本文關(guān)鍵詞：hPPARs天然配體篩選平臺(tái)的建立及應(yīng)用　出處：《重慶醫(yī)科大學(xué)》2008年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： MBP hPPARs RBA 轉(zhuǎn)錄激活 激動(dòng)劑

【摘要】： 目的建立人過(guò)氧化物酶體增殖物活化受體(human peroxisome prolifterator-activated receptors,hPPARs)配體的篩選平臺(tái),并從中藥單體中篩查hPPARs的天然配體。方法從人肝癌組織提取總RNA,RT-PCR擴(kuò)增出hPPARs配體結(jié)合區(qū)(ligand binding domain,LBD)cDNA,將其克隆入表達(dá)載體pMAL-p2x麥芽糖結(jié)合蛋白(maltose binding protein,MBP)編碼基因malE序列的下游,轉(zhuǎn)化入E coli.TB1。含重組質(zhì)粒的宿主菌經(jīng)最佳優(yōu)化條件誘導(dǎo)后,超聲破碎取上清。產(chǎn)物用多糖親和樹(shù)脂進(jìn)行純化,純化的MBP-hPPARsLBD用Xa因子酶切、多糖親和樹(shù)脂層析、DEAE-52陰離子交換層析純化回收。放射性標(biāo)記配基結(jié)合實(shí)驗(yàn)(radioligand binding assays,RBA)鑒定MBP-hPPARsLBD和hPPARsLBD的配體結(jié)合功能。高效液相色譜紫外檢測(cè)法(high performance liquid chromatography and ultraviolet detection,HPLC-UV)建立hPPARsLBD配體的篩選平臺(tái),并對(duì)待試中藥單體進(jìn)行篩選,用經(jīng)典的放射性標(biāo)記配基競(jìng)爭(zhēng)結(jié)合實(shí)驗(yàn)(radioligand binding competition assays,RBCA)法對(duì)HPLC-UV法篩選結(jié)果進(jìn)行檢驗(yàn)。反式激活報(bào)告基因法測(cè)定篩出藥物的功能活性。結(jié)果含重組質(zhì)粒的宿主菌經(jīng)最佳優(yōu)化條件(0.4 mmol/L,30℃振蕩培養(yǎng)6 h)誘導(dǎo),從1L培養(yǎng)物中可獲得約20 mg MBP-hPPARsLBD,約占胞質(zhì)總蛋白的31%。經(jīng)純化以及酶切回收,獲得了電泳純的MBP-hPPARsLBD和hPPARsLBD,其產(chǎn)量分別為14 mg/L和5 mg/L,得率分別為70%和45%。RBA顯示MBP-hPPARsLBD和hPPARsLBD的結(jié)合配體功能是等效的。根據(jù)分子排阻色譜分離(Molecular Exclusion Chromatography,MEC)理論建立了HPLC-UV法hPPARs配體篩選平臺(tái)。用該平臺(tái)對(duì)待試藥進(jìn)行了篩選,結(jié)果顯示小檗堿(berberine,Ber)和柚皮素(naringenin,Nar)能特異性結(jié)合hPPARα,大豆苷元(daidzein ,Dai)能特異性結(jié)合hPPARα和hPPARγ1。RCBA法結(jié)果與HPLC-UV法相同。用反式激活報(bào)告基因法測(cè)試待試中藥單體的功能活性,結(jié)果顯示,Dai、Ber和Nar能激活hPPARα,Dai、Nar和姜黃素(curcumin ,Cur)能激活hPPARγ1,而Dai、Ber、Nar、虎杖苷(polydatin, Pol)對(duì)hPPARδ有一定程度的激活作用。結(jié)論1)利用pMAL-p2x表達(dá)純化體系,獲得了大量、可溶的、電泳純的MBP-hPPARsLBD和hPPARsLBD。經(jīng)RBA鑒定,MBP的融合沒(méi)有影響hPPARsLBD的配體結(jié)合功能;2)HPLC-UV法為一種新的,簡(jiǎn)便、經(jīng)濟(jì)、環(huán)保、有效的受體配體篩選方法,可用于研究受體配體結(jié)合反應(yīng);3)確認(rèn)Ber、Nar是hPPARα的天然激動(dòng)劑,Dai為hPPARα和hPPARγ1的雙激動(dòng)劑。此外,Cur對(duì)hPPARγ1, Dai、Ber、Nar、Pol對(duì)hPPARδ有一定程度的激活作用。提示Dai、Ber、Nar、Pol和Cur等中藥單體至少通過(guò)激動(dòng)hPPARs,調(diào)節(jié)下游多個(gè)靶基因表達(dá),從而發(fā)揮調(diào)血脂、降血糖、抗腫瘤、抗炎等多種藥理效應(yīng)。
[Abstract]:Objective to establish a screening platform for human peroxisome prolifterator-activated receptors (hPPARs) ligand and screen the natural ligands of hPPARs from Chinese herbal monomers. Methods the total RNA was extracted from human liver cancer tissue, and the ligand binding domain (LBD) cDNA was amplified by RT-PCR. The cDNA was cloned into the downstream of the expression vector pMAL-p2x maltose binding protein (maltose binding hPPARs) coding gene sequence, and transformed into the binding. The host bacteria containing the recombinant plasmid were induced by optimal optimization, and the supernatant was broken by ultrasonic breakage. The product was purified by polysaccharide affinity resin, and purified MBP-hPPARsLBD was purified by Xa factor digestion, polysaccharide affinity resin chromatography and DEAE-52 anion exchange chromatography. The ligand binding function of MBP-hPPARsLBD and hPPARsLBD was identified by radioligand binding assays (RBA). The UV detection method of high performance liquid chromatography (high performance liquid chromatography and ultraviolet detection, HPLC-UV) and establish a screening system for hPPARsLBD ligands, and to try traditional Chinese medicine monomers were used, combined with the experimental radiolabeled ligand competition (radioligand binding competition assays classic, RBCA) method for HPLC-UV screening test results. The functional activity of the sifting drug was determined by the trans activation report gene method. Results the host strain containing recombinant plasmid was induced by optimum conditions (0.4 mmol/L, 30 h oscillating for 6 h), and about 20 mg MBP-hPPARsLBD was obtained from 1L culture, accounting for about 31% of total cytoplasmic protein. The purified MBP-hPPARsLBD and hPPARsLBD were obtained by purification and enzyme digestion. The yield was 14 mg/L and 5 mg/L, respectively, and the yield was 70% and 45%, respectively. RBA shows that the binding ligand function of MBP-hPPARsLBD and hPPARsLBD is equivalent. Based on the theory of molecular exclusion chromatography (Molecular Exclusion Chromatography, MEC), a HPLC-UV hPPARs ligand screening platform was established. This platform is used to treat reagents were screened, showed that berberine (berberine, Ber) and naringenin (naringenin, Nar) can specifically bind to hPPAR alpha, daidzein (daidzein, Dai) can specifically bind to hPPAR alpha and hPPAR gamma 1. The results of RCBA method are the same as that of HPLC-UV method. Trans activation of reporter gene method test of tested functional activity, traditional Chinese medicine monomer showed that Dai, Ber and Nar can activate hPPAR alpha, Dai, Nar and curcumin (curcumin, Cur) can activate hPPAR gamma 1, Dai, Ber, Nar, polydatin (polydatin, Pol) activity the degree of hPPAR. Conclusion 1) a large, soluble, electrophoretic pure MBP-hPPARsLBD and hPPARsLBD were obtained by using pMAL-p2x to express the purification system. Identified by RBA, MBP fusion hPPARsLBD did not affect the ligand binding function; 2) the HPLC-UV method is a new, simple and economical, environmental and effective receptor ligand screening method can be used to study the receptor ligand binding reaction; 3) confirm the Ber, Nar is hPPAR a natural agonist, Dai hPPAR alpha 1 double and hPPAR gamma agonist. In addition, the activation of hPPAR gamma 1, Dai, Ber, Nar, Pol on hPPAR delta is to a certain extent by Cur. It suggests that Dai, Ber, Nar, Pol and Cur can modulate the expression of multiple target genes at least through activating hPPARs, and play a variety of pharmacological effects such as lipid regulation, hypoglycemic, anti-tumor, anti-inflammatory and so on.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類號(hào)】：R346

