成熟T淋巴细胞分离培养及其慢病毒介导的基因转染的研究

发布时间：2018-05-24 21:34

  本文选题：诱导多能干细胞 + 慢病毒转染　； 参考：《第二军医大学》2011年硕士论文

【摘要】：诱导多能干细胞(induced pluripotent stem cells, iPSc)是利用病毒载体将特定转录因子组合转入分化细胞,使其重编程为类似胚胎干细胞的一类细胞。它不仅具有自我更新和多向分化潜能,而且在细胞形态、基因和蛋白表达、表观遗传修饰状态、类胚体和畸形瘤生成能力等方面都与胚胎干细胞相似。该技术是2006年由Yamanaka将重组有Oct3/4、Sox2、c-Myc和Klf4四个转录因子基因的病毒载体引入小鼠成纤维细胞,并成功诱导其重编程而最先创建的。在随后的研究中,如何选用来源便捷、数目充足的成熟体细胞为重编程的靶细胞,成为iPS研究的热点之一。而具备上述选材条件的成熟T淋巴细胞,却存在体外成活时间短和病毒转导效率极低等技术难题。 慢病毒(lentivirus)载体是以人类免疫缺陷Ⅰ型病毒(HIV-1)为基础发展起来的基因治疗载体。区别一般的逆转录病毒载体,它对分裂细胞和非分裂细胞均具有感染能力。在相关的细胞实验操作中,对于一些按常规方法难以转入甚至无法转入的细胞,如原代细胞、干细胞等未分化细胞,通过慢病毒介导的方法能够大大提高基因的转导效率,而且使目的基因整合到宿主细胞基因组的几率也明显增加,这为报告基因在细胞内的高效瞬时表达研究提供了有利途径。 本研究建立了适合小鼠成熟活化T淋巴细胞体外长时间培养,以及慢病毒转导的技术平台,获得的主要研究结果如下: 1、成熟T淋巴细胞的增殖与活化。采用Ficoll梯度离心法分离小鼠单个核细胞,分别用刀豆蛋白A(ConA)和anti-mouse CD3 anti-mouse CD28等方法活化成熟T淋巴细胞,并利用流式细胞法检测T细胞表面CD4~+CD44~(high)等活化标志;羧基荧光素双乙酸盐-琥珀酰亚胺酯(CFSE)示踪法检测活化细胞的增殖分裂状况。 结果显示:可从3月龄的雌性Balb/c小鼠(SPF级)的脾脏中分离到～2×10~7单个核细胞;96孔“U”型板经anti-mouse CD3 anti-mouse CD28(终浓度分别为2μg/mL、4μg/mL)包被后,可使2.0×10~5个细胞/孔在第三天(D3)迅速增殖至1.6×10~6个细胞/孔,经过anti-mouse CD3 anti-mouse CD28刺激活化的CD4+CD44high T淋巴细胞占活细胞总数的(74.0±1.8)%,且CFSE强阳性细胞由原来(92.3±2.8)%降至(16.34±1.3)%;该活化T淋巴细胞在含IL-2(终浓度30 ng/mL)CTL培养基中可维持生长(D14细胞数为1.5×10~6/孔);而经ConA刺激激活的T淋巴细胞在D7才能达到最高值1.4×10~6个细胞/孔,且随着培养时间延长,细胞凋亡增多,直至完全死亡。实验评价了小鼠成熟T淋巴细胞在体外培养条件下能否保持长期的增殖与活化状况的可行性,为下一步进行GFP重组慢病毒转导奠定基础。 2、慢病毒的包装及对成熟T淋巴细胞的转染效率。采用脂质体将GFP重组慢病毒质粒转染到包装细胞293T中,48小时后收集细胞上清液,进一步用超速离心的方法纯化GFP重组慢病毒,转导LEPC细胞D2后,采用流式细胞法检测其转染效率和病毒滴度。最后将纯化的GFP重组慢病毒分别按MOI=1,5,10等不同滴度,转染经anti-mouse CD3 anti-mouse CD28刺激激活的T淋巴细胞,并观测GFP重组慢病毒对T淋巴细胞的转染效率。 结果显示:经脂质体法将GFP重组慢病毒质粒转染到包装细胞293T,在48小时后,293T全部带有绿色荧光,转染效率为95%以上。未纯化的GFP重组慢病毒颗粒(500μL病毒/孔)转导LEPC细胞,经流式细胞仪分析GFP的阳性率为(85.9±3.6)%;而经超离纯化浓缩10倍的重组慢病毒组(50μL病毒/孔),其GFP阳性率仍达(74.3±1.8)%。纯化后的GFP重组慢病毒转染经anti-mouse CD3 anti-mouse CD28刺激激活的T淋巴细胞3天后,GFP阳性率分别为:MOI=1组(25.8±1.75)%,MOI=5组(36.0±9.5)%,MOI=10组(43.0±4.75)%;与同滴度的腺病毒相比,其转染T淋巴细胞的效率有较明显地提高;且随着GFP重组慢病毒滴度的增加,对T淋巴细胞的转染效率也愈高。有关进一步提高慢病毒对该T淋巴细胞转导效率的实验目前仍在进行中。 该技术体系的建立与不断完善,为下一步用肝特异性转录因子诱导成熟T淋巴细胞分化为肝干细胞准备了条件。
[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 .


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