發(fā)布時(shí)間：2018-03-28 14:46

  本文選題：細(xì)胞松弛素　切入點(diǎn)：細(xì)胞損傷　出處：《揚(yáng)州大學(xué)》2014年碩士論文

【摘要】：內(nèi)生真菌(EndophytEC fungi)是指其在生活史的某一段時(shí)期生活在植物組織內(nèi),對(duì)植物組織沒(méi)有引起明顯病害癥狀的真菌。內(nèi)生真菌種類繁多,能產(chǎn)生結(jié)構(gòu)多樣的活性代謝產(chǎn)物,近年來(lái)已成為國(guó)內(nèi)外資源開(kāi)發(fā)利用的研究熱點(diǎn)之一。 本文所研究的八種細(xì)胞松弛素(cytochalasin, Cyt)類化合物是從白茅內(nèi)生菌球毛殼菌Chaetomium globosum IFB-WZQ和青皮內(nèi)生菌擬莖點(diǎn)霉Phomopsis sp. IFB-E060固體發(fā)酵產(chǎn)物中分離獲得的。分別是：chaetoglobosin F (編號(hào)WQ-1)、chaetoglobosin Fex (WQ-2)、chaetoglobosin E (WQ-3)、cytoglobosin A (WQ-4)、penochalasin C (WQ-6)、 isochaetoglobosin D (WQ-9)、cytochalasin H (QS-1)和18-methoxycytochalasin J (QS-2)。它們都具有相似的氫化異吲哚-1-酮結(jié)構(gòu)母核,在10位有吲哚或苯環(huán)取代。早期在對(duì)編號(hào)為QS-1化合物抗癌活性研究時(shí),對(duì)其它活性也進(jìn)行了篩選,發(fā)現(xiàn)該化合物具有較好的抗氧化活性,且對(duì)PC12細(xì)胞損傷有一定潛在的保護(hù)作用。查閱文獻(xiàn)未見(jiàn)有類似的報(bào)道。鑒于該類化合物擁有相同的母核,其差異主要在母核和大環(huán)的取代基上。為此,本研究應(yīng)用體外抗氧化模型、H2O2和MPP+損傷細(xì)胞模型,考察八種Cyt類化合物是否具有抗氧化活性?其活性強(qiáng)度與結(jié)構(gòu)的關(guān)系如何?對(duì)自由基及神經(jīng)毒素引起的細(xì)胞損傷是否具有保護(hù)作用?通過(guò)對(duì)該類化合物抗氧化活性構(gòu)效關(guān)系規(guī)律的揭示,將為尋找具有強(qiáng)抗氧化活性的先導(dǎo)化合物提供實(shí)驗(yàn)資料,對(duì)植物內(nèi)生菌這一新型藥用資源的開(kāi)發(fā)利用提供有益思路。主要實(shí)驗(yàn)結(jié)果如下： 1.清除自由基能力測(cè)定 方法：(1)DPPH自由基測(cè)定：采用微量比色法測(cè)定DPPH自由基吸光度,計(jì)算清除率。Cyt類化合物的濃度為10-6,10-4,10-4,10-3,10-2,10-1,5×10-1mmol/L, VE為陽(yáng)性藥。(2)ABTS自由基測(cè)定：采用ABTS法微量比色測(cè)定ABTS自由基吸光度,計(jì)算清除率。以Trolox(維生素E的類似物)為標(biāo)準(zhǔn)對(duì)照,計(jì)算Trolox當(dāng)量總抗氧化能力(TEAC)。Cyt類化合物的濃度為10-6,10-5,10-4,10-3,10-2mmol/L。(3)應(yīng)用SPSS17.0計(jì)算半抑制濃度(EC50),反映藥物的效價(jià)強(qiáng)度,Scott法計(jì)算最大效應(yīng)(Emax),反映藥物的效能。采用單因素方差分析和SNK檢驗(yàn),進(jìn)行抗氧化能力排序。 結(jié)果：(1)DPPH自由基清除能力：最大清除率Emax90%的有：WQ-9、QS-1和WQ-1；Emax=35%～60%:WQ-2、WQ-4、WQ-3和QS-2；通過(guò)對(duì)ECso和Emax統(tǒng)計(jì)分析,八種Cyt類化合物的抗氧化作用效價(jià)強(qiáng)度排序?yàn)椋篧Q-9=QS-1=WQ-1WQ-2 WQ-4=WQ-3QS-2。效能排序與上述一致。WQ-6EC50無(wú)法計(jì)算,無(wú)清除自由基能力。 (2)ABTS自由基清除能力：依據(jù)EC50,八種化合物清除ABTS自由基能力排序?yàn)椋篞S-1=WQ-9=WQ-1WQ-2WQ-3QS-2WQ-4,WQ-6EC50無(wú)法計(jì)算；依據(jù)Emax,效能排序?yàn)椋篞S-1=WQ-9=WQ-1WQ-2=WQ-3=QS-2=WQ-4WQ-6。QS-1的TEAC最大,達(dá)到Trolox的746倍,其次是WQ-1和WQ-9為373倍,WQ-4最低為0.001倍,WQ-6無(wú)法計(jì)算。 結(jié)論：除WQ-6外,其余七種化合物均有不同程度的體外抗氧化活性,其中,QS- 1、WQ-9和WQ-1活性最強(qiáng)。 2.抗H202損傷細(xì)胞作用研究 方法：通過(guò)對(duì)不同濃度(50～800μmol/L)H2O2作用不同時(shí)間(2,3,4,5,6h)考察,確定H2O2制備PC12細(xì)胞損傷模型條件。MTT法測(cè)定細(xì)胞存活率；比色法測(cè)定乳酸脫氫酶(LDH)活性。分組：(1)對(duì)照組：含0.01%DMSO的DMEM溶液；(2)模型組：200μmol/LH2O2溶液；(3)陽(yáng)性藥物組：1000μmol/L VE溶液；(4)Cyt類化合物組：濃度為0.001,0.0025,0.005,0.01,0.05,0.1,0.2μmol/L。EC50(μmol/L)和Emax計(jì)算以及統(tǒng)計(jì)分析同上。 結(jié)果：(1)H2O2損傷PC12細(xì)胞模型：以200μmol/L H2O2溶液作用PC12細(xì)胞4h,細(xì)胞存活率為41.3%±3.6%(P0.001),培養(yǎng)基上清液中LDH活性出對(duì)照組16.9±1.4U/dl上升為109.1±2.5U/dl(P0.05)。表明H2O2模型制備成功。 (2)細(xì)胞存活率：除WQ-6外,其它化合物均可提高細(xì)胞存活率,Emax90%的有：WQ-9、WQ-1和QS-1；Emax=73%～77%:WQ-2,WQ-4和WQ-3。QS-2雖也能提高細(xì)胞存活率(P0.01,0.001),但沒(méi)有量效關(guān)系,WQ-6則無(wú)效。經(jīng)統(tǒng)計(jì),效價(jià)強(qiáng)度排序?yàn)椋篧Q-1=QS-1=WQ-9WQ-4=WQ-2WQ-3,QS-2和WQ-6無(wú)法計(jì)算EC50。效能排序?yàn)椋篧Q-9=WQ-1=QS-1WQ-2=WQ-4=WQ-3QS-2WQ-6。 (3)LDH活性：除WQ-6外,與模型組109.1±2.5U/dl相比,其余七種化合物(0.005μmol/L)均可使LDH活性下降(P0.05,0.01)。經(jīng)統(tǒng)計(jì),LDH作用強(qiáng)度排序?yàn)椋篧Q-9=QS-1=WQ-1=WQ-2WQ-4=WQ-3=QS-2WQ-6。 結(jié)論：除WQ-6外,其余七種化合物均可不同程度地抑制H202誘導(dǎo)的PC12細(xì)胞損傷,使細(xì)胞存活率提高,LDH釋放減少。其中WQ-9,QS-1和WQ-1作用最強(qiáng),WQ-2,WQ-4和WQ-3其次,QS-2無(wú)量效關(guān)系,WQ-6無(wú)效。 3.抗MPP+損傷細(xì)胞作用研究 方法：MTT法測(cè)定八種化合物細(xì)胞毒性,濃度分別為0.005,0.05,0.1,0.2,2.0,20μmol/L。應(yīng)用500μmol/L MPP+作用PC12細(xì)胞48h制備MPP+細(xì)胞損傷模型。給藥分組：(1)對(duì)照組：加含0.01%DMSO的DMEM溶液;(2)模型組：500μmol/L MPP+；(3)cyt類化合物組：濃度為0.005,0.01,0.025,0.05,0.1,0.15,0.2μmol/L。MTT法測(cè)定各組細(xì)胞OD值,計(jì)算細(xì)胞存活率；倒置顯微鏡觀察細(xì)胞形態(tài)；比色法測(cè)定LDH活性；流式細(xì)胞儀測(cè)定ROS含量。EC5o和Emax計(jì)算以及統(tǒng)計(jì)分析同上。 結(jié)果：(1)細(xì)胞毒性：八種化合物各濃度組細(xì)胞存活率均在90%以上,表明它們對(duì)PC12細(xì)胞增殖沒(méi)有明顯影響,無(wú)細(xì)胞毒性。 (2)細(xì)胞存活率：模型組細(xì)胞存活率明顯降低(P0.001)；QS-2不能抑制這種下降,WQ-6(0.005～0.05μmol/L)反增強(qiáng)抑制作用。其余化合物均能不同程度抑制細(xì)胞存活率的下降。效價(jià)排序?yàn)椋篧Q-1=WQ-9=QS-1WQ-2WQ-4WQ-3,WQ-6和QS-2ECso無(wú)法計(jì)算。效能強(qiáng)度排序?yàn)椋篞S-1=WQ-1=WQ-9WQ-2=WQ-4=WQ-3WQ-6=QS-2。 (3)LDH活性：除WQ-6、QS-2和WQ-4外,與模型組134.987U/dl相比,其余5種化合物(0.025μmol/L)均可使LDH活性下降(P0.01,0.001)。經(jīng)統(tǒng)計(jì),作用強(qiáng)度順序?yàn)椋篧Q-1=WQ-9-QS-1WQ-3=WQ-2WQ-4=QS-2WQ-6。 (4)細(xì)胞形態(tài)：對(duì)照組PC12細(xì)胞呈明顯的梭形,MPP+模型組細(xì)胞形態(tài)顯著改變,梭形細(xì)胞減少或消失,出現(xiàn)球形或圓形,八種化合物預(yù)處理后,WQ-1、WQ-2、WQ-9、QS-1能一定程度改善以上形態(tài)改變,其它4種化合物作用不明顯。 (5)ROS含量比值：與對(duì)照組ROS比值為1比較,模型組ROS比值(13.13±0.11)極顯著增高(P0.001)。除WQ-6、WQ-4和QS-2外,與模型組相比,WQ-1、WQ-9、 QS-1、WQ-2和WQ-3能顯著降低比值(P0.001,0.01)。作用強(qiáng)度順序?yàn)椋篧Q-1WQ-9 QS-1WQ-3WQ-2=WQ-4。 結(jié)論：除WQ-6和QS-2外,其余6種化合物均能不同程度的對(duì)抗MPP+誘導(dǎo)的細(xì)胞損傷,其中以WQ-1、WQ-9和QS-1作用最強(qiáng),WQ-2、WQ-3和WQ-4次之。WQ-6不僅不能對(duì)抗MPP+誘導(dǎo)的細(xì)胞損傷,反有促進(jìn)細(xì)胞損傷作用。 4.八種Cyt類化合物抗氧化作用構(gòu)效關(guān)系分析 根據(jù)上述三個(gè)實(shí)驗(yàn)中各指標(biāo)的EC50進(jìn)行構(gòu)效關(guān)系分析：(1)10-吲哚取代或10-苯基取代對(duì)抗氧化活性沒(méi)有影響；(2)化合物抗氧化作用的強(qiáng)弱與結(jié)構(gòu)中的羰基和羥基有關(guān)：21位的酯基比18位的甲氧基作用強(qiáng)；6,7位的環(huán)醚比醇羥基作用強(qiáng)；20位的羰基比醇羥基作用強(qiáng)；(3)大環(huán)內(nèi)含有吡咯環(huán)時(shí)不僅無(wú)抗氧化活性,甚至可增加細(xì)胞損傷。
[Abstract]:Endogenous fungi ( fungi ) refer to fungi which are living in plant tissues for a certain period of life and do not cause obvious disease symptoms to plant tissue . There are many kinds of endogenic fungi , which can produce various active metabolites , which have become one of the hot spots in the development and utilization of resources at home and abroad in recent years .

