过氧化物酶体增殖物激活受体γ激动剂抑制角膜移植免疫排斥反应的实验研究

发布时间：2018-07-25 11:26

【摘要】： 目的观察过氧化物酶体增殖物激活受体γ(Peroxisome Proliferator-Activated Receptor gamma, PPARγ)激动剂局部应用对鼠角膜的影响,判断其药物毒性。方法将PPARγ激动剂DK2配制成滴眼液,将0.1%,0.5%,1.0% PPARγ激动剂局部滴用于Wistar大鼠右眼,观察眼局部改变和荧光素钠染色情况,并按照制定的眼部刺激反应评分标准,评判其对眼部刺激性,并行角膜病理组织切片染色和ELISA检测房水PPARγ浓度,综合评价药物安全性。结果药物各组鼠眼部刺激反应评分均为1-3分,角膜切片HE和免疫组化染色示角膜结构完整,细胞无损伤浸润。ELISA测得给药后房水中PPARγ浓度明显增加,且PPARγ的表达具有浓度依赖性,但0.5%,1.0%两个给药组之间差异无统计学意义(P0.05)。结论 1.0%浓度的PPARγ激动剂DK2滴眼液对鼠眼无刺激,具有安全性。目的探讨过氧化物酶体增殖物激活受体γ(Peroxisome Proliferator-Activated Receptor gamma, PPARγ)激动剂对大鼠角膜移植排斥反应的影响。方法 SD大鼠为供体,Wistar大鼠为受体,建立SD-Wistar大鼠同种异体穿透性角膜移植模型。随机分A、B、C、D及E组,其中A组：对照组,术后予以无菌生理盐水点眼；B组：0.1%DK2滴眼液组；C组：1.0%DK2滴眼液组；D组：DK2口服灌胃给药组；E组：1.0%环孢霉素A(CsA))滴眼液组；F组：Wistar大鼠自体同基因移植对照组,术后生理盐水点眼。术后定期对各组角膜植片进行临床观察,以混浊、水肿、新生血管3项指标作为临床评估标准,记录排斥指数(RI)、新生血管指数(NI)、植片存活时间。实时定量荧光PCR检测鼠角膜内ICAM-1mRNA表达,流式细胞学检测各组大鼠颌下淋巴结MHC-Ⅱ、CD80的变化情况,免疫组织化学法检测大鼠颌下淋巴结CD86的表达。酶联免疫吸附试验(ELISA)检测鼠房水中IL-10的含量。结果 A-E组的角膜植片平均存活时间为9.2±1.5d、10.1±1.8d、18.3±1.1d、20.1±1.6d、18.1±1.5d,F组植片存活时间至观察期结束(28.0d)。除B组外,其他用药组植片存活时间与A组比较,差异均有统计学意义(P0.05)。C、E组植片存活时间两两比较差异无统计学意义(P0.05),D组植片存活时间与C组相比较有统计学差异。术后第9d角膜植片ICAM-1 mRNA显示,A、B组相对表达量明显高于C、D、E、F组；颌下淋巴结免疫指标检测,流式细胞学结果显示第9 dC、D、E组DC表面MHC-Ⅱ及CD80单阳性表达率低于对照A,B组(P0.05)；免疫组化染色显示,第9d时A、B组淋巴结高表达CD86,而C、D、E、F组未见有明显表达,至术后18d,C、D、E组CD86表达明显增加。ELISA检测术后第3、9、18天各组大鼠房水IL-10含量较低,各组平均含量无明显差异；第9 d后,随着排斥反应发生延迟,C、D、E组IL-10含量较A、B组增加,但变化幅度不大。结论 PPARγ激动剂能显著延长大鼠角膜植片的存活时间,其抑制角膜移植免疫排斥反应具有随浓度增加效应,并且全身用药效果强于局部滴用1.0%DK2滴眼液和1.0%环孢霉素A。PPARγ激动剂能有效抑制角膜植片炎性细胞浸润和ICAM-1 mRNA的表达,抑制局部淋巴结内树突状细胞的聚集和递呈功能,可能是其发挥抗排斥作用机制的一部分。