多能干細(xì)胞分化再生T細(xì)胞的體內(nèi)抗腫瘤評(píng)估及再生可移植髓系細(xì)胞研究
發(fā)布時(shí)間:2021-06-10 12:25
從多能干細(xì)胞誘導(dǎo)分化出可移植、有功能的血液及免疫細(xì)胞一直是再生醫(yī)學(xué)領(lǐng)域的研究熱點(diǎn)和難點(diǎn)。本文第一部分證明小鼠胚胎干細(xì)胞分化來源、體內(nèi)成熟的T細(xì)胞可以結(jié)合CAR-T技術(shù)改造實(shí)現(xiàn)體內(nèi)清除腫瘤的目的,第二部分初步實(shí)現(xiàn)誘導(dǎo)小鼠胚胎干細(xì)胞獲得可移植的髓系祖細(xì)胞種子,移植后可在體內(nèi)短期再生髓系細(xì)胞(包括粒細(xì)胞、單核-巨噬細(xì)胞)。本研究為再生T細(xì)胞以及可移植髓系細(xì)胞的轉(zhuǎn)化研究提供了技術(shù)借鑒。嵌合抗原受體 T 細(xì)胞(Chimeric Antigen Receptor T cells,CAR-T)療法已成為臨床上治療血液腫瘤的有效手段。通常CAR-T細(xì)胞是通過分離患者外周血來源的T細(xì)胞在體外進(jìn)行基因編輯而制備,但是患者自身的T細(xì)胞數(shù)量有限且經(jīng)常出現(xiàn)功能異;蚝慕,因此需要探索T細(xì)胞新的來源。本研究基于課題組已開發(fā)的定向誘導(dǎo)T細(xì)胞技術(shù),利用Runx1-Hoxa9條件性表達(dá)的多能干細(xì)胞系誘導(dǎo)分化出可植入免疫缺陷鼠(B-NDG)的造血祖細(xì)胞(induced hematopoietic progenitorcells,iHPCs),移植B-NDG小鼠5周后,從脾臟中分離出再生的T細(xì)胞(induced T cel...
【文章來源】:中國(guó)科學(xué)技術(shù)大學(xué)安徽省 211工程院校 985工程院校
【文章頁數(shù)】:126 頁
【學(xué)位級(jí)別】:博士
【部分圖文】:
圖1.1.1.1正在開發(fā)的753種細(xì)胞療法的全球地理分布??圖注:美國(guó)和中國(guó)分別以344種和203種細(xì)胞療法引領(lǐng)著細(xì)胞療法發(fā)展競(jìng)賽??
-DL1?基質(zhì)細(xì)??胞共培養(yǎng),采用造血分化培養(yǎng)基將iHECs誘導(dǎo)分化為可用于移植的誘導(dǎo)型造血??祖細(xì)胞(induced?hematopoietic?progenitor?cells,iHPCs?)(Lin—c-Kit+CD?127+/CD135+)??(圖?1.3.2)。??0?SOK?100K?150K?200K?260K?0?1〇4?1〇5?-1〇3?〇?1〇3?1〇4?1〇S??FSC-A???CD45-PerCP-Cy5.5???CD201-PE????圖1.3.1?iR9?mESCs多能干細(xì)胞定向誘導(dǎo)分化iHECs細(xì)胞day?11流式分選圖??圖注:將iR9mESCs通過2.5天的倒置培養(yǎng)形成擬胚體(Embryoidbodies,EBs),再將EBs??經(jīng)過誘導(dǎo)培養(yǎng)(第6天通過Dox誘導(dǎo)啟動(dòng)和FoxaP表達(dá))使其分化為誘導(dǎo)型生血內(nèi)??皮細(xì)胞iHECs,在第11天將分化的細(xì)胞進(jìn)行收集分選出iHECs?(CD3rCD41lDWCD45-c-??Kit+CDSOW),細(xì)胞分選前由CD31陽性富集,此圖為三次獨(dú)立實(shí)驗(yàn)的代表性圖。???7-AAD-???麵,1_??-1〇3?〇?103?104?105?-103?0?103?1〇4?105?-103?0?10??104?10*??CD45-APC???CD135-PE???CD127-BV421?-??27??
