紫杉醇和吡柔比星治療骨肉瘤的分子機(jī)制研究

1、紫杉醇和吡柔比星治療骨肉瘤的分子機(jī)制研究 河北醫(yī)科大學(xué) 碩士學(xué)位論文 紫杉醇和吡柔比星治療骨肉瘤的分子機(jī)制的研究 姓名：林亮 申請學(xué)位級別：碩士 專業(yè)：外科學(xué) 指導(dǎo)教師：劉思源 20090301 中文摘要 紫杉醇和吡柔比星治療骨肉瘤的分子機(jī)制的研究 摘 要 目的：骨肉瘤是最常見的原發(fā)性惡性骨腫瘤，好發(fā)于 10-20歲的青少年，惡性程度高，預(yù)后差【11，手術(shù)和化療在臨 床治療骨肉瘤提高患者生存期中發(fā)揮著重要和關(guān)鍵的作用 12。但是，在骨肉瘤復(fù)發(fā)患者的化療中發(fā)現(xiàn)存在高的失敗率 和較低的中位生存率【3】。研究骨肉瘤化療機(jī)制對骨肉瘤的治 療和提高病人的生存率是非常必要的。吡柔比星已臨床用于 治療骨肉瘤

2、，而紫杉醇只作為治療骨肉瘤的二線用藥而且應(yīng) 用不如吡柔比星【41。目前已有吡柔比星和紫杉醇在臨床上聯(lián) 合應(yīng)用的報(bào)道【571。然而，很少有對化療藥物誘導(dǎo)骨肉瘤細(xì) 胞凋亡的分子機(jī)制的研究。因此，為了找到更加合理和-彳了效 的綜合治療方箸：，本實(shí)驗(yàn)研究了吡柔比星和紫杉醇誘導(dǎo)凋1 活性的分子機(jī)制。 10DMEM培養(yǎng)基中常規(guī)培養(yǎng)，待細(xì)胞進(jìn)入對數(shù)生長期后月j 于實(shí)驗(yàn)。 l光鏡觀察：將不同濃度的紫杉醇和吡柔比星作用于 MG63細(xì)胞，在倒置相差顯微鏡下定時觀察細(xì)胞的生長情況 并照相。 2 培養(yǎng)于96孔培養(yǎng)板中，進(jìn)入對數(shù)生長期后采用四甲基偶氮 中文摘要 作用。 3流式細(xì)胞儀檢測細(xì)胞周期：采用不同濃度的紫杉醇和吡

3、 度為5106個，用PBS洗一次，離心去上清；加入冰預(yù)冷 TrintonX1001O 室溫避光 PropidiumIodide，PI：50mgL II型流式細(xì)胞儀檢測細(xì)胞凋亡和細(xì) 染色30min，用EpicsXL 胞周期分布，應(yīng)用軟件進(jìn)行分析。 4Western印跡法觀察紫杉醇及吡柔比星作用后的人成骨 的表達(dá)。 杉醇處理MG一63細(xì)胞12h后，細(xì)胞折光變強(qiáng)，部分細(xì)胞體 積變小變圓，24h后細(xì)胞開始皺縮，貼壁能力差，細(xì)胞體 ：； 更小，少量細(xì)胞漂浮，48h后細(xì)胞體積普遍變小，胞質(zhì)濃縮， 大量細(xì)胞變圓漂浮，72h后大部分細(xì)胞變圓，失去增殖能力 漂浮于液體中，出現(xiàn)明顯的藥物抑制現(xiàn)象。103nmolL

4、吡柔 比星作用12h后細(xì)胞增值數(shù)量減少，細(xì)胞體積增大胞膜破裂 胞質(zhì)外溢，細(xì)胞壞死漂浮，24h后分裂期細(xì)胞少見，胞膜破 裂細(xì)胞壞死，漂浮細(xì)胞增多，48h后大部分細(xì)胞形態(tài)不規(guī)則， 體積增大，壞死漂浮，存活細(xì)胞顏色晦暗，生長停滯，72h 中文摘要 顯示紫杉醇和吡柔比星均能明顯抑制MG63細(xì)胞增殖，并 后，G0G1期前面出現(xiàn)凋亡峰，G2M期所占比例隨藥物濃 度和時間增加呈上升趨勢，而G，期所占比例呈遞減趨勢。 48h、72h后未見明顯凋亡峰出現(xiàn)，S期所占比例隨藥物濃度 紫杉醇和吡柔比星處理過的骨肉瘤細(xì)胞與對照組相比均表 蛋白表達(dá)增強(qiáng)。 結(jié)論： 1 紫杉醇和吡柔比星均對骨肉瘤細(xì)胞MG63 存在抑制細(xì)胞增

