重组hFOXA2和hPDX1慢病毒载体诱导乳牙牙髓干细胞重编程为胰岛素生成样细胞

上海口腔医学 ›› 2013, Vol. 22 ›› Issue (6): 634-642.

重组hFOXA2和hPDX1慢病毒载体诱导乳牙牙髓干细胞重编程为胰岛素生成样细胞

石建峰，朱春晖，刘瑾，孙俊毅，饶国洲，李昂

(西安交通大学口腔医院医学研究中心，陕西西安 710004)

收稿日期:2013-03-15 修回日期:2013-05-30 出版日期:2013-04-12 发布日期:2013-04-12
通讯作者: 李昂，E-mail：drliang@mail.xjtu.edu.cn
作者简介:石建峰 (1973-)，男，硕士，主管技师，E-mail:921676599@qq.com
基金资助:
陕西省科技攻关计划项目(2009K17-06)；陕西省科技统筹创新工程计划资源主导型产业关键技术(链)项目(2011KTCL03-24)

Recombinant hFOXA2 and hPDX1 lentivirus induced dental pulp stem cells from deciduous teeth reprogramming for insulin-producing cells

SHI Jian-feng, ZHU Chun-hui, LIU Jin, SUN Jun-yi, RAO Guo-zhou, LI Ang

Medical Research Centre, Stomatological Hospital of Xi’an Jiaotong University. Xi’an 710004, Shaanxi Province, China

Received:2013-03-15 Revised:2013-05-30 Online:2013-04-12 Published:2013-04-12
Supported by:
Supported by Science and Technology Research Program of Shaanxi Province (2009K17-06) and Science and Technology Innovation as a Whole Plan Resources Leading Industry Key Technology (Chain) Project of Shaanxi Province (2011KTCL03-24).

摘要/Abstract

摘要： 目的：构建重组转录因子hFOXA2 和hPDX1慢病毒载体，转染乳牙牙髓干细胞，诱导其向胰岛素生成样细胞(insulin-producing cells，IPCs)分化。方法：酶消化法分离培养人牙髓干细胞(DPSCs)，有限稀释法纯化培养，流式细胞术分析细胞表面标志，并进行体外多向分化诱导验证；构建重组Lenti-hFOXA2、Lenti-hPDX1慢病毒载体，Lipofectamine2000转染293T细胞进行病毒包装，通过荧光细胞计数测定病毒感染效率和滴度；以最佳感染复数(MOI)感染牙髓干细胞，诱导其体外重编程为胰岛素生成样细胞，免疫荧光染色测定PDX1、FOXA2及胰岛素原表达，ELISA法检测胰岛素分泌情况。采用SPSS13.0软件包对数据进行统计学分析。结果：体外成功分离培养人DPSCs；细胞表面表达STRO-1(98.01%)、CDl46(98.51%)、CD34(99.54%)和CD45(24.08%)。体外成骨、成软骨、成脂肪诱导分化特异性染色阳性；成功构建重组Lenti-hFOXA2、Lenti-hPDX1慢病毒载体，双酶切和测序鉴定与GenBank的序列完全符合；病毒包装效率分别为(96.15±0.17)%和(95.49±0.21)%，感染滴度约为1.80×108 GTU/mL，最佳MOI为20；诱导后的细胞表达胰岛素原、FOXA2和PDX1，胰岛素分泌量为1.92 μmol/L，较未诱导组和对照组均显著增高(P<0.01)。结论：DPSCs经重组转录因子慢病毒载体Lenti-hFOXA2、Lenti-hPDX1转染诱导，可启动细胞重编程机制，形成胰岛素生成样细胞，可作为糖尿病基因治疗的种子细胞。

关键词: 乳牙牙髓干细胞, 表面标志, 诱导分化, 细胞编程, 胰岛素分泌

Abstract: PURPOSE: The purpose of this study was to culture and identify dental pulp stem cells(DPSCs) from deciduous teeth in vitro and construct the recombinant hFOXA2 and hPDX1 lentivirus vectors and transfect the DPSCs to induce insulin-producing cells (IPCs). METHODS: DPSCs were separated and cultured by enzyme digest method, and purified by limited dilution method. Flow cytometry was used to determine the surface marker expression of the DPSCs, and the ability of multiple differentiations was determined by specific staining. hFOXA2 and hPDX1 genes were amplified by PCR, and the recombinant hFOXA2 and hPDX1 lentivirus vectors were reconstructed and transfected into 293T cells by lipofectamine2000 for virus packaging. The viral infection efficiency and titer were determined through fluorescence cell count. The recombinant virus was used to infect the DPSCs cells via multiplicity of infection(MOI)and induce the DPSCs reprogramming for IPCs. Immunofluorescence staining was used to measure the expression of proinsulin, FOXA2 and PDX1. ELISA method was used to detect the insulin secretion. The data was analyzed Using SPSS13.0 software package. RESULTS: DPSCs were isolated and cultured successfully. Cell surface highly expressed STRO-1 (98.01%), CDl46 (98.51%), CD34 (99.54%) and CD45 (24.08%). The multi-lineage differentiation capacity into osteoblasts, chondrocytes, and adipose was achieved. The recombinant hFOXA2 and hPDX1 lentivirus vectors were successfully constructed. Double enzyme digestion and sequencing appraisal showed that the sequence was fully consistent with GenBank retrieval. Virus packing efficiency was (96.15±0.17) % and (95.49±0.21) % respectively, and the infection titer was about 1.80×108 GTU/mL. The best MOI of the virus was 20. After inducing the cells to express proinsulin, FOXA2 and PDX1, insulin secretion volume was about 1.92 μmol/L. Compared with the uninduced group and control group, insulin secretion increased significantly (P<0.01). CONCLUSIONS: The recombinant transcription factor virus can activate cell reprogramming mechanism, form insulin-producing cells, and can be used for gene therapy of diabetes seed cells.

Key words: Deciduous dental pulp stem cell, Surface marks, Induced differentiation, Cell programming, Insulin secretion

中图分类号:

R781.3

石建峰, 朱春晖, 刘瑾, 孙俊毅, 饶国洲, 李昂. 重组hFOXA2和hPDX1慢病毒载体诱导乳牙牙髓干细胞重编程为胰岛素生成样细胞[J]. 上海口腔医学, 2013, 22(6): 634-642.

SHI Jian-feng, ZHU Chun-hui, LIU Jin, SUN Jun-yi, RAO Guo-zhou, LI Ang. Recombinant hFOXA2 and hPDX1 lentivirus induced dental pulp stem cells from deciduous teeth reprogramming for insulin-producing cells[J]. Shanghai Journal of Stomatology, 2013, 22(6): 634-642.

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