本文選題：骨髓間充質(zhì)干細(xì)胞 + 表型　； 參考：《南京醫(yī)科大學(xué)》2010年博士論文

【摘要】：目的:分離、培養(yǎng)和鑒定骨髓間充質(zhì)干細(xì)胞(mesenchymal stem cells,MSCs),研究其特征和向心肌細(xì)胞分化的能力。 方法:嚴(yán)格無(wú)菌條件下從C57BL/10J小鼠的股骨獲取全骨髓細(xì)胞,進(jìn)行體外培養(yǎng),對(duì)貼壁細(xì)胞進(jìn)行傳代。觀察貼壁細(xì)胞的生長(zhǎng)狀況和形態(tài)特征,并應(yīng)用流式細(xì)胞儀對(duì)培養(yǎng)的第3-4代貼壁生長(zhǎng)細(xì)胞的表面抗原(CD)進(jìn)行表型分析,以鑒定是否為MSCs;并誘導(dǎo)MSCs向脂肪細(xì)胞和成骨細(xì)胞方向分化,檢驗(yàn)其多向分化能力。以10μmol/L 5-氮胞苷(5-Azacytidine , 5-Aza)與MSCs共孵育24小時(shí),繼續(xù)培養(yǎng)4周,采用免疫細(xì)胞化學(xué)法檢測(cè)MSCs經(jīng)5-氮胞苷誘導(dǎo)后心肌特異性蛋白(心肌肌鈣蛋白I)的表達(dá),研究體外培養(yǎng)的MSCs向心肌細(xì)胞分化的能力。 結(jié)果:從實(shí)驗(yàn)小鼠骨髓獲取的貼壁生長(zhǎng)的成纖維樣細(xì)胞可多次傳代,并保持其快速增殖特點(diǎn)。流式細(xì)胞術(shù)檢測(cè)顯示,培養(yǎng)的第3代貼壁生長(zhǎng)的成纖維樣細(xì)胞呈CD14(-),CD34(-),CD45(-),而CD29(+),CD44(+),CD105(+),符合骨髓間充質(zhì)干細(xì)胞特點(diǎn)。在體外,骨髓間充質(zhì)干細(xì)胞能向脂肪細(xì)胞、成骨細(xì)胞方向分化。以5-氮胞苷(10μmol/L)孵育24小時(shí)后再繼續(xù)培養(yǎng)4周,可分化為表達(dá)心肌特異性蛋白(心肌肌鈣蛋白I)的細(xì)胞。 結(jié)論:可通過(guò)對(duì)貼壁生長(zhǎng)細(xì)胞反復(fù)傳代培養(yǎng)的方法從全骨髓細(xì)胞中分離、純化骨髓間充質(zhì)干細(xì)胞。骨髓間充質(zhì)干細(xì)胞具有貼壁生長(zhǎng)、快速增殖的特性,在體外可多次傳代而保持其特性。體外培養(yǎng)的骨髓間充質(zhì)干細(xì)胞可被誘導(dǎo)向脂肪細(xì)胞和成骨細(xì)胞方向分化,并可被5-氮胞苷誘導(dǎo)分化為心肌樣細(xì)胞,提示骨髓間充質(zhì)干細(xì)胞有分化為心肌細(xì)胞的潛能,可作為急性心肌梗死后細(xì)胞移植治療的細(xì)胞資源。 目的:在細(xì)胞水平研究脂多糖(Lipoplysaccharide, LPS)對(duì)MSCs增殖以及對(duì)血管內(nèi)皮細(xì)胞生長(zhǎng)因子(vascular endothelial growth factor,VEGF)釋放的影響,并探討Toll樣受體4/核因子-κB ( toll-like receptor 4/nuclear factor—κB, TLR4/NF-κB)信號(hào)通路對(duì)MSCs增殖和分泌VEGF的作用。 方法:①取第3代野生型小鼠骨髓間充質(zhì)干細(xì)胞(wild-type MSCs, wMSCs)和TLR4基因缺陷型小鼠骨髓間充質(zhì)干細(xì)胞(TLR4 gene deleted MSCs, tMSCs)與不同濃度的LPS(0μg/ml,0.01μg/ml,0.1μg/ml,1.0μg/ml,10μg/ml)進(jìn)行共培養(yǎng),48小時(shí)后,用MTT法檢測(cè)MSCs的增殖。②取第3代wMSCs,與不同濃度的LPS(0μg/ml,0.01μg/ml,0.1μg/ml,1.0μg/ml,10μg/ml)進(jìn)行共培養(yǎng),48小時(shí)后,用ELISA法檢測(cè)細(xì)胞培養(yǎng)液上清中VEGF濃度。