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 段雯;李偉;靳紅衛(wèi);夏鑄;程訓(xùn)官;張昕宇;鄭小紅;余瑜;;基于脂代謝靶點(diǎn)蛋白hPPARγ-LBD的抗腫瘤藥物篩選模型的研究[J];重慶醫(yī)科大學(xué)學(xué)報(bào);2010年06期

相關(guān)碩士學(xué)位論文 前5條

1 陳加飛;小檗堿抗血管緊張素Ⅳ誘導(dǎo)的心肌肥大與PPAR-α/NO信號(hào)通路關(guān)系研究[D];重慶醫(yī)科大學(xué);2011年

2 王全華;小檗堿抗Ang Ⅳ誘導(dǎo)的血管平滑肌細(xì)胞增殖與PPARα-NO信號(hào)通路關(guān)系研究[D];重慶醫(yī)科大學(xué);2011年

3 王明豐;小檗堿抗高糖高胰島素誘導(dǎo)心肌肥大與PPARα-NO信號(hào)通路關(guān)系研究[D];重慶醫(yī)科大學(xué);2010年

4 段雯;可溶性hPPARγ-LBD重組蛋白的制備及功能表征[D];重慶醫(yī)科大學(xué);2010年

5 彭曉鳳;PPARγ-PI_3K/AKT-NO信號(hào)通路在姜黃素抗高糖高胰島素誘導(dǎo)心肌肥大中的作用[D];重慶醫(yī)科大學(xué);2012年

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本文編號(hào)：1345679