簡介：東北農業(yè)大學碩士學位論文中藥透皮劑對實驗性和臨床型乳腺炎治療效果研究姓名杜瓊申請學位級別碩士專業(yè)基礎獸醫(yī)學指導教師張秀英20080620ABSTRACTSTUDIESONTHERAPEUTICEFFICACYOFTRADITIONALCHINESEMEDICINEPERCUTANEOUSONMOUSETENTATIVEMASTITISANDCOWCLINICALMASTITISABSTRACTDAIRYCOWMASTITISISONEOFTHEMOSTCOMMONDISEASES；NCOWCULTIVATION．ITNOTONLYINFLUENCESTHEQUANTITYOFCOWMILKPRODUCTION，CAUSESSEVEREECONOMICLOSSES，BUTALSOINFLUENCESTHEQUALITYOFTHEMILK．PEOPLEHAVEPAYEDCLOSEATTENTIONTOTHEPREVENTIONOFTHECOWMASTITIS．ATPRESENTTHECOWMAINPATHOGENICBACTERIAOFMASTITISARESTAPHYLOCOCCI，STREPTOCOCCIANDESCHERICHIACOLI．USUALLYTHEANTIBIOTICSWEREUSEDTOCURETHECOWMASTITIS，HOWEVER,THEPROBLEMSOFRESIDUESANDBACTERIARESISTANCETOANTIBIOTICSHAVEBECOMINGMOREANDMORESERIOUSWHENANTIBIOTICWEREUSED，WHICHHAVEBEENWORRIEDBYTHEPEOPLEOFALLWORLD．CHINESEMEDICINALHERBDOESNOTYIELDTOLERANCEANDHAVETHENATUREOFLOWPOISONANDANTIINFLAMMATORYANDSOON．THETHERAPYOFCOWMASTITISWITHCHINESEMEDICINALHERBISFOLLOWEDINTERESTBYMANYSCHOLARS．TRADITIONALCHINESEMEDICINEPERCUTANEOUSISANEXTERNALPREPARATIONADVANCEDDEVELOPMENTBYOURLABORATORY，ITMAINCONTAINSDANDELION、FORSYTHIASUSPENSAANDBAICALSKULLCAPROOT，HASHEAT。CLEARINGANDDETOXICATING，ANTIBIOSISANDANTIPHLOGISTIC，DETUMESCENCEANDACESODYNEFUNCTION．INORDERTOESTIMATETHETHERAPEUTICEFFICACYOFTHISPREPARATIONONTHEMASTIFFS，WECONDUCTEDTHISTOPICRESEARCH．55BACTERIALSTRAINSWEREISOLATEDFROM41MILKSAMPLESOFCLINICALMASTITIS，INCLUDINGSTAPHYLOCOCCI20OF55，36．4％，STREPTOCOCCI24OF55，43．6％ANDGRAM．NEGATIVEBACILLI11OF55，20喲．THELEADINGPATHOGENSOFCLINICALMASTITISWERESTREPTOCOCCUSAGALACTIAE，STAPHYLOCOCCUSAUREUSANDESCHERICHIACOLIACCOUNTINGFOR30．9％．21．8％AND16．3％．BYUSINGTHEAPPROACHOFARTIFICIALCHALLENGEMOUSE，0．05ML106CFUML。1BACTERIUMLIQUIDWHICHCONTAINTHREEKINDSBACTERIUMWEREINJECTEDINTOMAMMARYGLAND．24HOURSLATER，MOUSEEMERGEDAPPARENTCLINICALSYMPTOMANDPATHOLOGICALCHANGE。THECONTENTOFTNFNANDACTIVITYOFNAGASEINMAMMARYGLANDTISSUERISEDOBVIOUSLY,ANDBACTERIUMSWEREISOLATEDFROMTHEMAMMARYGLANDWHICHWEINJECTEDBACTERIUMS．ITDEMONSTRATEDTHATWESUCCEEDMAKINGMOUSETENTATIVEMASTITIS．THROUGHTHETHERAPEUTICTESTONMOUSETENTATIVEMASTITIS，THETHERAPEUTICEFFICACYOFTRADITIONALCHINESEMEDICINEPERCUTANEOUSWASESTIMATED．THERESULTSSHOWEDTHECURERATEANDEFFECTIVEPOWEROFTRADITIONALCHINESEMEDICINEPERCUTANEOUSWERE60％AND80％，ITCOULDOBVIOUSLYLLL

簡介：東北農業(yè)大學博士學位論文奶山羊乳腺發(fā)育的形態(tài)學研究姓名曲波申請學位級別博士專業(yè)動物生物化學與分子生物學指導教師李慶章20080620東北農業(yè)大學農學博上學位論文到妊娠期迅速升高，整個泌乳期B酪蛋白表達水平比較平穩(wěn)，各時點無差異，而其基因表達水平卻有顯著差異，可見乳腺中P酪蛋白的表達調控可能涉及多種調節(jié)岡素的參與退化早期B酪蛋白及其基因表達水平開始逐漸下降，但仍顯著高于青春期，可能是為下一次妊娠做準備，更快進入分泌表達狀態(tài)。此外，本研究采用活細胞熒光標記法結合激光共聚焦顯微技術，在Ⅱ細胞水平觀察奶山羊乳腺發(fā)育中的結構變化。結果顯示奶山羊乳腺發(fā)育中，腺上皮細胞中內質網和線粒體的變化規(guī)律與乳腺組織超微結構和乳成分組化檢測結果基本相符。退化期乳腺上皮細胞內仍維持一定數(shù)量的細胞器，可能是維持細胞正常機能和活動所必需的。關鍵詞奶山羊；乳腺發(fā)育；P一酪蛋白；激光共聚焦顯微技術