③將培養(yǎng)的細(xì)胞分為4組:1. wMSCs組;2. wMSCs+1.0μg/ml LPS組;3. wMSCs+ 1.0μg/ml LPS+四氫吡咯二硫代氨基甲酸酯(pyrrolidine dithiocarbamate,PDTC)組;4. tMSCs+ 1.0μg/ml LPS組。用ELISA法檢測(cè)各組細(xì)胞培養(yǎng)液上清中VEGF濃度,RT-PCR法檢測(cè)各組細(xì)胞中VEGF的mRNA含量。 結(jié)果:①不同濃度的LPS均能夠促進(jìn)wMSCs增殖,其中1.0μg/ml的LPS作用最強(qiáng),LPS不能促進(jìn)tMSCs增殖。②不同濃度的LPS均能夠促進(jìn)wMSCs旁分泌VEGF,其中1.0μg/ml的LPS作用最強(qiáng)。③用TLR4基因缺陷的MSCs或用NF-κB抑制劑后,VEGF的mRNA和蛋白表達(dá)量均減少。 結(jié)論:1.0μg/ml的LPS能夠最大限度的促進(jìn)MSCs增殖,促進(jìn)MSCs旁分泌VEGF。用TLR4基因缺陷的MSCs或用NF-κB抑制劑后,VEGF分泌量減少。說(shuō)明LPS是通過(guò)TLR4/NF-κB信號(hào)通路促進(jìn)MSCs旁分泌VEGF的。 目的:研究C57BL/10J雄性小鼠的MSCs經(jīng)LPS預(yù)處理后,能否改善心肌梗死大鼠的心功能,并探討其中可能機(jī)制。 方法:用結(jié)扎冠狀動(dòng)脈前降支的方法制作大鼠的急性心肌梗死模型,80只Wistar雌性大鼠隨機(jī)分為4組,各組大鼠分別在心肌內(nèi)注射下列物質(zhì):30μlPBS(對(duì)照組),3×106個(gè)野生型MSCs/30μl (wMSCs移植組),3×106個(gè)1.0μg/ml LPS預(yù)處理的野生型MSCs/30μl (LPS-wMSCs移植組), 3×106個(gè)1.0μg/ml LPS預(yù)處理的TLR4基因缺陷型MSCs/30μl (LPS-tMSCs移植組)。3周后,用心臟超聲心動(dòng)圖測(cè)定檢測(cè)大鼠的心功能,TTC法檢測(cè)心肌梗死面積,Masson′s染色法檢測(cè)心肌纖維化程度,用real-time PCR法檢測(cè)移植細(xì)胞存活率,免疫組織化學(xué)法檢測(cè)新生血管密度和心肌細(xì)胞凋亡率,Western blot法分析VEGF和磷酸化Akt表達(dá)。 結(jié)果:細(xì)胞移植3周后,在4組中,經(jīng)1.0μg/ml LPS預(yù)處理的野生型MSCs移植組(LPS-wMSCs移植組)較其余各組LVDd、LVDs均顯著減少(P0.01),而LVEF和FS則顯著增加(P0.01)。與其余3組相比,LPS-wMSCs移植組心肌纖維化顯著減少(6.6±0.5%,P0.05),心肌細(xì)胞凋亡顯著減少(14.8±1.5%,P0.01),新生血管密度顯著增多(45.3±4.3,P0.01)。移植細(xì)胞的存活率在LPS-wMSCs移植組也顯著增加(為wMSCs組的1.89±0.10倍,P0.01)。心肌組織內(nèi)VEGF和磷酸化Akt表達(dá)在LPS-w MSCs移植組也增加。 結(jié)論:LPS預(yù)處理能夠增加移植MSCs的存活率,促進(jìn)VEGF的表達(dá),激活PI3K/Akt信號(hào)通路。MSCs在移植之前經(jīng)過(guò)LPS預(yù)處理后能夠更好的改善心臟功能,增加新生血管密度。LPS的預(yù)處理能夠作為增強(qiáng)MSCs生物學(xué)功能的一種新的手段。
[Abstract]:Objective: to isolate, culture and identify mesenchymal stem cells (MSCs) and study its characteristics and ability to differentiate into cardiomyocytes.
