高剂量pSin慢病毒载体介导的转染对诱导性多能干细胞影响的研究

doi:10.3877/cma.j.issn.2095-1221.2017.03.001

中华细胞与干细胞杂志(电子版) ›› 2017, Vol. 07 ›› Issue (03) : 125 -130. doi: 10.3877/cma.j.issn.2095-1221.2017.03.001

所属专题：文献；

论著

高剂量pSin慢病毒载体介导的转染对诱导性多能干细胞影响的研究

黄乙涓¹, 黄强², 吕欧³^,^†(

)

^1. 51000　广州，中山大学中山医学院基础医学系
^2. 51000　广州，国家知识产权局专利局专利审查协作广东中心
^3. 51000　广州润成化工材料有限公司

收稿日期:2016-12-30 出版日期:2017-06-01
通信作者: 吕欧

Effect of high dose pSin lentiviral vector-mediated transfection on pluripotent stem cells

Yijuan Huang¹, Qiang Huang², Ou Lyu³^,^†()

^1. Department of immunology, Sun Yat-sen University Zhongshan School of Medicine, Guangzhou 51000, China
^2. State Intellectual Property Office Patent Office Patent Examination Cooperation Guangdong Center, Guangzhou 51000, China
^3. Guangzhou Runcheng Chemical Materials Limited Company, Guangzhou 51000, China

Received:2016-12-30 Published:2017-06-01
Corresponding author: Ou Lyu
About author:
Corresponding author: Lyu Ou, Email: 441823577@qq.com

全文 ( ) 下载PDF ( ) 导出引用

目的

研究慢病毒载体pSin介导的基因修饰后的诱导性多能干细胞（iPS），细胞形态和传代的影响。

方法

通过包装慢病毒pSin-GFP感染iPS，分为实验组（细胞与病毒滴度比例1 : 200）和对照组（细胞与病毒滴度比例1 : 1）感染人iPS，经过嘌呤霉素药物筛选获得单克隆，重复3次实验观察细胞形态学变化记录细胞每代克隆数，采用方差分析及t检验进行统计学分析数据结果，用免疫荧光和werstern检测iPS标志蛋白表达。

结果

pSin-GFP病毒感染iPS后挑取24个单克隆细胞株，传十代后实验组（4.67±0.33）明显少于对照组（16.00±0.58）的细胞克隆数（t =17.00，P?<?0.01），实验组细胞状态稳定并正常表达Oct-4、Sox2、SSEA-1、Nanog和Tra-1-60。

结论

使用1?:?200的高比例慢病毒载体pSin介导的基因修饰后的iPS可传代次数减少，形态学并未发生变化，其正常表达的干细胞标志性蛋白。

关键词: 多能干细胞, 慢病毒属, 基因修饰

Objective

To study the effect of lentiviral vector pSin-mediated gene modification on pluripotent stem cell lines.

Methods

The lentivirus pSin-GFP was packaged, iPS cells were divided into experimental group (cell and virus titer ratio 1 : 200) and control group (cell and virus titer ratio 1 : 1) and infected with virus. Stem cells were harvested and the single cell clones were obtained by limit dilution method. The cell clones after infection was calculated by subculture and pluripotent stem cell markers were detected by Western blot and immunofluorescence staining. The results were analyzed by variance analysis and t test.

Results

Twenty-four monoclonal cell lines were selected after 10 generations. Cell cloning number of the experimental group (4.67±0.33) was significantly fewer than that of the control group (16.00±0.58) (t =17.00, P < 0.01) . Stable passaged cells expressed Oct-4, Sox2, Nanog, SSEA-1 and Tra-1-60.

Conclusion

After genetic modification, the proliferation potential of iPS decreases, but their morphology is not changed and they express common stem cell markers.

Key words: Pluripotent stem cells, Lentivirus, Genetically modified

图1 荧光显微镜下观察两组pSin-GFP病毒感染24h后iPS细胞形态（×100）

图2 倒置显微镜下观察两组病毒感染24 h后iPS细胞形态（×100）

表1 慢病毒pSin-GFP转染对细胞代数的影响（个， ± s）

分组	细胞代数
分组	1	2	3	4	5	6	7	8	9	10
实验组	24.00±0.00	12.67±0.33	11.00±0.58	9.00±0.58	6.33±0.88	5.33±0.33	4.67±0.33	4.67±0.33	4.67±0.33	4.67±0.33
对照组	24.00±0.00	20.67±0.67	20.67±0.67	19.67±0.33	18.00±1.16	16.67±0.67	16.67±0.67	16.00±0.58	16.00±0.58	16.00±0.58
空白组	24.00	24.00	24.00	24.00	24.00	24.00	24.00	24.00	24.00	24.00
F值	--	192.00	64.70	256.00	33.10	1156.00	432.00	289.00	289.00	289.00
P值	--	0.01	0.02	0.00	0.03	0.00	0.00	0.00	0.00	0.00

