本文選題：大鼠　切入點：骨髓間充質(zhì)干細胞　出處：《華中科技大學》2013年博士論文　論文類型：學位論文

【摘要】：目的：建立體外培養(yǎng)擴增、檢測鑒定SD大鼠骨髓間充質(zhì)干細胞(BMSCs)的方法，觀察其生物學特性； 方法：采取全骨髓培養(yǎng)法分離和純化大鼠BMSCs，用顯微鏡觀察細胞形體，用流式細胞儀檢測細胞純度，觀察BMSCs在誘導條件下向成骨細胞分化的能力； 結(jié)果：全骨髓培養(yǎng)法培養(yǎng)的BMSCs具有良好的貼壁性，流式細胞儀檢測：CD44(96.49%)、CD90(95.63%)、CD45(0.64%)、CD34(0.39%)；第3代細胞經(jīng)誘導劑誘導可向成骨細胞分化； 結(jié)論：本實驗用全貼壁篩選法培養(yǎng)出高純度的BMSCs，增殖穩(wěn)定，在條件誘導下具有多分化的潛能。 目的：利用GDNF和NT-3雙基因轉(zhuǎn)染誘導大鼠骨髓間充質(zhì)干細胞(BMSCs)分化為神經(jīng)樣細胞，為其治療神經(jīng)性疾病提供實驗基礎； 方法：全骨髓法分離培養(yǎng)BMSCs，流式細胞術及成骨誘導預實驗檢測BMSCs純度及特性。轉(zhuǎn)染GDNF和NT-3雙基因后，在顯微鏡下觀察細胞形態(tài)變化；利用RT-PCR和免疫熒光檢測神經(jīng)細胞特異性標志物表達； 結(jié)果：BMSCs能在體外成功分離培養(yǎng)，誘導分化后，BMSCs胞體變圓，伸出明顯突起，并可見多數(shù)細胞相互交織成網(wǎng)狀結(jié)構，呈神經(jīng)細胞樣形態(tài)。RT-PCR檢測GDNF、NT-3、NSE、nestin、MAP-2基因表達，免疫熒光標記檢測可見表達MAP-2和GFAP； 結(jié)論：GDNF和NT-3雙基因修飾誘導的BMSCs可分化為神經(jīng)樣細胞，并表達神經(jīng)元的標志，為基因治療神經(jīng)系統(tǒng)疾病如先天性巨結(jié)腸提供實驗基礎。 目的：本研究利用膜片鉗技術，比較MSCs及誘導后神經(jīng)樣細胞之間通道電流的情況，進一步確認分化后神經(jīng)樣細胞的電生理功能，將為腸神經(jīng)系統(tǒng)缺如疾病的治療提供前期工作基礎。 方法：應用膜片鉗技術，采用全細胞記錄方式，對由GDNF和NT-3雙基因誘導的分化前BMSCs和分化后的神經(jīng)樣細胞進行電生理功能檢測； 結(jié)果：分化前記錄到延遲整流樣鉀電流(IKDR)在+60mV時電流大小為583.6536±74.75945pA，電流密度為10.25393±1.313413pA/pF；鈣激活鉀通道電流（IKCa）在+60mV時記錄到電流峰值為370.775±49.57507pA，電流密度為6.513967±0.87096pA/pF；瞬時外向鉀通道電流（Ito）在+60mV時電流峰值為467.03±68.44461pA，電流密度為8.205025±1.20247pA/pF；分化后IKDR在+60mV時電流大小為850.32±53.5708pA，電流密度為18.72207±1.578505pA/pF； IKCa在+60mV時電流峰值為452.6455±13.4805pA，電流密度為8.058586±0.943178pA/pF； Ito電流峰值為621.194±66.039pA，電流密度為15.00152±1.918339pA/pF；分化前BMSCs的IKDR和分化后神經(jīng)樣細胞的IKDR在+60mV時電流峰值相比較具有明顯的統(tǒng)計學差異（t=2.721，P值=0.015）；分化前IKDRBMSCs電流密度和分化后神經(jīng)樣細胞IKDR電流密度相比較亦具有明顯的統(tǒng)計學差異（t=2.441，P值=0.030）； 結(jié)論：大鼠骨髓BMSCs誘導分化的神經(jīng)元樣細胞初步具有神經(jīng)元的電生理特性，，是BMSCs由神經(jīng)前體細胞向成熟神經(jīng)元這一終點轉(zhuǎn)化過程中由未成熟逐漸邁向成熟的過程。
[Abstract]:Objective: to establish a method for detection and identification of bone marrow mesenchymal stem cells (BMSCs) in SD rats in vitro, and to observe the biological characteristics of the bone marrow mesenchymal stem cells (BMSCs).
Methods: whole bone marrow culture was used to isolate and purify rat BMSCs. Cell morphology was observed by microscope. The purity of cells was detected by flow cytometry, and the ability of BMSCs to differentiate into osteoblasts under induction conditions was observed.
Results: BMSCs cultured in whole bone marrow culture showed good adherence. Flow cytometry showed that CD44 (96.49%), CD90 (95.63%), CD45 (0.64%), CD34 (0.39%), and third generation cells could differentiate into osteoblasts through inducers.
Conclusion: in this experiment, the highly purified BMSCs was cultured with full wall screening method, and the proliferation was stable, and it had the potential of multi differentiation under the condition of condition induced.
Objective: to induce rat bone marrow mesenchymal stem cells (BMSCs) to differentiate into neuron like cells by double gene transfection of GDNF and NT-3, and provide experimental basis for their treatment of neuropathic diseases.
