【摘要】： 第一部分:人胎兒骨髓間充質(zhì)干細(xì)胞分離純化及基本生物學(xué)特性研究 目的觀察人胎兒骨髓間充質(zhì)干細(xì)胞(FMSCs)體外分離、培養(yǎng)方法,并對(duì)其生物學(xué)特性進(jìn)行初步分析。 方法取4個(gè)月胎齡的流產(chǎn)胎兒,無(wú)菌條件下分離胎兒四肢骨,PBS沖洗骨髓腔;采用Ficoll法分離胎兒骨髓腔沖洗液,離心后取單個(gè)核細(xì)胞層培養(yǎng)、擴(kuò)增、傳代,觀察細(xì)胞生物學(xué)特性;采用MTT法觀察細(xì)胞生長(zhǎng)曲線及分裂指數(shù)曲線。取第3代細(xì)胞,在含新生牛血清、地塞米松、β-甘油磷酸鈉、抗壞血酸的DMEM-LG培養(yǎng)基中誘導(dǎo)其成骨分化;在含新生牛血清、地塞米松、胰島素的DMEM-LG培養(yǎng)基中誘導(dǎo)其成脂分化。 結(jié)果接種48h后,貼壁細(xì)胞呈梭形、多角形、成纖維細(xì)胞樣形態(tài)。10天后開始傳代,傳代培養(yǎng)的潛伏期為24-36h,對(duì)數(shù)增殖期約為2-3天,第6天進(jìn)入平臺(tái)期。傳代至15代后生長(zhǎng)速度變慢;FCM檢測(cè)顯示,細(xì)胞高表達(dá)CD29、CD44及CD49e分子,低表達(dá)CD106、CD54、CD11b,而不表達(dá)CD34。成骨、成脂培養(yǎng)基誘導(dǎo)3周后,Von Kossa染色呈黑色,油紅染色成橘紅色。 結(jié)論采用Ficoll密度梯度離心、貼壁篩選法及單克隆培養(yǎng)法可以分離、培養(yǎng)、擴(kuò)增出具有高度同源性的胎兒骨髓間充質(zhì)干細(xì)胞;并在體外成功的誘導(dǎo)其向成骨細(xì)胞、脂肪細(xì)胞分化。 第二部分:低劑量輻射對(duì)骨髓間充質(zhì)干細(xì)胞體內(nèi)分布和歸巢特性的影響 目的觀察骨髓間充質(zhì)干細(xì)胞治療脊髓損傷動(dòng)物模型及經(jīng)X線照射后的分布和歸巢特性。 方法取部分昆明小鼠做骨髓間充質(zhì)干細(xì)胞(BMMSCs)分離培養(yǎng);部分按照改良的Allen's打擊法建立脊髓損傷模型,并隨機(jī)分成照射組和對(duì)照組;CFDA SE標(biāo)記后的原代BMMSCs于照射組照射后30min時(shí),給所有脊髓損傷模型的損傷局部進(jìn)行細(xì)胞移植。分別于細(xì)胞移植后24h和96h在熒光顯微鏡下觀察BMMSCs移植后在受者體內(nèi)的分布及歸巢特性。 結(jié)果細(xì)胞移植24h后,所有脊髓損傷模型的損傷部位均可見(jiàn)被植入的BMMSCs,但照射組明顯多于對(duì)照組;96h后,照射組脊髓損傷部為仍可見(jiàn)被植入的BMMSCs,但其他組織未見(jiàn)該細(xì)胞;對(duì)照組沒(méi)有檢測(cè)到供體BMMSCs。 結(jié)論在脊髓損傷模型中,被移植后進(jìn)入循環(huán)的間充質(zhì)干細(xì)胞可以靶向分布到各損傷部位;經(jīng)X線照射后,BMMSCs可以通過(guò)增殖能力的增強(qiáng)來(lái)維持機(jī)體內(nèi)環(huán)境的穩(wěn)定。
[Abstract]:Part I: isolation and purification of human fetal bone marrow mesenchymal stem cells and their basic biological characteristics objective to observe the methods of isolation and culture of human fetal bone marrow mesenchymal stem cells (FMSCs) in vitro. The biological characteristics were analyzed preliminarily. Methods Fetal limb bone was isolated from aborted fetus at 4 months of gestational age, and PBS was used to wash the medullary cavity of fetus, and Ficoll method was used to separate the lavage fluid of fetal medullary cavity. The mononuclear cell layer was cultured, amplified and subcultured after centrifugation, and the cell biological characteristics were observed. Cell growth curve and mitotic index curve were observed by MTT method. The osteogenic differentiation was induced in the DMEM-LG medium containing newborn bovine serum, dexamethasone, sodium 尾 -glycerophosphate and ascorbic acid, and adipogenic differentiation was induced in DMEM-LG medium containing newborn bovine serum, dexamethasone and insulin. Results 48 h after inoculation, the adherent cells were fusiform, polygonal and fibroblast-like. 10 days later, the incubation period was 24-36 hours, the logarithmic proliferation period was 2-3 days, and the sixth day entered the plateau phase. After passage to 15 generations, the growth rate became slower. FCM detection showed that the cells expressed CD29,CD44 and CD49e molecules, but not CD34. but low expression of CD106,CD54,CD11b,. After 3 weeks of induction, Von Kossa staining was black and oil red was orange. Conclusion the fetal bone marrow mesenchymal stem cells with high homology can be isolated and cultured by Ficoll density gradient centrifugation, adherent screening and monoclonal culture, and can be successfully induced to differentiate into osteoblasts and adipocytes in vitro. Part two: effects of low dose radiation on the distribution and homing characteristics of bone marrow mesenchymal stem cells objective to observe the distribution and homing characteristics of bone marrow mesenchymal stem cells in the treatment of spinal cord injury. Methods (BMMSCs) of bone marrow mesenchymal stem cells was isolated from some Kunming mice, and the spinal cord injury model was established by modified Allen's attack method. The mice were randomly divided into irradiation group and control group, the primary BMMSCs labeled by; CFDA SE were treated with 30min after irradiation. All spinal cord injury models were transplanted with cells. The distribution and homing characteristics of BMMSCs were observed under fluorescence microscope at 24 h and 96 h after transplantation, respectively. Results 24 hours after transplantation, implanted BMMSCs, could be seen in all the injured sites of spinal cord injury model, but it was more in irradiation group than in control group, 96 hours later, the injured part of spinal cord in irradiation group was still implanted BMMSCs, but no cells were found in other tissues. No donor BMMSCs. was detected in the control group Conclusion in the model of spinal cord injury, mesenchymal stem cells entering the circulation after transplantation can be targeted to various injury sites, and BMMSCs can maintain the stability of the internal environment through the enhancement of proliferative ability after X-ray irradiation.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R329

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