表1 慢病毒pSin-GFP转染对细胞代数的影响（个， ± s）

分组	细胞代数
分组	1	2	3	4	5	6	7	8	9	10
实验组	24.00±0.00	12.67±0.33	11.00±0.58	9.00±0.58	6.33±0.88	5.33±0.33	4.67±0.33	4.67±0.33	4.67±0.33	4.67±0.33
对照组	24.00±0.00	20.67±0.67	20.67±0.67	19.67±0.33	18.00±1.16	16.67±0.67	16.67±0.67	16.00±0.58	16.00±0.58	16.00±0.58
空白组	24.00	24.00	24.00	24.00	24.00	24.00	24.00	24.00	24.00	24.00
F值	--	192.00	64.70	256.00	33.10	1156.00	432.00	289.00	289.00	289.00
P值	--	0.01	0.02	0.00	0.03	0.00	0.00	0.00	0.00	0.00

图3 流式检测药筛后实验组iPS细胞绿色荧光表达量

图4 werstern检测Oct-4蛋白和Sox2蛋白表达情况

图5 免疫荧光检测多能性基因SSEA-1和Nanog和Tra-1-60细胞的表达情况

1	Takahashi K, Tanabe K, Ohnuki M, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors[J]. Cell, 2007, 131(5):861-872.
2	Lerou PH, Daley GQ. Therapeutic potential of embryonic stem cells[J]. Blood Rev, 2005, 19(6):321-331.
3	Murry CE, Keller G. Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development[J]. Cell, 2008, 132(4):661-680.
4	Zhang D, Jiang W, Liu M, et al. Highly efficient differentiation of human ES cells and iPS cells into mature pancreatic insulin-producing cells[J]. Cell Res, 2009, 19(4):429-438.
5	Song ZH, Cai J, Liu YX, et al. Efficient Generation of hepatocyte-like cells from human induced pluripotent stem cells[J]. Cell Res, 2009, 19(11):1233-1242.
6	Wernig M, Zhao JP, Pruszak J, et al. Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson's disease[J]. Proc Natl Acad Sci U S A, 2008, 105(15):5856-5861.
7	Aoi T, Yae K, Nakagawa M, et al. Generation of pluripotent stem cells from adult mouse liver and stomach cells[J]. Science, 2008, 321(5889):699-702.
8	Stadtfeld M, Nagaya M, Utikal J, et al. Induced pluripotent stem cells generated without viral integration[J]. Science, 2008, 322(593):945-949.
9	Hanna J, Markoulaki S, Schorderet P, et al. Direct reprogramming of terminally differentiated mature B lymphocytes to pluripotency[J]. Cell, 2008, 133(2):250-264.
10	申红芬，姚志芳，肖高芳, 等. 诱导性多潜能干细胞(iPS cells)——现状及前景展望[J]. 生物化学与生物物理进展, 2009, 36(8):950-960.
11	Bhutani N, Brady JJ, Damian M, et al. Reprogramming towards pluripotency requires AID-dependent DNA demethylation[J]. Nature, 2010, 463(7284):1042-1047.
12	Nakagawa M, Takizawa N, Narita M, et al. Promotion of direct reprogramming by transformation-deficient Myc[J]. Proc Natl Acad Sci U S A, 2010, 107(32):14152-14157.
13	Cavazzana-Calvo M, Hacein-Bey S, De Saint Basile G, et al. Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease[J]. Science, 2000, 288(5466):669-672.
14	Hacein-Bey-Abina S, Von Kalle C, Schmidt M, et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1[J]. Science, 2003, 302(5644):415-419.
15	Hacein-Bey-Abina S, Garrigue A, Wang GP，et al. Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1[J]. J Clin Invest, 2008, 118(9):3132-3142.
16	Howe SJ, Mansour MR, Schwarzwaelder K, et al. Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients[J]. J Clin Invest, 2008, 118(9):3143-3150.
17	Stein S, Ott MG, Schultze-Strasser S, et al. Genomic instability and myelodysplasia with monosomy 7 consequent to EVI1 activation after gene therapy for chronic granulomatous disease[J]. Nat Med, 2010, 16(2):198-204.
18	Cattoglio C, Facchini G, Sartori D, et al. Hot spots of retroviral integration in human CD34⁺ hematopoietic cells[J]. Blood, 2007, 110(6):1770-1778.
19	Ronen K, Negre O, Roth S, et al. Distribution of lentiviral vector integration sites in mice following therapeutic gene transfer to treat β-thalassemia[J]. Mol Ther, 2011, 19(7):1273-1286.
20	Rittelmeyer I, Rothe M, Brugman MH, et al. Hepatic lentiviral gene transfer is associated with clonal selection, but not with tumor formation in serially transplanted rodents[J]. Hepatology, 2013, 58(1):397-408.