簡介：鄭州大學博士學位論文TPA乳腺特異性表達載體的構建及其瞬時表達姓名丁一申請學位級別博士專業(yè)病理與病理生理學指導教師張欽憲20040428鄭州人學2004脯博I例冗生論文TPA乳腺特異性衰叢戴怍的構建披』L瞬時表達乳腺生物反應器MAMMARYGLANDBIOREACTOR技術是利用哺乳動物乳腺特異性表達的啟動子元件構建轉基兇動物，指導外源基因在乳腺中表達，并從轉基因動物的奶液中獲取重組蛋白。利用乳腺生物反應器生產藥用蛋白具有表達體系簡前、町進行翻譯后修飾及費用低廉等優(yōu)點。乳腺生物反應器己成為生產重組蛋白的首選手段。建立乳腺生物反應器的關鍵在于構建乳腺特異性表達載體。乳腺特異性表達載體由乳腺特異性啟動子、目的基因和加POLYA信號三部分紕成。目前已克隆并用作構建載體的乳蛋白基因主要包括B一乳球蛋白基因BLG、酪蛋白基因和乳清酸蛋白基因WAP等。本研究中，首先將克隆的乳清酸蛋白基岡WAP24KB啟動子插入PBLUESCRIPT質粒，構建質粒PW；然后將克降的037KB加POLYA信號插入WAP啟動子的下游，構建I_LR指導外源基岡在乳腺特異性表達的載體PWA；最后將克隆的目的基因21KBTPACDP4A連接于UP啟動子和加POLYA信號之間，構建PA乳腺特異性表達載體PWTA。構建成功的PWTA表達載體通過脂質轉染胺試劑LIPOFECTAMINE?！薄癘O轉染乳腺癌細胞系，通過體外注射轉移至妊娠期小鼠乳腺，以原位雜交技術檢測TPA在乳腺癌細胞系和泌乳期小鼠乳腺中的瞬時表達，驗證PWTA表達載體的表達能力。材料和方法1載體PW的構建以含WAP啟動子的質粒WAP2H；H為模板，設計引物上、下游引物分別帶有NHEI和ITINDLII酶切位點，PCR擴增WAP啟動子DNA片段；以NHEL和HJNDLLL對擴增產物進行雙酶切，瓊脂糖凝膠電泳回收2，4KB酶切片段。以XBAI和HINDLII雙酶切PBLUESCRIPT質粒，瓊脂糖凝膠電泳回收297KB酶切片段。NHEI和XBAT粘往末端互補。以體外連接試劑盒連接24KBWAP肩動子片段和297KBPBLTLESCRPL質粒DNA片段，16。C，連接過夜。重組體轉化感受念JML09細菌。將轉化菌涂于含AMP的培養(yǎng)皿，37“C，培養(yǎng)過夜。隨機挑選10個克隆，接種于含AMP的LB培養(yǎng)基，37℃，培養(yǎng)過夜。小提質粒，分別進行TTINDIII單酶切和HINDIIL／SAC11雙酶切鑒定。構建成功的載體命名為PW。2載體PWA的構建以PCDNA30質粒為模板，設計引物T、卜游引物

簡介：軍事醫(yī)學科學院碩士學位論文牛ΑS1酪蛋白基因序列指導的人組織型纖溶酶原激活劑突變體微小基因在小鼠乳腺中的表達姓名譚曉紅申請學位級別碩士專業(yè)細胞生物學指導教師鄧繼先200061BOVINEASLCASEINGENESEQUENCESDIRECTEXPRESSIONOFAVARIANTOFHUMANTISSUEPLASMINOGENACTIVATORINTHEMILKOFTRANSGENICMICEABSTRACTTHEBIRTHOF“SUPERMICE”BROKETHEVALLATIONOFSPECIESINBIOSPHERE，ANDTHERESEARCHOFTRANSGENICANIMALACHIEVEDDRAMATICPROGRESSTRANSGENICPHARMACYSPDNGUPFOLLOWINGTHESTUDIESOFTHEMAMMARYGLANDBIOREACTORTHEMAMMARYGLANDOFTRANSGENICLIVESTOCKISLIKELYTOSERVEASTHEBIOREACTORFORPRODUCINGTHEBIOACTIVEPOLYPEPTIDEMEDICINEORPROTEINWITHSPECIALNUTRITIONINTLLISSTUDYTHEPROMOTEROFCASEINGENEWASUSEDTODIRECTTHETPAMINIGENETOEXPRESSINTHEMAMMARYGLANDSPECIFICALLYTHEMBTANTTPAWASDETECTEDINTHEMILKOFTHETRANSGENICMICE111ESEQUENCESOF58KBCOMPRISINGOFTHESEQUENCESFROMEXORT2TOEXON6OFTPAWEREAMPLIFIEDFROMHUMANGENOMICDNABYPCRLATPAMINIGENEWASGENERATEDBYLIGATINGTHEGENOMICSEQUENCESOFTPATOTHELATPACDNAATTHESITEOFNARIALPHASLCASEINVECTORWASMODIFIEDBYDELETINGTHESEQUENCESFROMTHESTARTINGSIGNALCODEOFTRANSLATIONANDAFTERITANXHOISITEWASINTRODUCEDATTHE3’ENDOFCASEINGENEOFTHEVECTORTOFACILITATETHESEPARATIONOFFUSIONGENEFROMTHEPROKARYOTICSEQUENCESFORMIRCOINJECTIONNLEMAMMARYGLANDSPECIFICEXPRESSIONCONSTRUCTWASOBTAINEDBYINSERTINGTHELATPAMINIGENEINTHEMODIFIEDCASEINVECTORNLEFUSIONGENECONTAININGTHEPROMOTEROFCASEINGENELATPAMINIGENEANDTHEDOWNSTREAMOFCASEINGENEWASINTRODUCEDINTOTHEFERTILIZEDEGGSOFMICEBYMICROINJECTIONTHEINJECTEDEGGSWEREFURTHERTRANSPLANTEDINTOTHEOVIDUCTSOFRECIPIENTMICEABONT2700FERTILIZEDEGGSWEREINJECTED，AND2200EGGSWERETRANSPLANTEDINTO81RECIPIENTMICEOF47OFFSPRING，F(xiàn)IVEWEREPOSITIVETRANSGENICMICEN心CONCENTRATIONOFLATPAINTHEMILKOFONEFEMALETRANSGENICMOUSEIS018UG／M1THISRESULTSHOWEDTHAT也ELATPAMINIGENECOULDCOITCCUYEXPRESSTHEBIOACTIVELATPAINTHEMILKOFTHETRANSGENICMOUSEUNDERTHECONTROLOFCASEINGENE2