目的探讨过氧化物酶体增殖物激活受体γ(Peroxisome Proliferator-Activated Receptor gamma, PPARγ)激动剂对体外诱导培养的大鼠骨髓来源树突状细胞(dendritic cells,DCs)分化成熟及免疫活性影响,探讨药物对DCs表达TLR4的影响。方法用GM-CSF和IL-4体外定向诱导Wistar大鼠骨髓细胞分化成树突状细胞,①分别加入两种不同的PPARγ激动剂DK2和马来酸罗格列酮,与细胞共培养12-72h后,MTT法检测树突状细胞生长抑制率,乳酸脱氢酶检测法(LDH)比较两种药物的药效；②细胞随机分成4组,即空白对照组,阳性对照组(脂多糖,LPS),低浓度药物组和高浓度药物组,倒置显微镜下观察DCs形态变化,流式细胞仪检测DCs表面免疫分子MHC-Ⅱ、CD80表达率,酶联免疫吸附试验(ELISA)检测DCs培养上清液中IL-10含量,混合淋巴细胞增殖反应(MLR)测定DCs刺激T淋巴细胞增殖能力的改变,实时定量PCR检测PPARγmRNA表达；③免疫荧光染色法检测药物对TLR-4表达的影响。结果 ①MTT结果显示两种PPARγ激动剂DK2和马来酸罗格列酮的IC50值分别为15.60±0.54μmol/L和53.8±1.94μmol/L,此时的LDH释放率分别为16.8%和27.4%。 ②经DK2干预后,流式细胞仪检测DC表面MHC-Ⅱ、CD80阳性率降低,差异均有统计学意义(P0.05)。ELISA显示LPS刺激后IL-10的含量增加,药物干预后表达量进一步增加,各组差异有统计学意义(P0.05)。MLR实验显示随着刺激细胞(DC)浓度下降,反应T细胞增殖能力随之下降,并且药物能降低T细胞增殖能力,差异有统计学意义(P0.05)。实时定量PCR结果显示脂多糖刺激后,DCs表达PPARγmRNA略有增加,加入DK2后,PPARγmRNA表达量随药物浓度增加而进一步增加,差异有统计学意义(P0.05),PPARγ受体拮抗剂能抑制DK2的激动作用。免疫荧光检测示DK2抑制DCs表达TLR4。结论 PPARγ激动剂DK2能抑制大鼠DC分化成熟及其免疫功能,抑制效率具有浓度依赖性。目的探讨过氧化物酶体增殖物激活受体γ(Peroxisome Proliferator-Activated Receptor gamma, PPARγ)激动剂对角膜新生血管的抑制作用及机制。方法 36只SD大鼠随机分成A、B、C及D组,每组9只大鼠,右眼为实验眼。B、C、D组大鼠行角膜囊袋法制作角膜新生血管模型。A组：空白对照组,正常大鼠角膜；B：阳性对照组,制作模型后生理盐水滴眼；C、D组：建模后分别滴用0.1%、1.0% PPARY激动剂滴眼液,均4次／d,共28d。裂隙灯显微镜下测量新生血管面积,Western-Blot检测角膜血管内皮生长因子(vascular endothelial growth factor, VEGF)表达。结果 D组大鼠角膜新生血管面积小于阳性对照B组和C组,差异有统计学意义(P0.05),而C组与B组之间差异无统计学意义(P0.05)。Western-Blot检测新生血管角膜表达VEGF明显增强,C、D组VEGF相对量有不同程度下降。结论局部应用PPARγ激动剂能有效抑制角膜新生血管生成,抑制VEGF的合成和分泌可能是阻止角膜新生血管生长机制的一部分。
[Abstract]:objective
The effects of the local application of the peroxisome proliferator activated receptor gamma (Peroxisome Proliferator-Activated Receptor gamma, PPAR gamma) agonist on the cornea of rats were observed to determine the drug toxicity.