?第1章多能干細(xì)胞分化再生的T細(xì)胞體內(nèi)抗腫瘤評(píng)估???圖1.3.2?iR9?mESCs多能干細(xì)胞定向誘導(dǎo)分化day?21流式分析iHPCs細(xì)胞??圖注:將第11天分選的iHECs與0P9-DL1基質(zhì)細(xì)胞共培養(yǎng)經(jīng)歷造血成熟,經(jīng)歷10天成熟??后即day?21時(shí)流式分析共培養(yǎng)成熟的誘導(dǎo)型造血祖細(xì)胞iHPCs,免疫表型限定為:??Lin?(CD2/CD3/CD4/CD8/CD1?lb/Grl/Terl?19/CD19/NK1???1/TCRyS)?-c-Kit+CD127+/CD135+。??1.3.?2?i?R9?mESCs來源的i?HPCs移植免疫缺陷鼠再生T淋巴細(xì)胞??接下來,我們利用重度免疫缺陷鼠(B-NDG,類似于NSG)作為iHPCs分??化產(chǎn)生iT細(xì)胞的反應(yīng)器,在iHECs進(jìn)行共培養(yǎng)的第10天,收集其分化形成的造??血祖細(xì)胞iHPCs?(2?x?1〇6/只)通過眼靜脈注射,移植到經(jīng)過X-ray照射處理(2.25??Gy)的B-NDG小鼠中,在移植后第5周,我們利用流式技術(shù)檢測(cè)到其脾臟有供??體來源的成熟iT細(xì)胞(CD45.2+CD3+CD4+或CD45.2+CD3+CD8+,注:再生的T??細(xì)胞命名為iT細(xì)胞)。流式數(shù)據(jù)顯示供體來源的細(xì)胞中CD4+細(xì)胞和CD8+細(xì)胞??比例分別為?63.2°/。、35.5%?(圖?1.3.3)。??CD45.2+CD3+??議It?? ̄|im'lI?fU'?i'V'i?I'm?1|?1???'?I?'!?。?I????J???103?0?103?1〇4?105??CD4-PE????圖1.3.3?iR9?mESCs來源的iHPCs移植受體鼠脾臟iT細(xì)胞流式分析?
【參考文獻(xiàn)】:
期刊論文
[1]Phase 1 clinical trial demonstrated that MUC1 positive metastatic seminal vesicle cancer can be effectively eradicated by modified Anti-MUC1 chimeric antigen receptor transduced T cells[J]. Fengtao You,Licui Jiang,Bozhen Zhang,Qiang Lu,Qiao Zhou,Xiaoyang Liao,Hong Wu,Kaiqi Du,Youcai Zhu,Huimin Meng,Zhishu Gong,Yunhui Zong,Lei Huang,Man Lu,Jirong Tang,Yafen Li,Xiaochen Zhai,Xiangling Wang,Sisi Ye,Dan Chen,Lei Yuan,Lin Qi,Lin Yang. Science China(Life Sciences). 2016(04)
[2]Treatment of solid tumors with chimeric antigen receptor-engineered T cells: current status and future prospects[J]. Shengmeng Di,Zonghai Li. Science China(Life Sciences). 2016(04)
[3]Gastric cancer and the epoch of immunotherapy approaches[J]. Elena Niccolai,Antonio Taddei,Domenico Prisco,Amedeo Amedei. World Journal of Gastroenterology. 2015(19)
本文編號(hào):3222366
【文章來源】:中國(guó)科學(xué)技術(shù)大學(xué)安徽省 211工程院校 985工程院校
【文章頁數(shù)】:126 頁
【學(xué)位級(jí)別】:博士
【部分圖文】:
圖1.1.1.1正在開發(fā)的753種細(xì)胞療法的全球地理分布??圖注:美國(guó)和中國(guó)分別以344種和203種細(xì)胞療法引領(lǐng)著細(xì)胞療法發(fā)展競(jìng)賽??