簡介：軍事醫(yī)學科學院博士學位論文牛Β乳球蛋白基因調控成分的克隆和用以制備小鼠乳腺生物反應器的研究姓名陳紅星申請學位級別博士專業(yè)遺傳學指導教師蘇國富200061達到12UG／ML，最低則無法檢測到TPA活性。這個結果顯示位點效應對TPA的表達具有重大的影響，也提示表達載體中用以對抗位點效應的MAR序列并未起作用。上述的結果表明／本文克隆的牛B乳球蛋白基因表達調控序列可以控制目的基因在轉基因小鼠中表達，并且產物能分泌到乳汁中，可以用以制備乳腺生物反應器。關鍵詞生物反應器TPAB乳球蛋白2

簡介：中國農業(yè)大學博士學位論文擬蜘蛛牽絲蛋白基因在大腸桿菌和小鼠乳腺表達研究姓名許紅韜申請學位級別博士專業(yè)生物化學與分子生物學指導教師李寧20040601ABSTRACTSPIDERDRAGLINESILKISALLIMPRESSIVEMATERIALWITHACOMBINATIONOFTENSILESTRENGTHANDELASTICITYTHATENDOWSITWITHUNIQUETOUGHNESSANDHIGHERENERGYTOBREAKTHANANYOTHERCOMMONMATERIAL，NATURALORARTIFICIALDRAGLINESILKISFIVETIMESSTRONGERBYWEIGHTANDLIGHTERTHANSTEELBECAUSEOFITSEXCELLENTMECHANICALPROPERTIESTHEDRAGLINESILKCOULDBEUSEFULFORINDUSTRIAL，MILITARYMATERIAL，WEAVEANDMEDICALPURPOSESTHISRESEARCHFOCUSESONACQUIRINGARTIFICIALSPIDERDRAGLINESILKPROTEINGENEWHICHCANEXPRESSTARGETPROTEINEFFICIENTLYANDCALLBEPERFORMEDEASILY，ANDTHENPRODUCESPIDERSILKINLARGEQUANTITIESFORINDUSTRIALUSEACCORDINGTOTHEKNOWNPARTIALCDNASEQUENCEOFDRAGLINESILKTWOARTIFICIALDRAGLINESILKGENEMONOMERS，A360BPSEQUENCEANDA390BPSEQUENCE，WEREDESIGNEDTOENCODEANALOGSOFTHEPROTEINOFNEPHILACLAVIPESDRAGLINESILKDNAMONOMERSEQUENCESWEREMULFIMERIZEDTOENCODEHIGHMOLECULARWEIGHTARTITICIAISPIDERDRAGLINESILKPROTEINTWOTIMERFOTTRTIMERSIXTIMEREIGHTTIMERANDSIXTEENTIMERWERERESPECTIVELYGAINEDACCORDINGTOTILEKNOWNPARTIALAMINOACIDSEQUENCEOFDRAGLINESILK，ANAN『LINOACIDSEQUENCEOF19AAWASSYNTHESIZEDANTISERUMAGAINSTDRAGLINESILKPROTEINWASRAISEDINRABBITSAGAINSTS3RNTHETIEPEPTIDESCONJUGATEDTOKEYHOLELIMPETHEMOCYANINEIGHTTIMERANDSIXTEENTIMEROFTWOARTIFICIALDRAGLINESILKGANEMONOMERSWERECLONEDINTOPET30AVECTORAPROKARYOTICEXPRESSIONVECTORANDINDUCEDWITHIPTGFOUREXPRESSIONVECTORPET30AX8、PET30AX16、PET30AF8ANDPET30AF16WERECONSTRUCTEDEACHSTRAINPRODUCEDACHARACTERISTICHETEROGENEOUSARRAYOFIMNLUNOREACTIVEBANDSTHEMASSESOFTHESTRONGESTBANDWERERESPECTIVELY85KD、165KD、85KDAND165KD，WHICHREPRESENTSTHEFULLLENGTHGENEPRODUCTINEACHPATCERNTHECONCENTRATIONSOFRECOMBINANTDRAGLINESILKPROTEINWERERESPECTIVELY800MGL、500MG／L、200MG／LAND200MG／LACCORDINGTOGOAT，EASEINSIGNALSEQUENCE，A125BPDNASEQUENCEWASDESIGNEDANDSYNTHESIZEDFOURTIMERANDSIXTIMEROFTWOARTIFICIALDRAGLINESILKGENEMONOMERSWERELIGATEDWITHTHESIGNALSEQUENCEANDTHENWERECLONEDINTOPBCLVECTOR，WHICHISASPECIFICMILKEXPRESSIONVECTORFOUREXPRESSIONVECTORPBCIF4，PBCIX4PBCLF6ANDPBCIX6WERECONSTRUCTEDTRANSGENIEMICEWEREGENERATEDBYMICROINJEETIONOFFERTILIZEDOOCYTEWITHTWOCONSTRUCTS，PBCIF4ANDPBCLX6FORPBCIX6VECTORTRANSGENICMICEWERESCREENEDBYPERANDSOUTHERNBLOTWHICHREVEALEDTHAT10MICE5毋5早AMONG繇MICEWERE打ANSGENICPOSITIVETHEINTEGRATIONRATEIS17％FIMICEFORMOSTOFFOUNDERLINEWEREALSODETECTEDBVPCRANDTHEPOSITIVETRANSGENICMICEWEREFOUNDMILKOFFIVEF0MICEANDEIGHTF1MICEWASANALYZEDBYWESTERNBLOTANDRADIOIMNMOASSAYTWOF0MICEANDSEVENFLMICEEXPRESSEDRECOMBINANTDRAGLINESILKPROTEINTHEMOLECULARNLASSESOFEXPRESSEDRECOMBINANTDRAGLINESILKPROTEINBYMOSTOFMICEWEREABOUT55KD，WHICHWASCONSISTENTWITHTHATOFTHEORETICCALCULATIONBUTSOMEMICEEXPRESSEDRECOMBINANTDRAGLINESILKPROTEINWHICHMOLECULARMASSESWERENOTCONSISTENTWITHTHATIⅡ