Method
PPAR gamma agonist DK2 was prepared into eye drops and 0.1%, 0.5%, 1% PPAR gamma agonists were applied locally to the right eye of Wistar rats. The local changes of the eye and the staining of fluorescein sodium were observed. In accordance with the standard of eye stimulation reaction, the eye irritation was evaluated, corneal pathological tissue section staining and ELISA detection of PPAR gamma concentration in aqueous humor were carried out. Degree, comprehensive evaluation of drug safety.
Result
The score of eye stimulation reaction in each group was 1-3 points. Corneal HE and immunohistochemical staining showed that the corneal structure was intact. The concentration of PPAR gamma in aqueous humor increased significantly after the cell without injury.ELISA, and the expression of PPAR gamma was concentration dependent, but there was no statistical difference between the 0.5% and 1% groups (P0.05).
conclusion
The 1.0% concentration of PPAR gamma agonist DK2 eye drops did not irritate the eyes of mice and were safe.
objective
To investigate the effect of peroxisome proliferator activated receptor gamma (Peroxisome Proliferator-Activated Receptor gamma, PPAR gamma) agonists on corneal graft rejection in rats.
Method
SD rats were the donors and Wistar rats were the receptors, and the SD-Wistar rat model of penetrating keratoplasty was established. The rats were randomly divided into A, B, C, D and E groups, and A group: the control group was given aseptic saline eyes after operation; B group: 0.1%DK2 eye drops group; C group: oral administration group; 1% cyclosporin A (CsA)) eye drops group; group F: Wistar rats autologous gene transplantation control group, after operation physiological saline eye. After the operation, regular corneal graft was observed. 3 indexes of turbidity, edema and neovascularization were used as clinical evaluation criteria, the rejection index (RI), neovascularization index (NI), graft survival time and real-time quantitative fluorescence were recorded. The expression of ICAM-1mRNA in the cornea of rats was detected by PCR. The changes of MHC- II and CD80 in submaxillary lymph nodes were detected by flow cytometry. The expression of CD86 in the submaxillary lymph nodes of rats was detected by immunohistochemistry. The enzyme linked immunosorbent assay (ELISA) was used to detect the content of IL-10 in the aqueous humor of rats.
The average survival time of corneal graft in group A-E was 9.2 + 1.5D, 10.1 + 1.8D, 18.3 + 1.1d, 20.1 + 1.6d, 18.1 + 1.5D. The survival time of the F group was at the end of the observation period (28.0d). The survival time of the other group was compared with the A group, and the difference was statistically significant (P0.05), and there was no significant difference in the survival time 22. 0.05) the survival time of the D group was significantly different from that of the C group. The post operation 9D corneal graft ICAM-1 mRNA showed that the relative expression of A, B group was significantly higher than that of C, D, E, F group, the submandibular lymph node immunological indexes and the flow cytology results showed ninth dC. There was no obvious expression of C, D, E, F in group C, D, E, F, and C, D, E, F, and C, C, D. The average content of the aqueous humor in each group was lower, and the average content of each group was no significant difference after the operation. After ninth, the content of the rejection was delayed. A, group B increased, but the change was small.
PPAR gamma agonist can significantly prolong the survival time of corneal graft in rats, and its inhibition of corneal transplantation immune rejection has the effect of increasing concentration, and the effect is stronger than that of local drops of 1.0%DK2 eyedrops and 1% cyclosporin A.PPAR gamma agonists, which can effectively inhibit the infiltration of corneal graft and the expression of ICAM-1 mRNA. It may be a part of the anti-rejection mechanism by which dendritic cells can aggregate and present in local lymph nodes.
objective
The effects of peroxisome proliferator activated receptor gamma (Peroxisome Proliferator-Activated Receptor gamma, PPAR gamma) agonists on the differentiation, maturation and immune activity of rat bone marrow derived dendritic cells (dendritic cells, DCs) induced in vitro were investigated, and the effects of drugs on DCs expression TLR4 were investigated.
