本文選題：和厚樸酚脂質(zhì)體 + 氧誘導(dǎo)視網(wǎng)膜病變��； 參考：《武漢大學(xué)》2012年博士論文

【摘要】：第一部分和厚樸酚脂質(zhì)體對(duì)氧誘導(dǎo)視網(wǎng)膜新生血管形成的抑制作用 目的：探討和厚樸酚脂質(zhì)體(honokiol liposome, HNK-L)對(duì)氧誘導(dǎo)視網(wǎng)膜病變模型(oxygen-induced retinoopathy, OIR)視網(wǎng)膜新生血管(retinal neovascularization, RNV)形成的作用。 方法：取72只鼠齡為7d(P7)的C57BL/6J小鼠,隨機(jī)分為6組：正常組、OIR模型組、空白脂質(zhì)體組和大、中、小劑量HNK-L干預(yù)組。正常組小鼠持續(xù)在常氧環(huán)境飼養(yǎng),其余五組置于密閉氧箱中,24h維持氧濃度在75%±2%,5d后(P12)將小鼠返回到常氧,繼續(xù)飼養(yǎng)至P17以建立OIR模型；其中HNK-L干預(yù)組再每日給予腹腔注射0.2ml不同劑量HNK-L：大劑量組(100mg/kg),中劑量組(50mg/kg),小劑量組(25mg/kg)。各組小鼠于P17斷頸處死,立即摘眼球制作視網(wǎng)膜石蠟切片HE染色以及CD105抗體免疫組化標(biāo)記血管內(nèi)皮細(xì)胞(endothelial cells, ECs),計(jì)算視網(wǎng)膜切片中突破內(nèi)界膜(internal limiting membrane, ILM)的ECs核數(shù)目。 結(jié)果：OIR模型組小鼠視網(wǎng)膜可見大量RNV形成,OIR模型小鼠突破ILM的EC核數(shù)目明顯多于正常組,差異有統(tǒng)計(jì)學(xué)意義(P0.01),同時(shí)該組運(yùn)用CD105抗體免疫組化標(biāo)記EC染色增強(qiáng)。不同劑量HNK-L干預(yù)組突破ILM的ECs核數(shù)目均有明顯減少(P0.05),且不同干預(yù)劑量的計(jì)數(shù)亦有顯著性差異,HNK-L劑量越大,突破ILM的ECs核數(shù)目越少(P0.05)。 結(jié)論：應(yīng)用C57BL/6J小鼠可建立OIR模型；CD105作為特異性ECs標(biāo)記物可以較好地顯示RNV形成情況；腹腔注射和厚樸酚脂質(zhì)體能顯著抑制OIR模型小鼠RNV形成。 第二部分和厚樸酚脂質(zhì)體對(duì)氧誘導(dǎo)視網(wǎng)膜病變VEGF-A, VEGFR-2及ES表達(dá)的影響 目的：檢測HNK-L抑制OIR模型中視網(wǎng)膜組織新生血管相關(guān)因子VEGF-A. VEGFR-2及ES的表達(dá)水平,從細(xì)胞因子水平探討HNK-L的作用機(jī)制。 方法：取P7C57BL/6J小鼠72只,隨機(jī)分為正常組、OIR模型組、空白脂質(zhì)體組和大、中、小劑量HNK-L干預(yù)組。正常組在常氧環(huán)境飼養(yǎng),其余五組按第一部分建立OIR模型,HNK-L干預(yù)組于P12返回到常氧,5d每日一次給予腹腔注射0.2ml不同劑量HNK-L：大劑量組(100mg/kg),中劑量組(50mg/kg),小劑量組(25mg/kg),P17斷頸處死各組小鼠,檢測VEGF-A, VEGFR-2及ES mRNA及蛋白在OIR模型視網(wǎng)膜組織的表達(dá)量。 結(jié)果：OIR模型組中VEGF-A及VEGFR-2mRNA及蛋白表達(dá)較正常組明顯增強(qiáng)(P0.05)；ES表達(dá)與正常組則無顯著性差異(P0.05)。HNK-L干預(yù)組VEGF-A、 VEGFR-2mRNA及蛋白表達(dá)明顯低于OIR模型組及空白脂質(zhì)體組(P0.05)；而HNK-L干預(yù)組ES mRNA及蛋白表達(dá)則較正常組/OIR模型組/空白脂質(zhì)體組明顯上調(diào)；HNK-L干預(yù)效應(yīng)與給藥劑量呈正相關(guān)。 結(jié)論：HNK-L抑制OIR模型中RNV形成的機(jī)制可能與HNK-L下調(diào)VEGF-A、 VEGFR-2的表達(dá),上調(diào)ES表達(dá)相關(guān)。 第三部分和厚樸酚脂質(zhì)體對(duì)氧誘導(dǎo)視網(wǎng)膜病變MAPK信號(hào)通路的影響 目的：觀察HNK-L在OIR模型RNV形成時(shí),對(duì)MAPK家族ERK1/2,p38MAPK及JNK信號(hào)通路的影響,從信號(hào)轉(zhuǎn)導(dǎo)通路角度探討HNK-L干預(yù)OIR模型中RNV形成的機(jī)制。 方法：取P7d C57BL/6J小鼠36只,隨機(jī)分為正常組,OIR模型組、空白脂質(zhì)體組和大、中、小劑量HNK-L干預(yù)組。正常組在常氧環(huán)境飼養(yǎng),其余組按第一部分方法建立OIR模型。HNK-L大、中、小劑量干預(yù)組在出氧箱后,每日分別給予0.2ml不同劑量(100mg/kg,50mg/kg,25mg/kg) HNK-L腹腔注射,P17斷頸處死各組小鼠,western blot法檢測視網(wǎng)膜組織p-ERK1/2、 p-p38MAPK及p-JNK的蛋白量。 結(jié)果：OIR模型組視網(wǎng)膜p-ERK1/2、 p-p38MAPK及p-JNK蛋白含量較正常組明顯增強(qiáng)(P0.05)。HNK-L干預(yù)組p-ERK1/2、 p-p38MAPK及p-JNK蛋白明顯低于OIR模型組／空白脂質(zhì)體組的水平(P0.05),且抑制效應(yīng)與給藥劑量呈正相關(guān)。 結(jié)論：OIR模型中RNV發(fā)生與MAPK家族ERK、 p38MAPK及JNK表達(dá)均相關(guān)；HNK-L抑制RNV形成的作用可能與影響MAPK家族信號(hào)通路有關(guān)。
[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.

