Isolation,culture,identification and immortalization of urine derived stem cell

Geng Ruizhi; He Yunfeng; Zhang Yao; Zhang Yuanyuan; Wu Xiaohou

doi:10.13406/j.cnki.cyxb.001257

您当前的位置：

首页 >

文章列表页 >

Isolation,culture,identification and immortalization of urine derived stem cell

更新时间：2024-01-17

- Isolation,culture,identification and immortalization of urine derived stem cell
- Journal of Chongqing Medical University Vol. 42, Issue 5, Pages: 526-531(2017)
- 作者机构：
  
  1. 重庆医科大学附属第一医院泌尿外科
  2. 维克森林大学再生医学中心
- 作者简介：
- 基金信息：
- DOI：10.13406/j.cnki.cyxb.001257
  CLC： R691
- Published：2017，
扫描看全文
Geng Ruizhi, He Yunfeng, Zhang Yao, et al. Isolation,culture,identification and immortalization of urine derived stem cell. [J]. Journal of Chongqing Medical University 42(5):526-531(2017)
DOI：

Geng Ruizhi, He Yunfeng, Zhang Yao, et al. Isolation,culture,identification and immortalization of urine derived stem cell. [J]. Journal of Chongqing Medical University 42(5):526-531(2017) DOI： 10.13406/j.cnki.cyxb.001257.

摘要

目的:分离、培养、鉴定尿液来源的干细胞并永生化

为后续下尿路组织重建的研究提供种子细胞。方法:收集人新鲜尿液

对得到的单个贴壁细胞进行培养。倒置显微镜观察尿源性干细胞(urine-derived stem cells

USC)形态

免疫荧光以及流式细胞术检测干细胞表面标记物。RT-PCR检测尿路上皮细胞和平滑肌相关基因。诱导USC向尿路上皮细胞和平滑肌细胞分化。将p SEB-h TERT逆转录病毒感染USC得到永生化的尿源性干细胞(immortalized urine-derived stem cells

i USC)。RT-PCR以及Western blot检测i USC和USC中h TERT基因和蛋白的表达并绘制增殖曲线。对i USC检测干细胞表面标记物CD73、CD90、CD146、SSEA-4及免疫荧光检测尿溶蛋白Ⅰa和肌间线蛋白。结果:成功分离培养得到USC

其外观呈"米粒状"

干细胞表面标记物CD73、CD90、CD146、SSEA-4均呈阳性;RT-PCR结果显示尿路上皮细胞表面标记物(尿溶蛋白Ⅰa和Ⅲ、细胞角蛋白-7和13)及平滑肌表面标记物(肌间线蛋白和α-平滑肌肌动蛋白)表达阳性。RT-PCR中h TERT基因表达量为41 636.00±4 134.42

同USC(25 452.67±1 586.32)比较具有统计学意义(P=0.032);Western blot中h TERT蛋白表达量为94 479.00±7 102.20

同USC(61 541.67±3 956.54)比较具有统计学意义(P=0.017)。i USC能连续多代培养

增殖曲线呈"S"形

且与USC对比其增殖能力增强(P0 d=0.272

P1 d=0.043

P3 d=0.000

P5 d=0.006

P7 d=0.001

P9 d=0.025)

细胞增殖与天数(P=0.000

F=219.572)和细胞类型(P=0.000

F=90.855)均相关;CD34、CD73、CD90、CD146、SSEA-4依次为0.3%、91.4%、15.3%、99.4%、95.3%

表达无变化;免疫荧光显示尿溶蛋白Ⅰa和肌间线蛋白呈阳性表达。结论:成功从尿液中分离、培养、鉴定尿源性干细胞并永生化

即i USC为再生医学和组织工程学提供稳定安全的种子细胞。

Abstract

Objective:To culture and characterize urine derived stem cells（USC）obtained from the urine

and to establish the immortalized stem cells. Methods:Fresh urine was collected. After urine samples being centrifuged

cell pellets were re-suspended and plated in 24-well tissue culture plates. Only single cells that attached to culture wells by day 2 after plating were used. The morphology of i USC was observed by inverted microscope. Stem cell surface mark CD73

CD90

CD146

SSEA-4 were detected with immunofluorescence and flow cytometry. Urothelial cell surface mark uroplakin Ⅰa

Ⅲ

cytokeratin（CK）-7 and 13 were detected with reverse transcription-polymerase chain reaction（RT-PCR）. Smooth muscle cell surface mark desmin and α-smooth muscle actin were detected with RT-PCR. Part of the cells were selected to induce differentiation of urothelial cells and smooth muscle cells. The human telomerase reverse transcriptase（h TERT）gene was transfected into urine derived stem cell to get the immortalized stem cell. RT-PCR and Western blot were used to detect h TERT. The morphology and the growth curve were observed. Uroplakin Ia and desmin were detected with immunofluorescence. Results:Single

small

compact "rice-grain"like cells was observed. The expression of stem cell surface markers

such as CD73

CD90

CD146

SSEA-4 was positive. RT-PCR findings confirmed that urothelial cell surface marker（uroplakinⅠa

cytokeratin（CK）-7

13）and smooth muscle cell surface marker（desmin

α-smooth muscle actin）were present in USC.The cells were induced to urothelial cells and smooth muscle cells successfully. The expression of h TERT gene detected by RT-PCR and Western blot confirmed that h TERT gene was transfected into USC successfully. The growth curves of USC and i USC showed a "S"shape

but i USC has a faster growth speed compared with USC

and has a statistically significant difference（P=0.043

P=0.000

P=0.006

P=0.001

P=0.025）. The expression of stem cell surface markers

such as CD73

CD90

CD146

SSEA-4 was positive as it presents in USC. i USC had the ability to differentiate into the cell lineages expressing urothelial and smooth muscle cell markers（uroplakin Ⅰa and Desmin）by immunofluorescence analysis. Conclusion:The urine-derived stem cells are successfully isolated and immortalized called immortalized urine-derived stem cells（i USC）. It provides seed cells of stable and safe for regeneration and tissue engineering in vitro studies.

关键词

干细胞泌尿系统细胞永生化

Keywords

stem cellUrinary systemcell immortalization

references

Views

387

下载量

CSCD

Alert me when the article has been cited

提交

Tools

Publicity Resources

Effect of artemisinin extract on the urinary and digestive system in rabbits

The constituent ratio of urinary system disease had changed among inpatient children in the Children’s Hospital Affiliated of Chongqing Medical University during 15 years

BMP13 on C₃H₁₀T_1/2 cells to improve cardiac function in myocardial infarction rats

Research progress in dental pulp regeneration strategy based on endogenous stem cell homing

Enhancement of radiosensitivity of glioma stem cells by shRNA-STAT3

Related Author

et al

ZHANG Hui-jun

阳海平

陶红

王莉

et al

LI Cui-cui

WEI Hao

Related Institution

Department of Physiology,College of Basic Medicine,Chongqing Medical University

Department of Kidney Immunity,the Children’s Hospital,Chongqing Medical University

Ministry of Education Key Laboratory of Child Development Disorders

Department of Orthodontics，Chongqing Healthy Center for Women and Children

Department of Endodontics，Chongqing Medical University Affiliated Stomatological Hospital

⁰