Methods: BMSCs was isolated and cultured in whole bone marrow, and the purity and characteristics of BMSCs were detected by flow cytometry and osteogenic induction. After transfection of GDNF and NT-3 double genes, morphological changes of cells were observed under microscope. RT-PCR and immunofluorescence were used to detect the expression of specific markers of nerve cells.
Results: BMSCs can be successfully isolated and cultured in vitro after induced differentiation, BMSCs cells became round, protruding obvious protrusions, and most visible cells interwoven into the mesh structure, a neural cell like morphology of.RT-PCR NT-3, NSE, detection of GDNF, nestin, MAP-2 gene expression, immunofluorescence detection showed the expression of MAP-2 and GFAP;
Conclusion: GDNF and NT-3 double gene modified BMSCs can differentiate into neuron like cells and express neuronal markers, providing experimental basis for gene therapy of nervous system diseases such as Hirschsprung's disease.
Objective: in this study, patch clamp technique was used to compare the channel currents between MSCs and induced neuron like cells. Further confirmation of the electrophysiological function of neural cells after differentiation will provide a preliminary basis for the treatment of diseases of the intestinal nervous system.
Methods: patch clamp technique and whole cell recording were used to detect the electrophysiological function of pre differentiated BMSCs and differentiated neuron like cells induced by GDNF and NT-3 double genes.
Results: before differentiation recorded delayed rectifier-like potassium current (IKDR) in +60mV when the current size is 583.6536 + 74.75945pA, the current density was 10.25393 + 1.313413pA/pF; calcium activated potassium current (IKCa) in the +60mV record to the current peak was 370.775 + 49.57507pA, the current density was 6.513967 + 0.87096pA/pF; transient outward potassium current (Ito) at +60mV peak current was 467.03 + 68.44461pA, the current density was 8.205025 + 1.20247pA/pF; differentiation after size IKDR in +60mV current was 850.32 + 53.5708pA, the current density was 18.72207 + 1.578505pA/pF; IKCa current at +60mV peak current density was 452.6455 + 13.4805pA, 8.058586 + 0.943178pA/pF; the peak current of Ito was 621.194 + 66.039pA and the current density was 15.00152 + 1.918339pA/pF; BMSCs IKDR before differentiation and differentiation of neuron like cells of the IKDR current in the +60mV when compared with the peak Significant statistical difference (t=2.721, P value =0.015). The IKDRBMSCs current density before differentiation and the IKDR current density after differentiation were also significantly different (t=2.441, P value =0.030).
Conclusion: the neuron like cells derived from rat bone marrow BMSCs have the electrophysiological characteristics of neurons. It is the process from immature BMSCs to mature neurons in the process of transformation from neural precursor cells to mature neurons.

【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2013
【分類號】：R725.7

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