脂多糖预处理对骨髓间充质干细胞移植效率的影响及机制探讨

发布时间：2018-05-16 08:38

本文选题：骨髓间充质干细胞 + 表型　；参考：《南京医科大学》2010年博士论文

【摘要】：目的:分离、培养和鉴定骨髓间充质干细胞(mesenchymal stem cells,MSCs),研究其特征和向心肌细胞分化的能力。方法:严格无菌条件下从C57BL/10J小鼠的股骨获取全骨髓细胞,进行体外培养,对贴壁细胞进行传代。观察贴壁细胞的生长状况和形态特征,并应用流式细胞仪对培养的第3-4代贴壁生长细胞的表面抗原(CD)进行表型分析,以鉴定是否为MSCs;并诱导MSCs向脂肪细胞和成骨细胞方向分化,检验其多向分化能力。以10μmol/L 5-氮胞苷(5-Azacytidine , 5-Aza)与MSCs共孵育24小时,继续培养4周,采用免疫细胞化学法检测MSCs经5-氮胞苷诱导后心肌特异性蛋白(心肌肌钙蛋白I)的表达,研究体外培养的MSCs向心肌细胞分化的能力。结果:从实验小鼠骨髓获取的贴壁生长的成纤维样细胞可多次传代,并保持其快速增殖特点。流式细胞术检测显示,培养的第3代贴壁生长的成纤维样细胞呈CD14(-),CD34(-),CD45(-),而CD29(+),CD44(+),CD105(+),符合骨髓间充质干细胞特点。在体外,骨髓间充质干细胞能向脂肪细胞、成骨细胞方向分化。以5-氮胞苷(10μmol/L)孵育24小时后再继续培养4周,可分化为表达心肌特异性蛋白(心肌肌钙蛋白I)的细胞。结论:可通过对贴壁生长细胞反复传代培养的方法从全骨髓细胞中分离、纯化骨髓间充质干细胞。骨髓间充质干细胞具有贴壁生长、快速增殖的特性,在体外可多次传代而保持其特性。体外培养的骨髓间充质干细胞可被诱导向脂肪细胞和成骨细胞方向分化,并可被5-氮胞苷诱导分化为心肌样细胞,提示骨髓间充质干细胞有分化为心肌细胞的潜能,可作为急性心肌梗死后细胞移植治疗的细胞资源。目的:在细胞水平研究脂多糖(Lipoplysaccharide, LPS)对MSCs增殖以及对血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)释放的影响,并探讨Toll样受体4/核因子-κB ( toll-like receptor 4/nuclear factor—κB, TLR4/NF-κB)信号通路对MSCs增殖和分泌VEGF的作用。方法:①取第3代野生型小鼠骨髓间充质干细胞(wild-type MSCs, wMSCs)和TLR4基因缺陷型小鼠骨髓间充质干细胞(TLR4 gene deleted MSCs, tMSCs)与不同浓度的LPS(0μg/ml,0.01μg/ml,0.1μg/ml,1.0μg/ml,10μg/ml)进行共培养,48小时后,用MTT法检测MSCs的增殖。②取第3代wMSCs,与不同浓度的LPS(0μg/ml,0.01μg/ml,0.1μg/ml,1.0μg/ml,10μg/ml)进行共培养,48小时后,用ELISA法检测细胞培养液上清中VEGF浓度。③将培养的细胞分为4组:1. wMSCs组;2. wMSCs+1.0μg/ml LPS组;3. wMSCs+ 1.0μg/ml LPS+四氢吡咯二硫代氨基甲酸酯(pyrrolidine dithiocarbamate,PDTC)组;4. tMSCs+ 1.0μg/ml LPS组。用ELISA法检测各组细胞培养液上清中VEGF浓度,RT-PCR法检测各组细胞中VEGF的mRNA含量。结果:①不同浓度的LPS均能够促进wMSCs增殖,其中1.0μg/ml的LPS作用最强,LPS不能促进tMSCs增殖。②不同浓度的LPS均能够促进wMSCs旁分泌VEGF,其中1.0μg/ml的LPS作用最强。③用TLR4基因缺陷的MSCs或用NF-κB抑制剂后,VEGF的mRNA和蛋白表达量均减少。结论:1.0μg/ml的LPS能够最大限度的促进MSCs增殖,促进MSCs旁分泌VEGF。用TLR4基因缺陷的MSCs或用NF-κB抑制剂后,VEGF分泌量减少。说明LPS是通过TLR4/NF-κB信号通路促进MSCs旁分泌VEGF的。目的:研究C57BL/10J雄性小鼠的MSCs经LPS预处理后,能否改善心肌梗死大鼠的心功能,并探讨其中可能机制。方法:用结扎冠状动脉前降支的方法制作大鼠的急性心肌梗死模型,80只Wistar雌性大鼠随机分为4组,各组大鼠分别在心肌内注射下列物质:30μlPBS(对照组),3×106个野生型MSCs/30μl (wMSCs移植组),3×106个1.0μg/ml LPS预处理的野生型MSCs/30μl (LPS-wMSCs移植组), 3×106个1.0μg/ml LPS预处理的TLR4基因缺陷型MSCs/30μl (LPS-tMSCs移植组)。3周后,用心脏超声心动图测定检测大鼠的心功能,TTC法检测心肌梗死面积,Masson′s染色法检测心肌纤维化程度,用real-time PCR法检测移植细胞存活率,免疫组织化学法检测新生血管密度和心肌细胞凋亡率,Western blot法分析VEGF和磷酸化Akt表达。结果:细胞移植3周后,在4组中,经1.0μg/ml LPS预处理的野生型MSCs移植组(LPS-wMSCs移植组)较其余各组LVDd、LVDs均显著减少(P0.01),而LVEF和FS则显著增加(P0.01)。与其余3组相比,LPS-wMSCs移植组心肌纤维化显著减少(6.6±0.5%,P0.05),心肌细胞凋亡显著减少(14.8±1.5%,P0.01),新生血管密度显著增多(45.3±4.3,P0.01)。移植细胞的存活率在LPS-wMSCs移植组也显著增加(为wMSCs组的1.89±0.10倍,P0.01)。心肌组织内VEGF和磷酸化Akt表达在LPS-w MSCs移植组也增加。结论:LPS预处理能够增加移植MSCs的存活率,促进VEGF的表达,激活PI3K/Akt信号通路。MSCs在移植之前经过LPS预处理后能够更好的改善心脏功能,增加新生血管密度。LPS的预处理能够作为增强MSCs生物学功能的一种新的手段。
[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.

【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R329

【共引文献】