5、殖作用且具有時間劑量依賴關(guān)系。 隨時間 延長不同濃度吡柔比星對細(xì)胞的最終抑制作用是一致的。凋 亡峰最高時的濃度可能是最適濃度。低濃度紫杉醇長時間和 高濃度紫杉醇短時間對細(xì)胞抑制作用致。濃度和時間不是 很重要的選擇，凋亡峰最高的時間和濃度效果最佳。 用，并且有一定的時間劑量依賴性，使細(xì)胞周期受到阻滯， 并進(jìn)一步誘導(dǎo)其凋亡。吡柔比星在細(xì)胞增殖周期中阻斷細(xì)胞 進(jìn)入G2期，使細(xì)胞停滯于S期，抑制DNA合成干擾細(xì)胞 分裂。在一定范圍內(nèi)存在時間劑量依賴性且對劑量關(guān)系更密 中文摘要 切。不同濃度紫杉醇作用骨肉瘤細(xì)胞后G0G1期比例整體呈 下降趨勢，表明紫杉醇對細(xì)胞增殖周期的G0G1期存在阻斷 作用，具體機(jī)制

6、有待進(jìn)一步研究。不同濃度吡柔比星作用骨 肉瘤細(xì)胞后細(xì)胞G0G1比例呈先下降后上升趨勢，表明吡柔 比星對骨肉瘤細(xì)胞增殖周期GOG1期延緩作用。 3 如果S期過大時停藥可導(dǎo)致腫瘤細(xì)胞繼續(xù)分裂， 且分裂很快，如果是G2M期，停藥后細(xì)胞很快進(jìn)入S期。 細(xì)胞被阻滯于GO期是最有效的。濃度過低時未見明顯凋亡 峰說明藥物只殺傷正常細(xì)胞達(dá)不到殺傷腫瘤細(xì)胞的目的。濃 度過高時仍無明顯凋亡峰出現(xiàn)，表明濃度過高時凋亡較少且 相對副作用較大。只有合適有效濃度時，即凋亡峰明顯時達(dá) 到殺傷腫瘤細(xì)胞的目的。吡柔比星還可能通過破壞骨肉瘤細(xì) 胞MG63細(xì)胞膜的結(jié)構(gòu)和功能直接殺死細(xì)胞。 4 紫杉醇可與吡柔比星聯(lián)合應(yīng)用治療骨肉瘤。

7、 關(guān)鍵詞：紫杉醇；吡柔比星；MG一63；細(xì)胞凋亡：MTT； 流式細(xì)胞；抗體 英文摘要 ofCell MolecularMechanism The Apoptosisby andPirarubicininaHuman Paclitaxel CellLine 0steosarcoma ABSTRACT in is suffered ective：Osteosarcoma，whichmainly Obj adolescentof1020 themostcommon old，is primarily years thelower witha and bonetumor highermalignancy

8、 malignancy and and playsignificant chemotherapy prognosis【l】Surgery to cancertreatment inclinicalantiosteosarcoma crucialroles failure achieve survival哆However,ahigh prolongedpatient are in ansurvivalrate observed rateandlOWmedi patients with intractable recurrent， chemotherapy undergoing the survi

9、val osteosarcomat31Topatient rate， improve ofthemechanismof for investigation chemotherapy treatmentiS hasbeen osteosarcomaveryimportantPirambicin the while usedforthetreatmentofosteosarcoma clinically wasusedasthe chemicalfor paclitaxel secondary drug andthe waslessthan osteosarcoma application The

10、antitumorof and havebeen paclitaxel potencypirarubicin inclinical few welldocumented of carriedoutonthe ofthemechanisms studieshave analysis findmore bonetumorcell orderto inducing deathTherefore，in andeffectivecombination we reasonable treatment，here 5 英文摘要 the investigatedpossible of apoptosisindu

11、cingactivity treatmentwith and pirarubicin was paclitaxelOur experiment aboutthemolecules mechanism ofthe of apoptosis osteosarcomawhich hadbeen might used and by pirarubicin clinicaltreatment paclitaxelfor Methods：MG?63cells wereculturedat37inDMEM with1 supplemented0heatinactivatedFBSinthetissue cu

12、lture flaskunderahumidified to 5C02 the when enteredthe experiment they logarithmicgrowth phase 1 Light observationofcell microscopy concentrations changes：differentof andTHPwere paclitaxel roledintheMG一63 cellsandtoobservatthe ofcell growth in invertedcontrast to regularly phase and microscope phot

