人尿源性干细胞的分离培养及向神经细胞定向分化的体内外实验研究

发布时间：2018-06-29 17:08

  本文选题：间充质干细胞 + 尿源性干细胞　； 参考：《南昌大学》2013年硕士论文

【摘要】：研究背景及研究目的： 中枢神经系统损伤的治疗一直是世界性难题，通过移植干细胞替代受损神经细胞继而重建神经功能的治疗方法越来越受到重视。研究表明移植神经干细胞（Neural Stem Cells，NSCs）治疗中枢神经系统损伤均具有一定疗效，但是NSCs来源有限、分离和培养均相当困难，移植NSCs又存在医学伦理限制、免疫排斥和存活率低等问题。而间充质干细胞（Mesenchymal Stem Cells，MSCs)来源广泛、分离培养技术成熟、无伦理限制和低免疫原性，但MSCs的获取手段多为有创的。 人尿源性干细胞(human urine-derived stem cells，hUSCs)是从尿液中分离出的多能干细胞，来源不受局限且安全无创，具有广阔的临床应用前景。现阶段国内外对其研究甚少，目前研究证实hUSCs具有MSCs的类似特性：表达类似的膜表面标志物及具有分化为成骨细胞、软骨细胞、平滑肌细胞和脂肪细胞的潜能。然而目前尚未见hUSCs向神经细胞定向分化的相关报道，本实验旨在体外分离培养hUSCs，观察其生物学特性，并于体外和体内鉴定其向神经细胞分化的能力，为将来利用干细胞移植治疗中枢神经系统损伤疾病寻找新的“种子细胞”来源。 方法： 1、建立体外分离培养hUSCs的技术并观察其生物学特性 1.1利用LMM101培养基贴壁筛选法分离培养hUSCs； 1.2MTT法观察细胞增殖能力； 1.3流式细胞术检测表面抗原CD29、CD90、CD44、CD45、CD34、CD73和CD133等的表达情况； 1.4采用成脂和成骨诱导培养基分别对hUSCs进行体外诱导，并采用油红O和茜素红染色进行鉴定； 2、体外诱导hUSCs向神经细胞定向分化 采用成神经诱导培养基对hUSCs进行神经定向诱导，实时定量荧光PCR（q-PCR）检测Nestin、Sox2、GFAP、β-tubulin III、NSE和MAP2mRNA的表达变化，细胞免疫荧光染色检测Nestin、Sox2的表达情况。 3、体内移植GFP-hUSCs，观察hUSCs在大鼠脑内增殖分化情况 利用水凝胶负载GFP-hUSCs移植到大鼠模型的脑损伤区，荧光显微镜下观察脑内GFP表达阳性细胞的分布情况，细胞免疫荧光染色检测Nestin、GFAP和β-tubulin III的表达情况。 结果： 1、成功建立了从人新鲜尿液中分离培养hUSCs的技术 hUSCs细胞于接种后3-7d开始贴壁，7-10d细胞呈集落生长，10-15d不同细胞克隆呈现4种不同形态：“铺路石”上皮细胞样、长梭形肌细胞样、扁圆形内皮细胞样和短梭形成纤维细胞样，经过2-3周的传代培养后P4代细胞形态呈较为均一的成纤维细胞样，细胞传至P10余代，仍然保持旺盛的增殖能力。 2、hUSCs的生物学特性 2.1MTT法检测细胞增殖能力的结果显示hUSCs生长曲线呈S形。 2.2流式细胞术检测结果显示分离得到的hUSCs CD44、CD29、CD90和CD73表达阳性，CD34、CD133和CD45表达阴性，表明其与间充质干细胞表达类似的细胞表面标志物。 2.3体外采用成脂和成骨诱导培养基分别对hUSCs诱导14d和21d后，油红O和茜素红染色均为阳性，表明hUSCs体外具有成脂和成骨的分化潜能。 3、体外诱导hUSCs向神经细胞定向分化 q-PCR结果显示hUSCs经神经定向诱导培养7d后Nestin、Sox2、MAP2、NSE、GFAP表达分别上调8.08±0.95倍、3.75±0.88倍、5.12±0.46倍、2.08±0.11倍和5.60±1.86倍，诱导12d后分别上调4.47±0.62倍、5.69±0.92倍、2.52±0.61倍、1.63±0.13倍和5.97±0.89倍(P＜0.05)；细胞免疫荧光染色检测结果显示经神经诱导7d后Nestin、Sox2阳性细胞率分别为17.2±0.58%和15.3±1.32%，，诱导12d时阳性细胞率分别为62.5±4.48%和33.7±2.76%（P＜0.05），结果表明hUSCs体外具有分化为神经细胞的潜能。 4、GFP-hUSCs脑内增殖分化状况 GFP-hUSCs于移植1W和3W后，发现宿主脑内存在GFP阳性细胞，同时部分GFP阳性细胞从脑损伤区迁移至海马区，并且表达GFAP和β-tubulin III，说明移植细胞能够于宿主脑内成功存活、迁移并分化为神经细胞。 结论：1、本研究成功建立了从人新鲜尿液中分离培养并大量扩增hUSCs的技术体系，且所分离培养的hUSCs具有间充质干细胞的类似特性；2、体外经神经定向诱导后hUSCs可分化为神经细胞；3、移植到脑损伤区，hUSCs能够存活、迁移，并向神经细胞分化；4、本研究成功寻找到了一种新的可供移植的“种子细胞”来源---hUSCS，为利用组织工程和再生医学技术治疗中枢神经系统疾病奠定了基础。
[Abstract]:Background and purpose of study :

The treatment of central nervous system injury has been a worldwide problem , and it has been paid more and more attention to the treatment of injured nerve cells by transplanting stem cells .

