B細(xì)胞型非霍奇金淋巴瘤細(xì)胞株中CHFR基因表達(dá)及甲基化的研究.pdf

1、B細(xì)胞型非霍奇金淋巴瘤細(xì)胞株中CHFR基因表達(dá)及甲基化的研究摘要目的檢測B細(xì)胞型非霍奇金淋巴瘤Raji細(xì)胞中CHFR基因的表達(dá)和甲基化狀態(tài)，以及去甲基化試劑5氮雜一2’一脫氧胞苷對淋巴瘤細(xì)胞增殖和凋亡的影響，為臨床應(yīng)用去甲基化藥物靶向治療非霍奇金淋巴瘤提供理論和實(shí)驗(yàn)依據(jù)。方法①體外培養(yǎng)非霍奇金淋巴瘤細(xì)胞株(Raji細(xì)胞)，分別以1t2mol／L，4umol／L，7pmol／L，10umol／L去甲基化試劑5一氮雜一2’一脫氧胞苷(5Az

2、a2’一de)處理Raji細(xì)胞；②采用RTPCR方法檢測反應(yīng)性增生淋巴結(jié)組織及非霍奇金淋巴瘤細(xì)胞株Raji細(xì)胞中CHFR基因的表達(dá)；③采用RTPCR方法檢測分別經(jīng)112mol／L，412mol／L，7umol／L，1011mol／L的5Aza一2’一de處理后的Raji細(xì)胞中CHFR基因的表達(dá)；④MS—PCR(甲基化特異性PCR)方法檢測Raji細(xì)胞株及經(jīng)1012mol／L去甲基化試劑處理后CHFR基因的甲基化狀態(tài)；@ccI(8檢測分別

3、經(jīng)112mol／L，412mol／L，7umol／L，10lamol／L去甲基化試劑處理后Raji細(xì)胞的增殖抑制率；⑥流式細(xì)胞術(shù)檢測分別經(jīng)分別經(jīng)1umol／L，411mol／L，712mol／L，1011mol／L去甲基化試劑處理后的Raji細(xì)胞凋亡率。結(jié)果①經(jīng)RTPCR檢測發(fā)現(xiàn)反應(yīng)性增生淋巴結(jié)組織中CHFR基因的相對表達(dá)量為0898007，Raji細(xì)胞中CHFR的相對表達(dá)量為O1214O013，提示Raji細(xì)胞中CHFR相對表達(dá)量明顯

4、低于正常細(xì)胞，差異有統(tǒng)計(jì)學(xué)意義(p005)。②RTPCR檢測經(jīng)不同濃度5Aza一2dc處理后CHFR基因相對表達(dá)量分別為01740012，02380015，031840019，038940009，經(jīng)5Aza一2’一dc處理后表達(dá)量上升，差異有統(tǒng)計(jì)學(xué)意義(p005)；③Raji細(xì)胞中CHFR基因啟動(dòng)子可見甲基化與非甲基化條帶共存的狀態(tài)，即部分甲基化，經(jīng)5Aza一2’一dc處理后甲基化條帶變暗，非甲基化條帶亮度增加；④分別以112mol／L

5、，4umol／L，7umol／L，1012mol／L的去甲基化試5一Aza一2’一dc處理細(xì)胞后，CCK一8檢測細(xì)胞的抑制率分別為(26o40)％，(88o96)％，(124o61)％，(16211)％，隨5Aza2’dc濃度的增高，細(xì)胞抑制率增加，差異有統(tǒng)計(jì)學(xué)意義(p005)。⑤流式細(xì)胞術(shù)檢測未經(jīng)5Aza一2j—dc處理的Raji細(xì)胞早期凋亡率為(172士01)％，經(jīng)1|lmol／L，4pmol／L，7lamol／L，1012mol／

6、L5Aza2’de處理細(xì)胞后，細(xì)胞的凋亡率分別為(3864O35)％，(6274O4)％，(9574033)％，(155046)％，細(xì)胞凋亡率增加，且在一定范圍內(nèi)凋亡率隨5Aza一2’一dc濃度的增高而增加，差異有統(tǒng)計(jì)學(xué)意義(p005)。TheExpressionandMehtyrlafionofCHFRinBcellNon—Hodgkin’sLymphomaCellsAbtractObjective：ToinvestigatemRNA

7、expressionlevelofCHFRgeneinB—cellNon—Hodgkin’sLymphomaCells，theeffectofCHFRgenemathylationandmethylationinhibitoronRaftcellsproliferationandapoptosis，whichcanprovideatheoreticalandexperimentalfoundationforLymphomaclinica

8、ltherapyMethods：④ThehumanBurkitt’SRajilymphomacellswerecultivatedinvitroRajicellsweretreated麗th1，4，7and10gmol／Lofmethylationinhibitor5一Aza一2’deoxycytidine(5Aza一2dC)；②TheCHFRgeneexpressionleveloflymphnodeofpatientswithnon

9、neoplasticdiseasesandRajicellswasdetectedbyRTPCR⑧TheCHFRgeneexpressionlevelaftertreatedwith1，4，7and10pmol／Lof5Aza一2’一dCwasdetectedbyRT—PCR；@MethylationofCHFRgeneofRajicellsandtreatedwith109mol／Lof5一Aza2dCwasanalyzedbyMS—

10、PCR；⑤ThecellsproliferationofRajicellstreatedwithdifferentconcentrationsof5Aza2dCwereanalyzedbyCCKassay⑥TheapoptosisofRajicellswasanalyzedbyflowcytometryResults：@TheCHFRmRNArelativeexpressionoflymphnodeandRaftcellswere089

11、8士007，0121士0013CHFRgenehadlow—expressioninRajicellsthedifferencewasstatisticallysignificant(P005)；②TherelativeexpressionofCHFRmRNAaftertreatedby5Aza2’dCatconcentrationsof1，4，7andl09mol／Lwere0174士0012，023840015，031840019，

12、0389士0009，respectively，thedifferencewasstatisticallysignificant(P005)5Aza一2dCCanup—regulatetheexpressionofCHFRmRNAbydemethylation(9InuntreatedRaftcellsCHFRgenewaspartlymethylationandmathylationstatewasdecreasedaftertreat

13、edwith5Aza一2dC@ThegrowthinhibitionrateofRajicellsaftertreatedwith5Aza2dCwere(26士040)％，(88096)％，(1244061)％，(162士11)％，respectivelyThegrowthofRajicellswasinhibitedWiththeelevationofthedrugsconcentration，theinhibitionrateinc

14、reasedThedifferencewasstatisticallysignificant(P005)@TheearlyapoptosisrateofRajicellsinthecontrolgroupwas(172士01)％Thesecellswhichweretreated謝th5Aza2dCwere(386035)％，(62704)％，(957士033)％，(155士046)％Withtheincreasingconcentra