簡介：鄭州大學碩士學位論文Β胡蘿卜素誘導人乳腺癌MCF7細胞凋亡與PPARΓ信號傳輸及活性氧產生的聯(lián)系姓名崔艷紅申請學位級別碩士專業(yè)應用化學指導教師趙文恩20060415使用50岍10L幾P一胡蘿卜素，當將處理時間從L天延長至Ⅱ4天時，細胞活力從71％降為42％。作為PPAR吖抑制劑的GW9662和／或GSH能夠削弱P．胡蘿H素誘導的細胞活力的下降，5HM01／LGW9662預處理能部分挽回P一胡蘿B素誘導的細胞活力的下降，而10肛M01幾GW9662預處理部分增強B．胡蘿H素誘導細胞活力的下降。GSH能呈劑量效應抑制P一胡蘿H素對細胞活力的抑制作用，用200岬OL，LGSH預處理細胞，能使細胞活力由50％恢復到70％，且5岬OL／LGW9662共同預處理能增加GSH對細胞的保護作用。50岫OL幾P．胡蘿H素處理3天能顯著增加細胞早期的凋亡率，用GW9662或GSH能削弱這種作用，且共同使用GW9662和GSH有協(xié)同作用。B一胡蘿H素對MCF一7細胞的周期沒有明顯的影響。4．B胡蘿H素誘導MCF．7細胞的細胞核形態(tài)的變化P一胡蘿H索處理的細胞，細胞核變小，濃縮，熒光加強和成核碎片等，細胞呈凋亡狀態(tài)。200¨MOL幾GSH預處理能明顯減少細胞核的形態(tài)變化，200啪。此GSH和5岬OL／LGW9662預處理能明顯減少細胞核的形態(tài)變化。單獨用GW9662預處理，對由P胡蘿N素誘導的細胞核的變化的影響不明顯。5．B一胡蘿B素使細胞內活性氧水平上升用50岬OI／LP一胡蘿H素處理3天后，細胞內活性氧水平明顯比對照組高，且用200肚M01幾GSH預處理能顯著抑制這種升高。用GW9662預處理不能降低由B．胡蘿H索引起的活性氧的增加，并且LO“MOL／LG、Ⅳ9662能使細胞內活性氧增加為對照組的120％。、6．B一胡蘿B素誘導細胞色素C的釋放50肛MOL／LP一胡蘿卜素處理3天后，紅色和綠色有所分離，說明細胞色素C從線粒體內釋放出來，跑到了胞漿里。通過WCSTEMBLOT檢測，進一步表明B．胡蘿H素誘導細胞色素C的釋放，200I_IMOI／LGSH或200肛MOL／LGSH5岬01／LGW9662能夠抑制細胞色素C的釋放。綜合前人的工作及我們的實驗數(shù)據，我們有理由認為，PPAR．T傳輸系統(tǒng)和活性氧的產生，可能在P胡蘿卜素抑制細胞生長和誘導凋亡方面的分子機制中都起到了重要的作用。并且，由于GW9662和GSH共同預處理作用效果更好，這兩個途徑可能有協(xié)同作用。II

簡介：Y977729分類號Q21UDC52Z密級壘玨級⑧西北農林針捉大學2006屆攻讀碩士學位研究生學位畢業(yè)論文人溶菌酶基因乳腺特異性表達載體的構建及其在小鼠乳腺癌細胞中的表達學科專業(yè)筮直生物堂研究方向喧乳動麴艇臉王程研究生值塵寶指導教師韭涵麴援完成時間2壁豎生查且CONSTRUCTION0FMA～口MARYGLANDSPECIFICEXPRESS10NALVECTORANDEXPRESS10NINMOUSEBREASTCARCINOMACELLSFORHUMANLYSOZYMEABSTRACTHUMANLYSOZYMEHLYZPARTICIPATEINTHEBODYDEFENSEMECHANISMITACTSASASHIELDAGAINSTBACTERIALINFECTIONANDITHASEFFECTSONMALIGNANTGROWTHANDIMMUNOLOREGULATIONSOITHASWIDEAPPLICATIONINMEDICINEANDFOODSTUFFINDUSTRYHLYZCANBEPURIFIEDFROMHUMANBREASTMILK，NEUTROPHILS，ANDURINEBECAUSEOFTHELIMITATIONOFITSSOURCEANDTHECOMPLEXITYOFITSEXTRACTION，ANDTHEPOTENTIALRISKOFTRANSMITTINGPATHOGENSTOTHENURSINGINFANTS，WECANNOTGETITBYLARGESCALESODEVELOPMENTOFSAFERRECOMBINANTHLYZISNEEDEDANIMALMAMMARYGLANDBIOREACTORISANOVELALTERNATIVESTRATEGYFORPRODUCTIONOFRECOMBINANTPROTEINSWITHMANYADVANTAGESINCLUDINGHIGHPRODUCTIONCAPACITIES，EASYPURIFICATION，F(xiàn)AITHFULTRANSLATIONALMODIFICATIONSANDAUTHENTICBIOLOGICALACTIVITIESOFTHEEXPRESSEDPRODUCTSINTHISRESEARCH，THEMAMMARYGLANDSPECIFICEXPRESSIONVECTORPEBHWASCONSTRUCTED，WHICHWASCONSISTEDOFREGULATINGELEMENTSREGULATIONREGIONOFBOVINE3CASEINANDEXPRESSIONSEQUENCEHUMANLYSOZYMECDNATHENPEBHWASTRANSFECTEDINTOMOUSEBREASTCARCINOMACELLSCULTURED加VITROANDTHEEXPRESSIONCELLSTRAINSWEREABTAINEDTHEEXPRESSEDPRODUCTSWEREANALYSEDBYSDSPAGEANDWESTERNBLOTTHERESULTSOFRESEARCHAREASFOLLOWING1HUMANLYSOZYMECDNAWASCLONEDFROMHUMANPLACENTATOTLERNAWITHRTPCR，WHICHWAS853BPANDCOMPRISEDINTACTCODINGREGIONSSEQUENCEANALYSISDEMONSTRATEDTHATITSHOMOLOGY、VITHTHERELATIVEREGIONOFHUMANLYSOZYMEONGENBANKWAS99％WITHINTHEOPENREADINGFRAME，THREESITEMUTATIONSOCCUREDWHICHCOULDNOTALTERPROTEIN’SFUNCTION2BYTHESTRATEGYOFDIRECTIONALCLONINGHLYZWASFUSEDWITHTHECOMMONLYPRIMARYEXPRESSIONVECTORPBCPTHENWASCLONEDDIRECTIONALLYINTOEXPRESSIONVECTORPEGFPCI，THEMAMMARYGLANDSPECIFICEXPRESSIONVECTORPEBHFORHLYZWASCONSTRUCTED，WHICHINCLUDEDRESISTANTGENEANDEGFPREPORTERGENEITCOULDALSOENSUREEGFPANDAIMEDGENEEXPRESSRESPECTIVELY3THEVECTORPEBHWASTRANSFECTEDINTOMOUSEBREASTCARCINOMACELLSCULTUREDINVITRO

簡介：山東大學碩士學位論文苯達莫司汀（BENDAMUSTINE）誘導小鼠乳腺癌細胞凋亡及其機制的研究姓名孔芳申請學位級別碩士專業(yè)細胞生物學指導教師孔慶忠20070506山東大學碩士學位論文中文摘要苯達莫司汀BENDAMUSTINE誘導小鼠乳腺癌細胞凋亡及其機制的研究背景乳腺癌是人類最常見的惡性腫瘤之一，其發(fā)病率近年來呈上升趨勢。在多種治療乳腺癌的方法中，化療仍是最為常用且效果較明顯的重要手段之一然而現(xiàn)有的化療藥物或多或少都會引起藥物耐受性的產生，因此，尋找有效殺傷乳腺癌細胞的化療藥物已成為研究者當前的主要任務之一臨床研究表明，作為氮芥類藥物且具多種功能的烷化劑，苯達莫司汀BENDAMUSTINE對乳腺癌有較好的療效然而，相關的研究數(shù)據有限，作用機制不清楚由于很多小分子化療藥物可通過影響腫瘤細胞周期和誘導細胞凋亡來抑制腫瘤細胞的生長，因此我們重點研究苯達莫司汀對體外培養(yǎng)的小鼠乳腺癌C127細胞周期和誘導凋亡的作用及作用機制，以期為腫瘤生物學和腫瘤化療提供理論依據目的觀察抗癌藥物苯達莫司汀對體外培養(yǎng)的小鼠乳腺瘤細胞系D127生長抑制及其作用機制，以期在細胞水平上為治療乳腺癌提供相關的理論依據方法；細胞增殖檢測方法采用形態(tài)學觀察和細胞記數(shù)法檢測苯達莫司汀對C127細胞形態(tài)和存活率的影響；細胞凋亡檢測方法用吖啶橙染色法，在熒光顯微鏡下觀察細胞核的形態(tài)變化和凋亡小體；TUNEL法檢測DNA的片段化；細胞凋亡和壞死的鑒別檢測乳酸脫氫酶CIDVI的釋放區(qū)分壞死和凋亡；P53蛋白表達測定免疫熒光結合激光共聚焦掃描顯微技術測定細胞中P53的表達情況；細胞周期的變化流式細胞術檢測苯達莫司汀對C127細胞周期的影響結果1相差顯微鏡觀察顯示苯達莫司汀20／ⅡG／ML作用C127細胞24H之后細胞形態(tài)無明顯變化，48H后細胞表現(xiàn)出凋亡的特征，細胞突起變少，細胞中顆粒和3

簡介：新疆農業(yè)大學碩士學位論文位點不依賴性乳腺表達載體的構建及在乳腺上皮細胞中的表達姓名胡艷秋申請學位級別碩士專業(yè)生化與分子生物學指導教師羅淑萍李文蓉20060601CONSTLLLCTIONANDCEUEXPRESSI蛐OFMAMMARYGLANDEXPRESSIONVBCTORWITHPOSITIONINDEPENDENTMANNERHUYANQIUABSTRACTTHEPURPOSEOFTHISPAPERWASTOCONSTMCTMAMMARYGLANDEXPRESSIONVECTORWITHPOSITIONINDEPENDENTMALLILER，WHICHCANPMBCTTRANSGENESFROMGENOMICPOSJFIONEFI色CTSANDEXPRESSINDEPENDENTLY．THEENDOGENOUS0一CASEINHADBEENAⅡALYZEDBYR1二PCRREVERSETRANSCRIPTIONPOIYMERASECHAINREACTIONFORTHEASSESSMENROFTHEFLLNCTIONOFMAMMARY印ITHELIALCELLSMECANDTHEEFFICACYOFHO蛐ONALINDUCTIONINVITMOPTIMIZEDVECTORPWHERE2V．01WASRECONSTMCTEDBYEXDSINGBCZGENEFIRSTLYANDTHEREAFTERTHEBLGVP2GENEWASINSERTEDIN枷LTIPLECLONESITEMCSFLANKINGBYINSULATORS．THUSAMAMMARYGLANDEXPRESSIONVECTORTHATTRIEDTOPROTECTTRANSGENESFROMGENOMICPOSITIONEFFECTSWASCONSTNLCTED．GOATM鋤MARYEPITLLELIALCELLSGMECWEREISOLATEDFROMLACTATINGGOATMAMMARYTISSUE．RT_PCRANDIMMUNOCHEMICALMETHODWEREUSEDTODETECTBCASEINEXPRESSION．THETRANSCRIPTIOⅡOFTWOMAMMARYEPITHELIALCEULINESC127ANDT47DWASALSOANALYZEDTOCOMPAREANDASSESSTHEPCASEINGENEEXPRESSIONINDI丘EFENTMAMMARY印ITHELIALCELLLINES．THERESULTSSHOWEDTHATPCASEINGEⅡEEXPRESSIONCANBEDETECTEDINPRIMARYCULTUREDGOATMAMMARYEPITHELIALCELLSGMEC1DERIVEDFROMLACTATING90ATASWELLASCONSEQUENNYPASSAGEDCELLSGMEC一3WITHTHEPRESENCEOFHORINOILESINCLUDINGPROLACTIN，INSULINANDHYDROCORTISONE2

