【摘要】：目的：通過將鞘氨醇-1-磷酸(S1P)受體調(diào)節(jié)劑FTY720眼用凝膠應(yīng)用于小鼠異基因角膜移植模型中，觀察FTY720眼用凝膠抑制小鼠角膜移植排斥的情況，并與臨床常用的眼用免疫抑制劑環(huán)孢霉素A（CsA）進行比較。最終明確FTY720眼用凝膠抑制小鼠角膜移植的排斥反應(yīng)的效果。通過流式細胞學、Realtime-PCR、Elisa、常規(guī)病理以及免疫組化方法對不同處理組小鼠的角膜、外周血、脾臟及頸部淋巴結(jié)標本進行檢測；以探索FTY720眼用凝膠局部點眼抑制小鼠異基因角膜移植排斥的作用機制。使用磁珠分選法分選出具有免疫調(diào)節(jié)功能的小鼠調(diào)節(jié)性T細胞，并將分選細胞分別與S1P受體調(diào)節(jié)劑（FTY720及FTY720-P）共培養(yǎng)。通過CCK-8法檢測共培養(yǎng)的調(diào)節(jié)性T細胞增殖情況，以及混合淋巴反應(yīng)結(jié)果；以確定S1P受體調(diào)節(jié)劑直接作用于調(diào)節(jié)性T細胞后的細胞增殖及免疫功能變化情況。通過Elisa法檢測共培養(yǎng)體系中與細胞功能相關(guān)的細胞因子(IL-10和TGF-β1)水平；Realtime-PCR法了解共培養(yǎng)細胞中相應(yīng)細胞因子mRNA的表達情況。最終以探索出S1P受體調(diào)劑直接作用于小鼠調(diào)節(jié)性T細胞后，其功能及表型發(fā)生變化的的機制。 方法： 1.制備0.1%、0.3%及0.5%三個濃度S1P受體調(diào)節(jié)劑FTY720溫度敏感型眼用凝膠。 2.取45雄性BALB/C小鼠隨機分為5組（空白對照組、低濃度、中濃度、高濃度FTY720眼用凝膠組以及1%環(huán)孢霉素眼液組），每只小鼠隨機選取一眼作為受體眼，分別接受雄性C57BL/6小鼠角膜植片，進行異基因穿透性角膜移植。術(shù)后即分別給予相應(yīng)方案處置，術(shù)后10-11天拆除角膜縫線；一月內(nèi)顯微鏡下觀察并記錄全部小鼠的角膜植片情況。至小鼠角膜發(fā)生排斥及觀察結(jié)束后處死實驗動物。 3.取30只雄性BALB/C小鼠同上隨機分為5組，每只小鼠隨機一眼作為受體眼，分別接受雄性C57BL/6小鼠角膜植片，進行異基因穿透性角膜移植。術(shù)后分別給予上述方案分組方案處置，術(shù)后10-11天拆除角膜縫線。術(shù)后14天時處死小鼠，分別進行實驗室檢查。其中取頸部引流淋巴結(jié)、外周血、脾臟進行流式細胞學檢查，了解CD4+T細胞及CD4+CD25+Foxp3+T細胞的分布情況。對外周血的標本使用Elisa法檢測外周血清中IL-2、IL-10、IFN-γ及TGF-β1的含量。每組取3只小鼠角膜分別采用RealtimePCR法檢測角膜植片中IL-2、IL-10、IFN-γ、TGF-β1及Foxp3mRNA表達情況。每組另取3只小鼠角膜分別進行常規(guī)HE染色，了解角膜排斥反應(yīng)情況；并對CD4+T細胞以及細胞因子IL-2、IL-10、IFN-γ及TGF-β1進行免疫組化檢查，了解CD4+T細胞在角膜植片浸潤情況以及上述有關(guān)細胞因子在角膜植片中含量的變化。 4.取30只雄性BALB/C小鼠；處死后應(yīng)用免疫磁珠分選法將脾臟中的CD4+CD25+以及CD4+CD25-T細胞分選出來。對分選后的細胞采用流式細胞技術(shù)明確分選效率。 5.對分選的細胞應(yīng)用CD3、CD28單抗聯(lián)合IL-2共同體外培養(yǎng)5天，充分激活上述分選細胞。 6.在激活的CD4+CD25+T細胞培養(yǎng)體系中分別加入10ng/ml、100ng/ml、1000ng/ml的FTY720以及FTY720-P，空白對照組加入10ulDMSO；繼續(xù)共培養(yǎng)48小時。CCK-8法檢測CD4+CD25+T細胞加入S1P受體調(diào)節(jié)劑前后細胞增殖情況（共計5天，第0天為加入S1P受體調(diào)節(jié)劑時、第1天及2天為加入S1P受體調(diào)節(jié)劑后時刻，第3及4天為洗滌上述細胞后繼續(xù)原培養(yǎng)條件進行培養(yǎng)的時刻）。 7.加入不同藥物后的48小時后，取共培養(yǎng)后的CD4+CD25+T細胞（調(diào)節(jié)性T細胞、Treg）與CD4+CD25-T細胞（效應(yīng)T細胞、Teff）按不同比例進行混合淋巴細胞培養(yǎng)，5天后CCK-8法檢測Teff細胞增殖情況。 8.加入不同藥物后的48小時后，取共培養(yǎng)CD4+CD25+T細胞上清液進行Elisa檢測，檢測IL-10、TGF-β1含量；從上述共培養(yǎng)細胞中提取總RNA，Realtime-PCR檢測IL-10、TGF-β1及Foxp3mRNA的表達情況。 結(jié)果： 1.成功制備0.1%、0.3%及0.5%三個濃度S1P受體調(diào)節(jié)劑FTY720溫度敏感型眼用凝膠。其在室溫環(huán)境下為液態(tài)，在近體溫狀態(tài)時迅速變?yōu)槟z凍狀。 2.觀察45只小鼠術(shù)后角膜植片情況，植片平均生存時間（MST）方面，0.5%FTY720組(24.11±1.58天)以及1%CsA組(25.00±1.91天)均較空白對照組（13.44±0.48天）明顯延長(P0.01)。 3.0.5%FTY720能顯著增加頸部淋巴結(jié)中CD4+T細胞以及Treg細胞的比例(P0.05和P0.01)。在0.5%FTY720組，角膜植片中TGF-β1mRNA表達明顯增高(p0.05),而在1%CsA組角膜植片中IL-2和IFN-γmRNA則明顯降低的(p0.01和p0.05)。免疫組化染色結(jié)果證實角膜植片中上述細胞因子含量變化情況與相應(yīng)mRNA表達基本一致。此外0.5%FTY720以及1%CsA點眼后，角膜植片中CD4+T細胞的浸潤明顯減少。 