本文選題：B細(xì)胞淋巴瘤 + 免疫球蛋白��； 參考：《中國人民解放軍軍醫(yī)進(jìn)修學(xué)院》2008年博士論文

【摘要】： 【目的】 應(yīng)用免疫球蛋白重鏈基因可變區(qū)家族的框架區(qū)基因與細(xì)胞因子編碼序列融合的DNA疫苗,體外轉(zhuǎn)染樹突細(xì)胞,刺激培養(yǎng)外周血淋巴細(xì)胞,以確定框架區(qū)DNA疫苗能否誘發(fā)抗原特異性細(xì)胞毒T淋巴細(xì)胞的產(chǎn)生,為該疫苗下一步應(yīng)用于主動(dòng)免疫或過繼性T細(xì)胞治療的臨床試驗(yàn)提供實(shí)驗(yàn)依據(jù)。 【方法】 鑒定已經(jīng)構(gòu)建的人免疫球蛋白重鏈基因可變區(qū)(IgHV)的框架區(qū)基因(FR)與粒-巨噬細(xì)胞集落刺激因子(GM-CSF)序列融合的家族性DNA疫苗,無內(nèi)毒素試劑盒方法提取質(zhì)粒。采集人外周血單個(gè)核細(xì)胞(PBMC),采用貼壁培養(yǎng)分離,細(xì)胞因子GM-CSF、IL-4誘導(dǎo),細(xì)胞因子“雞尾酒”促成熟的方法培養(yǎng)樹突細(xì)胞(DC),用流式細(xì)胞儀檢測(cè)DC的表型;基因槍方法將疫苗DNA質(zhì)粒轉(zhuǎn)染DC,反轉(zhuǎn)錄聚合酶鏈反應(yīng)(RT-PCR)及酶聯(lián)免疫吸附試驗(yàn)(ELISA)方法確認(rèn)轉(zhuǎn)染質(zhì)粒的表達(dá)。同時(shí)將懸浮的PBMC在IL-2等細(xì)胞因子誘導(dǎo)下培養(yǎng),用轉(zhuǎn)染后的DC刺激培養(yǎng)以誘導(dǎo)抗原特異性細(xì)胞毒T淋巴細(xì)胞(CTL)的產(chǎn)生。用流式細(xì)胞儀檢測(cè)培養(yǎng)的淋巴細(xì)胞表型,采用合成的MHC五聚體(MHCPentamer)方法檢測(cè)抗原特異性細(xì)胞毒T淋巴細(xì)胞,用熒光標(biāo)記的流式細(xì)胞術(shù)方法(FATAL)分析培養(yǎng)的T淋巴細(xì)胞對(duì)B細(xì)胞淋巴瘤細(xì)胞的殺傷作用,并采用基因測(cè)序儀方法對(duì)所培養(yǎng)的淋巴細(xì)胞進(jìn)行T細(xì)胞受體β鏈可變區(qū)(TCRBV)基因掃描分析,觀察培養(yǎng)前后T細(xì)胞各個(gè)克隆的變化情況。 【結(jié)果】 制備供轉(zhuǎn)染用的無內(nèi)毒素質(zhì)粒IgHV1-GM-CSF/pcDNA3.0,經(jīng)酶切、PCR鑒定,測(cè)序結(jié)果顯示包含IgHV1前導(dǎo)肽、FR區(qū)以及連接的GM-CSF cDNA序列,FR區(qū)序列包含受HLA-A*0201限制性的抗原九肽QLVQSGAEV的編碼序列。 外周血PBMC經(jīng)細(xì)胞因子誘導(dǎo)可以產(chǎn)生形態(tài)典型的DC,顯微鏡下計(jì)數(shù)典型形態(tài)的DC占85.0%～96.5%,獲得DC數(shù)量占PBMC的6.9%～11.3%;細(xì)胞因子促成熟后,DC表面CD80、CD83、CD86等標(biāo)志明顯增高,顯示獲得成熟的DC。比較4種方法轉(zhuǎn)染DC的效率,由高到低依次為Nucleofector電轉(zhuǎn)染、基因槍、Lipofectamine 2000和FuGENE 6,但電轉(zhuǎn)染后細(xì)胞死亡多,且失去多突觸結(jié)構(gòu)而變成圓形,因此選擇基因槍方法轉(zhuǎn)染DC,并對(duì)基因槍轉(zhuǎn)染DC條件進(jìn)行了優(yōu)化。經(jīng)RT-PCR及ELISA方法檢測(cè)轉(zhuǎn)染后的DC的GM-CSF水平增高,證明轉(zhuǎn)染的質(zhì)粒DNA能夠在DC中表達(dá)。 對(duì)2例正常人及2例B細(xì)胞淋巴瘤患者的PBMC進(jìn)行細(xì)胞培養(yǎng)及轉(zhuǎn)染或抗原肽刺激,FCM分析顯示體外培養(yǎng)的淋巴細(xì)胞主要以CD8+T淋巴細(xì)胞增生為主;Pentamer檢測(cè)結(jié)果顯示轉(zhuǎn)染IgHV1-GM-CSF融合疫苗的DC能夠誘導(dǎo)抗原特異性CTL產(chǎn)生,最高可達(dá)4.36%;相應(yīng)的抗原9肽也可以誘導(dǎo)CTL的產(chǎn)生,但時(shí)相要早于前者。