簡介：西北農林科技大學碩士學位論文STAT3基因轉染牛乳腺上皮細胞及轉染后細胞的生物學特性觀察姓名劉甲申請學位級別碩士專業(yè)發(fā)育生物學指導教師張涌20080501THEB10LOGICALCHARACTERISTICSOFBOVN呵EMAMMARYEPITHELIALCELLSTRANSFECTEDWITHSTAT3GENEABSTRACTSTATSARELATENTTRANSCRIPTIONFACTORS也ATMEDIATECUT拋NE鋤DGROWTHFACTORDIRETED仃蜀11LS謝PTIONKMANYHMNALLCANCERS吼D仃觚SFOMED∞LLLINES，STAT3ISPERSISTENTLYACTICATED，ANDINCENCULTⅧC，ACTIVESTAT3ISEITHERREQUIRED南R仃ANSFOMATION，E11HANCES仃孤SF0MATION，ORBLOCKSAPOPTOSISSTAT3GELLESWERE仃ALLSCRIPTIONFACTORSRALHERACTIVCLYSTLLDIEDINRECEILTYEARSMANYRESE∞CHRESULTSSHOWEDTHATSTAT3EXPRESSEDABNONNALLYINLOTSOF劬MORTISSUESANDCEUSYSTEMS，W，HICHW部CLOSELYRCLATCDT0MEPROLIFERATION鋤DDI任打E加『TIATIONOFTUMOR，CELLAPOPTOSIS，HNMUNEESCAPE，GEILESISOFBLOODVESSELS，ANDIILVASIONMDMETASTASISTHISEXPCRIMENT仃AILS斷EDTHEP1撇IDWHICHWAS誦THSTAT3GENETOBOVINEM鋤M哪EPITLLELIALCELLSN啪UGHTHE1IPOFACT鋤INE2000A11DSTUDIEDNLE111EBI010百CALCHARACT鰣STICSOFBOVINEM鋤MARREPITHELIALCELLS，PUTA劬D鋤EN謝ST印SFORPRODUCING仃ANSGELLICA11IMALSANDCELLIMMORTALIZATION1、BOVINEM鋤儺ARY印ITHELIALCELLSWEREIS01ATED鋤DP戚矗EDBYUSINGCOLLAGENASEDIGESTIONOFTHEM鋤MARR西AILDTISSUEFORMEPFIMA巧CELL伽LTURE，ANDSUBSCQU鋤TLYUSING仃YPSINSELECTEDDIGESTIONOFME饑1衄司CELLSFORODLP嘶FICATIONMO訕0109ICAL0BSERVATIONREVEALEDMATT11ECULTLLRCDCELLSPOSSESSEDTLLETYPICALCHARACTCROF印ITHELIALCELLS2、STAT3SI朗ALTRANSDUCERANDACTIVATOROFTRANS嘶PTION3GENEWASAMPLIFIED舶MPOTB7PLASMID訛CHCONTAINEDHUM鋤STAT3GCLLECDNA矗孵NEILT，TLLEILINS叭EDMOPEGFPCLVECTORTOCONS仃UCTREC0MBINANTPLASMID3、WBCULTILREDTHEBOVINEMAMMA巧EPITHELIALCELLSUSINGDMEMWITH15％FBS，也EN竹ANS斷EDTHESTAT3GEILETOCELLSOF廿LISTYPEMROU曲NLELIPOFACT鋤INE2000ANER48H，WEEX鋤INCDTLLEEXPRESSIONOFEGFPUSILLGNUORESCENTMICROSCOPE，24HOURSAREREXPO則RETOTHERECOMBINANTPL砌IDTLLE乜眥SFECTIONRALEOFTHEBEVCELLSW部L6％，SCREELLEDⅡLECLONEUSING600U咖LG418，也％MAKEDTHEDONEPFOPAGANDAUSING300UG／MLG4184、D舐VEDRNA舶MPOSITIVECLONECDLSALLDDESI弘EDTHEP枷CULARPRIMERTOGOT0THEIMPCRST印ATLAST，MEPOSITIVECELLSSHOWTHEAIMBAND2332BP，THENEGATIVECELLSHAVENOTHESAMEBAND5、FLOWCYTOME缸YWASUSEDT0ANDIYZEMECELLCYCLES、MULTIPLICATIONCAPACITYAND

簡介：中國農業(yè)大學博士學位論文摘要中文摘要哺乳動物的乳腺是唯一在動物出生后可以多次再生、退化的器官。不同發(fā)育時期的動物乳腺中都存在一定比例的干細胞，正是這些干細胞維持和保證了乳腺發(fā)育的特性。利用類乳腺干細胞再生乳腺是近兩年來興起的一項高新技術，具有以一下四個方面的重要意義為那些因乳腺腫瘤或癌癥而切除乳腺的婦女修復或再生功能性乳腺為那些需要隆胸的女性提供安全可行的填充新材料為將健康成年雌性家畜乳腺改造成為分泌藥用或保健用蛋白的乳腺提供一個實驗動物的技術平臺為其他器官的發(fā)育、再生或修復提供方法、思路和理論借鑒我們以小鼠為模型，運用組織化學、免疫熒光組織細胞化學、流式細胞儀分選方法FAGS以及分子生物學手段，研究了小鼠乳腺的發(fā)育規(guī)律小鼠乳腺織織中類乳腺干細胞小鼠乳腺細胞的分離、培養(yǎng)以及類乳腺干細胞的鑒定小鼠類乳腺干細胞分化的潛能小鼠乳腺類腺體體外短期培養(yǎng)富集類乳腺干細胞體系的優(yōu)化等。研究結果表明L3周齡的BALBC小鼠是去除乳腺腺體的最佳時期2不同發(fā)育時期乳腺組織中都存在類乳腺干細胞368周齡和懷孕期小鼠是獲得大量類乳腺干細胞的理想時期4首次證明體外短期培養(yǎng)的乳腺類腺體體系中，有一些細胞可以表達一定量的胚胎千細胞的特異性表達抗原基因OCT4REX1上皮干細胞的特異性表達抗原基因INTEGRIN31大部分已知干細胞共表達的抗原基因BCRPILACBG215小鼠類乳腺干細胞移植到去除乳腺腺體的小鼠乳脂墊中，可以再生功能性乳腺，因此，類乳腺干細胞在體內具有分化潛能6小鼠類乳腺干細胞在體外誘導泌乳體系中培養(yǎng)7犬，可以表達P一酪蛋自及其基因，表明類乳腺干細胞在體外具有分化潛能7首次建立了優(yōu)化的乳腺類腺體體外短期培養(yǎng)富集類乳腺干細胞體系，由此體系培養(yǎng)的類腺體獲得類乳腺干細胞SEAL1的比例遠遠高于文獻報道使用體系獲得類乳腺干細胞比例8小鼠乳腺類腺體結構體外短期培養(yǎng)獲得的細胞中有一定量的類乳腺干細胞存在優(yōu)化體系培養(yǎng)22小時獲得的細胞的類乳腺干細胞SEAL1比例遠遠高于培養(yǎng)72小時獲得細胞的比例相反，表達造血干細胞特異性抗原CD34的細胞CD34十比例在培養(yǎng)72小時比培養(yǎng)22小時高同時，培養(yǎng)的體系中還有一定比例的SEAL1CD34細胞存在?？