-DL1?基質(zhì)細(xì)??胞共培養(yǎng),采用造血分化培養(yǎng)基將iHECs誘導(dǎo)分化為可用于移植的誘導(dǎo)型造血??祖細(xì)胞(induced?hematopoietic?progenitor?cells,iHPCs?)(Lin—c-Kit+CD?127+/CD135+)??(圖?1.3.2)。??0?SOK?100K?150K?200K?260K?0?1〇4?1〇5?-1〇3?〇?1〇3?1〇4?1〇S??FSC-A???CD45-PerCP-Cy5.5???CD201-PE????圖1.3.1?iR9?mESCs多能干細(xì)胞定向誘導(dǎo)分化iHECs細(xì)胞day?11流式分選圖??圖注:將iR9mESCs通過2.5天的倒置培養(yǎng)形成擬胚體(Embryoidbodies,EBs),再將EBs??經(jīng)過誘導(dǎo)培養(yǎng)(第6天通過Dox誘導(dǎo)啟動(dòng)和FoxaP表達(dá))使其分化為誘導(dǎo)型生血內(nèi)??皮細(xì)胞iHECs,在第11天將分化的細(xì)胞進(jìn)行收集分選出iHECs?(CD3rCD41lDWCD45-c-??Kit+CDSOW),細(xì)胞分選前由CD31陽性富集,此圖為三次獨(dú)立實(shí)驗(yàn)的代表性圖。???7-AAD-???麵,1_??-1〇3?〇?103?104?105?-103?0?103?1〇4?105?-103?0?10??104?10*??CD45-APC???CD135-PE???CD127-BV421?-??27??
?第1章多能干細(xì)胞分化再生的T細(xì)胞體內(nèi)抗腫瘤評(píng)估???圖1.3.2?iR9?mESCs多能干細(xì)胞定向誘導(dǎo)分化day?21流式分析iHPCs細(xì)胞??圖注:將第11天分選的iHECs與0P9-DL1基質(zhì)細(xì)胞共培養(yǎng)經(jīng)歷造血成熟,經(jīng)歷10天成熟??后即day?21時(shí)流式分析共培養(yǎng)成熟的誘導(dǎo)型造血祖細(xì)胞iHPCs,免疫表型限定為:??Lin?(CD2/CD3/CD4/CD8/CD1?lb/Grl/Terl?19/CD19/NK1???1/TCRyS)?-c-Kit+CD127+/CD135+。??1.3.?2?i?R9?mESCs來源的i?HPCs移植免疫缺陷鼠再生T淋巴細(xì)胞??接下來,我們利用重度免疫缺陷鼠(B-NDG,類似于NSG)作為iHPCs分??化產(chǎn)生iT細(xì)胞的反應(yīng)器,在iHECs進(jìn)行共培養(yǎng)的第10天,收集其分化形成的造??血祖細(xì)胞iHPCs?(2?x?1〇6/只)通過眼靜脈注射,移植到經(jīng)過X-ray照射處理(2.25??Gy)的B-NDG小鼠中,在移植后第5周,我們利用流式技術(shù)檢測(cè)到其脾臟有供??體來源的成熟iT細(xì)胞(CD45.2+CD3+CD4+或CD45.2+CD3+CD8+,注:再生的T??細(xì)胞命名為iT細(xì)胞)。流式數(shù)據(jù)顯示供體來源的細(xì)胞中CD4+細(xì)胞和CD8+細(xì)胞??比例分別為?63.2°/。、35.5%?(圖?1.3.3)。??CD45.2+CD3+??議It?? ̄|im'lI?fU'?i'V'i?I'm?1|?1???'?I?'!?。?I????J???103?0?103?1〇4?105??CD4-PE????圖1.3.3?iR9?mESCs來源的iHPCs移植受體鼠脾臟iT細(xì)胞流式分析?
【參考文獻(xiàn)】:
期刊論文
[1]Phase 1 clinical trial demonstrated that MUC1 positive metastatic seminal vesicle cancer can be effectively eradicated by modified Anti-MUC1 chimeric antigen receptor transduced T cells[J]. Fengtao You,Licui Jiang,Bozhen Zhang,Qiang Lu,Qiao Zhou,Xiaoyang Liao,Hong Wu,Kaiqi Du,Youcai Zhu,Huimin Meng,Zhishu Gong,Yunhui Zong,Lei Huang,Man Lu,Jirong Tang,Yafen Li,Xiaochen Zhai,Xiangling Wang,Sisi Ye,Dan Chen,Lei Yuan,Lin Qi,Lin Yang. Science China(Life Sciences). 2016(04)
[2]Treatment of solid tumors with chimeric antigen receptor-engineered T cells: current status and future prospects[J]. Shengmeng Di,Zonghai Li. Science China(Life Sciences). 2016(04)
[3]Gastric cancer and the epoch of immunotherapy approaches[J]. Elena Niccolai,Antonio Taddei,Domenico Prisco,Amedeo Amedei. World Journal of Gastroenterology. 2015(19)
本文編號(hào):3222366
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