本文關(guān)鍵詞： 骨質(zhì)疏松癥 龜鹿二仙膠 斑馬魚 TGF-β NF-κB　出處：《南京中醫(yī)藥大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:運用龜鹿二仙膠干預(yù)斑馬魚糖皮質(zhì)激素誘導(dǎo)骨質(zhì)疏松(Glucocorticoid induced osteoporosis,GIOP)模型,觀察其治療效果,分析其作用機(jī)制。方法:1、采用成年斑馬魚建立GIOP模型。運用Masson和HE觀察斑馬魚魚鱗微觀結(jié)構(gòu),Western blot法檢測斑馬魚TGF-β、Smad3、P-Smad3蛋白的表達(dá),分析骨質(zhì)疏松(Osteoporosis,OP)和TGF-β/Smad信號通路之間的聯(lián)系。2、運用龜鹿二仙膠對該模型進(jìn)行干預(yù)。采用Masson和HE觀察斑馬魚魚鱗的微觀結(jié)構(gòu),檢測斑馬魚炎癥因子IL-6、TNF-α、IL-1β的含量,RT-PCR檢測斑馬魚TGF-β、Smad3、AP-1、NF-κBP65 基因表達(dá),Western blot 法檢測斑馬魚 TGF-β、Smad3、P-Smad3、AP-1、P-NF-KBP65、NF-κBP65、P-IκBα、IκBa蛋白的表達(dá),分析龜鹿二仙膠基于TGF-β信號和NF-κBP信號治療GIOP斑馬魚的作用機(jī)制。結(jié)果:1、建立GIOP斑馬魚模型。Masson及HE染色觀察:空白組Ⅰ型膠原致密規(guī)整,地塞米松低、中劑量組與空白組相比沒有差異;地塞米松高劑量組Ⅰ型膠原松散紊亂,與空白組相比有差異。檢測蛋白表達(dá):與空白組相比,地塞米松高劑量組TGF-β與P-Smad3蛋白降低(P0.01),地塞米松低劑量和中劑量組沒有差異(P0.05)。2、Masson及HE染色觀察:空白組斑馬魚Ⅰ型膠原致密規(guī)整,地塞米松組Ⅰ型膠原松散紊亂;給予龜鹿二仙膠低、中、高劑量和骨化三醇后,四組均能顯著改善地塞米松組Ⅰ型膠原松散紊亂的情況。3、檢測炎癥因子:地塞米松組較空白組IL-6、TNF-α、IL-1β含量升高(P0.01),骨化三醇與龜鹿二仙膠低、中、高劑量組較地塞米松組IL-6、TNF-α、IL-1β含量降低(P0.01)。4、檢測mRNA表達(dá):地塞米松組較空白組TGF-β、Smad3含量降低(P0.01),NF-κBP65、AP-1含量升高(P0.01),骨化三醇與龜鹿二仙膠低、中、高劑量組較地塞米松組 TGF-β、Smad3 含量升高(P0.01),NF-κBP65、AP-1 含量降低(P0.01)。5、檢測蛋白表達(dá):地塞米松組較空白組TGF-β、P-Smad3含量降低(P0.01),NF-KBP65、IκBα、AP-1含量升高(P0.01),骨化三醇與龜鹿二仙膠低、中、高劑量組較地塞米松組TGF-β、P-Smad3含量升高(P0.01),NF-κBP65、IκBα、AP-1 含量降低(P0.01)。結(jié)論:1、成功建立斑馬魚GIOP模型。2、龜鹿二仙膠經(jīng)TGF-β/Smad3信號通路促進(jìn)膠原合成治療OP。3、龜鹿二仙膠調(diào)控核轉(zhuǎn)錄因子NF-κB和AP-1抑制炎癥反應(yīng)治療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- 偽 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 偽, 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- 偽 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- 偽. 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 偽 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 偽 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.
【學(xué)位授予單位】：南京中醫(yī)藥大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R285.5

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