Methods: under strict aseptic conditions, all bone marrow cells were obtained from the femur of C57BL/10J mice and cultured in vitro. The adherent cells were subcultured in vitro. The growth and morphological characteristics of adherent cells were observed. The phenotype of the surface antigen of the cultured 3-4 generation adherent growth cells (CD) was analyzed by flow cytometry in order to identify whether it was MSCs. The differentiation of MSCs to adipocytes and osteoblasts was induced and its multidirectional differentiation was tested. 10 mol/L 5- azytidine (5-Azacytidine, 5-Aza) was incubated with MSCs for 24 hours and continued to be cultured for 4 weeks. The expression of cardiac specific protein (cardiac troponin I) induced by MSCs after 5- azocytosine was detected by immunocytochemical method, and the expression of cardiac troponin I was studied in vitro. The ability of the cultured MSCs to differentiate into cardiomyocytes.
Results: fibroid cells derived from the bone marrow of experimental mice can be passaged for many times and maintain their rapid proliferation characteristics. Flow cytometry showed that the third generation of cultured fibroid cells were CD14 (-), CD34 (-), CD45 (-), and CD29 (+), CD44 (+), CD105 (+), in line with the characteristics of bone marrow mesenchymal stem cells. In vitro, bone Medullary mesenchymal stem cells can differentiate into adipocytes and osteoblasts. After incubating 5- azytidine (10 mu mol/L) for 24 hours, the cells can continue to be cultured for 4 weeks and differentiate into cells expressing cardiac specific protein (cardiac troponin I).
Conclusion: bone marrow mesenchymal stem cells can be isolated and purified from all bone marrow cells by the method of repeated subculture of adherent growth cells. Bone marrow mesenchymal stem cells have the characteristics of adherent growth and rapid proliferation. The bone marrow mesenchymal stem cells can be induced to adipocytes in vitro. It can differentiate into osteoblasts and can be induced by 5- azacytidine to differentiate into myocardial like cells, suggesting that bone marrow mesenchymal stem cells have the potential to differentiate into cardiomyocytes. It can be used as a cell resource for the treatment of cell transplantation after acute myocardial infarction.
Objective: To study the effect of Lipoplysaccharide (LPS) on the proliferation of MSCs and the release of vascular endothelial growth factor (VEGF), and to explore the proliferation and secretion of the Toll like receptor 4/ nuclear factor kappa B pathway. The role of VEGF.
