重組諾氏梭菌對(duì)HER2-neu陽性小鼠乳腺癌的治療作用.pdf

1、中文摘要目的：評(píng)價(jià)減毒諾氏梭菌(CnovyiNT)作為基因治療載體在小鼠體內(nèi)應(yīng)用的靶向性與安全性。構(gòu)建HER2／neuILl2融合基因修飾的CnovyiNT(HER2／neuILl2CnovyiNT)。應(yīng)用HER2／neu陽性小鼠乳腺癌模型探討其對(duì)實(shí)體腫瘤的治療作用。方法：(1)CnovyiNT在正常小鼠體內(nèi)分布：將CnovyiNT芽孢懸液05ml(5107個(gè))經(jīng)尾靜脈注射給正常小鼠，分別于注射后l、2、4、7天處死4只小鼠，取其肝、腎

2、、肺勻漿并厭氧培養(yǎng)，計(jì)算各器官中的細(xì)菌數(shù)。(2)CnovyiNT在荷瘤小鼠體內(nèi)分布：制備EMT6乳腺癌荷瘤小鼠模型，將CnovyiNT芽孢懸液05ml(5lO7個(gè))經(jīng)尾靜脈注射荷瘤鼠體內(nèi)，分別于注射后l、2、4、7天處死小鼠，無菌剝?nèi)×鲶w、肝、腎和肺，勻漿并厭氧培養(yǎng)，計(jì)算瘤組織和各個(gè)器官中的細(xì)菌數(shù)。(3)CnovyiNT的直接溶瘤作用：將CnovyiNT芽孢懸液05ml(5107個(gè))經(jīng)尾靜脈注射荷瘤鼠體內(nèi)，于注射后1、2、4、7天分別處

3、死4只小鼠，無菌剝?nèi)×鲶w、肝、腎和肺，組織經(jīng)甲醛固定制作石蠟切片，HE染色觀察腫瘤組織壞死區(qū)大??；革蘭染色觀察CnovyiNT在腫瘤和各器官中的分布。(4)CnovyiNT在小鼠體內(nèi)應(yīng)用安全性評(píng)價(jià)：將CnovyiNT以靜脈注射的方式給予正常小鼠(510‘7個(gè)／只)，注射后每天觀察小鼠的攝食、飲水等情況，于第7天處死小鼠，剝?nèi)「?、。腎和肺勻漿后做厭氧培養(yǎng)，計(jì)數(shù)各個(gè)器官中的細(xì)菌數(shù)；探討CnovyiNT在小鼠體內(nèi)應(yīng)用的安全性。(5)HER2／

4、neuILl2CnovyiNT制備：以超聲(40kHz)介導(dǎo)的方式，將HER2／neuIL12融合基因穿梭質(zhì)粒(pEHl)導(dǎo)入CnovyiNT，經(jīng)紅霉素抗性篩選，獲得HER2／neuILl2CnovyiNT。(6)HER2／neu陽性乳腺癌模型制備：采用PEI轉(zhuǎn)染的方法將質(zhì)粒pcDNA6HER2／neu轉(zhuǎn)染小鼠乳腺癌細(xì)胞EMT6，經(jīng)殺稻瘟菌素(Blasticidin)篩選獲得HER2／neuEMT6細(xì)胞，將其注射于小鼠右前肋部皮下，建立

5、HER2／neU陽性乳腺癌模型。(7)HER2／neuILl2CnovyiNT對(duì)HER2／neu陽性乳腺癌的治療作用：HER2／neu陽性乳腺癌荷瘤小鼠24只，隨機(jī)分為4組。每組6只。I組：CnovyiNT組；II組：pEHl組；III組：HER2／neuILl2CnowfⅣ丁組；Ⅳ組：PBS組。I組注射CnovyiNT懸液01ml(1107個(gè))；II組注射pEHl01ml(100ug)；III組注射HER2／neuILl2Cnovyi

6、NT懸液01ml(1107個(gè))組；Ⅳ組注射01mlPBS。并于接種腫瘤細(xì)胞后第4、7、9、11、13、15天測(cè)量腫瘤的大小，繪制腫瘤生長(zhǎng)曲線；第15天處死小鼠，剝?nèi)×鲶w和脾臟稱重，計(jì)算脾指數(shù)。評(píng)價(jià)HER2／neuILl2CnovyiNT對(duì)HER2／neu陽性小鼠乳腺癌的治療作用。結(jié)果：(1)CnovyiNT靶向定位于腫瘤組織，并在其中持續(xù)大量增殖，單獨(dú)應(yīng)用即ofHER2／neuILl2—CnovyiNT■■●●／neiiOositive

7、DreastCancermlCeAbstractObjectiveToevaluatethefeasibilityofattenuatedCnovyi(CnovyiNT)asavectorforgendtherapy；preparedchimericgeneofHER2／neu—ILl2modifiedCnovyiNT(HER2／neuILl2一CnovyiNT)forthetherapyofHER2／neu—positivebreas

8、tcancerinmicemodelMethods(1)ThedistributionofCnovyiNTinnormalmiceThesuspensionofCnovyiNTO5ml(5x107)wasinjectiedintravenouslytonormalmiceofKunmingstrain，theywerekilledin1，2，4，7daysrespectivelyafterinjectionTheliverkidneya

9、ndlungweretakenandhomogenatedAfteranaerobicinoculating，thenumberofbacteriawascalculatedintheorgans(2)ThedistributionofCnovyiNTintumorbearingmiceEMT6breastcancerbearingmicemodelwereprepared，thenCnovyiNTsporesuspension05ml

10、(5x107)wasintravenouslyinjectedintothetumorbearingmiceThemiceweresacrificedat1，2，4and7daysrespectivelyafterinjectionThetumorsaswellastheirorgansweretakenandthefollowingprocesswasperformedasdescribedinthemethodof(1)(3)One

11、olyticeffectofCnovyiNTCnovyiNTsporeswereinjectedintravenouslyintothetumorbeatingmiceasthesamequantityandprocessaselucidatedinmethod(2)Afterparaffinslicefromtheorganswereprepared，HEandGram’Sdyeingwereperformedrespectively

12、forobservingthesizeofnecrosisareaintheformerandthebacterialdistributionin1atter(4)SecurityevaluatingCnovyiNTsuspensionwasintravenouslyinjectedtonormalmice(5x107／each)，theirfeeding，drinkingandgeneralconditionwasobservedda

13、ilyThemicewerekilledatthe7thdays，theirorgansweretakenandthefollowingprocesswasperformedasthemetod(1)(5)PreparationofHER2／neuIL12CnovyiNTThemethodofultrasoundDNAdelivery(UDD)Wasusedtotransformtherecombinantshuttleplasmido

14、fHER2／neu—ILl2chimericgene(pEH1)toCnov／糾嘎andtheHER2／neu—ILl2CnovyiNTwasselectedbyerythromycin(6)PreparationofHER2／neu—EMT6PElwasusedtotransfertherecombinantplasmidofpcDNA6HER2／neuintoEMT6breastcancercells，theHER2／neu—EMT

15、6wereselectedbyblasticidinThen，HER2／neu—positivebreastcancerbearingmicewerepreparedbyinjectingtheHER2／neu—EMT6modifiedcellsintherightprothoraxofmice(7)TheraputiceffectofHER2／neuILl2CnovyiNT24HER2／neupositivebreastcancerb