本文選題：間充質(zhì)干細胞 + 尿源性干細胞　； 參考：《南昌大學》2013年碩士論文

【摘要】：研究背景及研究目的： 中樞神經(jīng)系統(tǒng)損傷的治療一直是世界性難題，通過移植干細胞替代受損神經(jīng)細胞繼而重建神經(jīng)功能的治療方法越來越受到重視。研究表明移植神經(jīng)干細胞（Neural Stem Cells，NSCs）治療中樞神經(jīng)系統(tǒng)損傷均具有一定療效，但是NSCs來源有限、分離和培養(yǎng)均相當困難，移植NSCs又存在醫(yī)學倫理限制、免疫排斥和存活率低等問題。而間充質(zhì)干細胞（Mesenchymal Stem Cells，MSCs)來源廣泛、分離培養(yǎng)技術成熟、無倫理限制和低免疫原性，但MSCs的獲取手段多為有創(chuàng)的。 人尿源性干細胞(human urine-derived stem cells，hUSCs)是從尿液中分離出的多能干細胞，來源不受局限且安全無創(chuàng)，具有廣闊的臨床應用前景。現(xiàn)階段國內(nèi)外對其研究甚少，目前研究證實hUSCs具有MSCs的類似特性：表達類似的膜表面標志物及具有分化為成骨細胞、軟骨細胞、平滑肌細胞和脂肪細胞的潛能。然而目前尚未見hUSCs向神經(jīng)細胞定向分化的相關報道，本實驗旨在體外分離培養(yǎng)hUSCs，觀察其生物學特性，并于體外和體內(nèi)鑒定其向神經(jīng)細胞分化的能力，為將來利用干細胞移植治療中樞神經(jīng)系統(tǒng)損傷疾病尋找新的“種子細胞”來源。 方法： 1、建立體外分離培養(yǎng)hUSCs的技術并觀察其生物學特性 1.1利用LMM101培養(yǎng)基貼壁篩選法分離培養(yǎng)hUSCs； 1.2MTT法觀察細胞增殖能力； 1.3流式細胞術檢測表面抗原CD29、CD90、CD44、CD45、CD34、CD73和CD133等的表達情況； 1.4采用成脂和成骨誘導培養(yǎng)基分別對hUSCs進行體外誘導，并采用油紅O和茜素紅染色進行鑒定； 2、體外誘導hUSCs向神經(jīng)細胞定向分化 采用成神經(jīng)誘導培養(yǎng)基對hUSCs進行神經(jīng)定向誘導，實時定量熒光PCR（q-PCR）檢測Nestin、Sox2、GFAP、β-tubulin III、NSE和MAP2mRNA的表達變化，細胞免疫熒光染色檢測Nestin、Sox2的表達情況。 3、體內(nèi)移植GFP-hUSCs，觀察hUSCs在大鼠腦內(nèi)增殖分化情況 利用水凝膠負載GFP-hUSCs移植到大鼠模型的腦損傷區(qū)，熒光顯微鏡下觀察腦內(nèi)GFP表達陽性細胞的分布情況，細胞免疫熒光染色檢測Nestin、GFAP和β-tubulin III的表達情況。 結(jié)果： 1、成功建立了從人新鮮尿液中分離培養(yǎng)hUSCs的技術 hUSCs細胞于接種后3-7d開始貼壁，7-10d細胞呈集落生長，10-15d不同細胞克隆呈現(xiàn)4種不同形態(tài)：“鋪路石”上皮細胞樣、長梭形肌細胞樣、扁圓形內(nèi)皮細胞樣和短梭形成纖維細胞樣，經(jīng)過2-3周的傳代培養(yǎng)后P4代細胞形態(tài)呈較為均一的成纖維細胞樣，細胞傳至P10余代，仍然保持旺盛的增殖能力。 2、hUSCs的生物學特性 2.1MTT法檢測細胞增殖能力的結(jié)果顯示hUSCs生長曲線呈S形。 2.2流式細胞術檢測結(jié)果顯示分離得到的hUSCs CD44、CD29、CD90和CD73表達陽性，CD34、CD133和CD45表達陰性，表明其與間充質(zhì)干細胞表達類似的細胞表面標志物。 2.3體外采用成脂和成骨誘導培養(yǎng)基分別對hUSCs誘導14d和21d后，油紅O和茜素紅染色均為陽性，表明hUSCs體外具有成脂和成骨的分化潛能。 3、體外誘導hUSCs向神經(jīng)細胞定向分化 q-PCR結(jié)果顯示hUSCs經(jīng)神經(jīng)定向誘導培養(yǎng)7d后Nestin、Sox2、MAP2、NSE、GFAP表達分別上調(diào)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，誘導12d后分別上調(diào)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)；細胞免疫熒光染色檢測結(jié)果顯示經(jīng)神經(jīng)誘導7d后Nestin、Sox2陽性細胞率分別為17.2±0.58%和15.3±1.32%，，誘導12d時陽性細胞率分別為62.5±4.48%和33.7±2.76%（P＜0.05），結(jié)果表明hUSCs體外具有分化為神經(jīng)細胞的潛能。 4、GFP-hUSCs腦內(nèi)增殖分化狀況 GFP-hUSCs于移植1W和3W后，發(fā)現(xiàn)宿主腦內(nèi)存在GFP陽性細胞，同時部分GFP陽性細胞從腦損傷區(qū)遷移至海馬區(qū)，并且表達GFAP和β-tubulin III，說明移植細胞能夠于宿主腦內(nèi)成功存活、遷移并分化為神經(jīng)細胞。 結(jié)論：1、本研究成功建立了從人新鮮尿液中分離培養(yǎng)并大量擴增hUSCs的技術體系，且所分離培養(yǎng)的hUSCs具有間充質(zhì)干細胞的類似特性；2、體外經(jīng)神經(jīng)定向誘導后hUSCs可分化為神經(jīng)細胞；3、移植到腦損傷區(qū)，hUSCs能夠存活、遷移，并向神經(jīng)細胞分化；4、本研究成功尋找到了一種新的可供移植的“種子細胞”來源---hUSCS，為利用組織工程和再生醫(yī)學技術治療中樞神經(jīng)系統(tǒng)疾病奠定了基礎。
[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 鹵 0.95 , 3.75 鹵 0.88 , 5.12 鹵 0.46 , 2.08 鹵 0.11 and 5.60 鹵 1.86times , respectively , and after 12 days , the expression of Nestin , Sox2 , MAP2 , NSE and GFAP increased 4.47 鹵 0.62 , 5.69 鹵 0.92 , 2.52 鹵 0.61 , 1.63 鹵 0.13 and 5.97 鹵 0.89 , respectively ( P < 0.05 ) .
The results showed that the rates of Nestin and Sox2 positive cells were 17.2 鹵 0.58 % and 15.3 鹵 1.32 % , respectively , and the positive cell rates were 62.5 鹵 4.48 % and 33.7 鹵 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 .
【學位授予單位】：南昌大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R329.2

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