The eight kinds of cytochalasis ( Cyt ) compounds studied in this paper were Phomopsis sp . from Chaetomium llum sum IFB - WZQ and Penicillium sp . In this study , it was found that the compounds possess the same antioxidant activity , and have some potential protective effects on PC12 cell injury .

1 . Determination of free radical capacity

Methods : ( 1 ) Free radical determination : The concentration of Cyt compound was 10 - 6 , 10 - 4 , 10 - 4 , 10 - 3 , 10 - 2 , 10 - 1 , 5 脳 10 - 1 mmol / L , VE as positive drug . The concentration of Cyt compound was 10 - 6 , 10 - 5 , 10 - 4 , 10 - 3 , 10 - 2mmol / L . ( 3 ) The maximal effect ( Emax ) was calculated by using SPSS 17.0 .

Results : ( 1 ) Free radical scavenging ability : The maximum clearance rate of Emax90 % was WQ - 9 , QS - 1 and WQ - 1 ;
Emax = 35 % ~ 60 % : WQ - 2 , WQ - 4 , WQ - 3 and QS - 2 ;
Through the statistical analysis of ECso and Emax , the effect valence of eight Cyt compounds was ranked as WQ - 9 = QS - 1 = WQ - 1WQ - 2 WQ - 4 = WQ - 3QS - 2 . The efficacy sequencing was consistent with that above . WQ - 6EC50 could not be calculated without scavenging free radicals .

( 2 ) ABTS free radical scavenging ability : According to EC50 , the ability of scavenging free radicals ABTS from eight compounds was : QS - 1 = WQ - 9 = WQ - 1WQ - 2WQ - 3QS - 2WQ - 4 , WQ - 6EC50 could not be calculated ;
According to Emax , the efficiency ranking is : QS - 1 = WQ - 9 = WQ - 1WQ - 2 = WQ - 3 = QS - 2 = WQ - 4WQ - 6 . QS - 1 has a maximum TEAC of 746 times , followed by WQ - 1 and WQ - 9 373 times , WQ - 4 is at least 0.001 times , and WQ - 6 cannot be calculated .

Conclusion : In addition to WQ - 6 , the other seven compounds have different levels of antioxidant activity in vitro , including QS - 6 .

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