基于TGF-β与NF-κB信号探讨龟鹿二仙胶对GIOP斑马鱼治疗作用机制

发布时间：2018-01-26 11:59

本文关键词： 骨质疏松症龟鹿二仙胶斑马鱼 TGF-β NF-κB　出处：《南京中医药大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:运用龟鹿二仙胶干预斑马鱼糖皮质激素诱导骨质疏松(Glucocorticoid induced osteoporosis,GIOP)模型,观察其治疗效果,分析其作用机制。方法:1、采用成年斑马鱼建立GIOP模型。运用Masson和HE观察斑马鱼鱼鳞微观结构,Western blot法检测斑马鱼TGF-β、Smad3、P-Smad3蛋白的表达,分析骨质疏松(Osteoporosis,OP)和TGF-β/Smad信号通路之间的联系。2、运用龟鹿二仙胶对该模型进行干预。采用Masson和HE观察斑马鱼鱼鳞的微观结构,检测斑马鱼炎症因子IL-6、TNF-α、IL-1β的含量,RT-PCR检测斑马鱼TGF-β、Smad3、AP-1、NF-κBP65 基因表达,Western blot 法检测斑马鱼 TGF-β、Smad3、P-Smad3、AP-1、P-NF-KBP65、NF-κBP65、P-IκBα、IκBa蛋白的表达,分析龟鹿二仙胶基于TGF-β信号和NF-κBP信号治疗GIOP斑马鱼的作用机制。结果:1、建立GIOP斑马鱼模型。Masson及HE染色观察:空白组Ⅰ型胶原致密规整,地塞米松低、中剂量组与空白组相比没有差异;地塞米松高剂量组Ⅰ型胶原松散紊乱,与空白组相比有差异。检测蛋白表达:与空白组相比,地塞米松高剂量组TGF-β与P-Smad3蛋白降低(P0.01),地塞米松低剂量和中剂量组没有差异(P0.05)。2、Masson及HE染色观察:空白组斑马鱼Ⅰ型胶原致密规整,地塞米松组Ⅰ型胶原松散紊乱;给予龟鹿二仙胶低、中、高剂量和骨化三醇后,四组均能显著改善地塞米松组Ⅰ型胶原松散紊乱的情况。3、检测炎症因子:地塞米松组较空白组IL-6、TNF-α、IL-1β含量升高(P0.01),骨化三醇与龟鹿二仙胶低、中、高剂量组较地塞米松组IL-6、TNF-α、IL-1β含量降低(P0.01)。4、检测mRNA表达:地塞米松组较空白组TGF-β、Smad3含量降低(P0.01),NF-κBP65、AP-1含量升高(P0.01),骨化三醇与龟鹿二仙胶低、中、高剂量组较地塞米松组 TGF-β、Smad3 含量升高(P0.01),NF-κBP65、AP-1 含量降低(P0.01)。5、检测蛋白表达:地塞米松组较空白组TGF-β、P-Smad3含量降低(P0.01),NF-KBP65、IκBα、AP-1含量升高(P0.01),骨化三醇与龟鹿二仙胶低、中、高剂量组较地塞米松组TGF-β、P-Smad3含量升高(P0.01),NF-κBP65、IκBα、AP-1 含量降低(P0.01)。结论:1、成功建立斑马鱼GIOP模型。2、龟鹿二仙胶经TGF-β/Smad3信号通路促进胶原合成治疗OP。3、龟鹿二仙胶调控核转录因子NF-κB和AP-1抑制炎症反应治疗OP。
[Abstract]:Objective: to study the effect of Guilu Erxian gum on glucocorticoid induced osteoporosis induced by glucocorticoid in zebrafish. Methods the GIOP model was established by adult zebrafish and the microscopic structure of zebrafish scales was observed by Masson and HE. The expression of P-Smad3 protein in zebrafish TGF- 尾 Smad3 was detected by Western blot, and Osteoporosis was analyzed. The relationship between OP2 and TGF- 尾 / Smad signaling pathway was observed by Masson and HE. The levels of IL-6 TNF- 伪 and IL-1 尾 in zebrafish were detected by RT-PCR. The levels of TGF- 尾 and Smad3AP-1 in zebrafish were detected by reverse transcription-polymerase chain reaction (RT-PCR). The expression of NF- 魏 BP65 gene was detected by Western blot in zebrafish TGF- 尾 Smad3AP-1 and P-NF-KBP65. NF- 魏 BP65, P-I 魏 B 伪, I 魏 Ba protein expression. The mechanism of the treatment of GIOP zebrafish based on TGF- 尾 signal and NF- 魏 BP signal was analyzed. GIOP zebrafish model. Masson and HE staining: type I collagen in blank group was compact and regular, dexamethasone was low, there was no difference between middle dose group and blank group. High dose of dexamethasone group I collagen loose disorder, compared with the blank group is different. Detection of protein expression: compared with the blank group. The levels of TGF- 尾 and P-Smad3 protein decreased in the high dose group of dexamethasone, but there was no difference between the low and middle dose groups of dexamethasone. Observation of Masson and HE staining: type I collagen of zebrafish in blank group was compact and regular, while type I collagen in dexamethasone group was loose and disorder. After giving Guelu Erxian gum low, medium and high dose and ossifying triol, all the four groups could significantly improve the loose disorder of type I collagen in dexamethasone group. The inflammatory factor: dexamethasone group was more than blank group IL-6. The content of TNF- 伪 and IL-1 尾 increased P0.01A, and the ossification triol and Guilu Erxian gum were lower than that of dexamethasone group, and the middle dose group was higher than that of dexamethasone group (IL-6) TNF- 伪. The content of IL-1 尾 decreased (P 0.01). 4. The expression of mRNA: compared with the control group, the content of TGF- 尾 Smad3 in dexamethasone group was lower than that in control group (P 0.01), and NF- 魏 BP65 was decreased in dexamethasone group. The content of AP-1 was higher than that of dexamethasone group (P 0.01), and the content of ossification triol and Guilu Erxian gum was lower than that of dexamethasone group (P 0.01). The content of TGF- 尾 Smad3 in high dose group was higher than that in dexamethasone group. The content of NF- 魏 BP65 AP-1 was lower than that of control group (P 0.01). The protein expression of Dexamethasone group was lower than that of control group (P 0.01). The content of NF-KBP65 and I 魏 B 伪 AP-1 increased, and the ossification triol was lower than that of the dexamethasone group, and the middle and high dose groups were lower than that of dexamethasone group TGF- 尾. The content of P-Smad3 was increased and the content of NF- 魏 B P65, I 魏 B 伪 and AP-1 was decreased. Conclusion: 1. The GIOP model of zebrafish was successfully established. 2. The Glucoderma vulgaris promoted collagen synthesis in the treatment of OP.3 through TGF- 尾 / Smad3 signaling pathway. Guilu Erxian Gluten regulates nuclear transcription factors NF- 魏 B and AP-1 to inhibit inflammatory response in the treatment of OPP.
【学位授予单位】：南京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5

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