13、o cultures 2 MTTessay：Thewereinitiatedi n96well plates x ata of5 104Cellswere for treated72hwith density various o 02 03 04 concentrations 1nmolL，1nmolL，1 nmolL，1nmolL of 03 04 05 paclitaxelor 102nmolL，1nmolL，1 nmo nmolL，1 lL of with three wellsfor each pirarubicin replicate concentration The relati

14、vecellnumber ofadherentcellswas thendetermined theMTTmethod by cellswere with 3 Flow treated cytometryanalysis：The various concentrationsof and for24h paclitaxelpirarubicin Thenwe cellsfromcontrol gathered group withoutmedication ， identical concentrations 1 0znmolL paclitaxel group，and identical 1

15、on concentrations 10jnmolL pirarubicin2h， group 6 英文摘要 48hand72h 5100cells respectivelyWegot and attherateof1 washedoncewithPBSand 000rmin，then harvested in 1bovineserum bytrypsinization，washed fixedwith 75ethanol O5 albumin BSA and containing between20forat1east1 hat4Thecellswerewashedinl BSAand in

16、acold Pirarubicinsolution resuspended staining 00 RnaseAand10 Pirarubicinin mlfor 1 Itgml ggml PBS 1 minat 40 4Data and werecarriedout acquisitionanalysis by aflow erton， using cytometrysystem BeckmanCoulter,Inc，F(xiàn)ull CA，USA blot observedthe of 4 Westernanalysis：Wre expression andbaxin and PCNA，cycli

17、nD1，cyclinE，Bcl一2 paclitaxel PirarubicintreatedMG一63westernblot by theMG一63cellstreated Paclitaxel Results： 1 After by 1 about observedthatundertheinvertedcontrast 2h，we phase became cellsrefraction volumeof microscope，the strong，the somecellsbecameround after and small，and 24h，cellsbegan to adhere

18、became ofthem shrink，theability weak，volume fewcells volumeofcells smaller,a floated，after48h，the becamesmaller lotofcells concentrated，a universally,cytoplast roundand came the became whenit to of floated，and 72h，most floatedandthe cells became treated change significantAfterby 105nmolLPirarubicin1

19、2hthe numberofcells increasing decreasedandvolume membrane enlarged，cell broke，and floatedAfter at cells overflew,necrotic 24h，cells cytoplast fission could be membrane stage seen，cell broke，more rarely 7 英文摘要 necroticcells after ofmostcell floated，and48h，thetype became ofthembecame cells irregular,

20、volume larger,necrotic the cellswere floated，even tarnish，stoppedgrowing living After cellsfloated MTT 72h，necrotic increasingly 2 The thatafter MG63cellsindifferent resultsshowed wetreated with indifferent time12h，24h、48hand72hand drugs 0 02 03 04nmolL concentration， 1nmolL，1nmolL，1nmolL，1 02 03 04

21、 05nmolL Paclitaxel， 1nmolL，1nmolL，1nmolL，1 ofthe could retrainthe Pirarubicin，both significantly drugs of theeffectivenesson MG一63，and depended multiplication time anddose I-CM ofexperiment thatwhenthecellsweretreatedindifferent found analysis，we concentrat 1OnmolL，10nmolL，10nmolL，1 andthen identic

22、al at by concentrations 1 12h、24h、48hand of cameoutbefore 72h，thepeakapoptosis G0G1 ofG2M increasedassociated percentagestage stage，the oftimeand the of with concentration，and increasing percentage G1 hadthe ofdecreaseAndwhenthecells stage tendency treated in different were byPirarubicin 103nmolL、10

23、4 102nmolL、 nmolL、105 10nm01L、 andthen identical 03nmolL nmolL 24h by concentrations 1 ous at1 and wasnotobvi Pirarubicin2h，24h，48h72h，there the ofS hadthe percentagestage tendency apoptosispeak，and with oftimeand ofincrease increasing withthe of control Western-blotting：Comparedgroup，the Bcl一2 of a

24、nd expression protein 8 英文摘要 weredown in andPirarubicin regulatedpacitaxel groups，and bax proteinexpressionupregulated MG一63osteoscarcomacellcouldbe Conclusion： 1 The inhibitedPaclitaxelandPirarubicin ontimeand by depended inhibit doseThefinal ratewasthesamein different concentration thetimeTheconcentrationat byincreasing bethesuitableconcentrationTheinhibit apoptosispeakmight rateofPaclitaxelinlowconcentrationand time treating long wassameasthatin concentrationandshort high treatin