殺傷試驗(yàn)顯示轉(zhuǎn)染DNA疫苗DC刺激的T細(xì)胞對(duì)具有同樣IgHV1家族特征的B細(xì)胞淋巴瘤細(xì)胞具有增強(qiáng)的殺傷作用,2次刺激后的患者的T細(xì)胞對(duì)腫瘤細(xì)胞殺傷率為32.4±4.9%,而抗原9肽誘導(dǎo)的殺傷率為18.1±3.5%。TCRBV基因掃描顯示淋巴瘤、白血病患者治療后T細(xì)胞各個(gè)TCRβ家族中一些淋巴細(xì)胞消失,呈寡克隆分布或完全缺失,僅少數(shù)保持多克隆分布,而經(jīng)過DC及細(xì)胞因子的刺激培養(yǎng)后,TCRβ則呈現(xiàn)完整的多克隆分布;經(jīng)過DNA疫苗刺激的則呈現(xiàn)部分家族個(gè)別優(yōu)勢(shì)克隆特征,提示抗原誘導(dǎo)的CTL克隆可能存在于其中。 【結(jié)論】 1.采用基因槍方法成功轉(zhuǎn)染由人PBMC誘導(dǎo)培養(yǎng)的原代DC,制備家族性IgHV框架區(qū)DNA疫苗免疫的DC。 2.家族性IgHV框架區(qū)DNA疫苗體外能夠誘導(dǎo)抗原特異性CTL細(xì)胞,對(duì)同IgH家族的B細(xì)胞淋巴瘤細(xì)胞具有殺傷作用。
[Abstract]:Purpose of the project


In order to determine whether the DNA vaccine of the framework region can induce the generation of antigen - specific cytotoxic T - lymphocytes , it provides an experimental basis for the next step of the vaccine in active immunization or adoptive T cell therapy .


Methodology


Human peripheral blood mononuclear cells ( PBMC ) were isolated from human peripheral blood mononuclear cells ( PBMC ) , and the cells were cultured under the induction of cytokines such as IL - 2 . The cells were transfected with DCs , reverse transcription polymerase chain reaction ( RT - PCR ) and enzyme linked immunosorbent assay ( ELISA ) .


The result is not valid .


A non - endotoxin plasmid IgHV1 - GM - CSF / pcDNA3.1 for transfection was prepared and identified by enzyme digestion and PCR . The results showed that the sequence of the IgHV1 leader peptide , the FR region and the linked GM - CSF cDNA sequence , the FR region sequence contained the coding sequence of the antigen 9 peptide QLVQSGAEV which was restricted by HLA - A * .


A typical DC was produced by cytokine induction in peripheral blood mononuclear cells ( PBMC ) . The typical morphology of DC was 85.0 % 锝，

本文編號(hào)：1947087