【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R774.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前8條

1 林桂蕓,謝生發(fā),謝鴻,鷗俊;和厚樸酚抑菌作用的研究[J];成都大學(xué)學(xué)報(bào)(自然科學(xué)版);2003年02期

2 許玉花;劉燕;張金玲;;p38絲裂原活化蛋白激酶在大鼠視網(wǎng)膜缺血再灌注損傷后的磷酸化及其意義[J];第四軍醫(yī)大學(xué)學(xué)報(bào);2007年08期

3 黎增輝;廖愛軍;;JNK信號(hào)通路[J];國際病理科學(xué)與臨床雜志;2010年03期

4 王冬娥;陸羨;;和厚樸酚的藥理作用及其抗腫瘤的機(jī)制研究進(jìn)展[J];醫(yī)學(xué)綜述;2009年11期

5 雷曉溪;劉蘇;鄢秀菊;王強(qiáng);;和厚樸酚抑制視網(wǎng)膜新生血管形成的實(shí)驗(yàn)研究[J];中成藥;2007年05期

6 王立青,江榮高,陳蕙芳;厚樸酚與和厚樸酚藥理作用的研究進(jìn)展[J];中草藥;2005年10期

7 李杰萍,梁統(tǒng),周克元;厚樸酚對(duì)趨化三肽激活的大鼠中性粒細(xì)胞功能的影響[J];中國藥科大學(xué)學(xué)報(bào);2003年03期

8 王嘉;王_";王自強(qiáng);陳菲;覃文新;;和厚樸酚抗血管生成作用的實(shí)驗(yàn)研究[J];腫瘤;2007年07期

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