4.全部各組小鼠外周血清中IL-2、IL-10、IFN-γ及TGF-β1含量未見變化。 5.自小鼠脾臟細胞中成功分選CD4+CD25+T細胞，流式細胞檢測結(jié)果顯示CD4+CD25+T細胞的分選效率在93%以上，分選出細胞中Foxp3+比例在94%以上。CD4+CD25-T細胞分選率接近96%其Foxp3+比例僅為1.45%左右。 6.對上述分選出的細胞應(yīng)用CD3、CD28單抗聯(lián)合IL-2共同體外培養(yǎng)5天后，上述細胞被充分激活。 7.對激活的的CD4+CD25+T細胞分別給予三個不同劑量的FTY720及FTY720-P共培養(yǎng)后，，經(jīng)過CCK-8檢測后發(fā)現(xiàn)與空白對照組比較，給予S1P受體調(diào)節(jié)劑后CD4+CD25+T細胞的增殖活性未見變化（P0.05）。 8.共培養(yǎng)的Treg細胞與Teff細胞進行混合淋巴細胞培養(yǎng)5天后，結(jié)果顯示在Treg:Teff為1:1時，高劑量FTY720、中等劑量及高劑量的FTY720-P組中Teff細胞增殖能力被明顯抑制（P0.05）；在Treg:Teff為1:4時，僅有高劑量的FTY720-P組中Teff細胞增殖能力被抑制（P0.05）；而在Treg:Teff為1:8時，各組之間未見顯著性差異（P0.05）。 9.共培養(yǎng)48小時后，取共培養(yǎng)CD4+CD25+T細胞上清液進行Elisa檢測。結(jié)果顯示檢測各組中IL-10含量未見變化；對于TGF-β1含量，在高劑量FTY720組、中等劑量和高劑量FTY720-P組的含量均顯著高于對照組（P0.05）。對上述共培養(yǎng)細胞進行RealtimePCR檢測，結(jié)果顯示三個劑量FTY720組細胞中IL-10、TGF-β1及Foxp3mRNA水平未見顯著變化；而在高劑量FTY720-P組細胞中TGF-β1及Foxp3mRNA的表達明顯增高（P0.05）。 結(jié)論： 1.0.5%FTY720眼用凝膠以及1%CsA眼液均能夠顯著延長異基因小鼠角膜移植術(shù)后免疫排斥反應(yīng)的發(fā)生時間。 2.0.5%FTY720眼用凝膠能夠增加頸部引流淋巴結(jié)中的CD4+T細胞的比率，且更為顯著地增加了Treg細胞在局部淋巴結(jié)中的比率。0.5%FTY720能夠增加角膜植片中TGF-β1mRNA及其蛋白的表達；并且能夠減少CD4+T細胞在角膜植片中的浸潤。 3.1%CsA眼液也能夠顯著減少CD4+T細胞在角膜植片中的浸潤；但是與0.5%FTY720眼用凝膠不同，其角膜植片中IL-2及IFN-γ的含量顯著減少。 4.各組血清中有關(guān)細胞因子的含量未見變化（局部點眼劑型未能影響到外周血中細胞因子的含量）。 5.三個濃度的FTY720及FTY720-P均不能影響共培養(yǎng)Treg細胞的增殖能力。 6.高劑量FTY720、中等及高劑量的FTY720-P能夠顯著增加共培養(yǎng)Treg細胞針對Teff細胞的抑制能力。其作用機制為增加了共培養(yǎng)細胞中CD25+及Foxp3+表型的比例，并且上調(diào)了共培養(yǎng)細胞中TGF-β1mRNA及相應(yīng)蛋白的表達；此外高劑量FTY720-P也能夠增加共培養(yǎng)Treg細胞中Foxp3mRNA表達。
[Abstract]:Objective: To observe the effect of FTY720 ophthalmic gel on corneal graft rejection in mouse and compare with the commonly used ocular immunosuppressant cyclosporin A (CsA). Finally, the effect of FTY720 ophthalmic gel on the rejection of corneal transplantation in mice was determined. The corneal, peripheral blood, spleen and neck lymph node specimens of different treatment group mice were tested by flow cytometry, Realtime-PCR, Elisa, routine pathology and immunohistochemistry. The regulatory T cells with immunomodulatory function were selected by magnetic bead sorting and the cells were co-cultured with S1P receptor modulators (FTY720 and FTY720-P), respectively. The proliferation of the co-cultured regulatory T cells was detected by the CCK-8 method, and the results of the mixed lymph reaction were determined to determine the cell proliferation and the change of the immune function of the S1P receptor modulator directly after the regulatory T cells. The level of cytokines (IL-10 and TGF-1 1) related to cell function in co-culture system was detected by Elisa method, and the expression of corresponding cytokine mRNA in co-cultured cells was obtained by Realtime-PCR. Finally, the mechanism of the change of the function and phenotype of the S1P receptor after the regulatory T cell of the mouse was explored. square Method:1. The temperature-sensitive type of FTY720, which is 0.1%, 0.3% and 0.5%, has three concentrations of S1P receptor modulator, FTY720, is prepared. 