發(fā)布時間：2018-05-24 21:34

  本文選題：誘導(dǎo)多能干細胞 + 慢病毒轉(zhuǎn)染�。� 參考：《第二軍醫(yī)大學》2011年碩士論文

【摘要】：誘導(dǎo)多能干細胞(induced pluripotent stem cells, iPSc)是利用病毒載體將特定轉(zhuǎn)錄因子組合轉(zhuǎn)入分化細胞,使其重編程為類似胚胎干細胞的一類細胞。它不僅具有自我更新和多向分化潛能,而且在細胞形態(tài)、基因和蛋白表達、表觀遺傳修飾狀態(tài)、類胚體和畸形瘤生成能力等方面都與胚胎干細胞相似。該技術(shù)是2006年由Yamanaka將重組有Oct3/4、Sox2、c-Myc和Klf4四個轉(zhuǎn)錄因子基因的病毒載體引入小鼠成纖維細胞,并成功誘導(dǎo)其重編程而最先創(chuàng)建的。在隨后的研究中,如何選用來源便捷、數(shù)目充足的成熟體細胞為重編程的靶細胞,成為iPS研究的熱點之一。而具備上述選材條件的成熟T淋巴細胞,卻存在體外成活時間短和病毒轉(zhuǎn)導(dǎo)效率極低等技術(shù)難題。 慢病毒(lentivirus)載體是以人類免疫缺陷Ⅰ型病毒(HIV-1)為基礎(chǔ)發(fā)展起來的基因治療載體。區(qū)別一般的逆轉(zhuǎn)錄病毒載體,它對分裂細胞和非分裂細胞均具有感染能力。在相關(guān)的細胞實驗操作中,對于一些按常規(guī)方法難以轉(zhuǎn)入甚至無法轉(zhuǎn)入的細胞,如原代細胞、干細胞等未分化細胞,通過慢病毒介導(dǎo)的方法能夠大大提高基因的轉(zhuǎn)導(dǎo)效率,而且使目的基因整合到宿主細胞基因組的幾率也明顯增加,這為報告基因在細胞內(nèi)的高效瞬時表達研究提供了有利途徑。 本研究建立了適合小鼠成熟活化T淋巴細胞體外長時間培養(yǎng),以及慢病毒轉(zhuǎn)導(dǎo)的技術(shù)平臺,獲得的主要研究結(jié)果如下: 1、成熟T淋巴細胞的增殖與活化。采用Ficoll梯度離心法分離小鼠單個核細胞,分別用刀豆蛋白A(ConA)和anti-mouse CD3 anti-mouse CD28等方法活化成熟T淋巴細胞,并利用流式細胞法檢測T細胞表面CD4~+CD44~(high)等活化標志;羧基熒光素雙乙酸鹽-琥珀酰亞胺酯(CFSE)示蹤法檢測活化細胞的增殖分裂狀況。 結(jié)果顯示:可從3月齡的雌性Balb/c小鼠(SPF級)的脾臟中分離到～2×10~7單個核細胞;96孔“U”型板經(jīng)anti-mouse CD3 anti-mouse CD28(終濃度分別為2μg/mL、4μg/mL)包被后,可使2.0×10~5個細胞/孔在第三天(D3)迅速增殖至1.6×10~6個細胞/孔,經(jīng)過anti-mouse CD3 anti-mouse CD28刺激活化的CD4+CD44high T淋巴細胞占活細胞總數(shù)的(74.0±1.8)%,且CFSE強陽性細胞由原來(92.3±2.8)%降至(16.34±1.3)%;該活化T淋巴細胞在含IL-2(終濃度30 ng/mL)CTL培養(yǎng)基中可維持生長(D14細胞數(shù)為1.5×10~6/孔);而經(jīng)ConA刺激激活的T淋巴細胞在D7才能達到最高值1.4×10~6個細胞/孔,且隨著培養(yǎng)時間延長,細胞凋亡增多,直至完全死亡。實驗評價了小鼠成熟T淋巴細胞在體外培養(yǎng)條件下能否保持長期的增殖與活化狀況的可行性,為下一步進行GFP重組慢病毒轉(zhuǎn)導(dǎo)奠定基礎(chǔ)。 2、慢病毒的包裝及對成熟T淋巴細胞的轉(zhuǎn)染效率。采用脂質(zhì)體將GFP重組慢病毒質(zhì)粒轉(zhuǎn)染到包裝細胞293T中,48小時后收集細胞上清液,進一步用超速離心的方法純化GFP重組慢病毒,轉(zhuǎn)導(dǎo)LEPC細胞D2后,采用流式細胞法檢測其轉(zhuǎn)染效率和病毒滴度。最后將純化的GFP重組慢病毒分別按MOI=1,5,10等不同滴度,轉(zhuǎn)染經(jīng)anti-mouse CD3 anti-mouse CD28刺激激活的T淋巴細胞,并觀測GFP重組慢病毒對T淋巴細胞的轉(zhuǎn)染效率。 結(jié)果顯示:經(jīng)脂質(zhì)體法將GFP重組慢病毒質(zhì)粒轉(zhuǎn)染到包裝細胞293T,在48小時后,293T全部帶有綠色熒光,轉(zhuǎn)染效率為95%以上。未純化的GFP重組慢病毒顆粒(500μL病毒/孔)轉(zhuǎn)導(dǎo)LEPC細胞,經(jīng)流式細胞儀分析GFP的陽性率為(85.9±3.6)%;而經(jīng)超離純化濃縮10倍的重組慢病毒組(50μL病毒/孔),其GFP陽性率仍達(74.3±1.8)%。純化后的GFP重組慢病毒轉(zhuǎn)染經(jīng)anti-mouse CD3 anti-mouse CD28刺激激活的T淋巴細胞3天后,GFP陽性率分別為:MOI=1組(25.8±1.75)%,MOI=5組(36.0±9.5)%,MOI=10組(43.0±4.75)%;與同滴度的腺病毒相比,其轉(zhuǎn)染T淋巴細胞的效率有較明顯地提高;且隨著GFP重組慢病毒滴度的增加,對T淋巴細胞的轉(zhuǎn)染效率也愈高。有關(guān)進一步提高慢病毒對該T淋巴細胞轉(zhuǎn)導(dǎo)效率的實驗?zāi)壳叭栽谶M行中。 該技術(shù)體系的建立與不斷完善,為下一步用肝特異性轉(zhuǎn)錄因子誘導(dǎo)成熟T淋巴細胞分化為肝干細胞準備了條件。
[Abstract]:Induction of pluripotent stem cells ( iPSc ) is a kind of cell which uses viral vectors to transfer specific transcription factors into differentiated cells , which are reprogrammed to resemble embryonic stem cells .