傊?，隨著對類乳腺干細胞研究的深入，我們急需找到類乳腺干細胞的特異性表達抗原。在此之前，仍然只能以造血干細胞的特異性表達抗原SEALI作為一個標記，進行類乳腺干細胞的分析和分選研究。進一步研究類乳腺干細胞的特異性表達譜，以期能夠找到類乳腺干細胞的特異性表達抗原，分離得到類乳腺干細胞，作為發(fā)育生物學和再生生物學研究的模FO另外，如果能對乳腺類腺體體外短期培養(yǎng)富集類乳IW干細胞體系進行優(yōu)化，或許可以摸索適合類乳腺干細胞體外擴增的培養(yǎng)體系。關鍵詞小鼠乳腺類乳腺干細胞SEALLCYTEKERATIN分化培養(yǎng)流式細胞儀中國農業(yè)大學博士學位論文八T3STRACTINSUMMARYWESHOULDINVESTIGATETHEMOREEFFICIENTCULTURALSYSTEMFORMAMMARYORGNOIDSINVITROFORENRICHEDMOREMAMMARYEPITHELIALSTEMCELLSANDTOSCREENTHEEXPRESSIONPROFILESINMAMMARYEPITHELIALSTEMCELLSTODISCOVERTHESPECIFICMARKERFORMAMMARYEPITHELIALSTEMCELLSINORDERTOUTILIZETHEMODELOFMAMMARYEPITHELIALSTEMCELLSTOREGENERATETRANSGENICMAMMARYGLANDSINCOWORSHEEPFORHUMANHEALTHTOUNCOVERTHEMECHANISMOFORGANOGENESISANDDIFERENTIATIONKEYWORDSMOUSEMAMMARYGLANDSTEMCELLSSCALLCYTOKERATINDIFERENTIATIONCULTUREFLOWCYTOMETRYANALYSISSORTINGVI

簡介：碩士學位論文SIRNASIRNA干擾干擾AIB3AIB3對乳腺癌細胞對乳腺癌細胞MDAMDAMBMB231231侵襲和轉移的影響侵襲和轉移的影響碩士研究生畢磊導師張灝教授專業(yè)生物化學與分子生物學研究方向腫瘤的發(fā)生與轉移入學時間2009年9月中國汕頭二零一二年五月學位論文原創(chuàng)性聲明本論文是我個人在導師指導下進行的工作研究及取得的研究成果。論文中除了特別加以標注和致謝的地方外，不包含其他人或其它機構已經發(fā)表或撰寫過的研究成果。對本文的研究做出貢獻的個人和集體，均已在論文中以明確方式標明。本人完全意識到本聲明的法律責任由本人承擔。作者簽名日期年月日學位論文使用授權聲明本人授權汕頭大學保存本學位論文的電子和紙質文檔，允許論文被查閱和借閱；學?？蓪⒈緦W位論文的全部或部分內容編入有關數(shù)據庫進行檢索，可以采用影印、縮印或其它復制手段保存和匯編論文；學?？梢韵驀矣嘘P部門或機構送交論文并授權其保存、借閱或上網公布本學位論文的全部或部分內容。對于保密的論文，按照保密的有關規(guī)定和程序處理。作者簽名導師簽名日期年月日日期年月日

簡介：西北農林科技大學碩士學位論文人乳鐵蛋白基因乳腺表達載體構建及細胞表達姓名宋紅衛(wèi)申請學位級別碩士專業(yè)臨床獸醫(yī)指導教師張涌20050601CONSTRUCTL0NANDCELLEXPRESSIONOFMAMMARYGLANDSPECIFICEXPRESSIONALVECTOROFHUMANLACTOFEERINGENEABSTRACTTHEKEYRESOLUTIONFORIMPROVINGTHEEXPRESSIONALLEVELOFFOREIGNGENEINTRANSGENICANIMALISTOSELECTAPPROPRIATEREGULATIONELEMENTS，WHICHCALLREGULATEFOREIGNGENESPECIFICALLYANDHIGHEFFICIENTLYEXPRESSIONINGLANDEPITHELIALCEILINTHISRESEARCHWEUSEDTHE5’FLANKINGREGULATIONELEMENTOFGOATBETA1ACT0910BULINGENEASPROMOTERANDHUMANMATRIXATTACHMENTREGIONASENHANCERTOCONSTRUCTMAMMARYGLANDSPECIFICEXPRESSIONALVECTOROFHUMANLACTOFERFINGEUEANDTRANSFECTEDINTOMAMMARYGLANDEPITHELIALCELLSCULTUREDINVITROWITHIIPOFEMINTINPOSTIVECELLSWERESELECTEDANDPURIFIEDBYTHEEXPRESSIONOFANTIBIOTICSGENENEOSOTHECELLSCANBEUSEDASSOBRCCSTOCREATETRANSGENICANIMALBYNUCLEARTRANSPLANTTECHNIQUESTHERESULTSOFRESEARCHISFOLLOWING1FORTHEPURPOSEOFCONSTRUCTINGMAMMARYGLANDSPECIFICEXPRESSIONALVECTORTHEGOATPLACTOGLOBULINGENE5FLANKINGFRAGMENTWASCLONEDBYPERAMPLIFICATIONITCONSISTEDINPARTOFTHEGENEUPSTREAMREGION、THEFIRSTEXONANDTHEFIRSTINTRONAFTERPERPRODUCTWASRECOVEREDANDPURIFIED，THEAIMFRAGMENTWASCLONEDATTSITEOFPMD18TVECTORTHEFRAGMENTWASSEQUENCED，ANDITWASCOMPAREDWITHORIGINALGENE，THERESULTSINDICATEDTHEHOMOLOGYWERE9970％INADDITION，BASECWASMISSEDAT一684ANDBASEGWASMUTATEDAITWASCOMPAREDWITHBOVINEBLGGENEVARIANTA，BOVINEBLGGENEVARIANTBANDSHEEPBLGGENEBYBLASTTHERESULTSINDICATEDTHEHOMOLOGYWERE9055％，9179％AND9609％RESPECTIVELY2THECLONEDFRAGMENTWASCLONEDINEXPRESSIONVECTORPEGFPCLANDCONSTRUCTEDVECTORPBLGGFEITWANTRANSFECTEDINTOMAMMARYGLANDEPITHELIALCELLSCULTUREDINVITROWITHLIPOFEMINTINANDSCREENEDCELLUNDERUVMICROSCOPEAFTER48HWEFOUNDTHATGFPGENEEXPRESSEDWELLWHICHINDICATEDTHECLONEDFRAGMENTISUSEFUL3VECTORPBLGANDPEGFPC1WEREDIGESTEDBYASEIANDHINDIIIAFTERPRODUCTSWERERECOVEREDANDPURIFIED，THEAIMFRAGMENTSWERECONNECTEDWITHT4DNATIGASEANDORIGINALEXPRESSIONVECTORPEBWASACQUIREDVECTRORPEBANDPLFWEREDIGESTEDBYHINDIIIANDSAL1MIDDLEEXPRESSIONVECTORPBLWASGOTWITH

簡介：J蜊‘惦』日蝕自￡L世虬UDC』2L￡L≈女J＆EL西貯農林聿凝大學2006屆攻讀碩士學位研究生學位畢業(yè)論文人乳鐵蛋白在山羊乳腺上皮細胞中的表達及核移植研究學科專業(yè)喳莊獸醫(yī)堂研冤方阿噓基鹽塑延臉王捏研咒生韭晶晶指導教師韭涵煎援完成時同墊些生且十目陜日楊＆LOSTHEEPRESSIONOFHUMANLACTOFERR【NINGOATMA們ⅥARYEPITHILIALCELLSANDTHESTUDYOFNUCLEARTRANSFERABSTRACTINTHISPAPER，THEHUMANLACTOFERRINGENEEXPRESSEDVECTORPBLMC1WHICHWASTRANSFECTEDINTOGOATMAMMARYGLANDEPITHELIALCELLWITHLIPOSOME。POSITIVECLONECELLSWERESELECTEDWITHG418ANDPCR。THENTHESECELLSWEREINDUCEDBYHORMONEPROLACTININSULINHYDROCORTISONETOEXPRESSHUMANLACTOFERRINGENE。THERESULTOFWESTERNBLOTANALYSISONCULTUREDCELLSUPEMATANTSHOWEDTHATTRANSFECTEDCELLSCANEXPRESSHUMANLACTOFERRINGENE。THEPOSITIVECELLSWEREUSEDASNUCLEUSDONORTOCREATETRANSGENICCLONEDEMBRYOSBYNUCLEARTRANSFERTECHNIQUES。THEFOREIGNGENEINTRANSGENICCLONEDEMBRYOSWASDETECTEDBYPCR，LAYSASOLIDFOUNDATIONFORCONSTRUCTINGTRANSGENICANIMALSANDITSMAMMARYGLANDBIOREACTOR。THERESULTSOFTHISRESEARCHWEREASFOLLOWING1THEPRIMARYCULTUREDGOATMAMMARYGLANDEPITHELIALCELL，MAJORITYOFTHECELLSWERESHORTSHUTTLELIKEORGUBOIDALWHICHLOOKEDLIKEBEEHIVE；PARTOFTHECELLSLOOKEDLIKEBIG、ROUNDFLATCAKESPARTOFTHECELLSWERELONG。THECELLSCOULDFORMISLANDLIKEORDOMELIKESTRUCTUREINMONOLAYERCULTURE。THETRANSFECTEDEXPERIMENTWASEXECUTEDWHENCELLSWERESUBCULTUREDTOTHE3THPASSAGEA2THEPUFF母IEDVECTORSWEREMIXEDINPROPERATIOWITHLIPOSOME，WHICHWASUSEDTOTRANSFECTEDCULTUREDOFGOATMAMMARYGLANDEPITHELIALCELLS。HIGHCLONERATECANBEACQUIREDBYTHELIPOSOMETRANSFECTEDMETHOD，THEISLANDLIKESTRUCTURECOULDFORMEDBYPOSITIVECELLS。3THETRANSFECTEDGOATMAMMARYGLANDEPITHELIALCELLSWERESELECTEDBYG418400PG／MLFORTWOWEEKS，THENWERECULTUREDING418200PG／ML。THETRANSFECTEDCELLSDNAWEREABSTRACTEDANDBEIDENTIFYIEDBYPCR，THERESULTSREVEALEDTHATTHISVECTORWASTRANSFECTEDINTOCELLSSUCCESSFULLY。4THETRANSFECTEDCELLSWEREINDUCEDBYHORMONEPROLACTININSULINHYDROCORTISONETOEXPRESSHUMANLACTOFERRINGENE。THESUPERUATANTWASCOLLECTED1TIMEEVERY6HS，THENWASCENTRIFUGEDFOR10～15MIN1000R／MINWHICHWASSTOREDIN20℃。THERESULTOFWESTERNBLOTANALYSISONCULTUREDCELLSSUPEMATANTSHOWEDTHATTRANSFECTEDCELLSCANEXPRESSHUMANLACTOFERRINPROTEIN，THEMOLECULARWEIGHTIS42KU。5THETRANSGENICCLONEDEMBRYOSWERECREATEDEFFECTIVELYBYMEANSOFELECTROFUSION，