2.45 male BALB/ C mice were randomly divided into 5 groups (blank control group, low concentration, medium concentration, high-concentration FTY720 ophthalmic gel group and 1% cyclosporin eye group), and each mouse was randomly selected as the receptor eye, and the male C57BL/6 mice were respectively accepted. mouse corneal graft The corresponding treatment was given after operation, and the corneal suture was removed for 10-11 days post-operation, and the angle of all mice was observed and recorded in a 1-month internal microscope. in that case of membrane-graft, at the end of the rejection of the cornea of the mouse and the end of the observation 3.30 male BALB/ C mice were randomly divided into 5 groups, and each mouse was given a random glance as the receptor eye to receive the male C57BL/6 mouse corneal graft and to carry out the isogenic gene. Penetrating keratoplasty, the above-mentioned protocol group protocol was given after operation, and the procedure was 10-11 after operation. The corneal suture was removed on day. The mice were sacrificed at 14 days post-operation, respectively Conduct the laboratory test. The lymph node, peripheral blood and spleen of the neck were taken for flow cytometry to understand the CD4 + T cells and the CD4 + CD25 + Foxp3 + T. The distribution of IL-2, IL-10, IFN-1 and TG in peripheral blood were detected by Elisa method. The levels of IL-2, IL-10, IFN-1, TGF-CD1 and Foxp3m were detected by using the RealtimePCR method. In each group, three mouse corneas were used for routine HE staining to understand the corneal rejection, and CD4 + T cells as well as the cytokines IL-2, IL-10, IFN-1 and TGF-CD1 were fed into each group. Immunohistochemical examination was performed to understand the infiltration of CD4 + T cells in the cornea and the related cytokines in the cornea. Changes in the content of the tablets.4.30 male BALB/ C mice were taken; the CD4 + CD25 + and CD4 + CD2 in the spleen were treated with the immunomagnetic bead sorting method after termination 5-T cells are sorted out. 5. The cells were treated with CD3, CD28 and IL-2, and cultured for 5 days. 6. In the activated CD4 + CD25 + T cell culture system,10 ng/ ml,100 ng/ ml,1000 ng/ ml of FTY720 and FTY720-P were