[1]	符莞孟, 王晓黎, 刘玉, 张潍, 张菊. 干细胞治疗多囊卵巢综合征的研究进展[J]. 中华细胞与干细胞杂志(电子版), 2023, 13(02): 108-114.
[2]	周逸凡, 金颖. ERK信号通路在人多能干细胞的多能性状态调控中的作用[J]. 中华细胞与干细胞杂志(电子版), 2023, 13(01): 27-35.
[3]	武玉康, 康九红. 多能干细胞在心脏发育和疾病研究中的应用[J]. 中华细胞与干细胞杂志(电子版), 2022, 12(06): 378-382.
[4]	高原, 盛伟, 黄国英. 多能干细胞在体外心脏模型构建研究中的应用[J]. 中华细胞与干细胞杂志(电子版), 2022, 12(05): 314-318.
[5]	柯敏霞, 杨黄恬. 心肌微组织的构建及其在心肌损伤修复中的研究进展[J]. 中华细胞与干细胞杂志(电子版), 2022, 12(04): 224-229.
[6]	胡敏洁, 王思贤, 王永煜. 人诱导多能干细胞及其在血管相关疾病模型中的应用[J]. 中华细胞与干细胞杂志(电子版), 2022, 12(03): 167-175.
[7]	陈妙纯, 吴高椿, 刘韬. 人诱导性多能干细胞向红系分化的研究进展[J]. 中华细胞与干细胞杂志(电子版), 2022, 12(02): 115-120.
[8]	张小佐, 霍海芹, 谭建新, 张芳, 冯浩洋, 许争峰. 多能干细胞体外分化为类神经管模型的研究进展[J]. 中华细胞与干细胞杂志(电子版), 2022, 12(01): 45-50.
[9]	杜为, 崔丽娟, 徐迎, 张华, 杜宏伟, 张金美, 刘容志, 王征宇, 杨文玲, 张宇. 脐带血单个核细胞诱导多能干细胞来源自然杀伤细胞的生物学特性[J]. 中华细胞与干细胞杂志(电子版), 2021, 11(06): 329-336.
[10]	刘锴. 无异源培养条件下人多能干细胞系H1非病毒转染方法的比较和优化[J]. 中华细胞与干细胞杂志(电子版), 2021, 11(05): 305-310.
[11]	马恩奇, 张倩, 陈雪梅, 刘韬. iPSCs诱导的造血干细胞对内皮细胞血管生成作用的影响及相关作用机制研究[J]. 中华细胞与干细胞杂志(电子版), 2021, 11(04): 193-199.
[12]	吴倩, 张梅. 胰岛类器官研究进展[J]. 中华细胞与干细胞杂志(电子版), 2021, 11(02): 120-124.
[13]	刘艺霖, 吴志鹏, 邱江. 多能干细胞诱导分化为肾脏类器官的研究进展与挑战[J]. 中华细胞与干细胞杂志(电子版), 2021, 11(01): 57-62.
[14]	汪若凤, 郭永洪, 田勤, 段维鸽. 多能性胚胎干细胞与DNA甲基化的关系[J]. 中华细胞与干细胞杂志(电子版), 2019, 09(03): 188-192.
[15]	汪东生, 吴理达, 顾雨春. 细胞基因疗法在视网膜退行性疾病中的应用和挑战[J]. 中华眼科医学杂志(电子版), 2022, 12(03): 129-133.

阅读次数

全文

摘要

选择文件类型/文献管理软件名称

选择包含的内容

Effect of high dose pSin lentiviral vector-mediated transfection on pluripotent stem cells

模态框（Modal）标题

选择文件类型/文献管理软件名称

选择包含的内容

Effect of high dose pSin lentiviral vector-mediated transfection on pluripotent stem cells