Methods: (1) the third generation wild type mouse bone marrow mesenchymal stem cells (wild-type MSCs, wMSCs) and TLR4 gene deficient mice bone marrow mesenchymal stem cells (TLR4 gene deleted MSCs, tMSCs) were co cultured with different concentrations of LPS (0 mu g/ml, 0.01 micron g/ml, 0.1 micron, 1 micron, 10 micron). After 48 hours, the proliferation was detected by the method. The third generation wMSCs was co cultured with different concentrations of LPS (0 mu g/ml, 0.01 mu g/ml, 0.1 mu g/ml, 1 mu g/ml, 10 mu g/ml). After 48 hours, the VEGF concentration in the supernatant of cell culture liquid was detected by ELISA method. (3) the cultured cells were divided into 4 groups, 1. wMSCs group, 2. wMSCs+1.0 micron g/ml group, 3. 1 mu four hydrogen pyrrole two thiocarbamate. Lidine dithiocarbamate, PDTC group; 4. tMSCs+ 1 mu g/ml LPS group. ELISA method was used to detect the concentration of VEGF in the supernatant of cell culture liquid, and RT-PCR method was used to detect mRNA content of VEGF in each cell.
Results: (1) the LPS of different concentrations could promote the proliferation of wMSCs, of which the LPS effect of 1 mu g/ml was the strongest, and LPS could not promote the proliferation of tMSCs. (2) the LPS in different concentrations could promote the secretion of VEGF beside wMSCs, and the LPS action of 1 micron g/ml was the strongest.
Conclusion: 1 g/ml LPS can promote the proliferation of MSCs to the maximum extent, promote the secretion of TLR4 gene defective MSCs by MSCs or NF- kappa B inhibitor, and decrease the secretion of VEGF, indicating that LPS is through the TLR4/NF- kappa signaling pathway to promote secretion.
Objective: To investigate whether MSCs pretreatment in C57BL/10J male mice can improve cardiac function after myocardial infarction in rats and explore the possible mechanism of LPS.
Methods: the rat model of acute myocardial infarction was made by ligating the anterior descending coronary artery. 80 Wistar female rats were randomly divided into 4 groups. The rats were injected with the following substances in the myocardium: 30 mu lPBS (control group), 3 x 106 wild type MSCs/30 Mu L (wMSCs transplantation group), 3 x 106 1 micron g/ml LPS pretreated wild MSCs/30 u l (LPS). -wMSCs transplantation group), after 3 x 106 1 mu g/ml LPS pretreated TLR4 gene defective MSCs/30 Mu L (LPS-tMSCs transplantation group), cardiac function was detected by cardiac echocardiography, myocardial infarction area was detected by TTC method, Masson 's staining method was used to detect myocardial fibrosis, and the survival rate of transplanted cells was detected by real-time assay, and immunization was used to detect the survival rate of the transplanted cells. The neovascularization density and myocardial cell apoptosis rate were detected by histochemical method. The expression of VEGF and phosphorylated Akt was analyzed by Western blot.
Results: after 3 weeks of cell transplantation, in the 4 groups, 1 mu g/ml LPS pretreated wild type MSCs transplantation group (LPS-wMSCs transplantation group) significantly decreased LVDd and LVDs (P0.01), while LVEF and FS increased significantly (P0.01). Compared with the other 3 groups, the cardiac fibrosis in the LPS-wMSCs transplantation group was significantly decreased (6.6 + 0.5%, P0.05), and the apoptosis of cardiac myocytes was significant The decrease (14.8 + 1.5%, P0.01) increased significantly (45.3 + 4.3, P0.01). The survival rate of transplanted cells was also significantly increased in the LPS-wMSCs transplantation group (1.89 + 0.10 times, P0.01). The expression of VEGF and phosphorylated Akt in the myocardium was also increased in the LPS-w MSCs transplantation group.
Conclusion: LPS preconditioning can increase the survival rate of the transplanted MSCs, promote the expression of VEGF, and activate the PI3K/Akt signal pathway.MSCs to improve the cardiac function better before the transplantation of LPS, and the preconditioning of the new vascular density.LPS can be used as a new means to enhance the biological function of MSCs.

【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R329

【共引文獻(xiàn)】

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