added, respectively, and the blank control group was added with 10 ulD. MSO; continued co-culture for 48 h. CCK-8 method for the detection of the proliferation of CD4 + CD25 + T cells before and after the addition of S1P receptor modulators (total 5 days, day 0, with S1P receptor modulator, day 1 and 2) after the S1P receptor modulator is added, the cells are washed after the cells are washed in days 3 and 4 7. After 48 hours after the addition of different drugs, the co-cultured CD4 + CD25 + T cells (regulatory T cells, Tregs) and the CD4 + CD25-T cells (effector T cells, Teff) were mixed for lymphocyte culture in different proportions, and CCK was used after 5 days. 8. After 48 hours after the addition of different drugs, the supernatant of the co-cultured CD4 + CD25 + T cells was taken for Elisa test to detect the content of IL-10 and TGF-CD1; total RNA was extracted from the co-cultured cells, and the real time-PCR was used to detect the IL-10 and TGF- the first and the second Results:1. The expression of Foxp3mRNA was 0.1%, 0.3% and 0.5%, respectively. P receptor modulator FTY720 temperature sensitive ophthalmic gel at room temperature In the case of 45 mice, the average survival time (MST), 0.5% FTY720 group (24.11-1.58 days) and 1% CsA (25.00-1.91 days) were compared with the blank control group (25.00-1.91 days). 13.44 (0.48 days) significantly prolonged (P0.01). 3.0.5% FTY720 significantly increased the CD4 + T fine in the cervical lymph node In the 0.5% FTY720 group, the expression of TGF-CD1 mRNA in the corneal grafts was significantly higher (p0.05), while IL-2 and IFN in the 1% CsA group were significantly higher (p0.05). -The results of the immunohistochemical staining showed that the expression of the mRNA in the cornea was significantly lower (p0.01 and p0.05). in addition, 0.5% FTY720 and 1% C 4. There was a significant decrease in the infiltration of CD4 + T cells in the corneal grafts after the eyes of the sA. 5. CD4 + CD25 + T cells were successfully sorted from the spleen cells of mice. The results of flow cytometry showed that the CD4 + CD25 + T cells were sorted. The efficiency is over 93%, and the ratio of Foxp3 + in the selected cells is over 94%. CD4 + CD25- The cell sorting rate of T cells was close to 96%, and the ratio of Foxp3 + was only about 1.45%. After three different doses of FTY720 and FTY720-P, the activated CD4 + CD25 + T cells were co-cultured with three different doses of FTY720 and FTY720-P. The proliferation activity of CD4 + CD25 + T cells was not changed after body conditioning (P0.05).8. The co-cultured Treg cells were