和厚朴酚脂质体对氧诱导视网膜病变的作用及机制研究

发布时间：2018-04-19 20:59

本文选题：和厚朴酚脂质体 + 氧诱导视网膜病变　；参考：《武汉大学》2012年博士论文

【摘要】：第一部分和厚朴酚脂质体对氧诱导视网膜新生血管形成的抑制作用目的：探讨和厚朴酚脂质体(honokiol liposome, HNK-L)对氧诱导视网膜病变模型(oxygen-induced retinoopathy, OIR)视网膜新生血管(retinal neovascularization, RNV)形成的作用。方法：取72只鼠龄为7d(P7)的C57BL/6J小鼠,随机分为6组：正常组、OIR模型组、空白脂质体组和大、中、小剂量HNK-L干预组。正常组小鼠持续在常氧环境饲养,其余五组置于密闭氧箱中,24h维持氧浓度在75%±2%,5d后(P12)将小鼠返回到常氧,继续饲养至P17以建立OIR模型；其中HNK-L干预组再每日给予腹腔注射0.2ml不同剂量HNK-L：大剂量组(100mg/kg),中剂量组(50mg/kg),小剂量组(25mg/kg)。各组小鼠于P17断颈处死,立即摘眼球制作视网膜石蜡切片HE染色以及CD105抗体免疫组化标记血管内皮细胞(endothelial cells, ECs),计算视网膜切片中突破内界膜(internal limiting membrane, ILM)的ECs核数目。结果：OIR模型组小鼠视网膜可见大量RNV形成,OIR模型小鼠突破ILM的EC核数目明显多于正常组,差异有统计学意义(P0.01),同时该组运用CD105抗体免疫组化标记EC染色增强。不同剂量HNK-L干预组突破ILM的ECs核数目均有明显减少(P0.05),且不同干预剂量的计数亦有显著性差异,HNK-L剂量越大,突破ILM的ECs核数目越少(P0.05)。结论：应用C57BL/6J小鼠可建立OIR模型；CD105作为特异性ECs标记物可以较好地显示RNV形成情况；腹腔注射和厚朴酚脂质体能显著抑制OIR模型小鼠RNV形成。第二部分和厚朴酚脂质体对氧诱导视网膜病变VEGF-A, VEGFR-2及ES表达的影响目的：检测HNK-L抑制OIR模型中视网膜组织新生血管相关因子VEGF-A. VEGFR-2及ES的表达水平,从细胞因子水平探讨HNK-L的作用机制。方法：取P7C57BL/6J小鼠72只,随机分为正常组、OIR模型组、空白脂质体组和大、中、小剂量HNK-L干预组。正常组在常氧环境饲养,其余五组按第一部分建立OIR模型,HNK-L干预组于P12返回到常氧,5d每日一次给予腹腔注射0.2ml不同剂量HNK-L：大剂量组(100mg/kg),中剂量组(50mg/kg),小剂量组(25mg/kg),P17断颈处死各组小鼠,检测VEGF-A, VEGFR-2及ES mRNA及蛋白在OIR模型视网膜组织的表达量。结果：OIR模型组中VEGF-A及VEGFR-2mRNA及蛋白表达较正常组明显增强(P0.05)；ES表达与正常组则无显著性差异(P0.05)。HNK-L干预组VEGF-A、 VEGFR-2mRNA及蛋白表达明显低于OIR模型组及空白脂质体组(P0.05)；而HNK-L干预组ES mRNA及蛋白表达则较正常组/OIR模型组/空白脂质体组明显上调；HNK-L干预效应与给药剂量呈正相关。结论：HNK-L抑制OIR模型中RNV形成的机制可能与HNK-L下调VEGF-A、 VEGFR-2的表达,上调ES表达相关。第三部分和厚朴酚脂质体对氧诱导视网膜病变MAPK信号通路的影响目的：观察HNK-L在OIR模型RNV形成时,对MAPK家族ERK1/2,p38MAPK及JNK信号通路的影响,从信号转导通路角度探讨HNK-L干预OIR模型中RNV形成的机制。方法：取P7d C57BL/6J小鼠36只,随机分为正常组,OIR模型组、空白脂质体组和大、中、小剂量HNK-L干预组。正常组在常氧环境饲养,其余组按第一部分方法建立OIR模型。HNK-L大、中、小剂量干预组在出氧箱后,每日分别给予0.2ml不同剂量(100mg/kg,50mg/kg,25mg/kg) HNK-L腹腔注射,P17断颈处死各组小鼠,western blot法检测视网膜组织p-ERK1/2、 p-p38MAPK及p-JNK的蛋白量。结果：OIR模型组视网膜p-ERK1/2、 p-p38MAPK及p-JNK蛋白含量较正常组明显增强(P0.05)。HNK-L干预组p-ERK1/2、 p-p38MAPK及p-JNK蛋白明显低于OIR模型组／空白脂质体组的水平(P0.05),且抑制效应与给药剂量呈正相关。结论：OIR模型中RNV发生与MAPK家族ERK、 p38MAPK及JNK表达均相关；HNK-L抑制RNV形成的作用可能与影响MAPK家族信号通路有关。
[Abstract]:Part I and inhibition of magnolol liposomes on oxygen induced retinal neovascularization
Objective: To investigate the effect of honokiol liposome (HNK-L) on the formation of retinal neovascularization (retinal neovascularization, RNV) in the oxygen induced retinopathy of retinopathy (oxygen-induced retinoopathy, OIR).
Methods: 72 C57BL/6J mice aged 7d (P7) were randomly divided into 6 groups: normal group, OIR model group, blank liposome group and large, medium, and small dose HNK-L intervention group. The normal group was kept in the normal oxygen environment, the other five groups were placed in the closed oxygen tank, the 24h maintained oxygen concentration of 75% + 2%, and 5D after 5D (P12) returned the mice to the normal oxygen and continued to feed. To P17 to establish a OIR model, the HNK-L intervention group was given a daily intraperitoneal injection of HNK-L with different doses of 0.2ml: a large dose group (100mg/kg), a medium dose group (50mg/kg) and a small dose group (25mg/kg). The mice in each group were executed at P17 broken neck, immediately picked up the eyeball to make the retina paraffin slice HE staining, and the CD105 antibody immunized to mark vascular endothelium. Cells (endothelial cells, ECs) calculate the number of ECs nuclei that break through the internal limiting membrane (ILM) in retina slices.
Results: a large number of RNV formed in the retina of the OIR model group, and the number of EC nuclei of the OIR model mice to break through the ILM was significantly more than that of the normal group. The difference was statistically significant (P0.01). At the same time, the group used the CD105 antibody immuno histochemical labeling EC staining. The number of ECs nuclei that broke through ILM in the HNK-L intervention group was significantly reduced (P0.05) and different. There was also a significant difference in the number of intervention doses. The greater the dose of HNK-L, the less the number of ECs nuclei that broke through ILM (P0.05).