Human urine - derived stem cells ( hUSCs ) are pluripotent stem cells isolated from urine , and their sources are not limited and non - invasive and have broad clinical application prospects .

Method :

1 . Establish the technique of in vitro isolation and culture of hUSCs and observe their biological characteristics

1.1 separating and culturing hUSCs by using LMM101 culture medium adherent screening method ;


1 . MTT assay was used to observe the cell proliferation ability .


1.3 Flow cytometry was used to detect the expression of CD29 , CD90 , CD44 , cd45 , CD34 , CD73 and CD133 .


1.4 In vitro induction of hUSCs was carried out by using fat - forming and osteogenic induction culture medium , and oil - red O and alizarin red staining were used for identification ;


2 . Differentiation of hUSCs into neural cells in vitro

The expression of Nestin , Sox2 , GFAP , 尾 - erbB - III , NSE and MAP2mRNA was detected by real - time quantitative fluorescence PCR ( q - PCR ) . The expression of Nestin and Sox2 was detected by immunofluorescence staining .

3 . GFP - hUSCs were transplanted in vivo , and the proliferation and differentiation of hUSCs in rat brain were observed .

GFP - hUSCs were transplanted into the brain injury region of the rat model by using hydrogel - loaded GFP - hUSCs . The distribution of GFP - positive cells in the brain was observed under the fluorescence microscope , and the expression of Nestin , GFAP and 尾 - GFP III was detected by immunofluorescence staining .

Results :

1 . The technique of separating and culturing hUSCs from fresh human urine was successfully established .

After 3 - 7 days after inoculation , the cells of hUSCs began to adhere to the wall , 7 - 10d cells were colony - growing , and 10 - 15d different cell clones presented four different forms : " paving stones " epithelial cells , long shuttle - shaped muscle cells , flat circular endothelial cells and short shuttle fibroblasts .

2 . Biological characteristics of hUSCs

2 . The results of MTT assay showed that the growth curve of hUSCs was S - shaped .

2.2 The expression of CD44 , CD29 , CD90 , and CD73 in isolated hUSCs was positive , CD34 , CD133 and CD73 were negative , indicating similar cell surface markers as mesenchymal stem cells .

2.3 After 14 and 21 days of induction of hUSCs in vitro , the staining of oil red O and alizarin red were positive , indicating that hUSCs had the potential of adipogenesis and osteogenic differentiation in vitro .

3 . Differentiation of hUSCs into neural cells in vitro

The results showed that the expression of Nestin , Sox2 , MAP2 , NSE and GFAP were up - regulated by 8.08 卤 0.95 , 3.75 卤 0.88 , 5.12 卤 0.46 , 2.08 卤 0.11 and 5.60 卤 1.86times , respectively , and after 12 days , the expression of Nestin , Sox2 , MAP2 , NSE and GFAP increased 4.47 卤 0.62 , 5.69 卤 0.92 , 2.52 卤 0.61 , 1.63 卤 0.13 and 5.97 卤 0.89 , respectively ( P < 0.05 ) .
The results showed that the rates of Nestin and Sox2 positive cells were 17.2 卤 0.58 % and 15.3 卤 1.32 % , respectively , and the positive cell rates were 62.5 卤 4.48 % and 33.7 卤 2.76 % ( P < 0.05 ) . The results showed that hUSCs had the potential to differentiate into neural cells in vitro .

4 . Status of proliferation and differentiation of GFP - hUSCs

GFP - hUSCs were transplanted 1 W and 3 W , GFP - positive cells were found in the host brain , while some GFP - positive cells migrated from the brain injury area to the hippocampus , and GFAP and 尾 - carotene III were expressed , suggesting that the transplanted cells were able to survive , migrate and differentiate into neural cells in the host brain .

Conclusion : 1 . This study successfully established a technical system for the isolation and culture of hUSCs from human fresh urine , and the isolated cultured hUSCs have similar characteristics of mesenchymal stem cells .
2 . The hUSCs can be differentiated into neural cells after the induction of neural orientation in vitro .
3 . The hUSCs can survive , migrate and differentiate into neural cells .
4 . This study successfully finds a new source of transplanted " seed cells " - - hUSCS , which lays a foundation for the use of tissue engineering and regenerative medicine in the treatment of central nervous system diseases .
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R329.2

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