The lentivirus vector is a kind of gene therapy vector developed on the basis of human immunodeficiency virus type I virus ( HIV - 1 ) . It has the ability to infect both the dividing cell and the non - dividing cell . In the related cell experiment operation , it is difficult to transfer into cells , such as primary cells , stem cells and the like , which are difficult to be transferred to in the conventional method , and the probability of integration of the target gene into the genome of the host cell also increases obviously , which provides an advantageous way for the efficient transient expression study of the reporter gene in the cell .


This study established a technical platform suitable for long - term culture of mature activated T lymphocytes in mice and slow virus transduction , and the main results obtained are as follows :


1 . The proliferation and activation of mature T lymphocytes were studied by Ficoll gradient centrifugation . The mature T lymphocytes were activated by means of A ( Con A ) and anti - mouse CD3 anti - mouse CD28 , respectively , and activated markers such as CD4 ~ + CD44 ~ ( high ) were detected by flow cytometry , and the proliferative division of activated cells was detected by carboxyfluorescein diacetate - succinimide ester ( CFSE ) tracing method .


The results showed that the spleen of Balb / c mice ( SPF ) could be isolated from the spleen of 3 - month - old female Balb / c mice ( SPF ) .


2 . The transfection efficiency of the slow virus and the transfection efficiency of the mature T lymphocytes were studied . After 48 hours , the GFP recombinant lentivirus was transfected into the packed cells . After 48 hours , the supernatant of the cells was collected and the transfection efficiency and virus titer were detected by flow cytometry . Finally , the purified GFP recombinant lentivirus was transfected with the anti - mouse CD3 anti - mouse CD28 and activated T lymphocytes , and the transfection efficiency of GFP recombinant lentivirus on T lymphocytes was observed .


緇撴灉鏄劇ず:緇忚剛璐ㄤ綋娉曞皢GFP閲嶇粍鎱㈢梾姣掕川綺掕漿鏌撳埌鍖呰緇嗚優(yōu)293T,鍦，

本文編號：1930621