Conclusion: the OIR model can be established in C57BL/6J mice. As a specific ECs marker, CD105 can show the formation of RNV. Intraperitoneal injection and honokiol liposome can significantly inhibit the formation of RNV in the OIR model mice.
The second part is the effect of honokiol liposome on the expression of VEGF-A, VEGFR-2 and ES in oxygen induced retinopathy.
Objective: to detect the expression level of neovascular related factors VEGF-A. VEGFR-2 and ES in the retina tissue of HNK-L and to investigate the mechanism of HNK-L from the level of cytokine in the retina tissue of OIR.
Methods: 72 P7C57BL/6J mice were randomly divided into normal group, OIR model group, blank liposome group and large, medium and small dose HNK-L intervention group. The normal group was raised in the normal oxygen environment, the other five groups were set up the OIR model in the first part, the HNK-L intervention group was returned to the normal oxygen in P12, and the 5D was given a large dose of HNK-L in the abdominal cavity once a day: large dose of HNK-L. Group (100mg/kg), medium dose group (50mg/kg), small dose group (25mg/kg), and P17 broken neck were executed in each group. The expression of VEGF-A, VEGFR-2, ES mRNA and protein in the retinal tissue of OIR model was detected.
Results: the expression of VEGF-A, VEGFR-2mRNA and protein in the OIR model group was significantly higher than that in the normal group (P0.05), and there was no significant difference between the ES expression and the normal group (P0.05).HNK-L intervention group VEGF-A, and the expression of VEGFR-2mRNA and protein was significantly lower than the OIR model group and the blank liposome group (P0.05), while the HNK-L intervention group was more than the normal group. The /OIR model group / blank liposome group was significantly up-regulated, and the intervention effect of HNK-L was positively correlated with the dose.
Conclusion: the mechanism of HNK-L inhibiting the formation of RNV in OIR model may be related to the down-regulation of VEGF-A, VEGFR-2 expression and up regulation of ES expression in HNK-L.
The third part and the effect of magnolol liposome on MAPK signaling pathway in oxygen induced retinopathy
Objective: To observe the effect of HNK-L on the MAPK family ERK1/2, p38MAPK and JNK signaling pathways during the formation of OIR model RNV, and to explore the mechanism of RNV formation in the OIR model of HNK-L intervention from the point of signal transduction pathway.
Methods: 36 P7d C57BL/6J mice were randomly divided into normal group, OIR model group, blank liposome group and large, medium and small dose HNK-L intervention group. The normal group was raised in the normal oxygen environment, and the other groups were set up OIR model.HNK-L large according to the first part method, and the small dose intervention group was given the different doses of 0.2ml (100mg/kg, 50mg/k) daily, respectively, after the oxygen delivery box. G, 25mg/kg) HNK-L was injected intraperitoneally, and P17 was sacrificed to kill each group of mice. Western blot assay was used to detect the protein content of p-ERK1/2, p-p38MAPK and p-JNK in retina.
Results: the content of p-ERK1/2, p-p38MAPK and p-JNK protein in the retina of the OIR model group was significantly enhanced (P0.05).HNK-L intervention group p-ERK1/2, p-p38MAPK and p-JNK protein was significantly lower than the OIR model group / blank liposome group (P0.05), and the inhibitory effect was positively correlated with the dosage.
Conclusion: the occurrence of RNV in the OIR model is related to the expression of ERK, p38MAPK and JNK in the MAPK family, and the effect of HNK-L on the formation of RNV may be related to the influence of the MAPK family signal pathway.

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R774.1

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