透明質(zhì)酸修飾-共載金絲桃素和乏氧誘導因子1α--siRNA的雙靶向納米粒的制備及評價.pdf

1、張俊玲透明質(zhì)酸修飾／共載金絲桃素和乏氧誘導因子lnsiRNA的雙靶向納米粒的制備及評價中文摘要本研究擬構(gòu)建共載針對乏氧誘導因子1cc(HIFla)的siRNA及具有壞死組織親和性的金絲桃素的PLGAPEI納米粒，并進行透明質(zhì)酸(HA)修飾，研究其理化性質(zhì)的同時評價該載體對腫瘤的體內(nèi)外靶向性，以期研制出一種高效、低毒、穩(wěn)定的靶向放療增敏藥物。第一部分乏氧細胞模型制作及針對乏氧誘導因子1儀(HIF1ct)的siRNA的構(gòu)建和篩選：利用wes

2、ternblot方法檢測出最佳的使鼻咽癌細胞CNE2乏氧的二氯化鈷濃度，以模擬乏氧細胞的狀態(tài)，為接下來的實驗創(chuàng)造最佳條件。westernblot結(jié)果顯示細胞內(nèi)加入150lamol／1的二氧化鈷對鼻咽癌細胞CNE2的HIF1a表達抑制效果最佳。同時，設計針對乏氧誘導因子1a(HIFIⅡ)表達的6對(siRNAl217，siRNAl6162，siRNA2258，siRNA2752，siRNA3724，siRNA3168)及一條陰性對照siR

3、NA(稱為NC)。在Lipofectamine2000介導下轉(zhuǎn)染人鼻明癌細胞系CNE2。同時采用RTPCR法和Westemblot方法檢測細胞中HIF—l(z含量，以Bactin蛋白做內(nèi)參照。結(jié)果顯示：當2pL的Lipofectamin2000(40pmol／L)轉(zhuǎn)染細胞時，能達到最佳轉(zhuǎn)染效率。與其他各組相比，轉(zhuǎn)染了siRNA3724，siRNA3168基因序列的人鼻咽癌細胞中，編碼HIF一1ctmRNA的含量明顯減少。成功設計了針對H

4、IF10【的siRNA，并從中篩選出siRNAl612，siRNA3724，siRNA3168都能夠有效抑制人鼻咽癌表達。第二部分載金絲桃素的PLGAPEI納米粒的構(gòu)建、修飾、毒性及穩(wěn)定性評價：利用乳化溶劑揮發(fā)法制備載金絲桃素的PLGA—PEI納米粒，然后對其表面進行不同比例的透明質(zhì)酸修飾，從而使之在水中能形成粒樣結(jié)構(gòu)，最終制備并評價得出穩(wěn)定性最高的疏水改性陽離子型透明質(zhì)酸修飾納米粒。其次，采用透射電鏡和原子力顯微鏡考察納米粒的外觀，均

5、顯示為類圓形，大小200nm左右，zeta電位為20mv，均符合相關(guān)納米粒要求。HA的投藥量對載金絲桃素的PLGAPEI納米粒的考察結(jié)果顯示當N／COOH大于等于4開始，納米粒的粒徑和電位都發(fā)生較大的變化，考慮是由于加入的HA本身帶有負電荷的作用。再者，MTT實驗結(jié)果示HA—PLGA—PEI—HypexicinNPs則比PLGA—PEI—HypericinNPs有較小的細胞毒性，這讓我們更加相信透明質(zhì)酸在不影響納米粒理化性質(zhì)的同時還可以

6、降低陽離子脂質(zhì)體的細胞毒性；利用瓊脂糖凝膠電泳的方法證明了不論是經(jīng)過HA修飾的或是未經(jīng)HA修飾的樣品都可以與呈陰離子的siRNA形成更牢固的結(jié)合；肝素解離實驗和抗核酸酶實驗證明了所制備出的納米粒載體具有較好的保護RNA能力，使得RNA免受RNAase的降解，有利于siRNA在體內(nèi)的穩(wěn)定。最后本課題利用細胞轉(zhuǎn)染這一張俊玲透明質(zhì)酸慘飾，共載金絲桃素和乏氧誘導因子lⅡ一siRNA的雙靶向納米粒的制備及評價ABSTRACT3Inthisarti

7、cle，wesynthesizedHyaluronieacid(HA)graftedPLGAPEInanoparticlethatloadedHypericin，smallmoleculedrugandgeneEventuallyitformedaechelontargetnanoscaledrugwegottheresearchofthenanoparticle’sphysicalandchemicalpropertiesatthes

8、ametimeofitstumortargetinginvivoandinvitroSOthatitcansolvetheproblemofradioresistanceInthefirstsectionweusedwesternblotmethodtodetectthecellmodelwhich1ackofoxygenWesternblotresultshowedthat150pmol／Lcobaltoxidecouldinhibi

9、tHIF一1alpha’SexpressioninnasopharyngealcarcinomaCNE2cellsAfterthat，westudiedwhetherthetechnologyofRNAinterferencecouldbeawayofanticancergenetherapysiRNAcontainingthesequenceofhumanHIFlamRNAcodingregionwasobtainedWealsode

10、tectedtheeffectofinhibitionbyRTPCRandWesternblotaftertransfectinghumangastriccancercelllineSGC一7901withliposomesTheresultsindicatedthattheexpressionofhumanHIFlagenewassuppressedspecificandeffectivelybysiRNA3724andsiRNA31

11、6scomparedtoothersForthesecondpart，atfirstwepreparedPLGAPEInanoparticlesthatloadedHypericinbyemulsionsolventevaporationmethodandthentookdifferentproportionsofhyaluronicacidontlleirS11lfacemodificationinordertomakeitforms

12、pontaneouslymicellesstructureinthewaterFinallywepreparedandevaluatedthestablehyaluronicacidcoatingnanoparticlesthathavehydrophobiccationicmodificationAfterthatWeusedtransmissionelectronmicroscopy(SEM)andatomicforcemicros

13、cope(AFM)toinvestigatetheappearanceofthenanoparticlesThoseresultsshowedthatnanoparticleisconformtotherelevantrequirements，around200hillsize，zetapotentialisplusorminus20myThirdlyTheresultsshowedthatwhentheN／COOHisgreatert

14、hanorequalto4，nanoparticlessizeandpotentialcouldhavesomechangesThesemayduetotheHAisanegativeroleMoreoverdeterminedbyMTTexperimentresultshowedtheHAHypericinPLGAPEInanoparticlesthanPLGAHypericinPEIhaslowcytotoxicitywecanbe

15、lievethathyaluronieacidaffectedthephysicalandchemicalpropertiesofnanoparticlesaswellasreducethecytotoxicityofcationicliposomesFourthly，agarosegelelectrophoresismethodprovedeitherthroughtheHAmodifiedorwithoutHAmodifiedsam

16、pleCanbecombinedstronglywiththeanionicsiRNAHeparinsolutionfromtheexperimentandnucleaseresistanceexperimentprovedthatthepreparationofnanoparticlesbycarrierhasbetterabilitytoprotectRNA，makesthedegradationofRNAfromRNAase，st