mixed with Teff cells for 5 days, and the results showed that Tef in the high dose of FTY720, medium dose and high dose of FTY720-P group at 1:1 in Treg: Teff The proliferation ability of the f cells was significantly inhibited (P0.05); in the case of Treg: Teff of 1:4, the proliferation ability of Teff cells in the FTY720-P group with only high dose was inhibited (P0.05); However, there was no significant difference between the groups at the time of Treg: Teff (P0.05). After the co-culture for 48 hours, the supernatant of the co-cultured CD4 + CD25 + T cells was taken for Elisa detection. The results showed no change in the content of IL-10 in each group; for the TGF-1 content, at high dose of FTY720 The results showed that the levels of IL-10, TGF-CD1 and Foxp3mRNA in the three-dose FTY720 group did not change significantly in the group, middle dose and high-dose FTY720-P group, while at the high dose of FTY72, the levels of IL-10, TGF-1 and Foxp3mRNA in the three-dose FTY720 group were not significantly changed. 0-P The expression of TGF-CD1 and Foxp3mRNA in the group was significantly higher (P0.05). Conclusion:1.5% FTY720 eyes 2.0.5% of FTY720 ophthalmic gel can increase the ratio of CD4 + T cells in the lymph nodes of the neck, and increase the ratio of the Treg cells to the local lymph nodes more significantly. 0.5% of the FTY720 can increase the cornea. The expression of TGF-CD1 mRNA and its protein in the implanted tablet can be reduced, and the infiltration of CD4 + T cells in the corneal implant can be reduced. 3.1% CsA can also significantly reduce the infiltration of CD4 + T cells in the corneal implant. The results showed that the content of IL-2 and IFN-1 in the corneal implant was significantly lower than that of the 0.5% FTY720 ophthalmic gel. .4. There was no change in the content of the relevant cytokines in each group (the local point-to-eye dosage form failed to affect the cells in the peripheral blood). 5. The three concentrations of FTY720 and FTY720-P could not affect the proliferation of the co-cultured Treg cells. The amount of FTY720, medium and high doses of FTY720-P can significantly increase the inhibitory capacity of co-cultured Treg cells against Teff cells. The mechanism of action is to increase the ratio of CD25 + and Foxp3 + phenotype in co-cultured cells and up-regulate the TGF-+ 1 m in co-cultured cells.
【學位授予單位】：中國人民解放軍軍醫(yī)進修學院
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R779.65

【共引文獻】

相關(guān)碩士學位論文 前1條

1 金卉;大腸腺癌中鞘氨醇激酶-1與1-磷酸鞘氨醇受體mRNA和蛋白表達的研究[D];廣西醫(yī)科大學;2010年

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