d时处死小鼠，取颈部引流淋巴结、腹腔肠系膜淋巴结、外周血清、脾脏等行流式细胞学检测，探讨CD4+T淋巴细胞和CD4+CD25+Foxp3+调节性T细胞的分布和聚集状态。 4、BALB/C小鼠同上随机分为七组，每组10只，建立C57BL/6-BALB/C小鼠角膜移植模型。术后分别给予上述分组方案注药，连续腹腔注射14d。术后14d时处死小鼠，所有处死小鼠取外周血清行Elisa法检测IL-2、IL-10、IFN-及TGF-1细胞因子含量。每组取5只小鼠角膜标本行RealtimePCR法检测小鼠角膜植片中IL-2、IL-10、IFN-、TGF-1及Foxp3mRNA表达情况，另对5只小鼠移植后角膜植片进行常规HE染色，并对CD4+T细胞以及细胞因子IL-2、IL-10、IFN-、TGF-1进行免疫荧光染色。结果：1、同种异体角膜移植安慰剂对照组角膜植片平均存活时间仅为（15.14±2.5d）；S1P1联合雷帕霉素组移植角膜植片出现排斥时间明显延长为（38.71±9.2d）；S1P1联合环孢霉素组角膜植片存活时间为（32.71±7.2d）；S1P1单独用药组角膜植片存活时间为（37.85±0.8d），三组用药组与对照组比较均具有统计学差异（p＜0.01)。 2、流式细胞仪结果显示S1P1单独用药组能显著增加颈部淋巴结和肠系膜淋巴结中CD4+T细胞及Treg细胞的比例（p＜0.01)，而在S1P1联合雷帕霉素组检出的颈部淋巴结和肠系膜淋巴结中CD4+T细胞及Treg细胞的比例，与对照组相比，也有统计学意义（p＜0.05)。 3、S1P1联合雷帕霉素组小鼠角膜植片中TGF-β1和IL-10mRNA表达较对照组明显增高(p0.01),体现出协同抑制作用；而在CsA单独用药组中IL-2和IFN-γmRNA表达降低(p0.05)。免疫荧光染色证实角膜植片中上述细胞因子含量同mRNA表达是基本一致的。S1P1单独用药组和S1P1联合雷帕霉素组均能明显减少CD4+T细胞在角膜植片中的浸润。 4、Elisa血清学检测结果发现，，在S1P1单独用药组，IL-10和TGF-β1含量较对照组有明显升高（p＜0.01)。在S1P1联合雷帕霉素组，未发现有统计学差异（p＞0.05)，在S1P1单独用药组，IFN-γ含量升高有统计学意义（p＜0.05)，IL-2的表达则各组之间未见显著性差异（p＞0.05)。结论：1、S1P1联合雷帕霉素、S1P1联合环孢霉素均能够显著延长小鼠角膜移植术后植片的存活时间，体现出S1P1联合用药对小鼠角膜移植植片发生免疫排斥具有协同抑制作用。 2、S1P1能够显著影响淋巴细胞的分布，增加颈部引流淋巴结和腹腔肠系膜淋巴结中CD4+T细胞和Treg细胞的比率。S1P1联合雷帕霉素亦能够增加引流至局部淋巴结之淋巴细胞，但程度不及前者。 3、S1P1联合雷帕霉素能够显著增加角膜植片中细胞因子TGF-β1和IL-10mRNA和蛋白的表达，体现出协同抑制作用。S1P1单独用药或S1P1联合雷帕霉素均能够减少CD4+T细胞在角膜植片中的浸润。 4、S1P1能够显著升高血清中细胞因子IL-10和TGF-β1的表达水平，IFN-γ的表达也有升高。
[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.

【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R779.65

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