Method
The bone marrow cells of Wistar rats were induced by GM-CSF and IL-4 in vitro to differentiate into dendritic cells. (1) two different PPAR gamma agonists, DK2 and rosiglitazone, were co cultured with 12-72h, and the growth inhibition rate of dendritic cells was detected by MTT method. The efficacy of two drugs was compared with the lactate dehydrogenase assay (LDH); 2. The machine was divided into 4 groups, namely the blank control group, the positive control group (lipopolysaccharide, LPS), the low concentration drug group and the high concentration drug group. The morphological changes of DCs were observed under the inverted microscope. The flow cytometry was used to detect the DCs surface immuno molecule MHC- II, the CD80 expression rate, and the enzyme linked immunosorbent assay (ELISA) was used to detect the IL-10 content in the supernatant of DCs culture, and the mixed lymphocyte increased. The proliferation of T lymphocytes stimulated by DCs was measured by colonization (MLR), and the expression of PPAR gamma mRNA was detected by real-time quantitative PCR, and the effect of the drug on the expression of TLR-4 was detected by immunofluorescence staining.
Result
The results of MTT showed that the IC50 values of two PPAR gamma agonists DK2 and rosiglitazone were 15.60 + 0.54 mu mol/L and 53.8 + 1.94 micron mol/L respectively, and the LDH release rates were 16.8% and 27.4%., respectively.
(2) after DK2, the flow cytometry was used to detect MHC- II on the surface of DC, and the positive rate of CD80 decreased. The difference was statistically significant (P0.05).ELISA showed that the content of IL-10 increased after LPS stimulation, and the expression amount of drug dry prognosis was further increased, and the differences in each group were statistically significant (P0.05).MLR experiments showed that with the decrease of the concentration of stimulated cells (DC), the reaction T cells increased. The colonization ability decreased, and the drug could reduce the proliferation ability of T cells. The difference was statistically significant (P0.05). Real-time quantitative PCR results showed that DCs expression PPAR gamma mRNA increased slightly after lipopolysaccharide stimulation. After adding DK2, PPAR gamma mRNA expression was further increased with the increase of drug concentration, the difference was statistically significant (P0.05), PPAR gamma receptor antagonist. DK2 inhibited the expression of TLR4 in DCs by immunofluorescence assay.
conclusion
PPAR-gamma agonist DK2 can inhibit the differentiation and maturation of DC and its immune function in rats, and the inhibition efficiency is concentration-dependent.
objective
To investigate the inhibitory effect and mechanism of peroxisome proliferator activated receptor gamma (Peroxisome Proliferator-Activated Receptor gamma, PPAR gamma) agonist on corneal neovascularization.
Method
36 SD rats were randomly divided into A, B, C and D groups, 9 rats in each group, the right eye was the experimental eye.B, C, and D group rats were made corneal neovascularization model.A group: blank control group, normal rat cornea; B: positive control group, after making model physiological saline drops eye; C, D group: after modeling, 0.1%, 1% agonist drops of eye drops respectively, drop eyes after modeling respectively drops of 0.1%, 1% of agonist drops of the eye drops respectively after the model drops of eye drops respectively drip eye drops, respectively drip 0.1%, 1% of agonist drops of the eye drops respectively after the model drops of eye drops, respectively drops of the eye drops of an agonist drops of eye drops respectively after the modeling drops of 0.1%, 1% agonists drop the eye respectively after modeling A total of 4 times / D, a total of 28d. slit lamp microscope was used to measure the area of neovascularization, and the expression of corneal vascular endothelial growth factor (vascular endothelial growth factor, VEGF) was detected by Western-Blot.
Result
The area of corneal neovascularization in D group was less than that of the positive control group B and C group, the difference was statistically significant (P0.05), but there was no significant difference between the C group and the B group (P0.05), and the expression of corneal expressed in the cornea of the neovascularization was significantly enhanced by P0.05.Western-Blot, and the VEGF relative quantity of the C and D groups decreased.
Local application of PPAR gamma agonist can effectively inhibit the formation of corneal neovascularization, and inhibit the synthesis and secretion of VEGF may be part of the mechanism of inhibiting the growth of corneal neovascularization.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R779.65

【相似文献】