成骨细胞—血管内皮细胞共培养体系对IGF-1表达及血管内皮细胞生物学特性的影响研究

发布时间：2018-06-20 00:25

  本文选题：成骨细胞 + 血管内皮细胞　； 参考：《泸州医学院》2014年硕士论文

【摘要】：目的：近年来，组织工程骨已成为社会研究的热点，而成骨细胞和血管内皮细胞共培养是组织工程骨研究的首要条件。本实验将从共培养体系的建立入手，进而研究共培养体系对胰岛素生长因子1（insulin-like growthfactor-1,IGF-1）表达的影响及对血管内皮细胞生物学特性的影响。方法：本实验分为三部分，第一部分以成骨样肉瘤细胞株（Human osteosarcoma MG-63cell, MG63）及血管内皮细胞株（Vascularendothelial cells, VECs）为研究对象，细胞传代培养至第六代备用，通过5种不同的比例建立MG63-VECs共培养体系，通过倒置显微镜下细胞形态的观察来确定两种细胞的最佳共培养比例；第二部分，以第一部分的最佳共培养比例建立MG63-VECs共培养体系作为实验组，MG63及VECs单独培养作为对照组，分别在12h,24h,36h及48h提取各组上清液，利用ELISA实验方法对实验组和对照组各个时间点进行IGF-1表达值的检测；再利用Transwell小室将VECs及MG63按最佳比例进行间接共培养，实验分为4组，共培养A组，共培养B组，MG63组，VECs组，分别在12h,24h,36h及48h对各组进行RNA提取，再用荧光定量PCR检测IGF-1mRNA的表达。第三部分观察共培养体系对血管内皮细胞生物学特性的影响，一方面通过DIi染色示踪法观察，将VECs进行DIi染色，与MG63按1：1进行直接共培养为实验组，VECs进行DIi染色后单独培养为对照组，96h后在荧光显微镜下观察两组的管型情况；另一方面通过鼠尾胶原实验观察。利用鼠尾胶原制备三维培养环境，实验组为MG63与VECs按1：1进行直接共培养，对照组为VECs单独培养。72h后在倒置显微镜下观察管型的情况。以上原始实验数据均使用SPSS13.0软件分析，数据用均数±标准差（x±s）表示，两样本均数比较用独立样本t检验；多个样本两两比较先用方差分析（ANOVA），再用多样本两两比较中的SNK法进行比较，p0.05及p0.01表示差异有显著性和非常显著性意义。结果：第一部分，MG63培养至第五代在倒置显微镜下观察，细胞形态不规则，多为三角形及多角形。VECs呈圆形或椭圆形，呈铺路石样生长；直接共培养中MG63和VECs细胞按1:1培养时两种细胞生长最为良好，细胞间杂生长，其中血管内皮细胞呈结节样生长，成骨细胞生长于血管内皮细胞结节周围。第二部分，ELISA结果显示：共培养组IGF-1表达值较对照组明显增加(P0.05)。荧光定量PCR结果显示：12h-48h共培养A组及MG63组IGF-1mRNA表达值随着时间呈上升趋势，共培养A组较MG63组IGF-1mRNA表达值明显增加(P0.05)；共培养B组和VECs组IGF-1mRNA表达值随着时间出现不稳定变化趋势，在12h和36h呈上升趋势，，24h和48h时呈下降趋势；共培养A组各时间点IGF-1mRNA表达值明显高于共培养B组养(P0.01)。第三部分，体外管型实验，DIi示踪法96h荧光显微镜下观察实验组VECs出现类管型样结构，对照组VECs上层未见明显管型样结构形成。鼠尾胶实验72h倒置显微镜下观察，对照组形成类血管腔样结构；实验组少量细胞坏死，未见明显的血管腔样结构形成。结论：1、直接共培养中MG63和VECs按1:1培养时两种细胞生长最为良好，细胞间杂生长，其中VECs呈结节样生长，MG63生长于VECs结节周围。2、在MG63-VECs共培养体系中IGF-1的表达值优于二者的单独培养；间接共培养条件下共培养A组IGF-1mRNA表达明显高于共培养B组。3、通过共培养体系的管型实验证明在成骨细胞的参与下血管内皮细胞更易形成管型验证了在骨组织血管化中成骨细胞不仅仅构建了骨组织基本结构，还作为一种管型诱导功能细胞，参与了VECs的迁移和管型形成。
[Abstract]:Objective: in recent years, tissue engineering bone has become a hot spot in social research, and co culture of osteoblasts and vascular endothelial cells is the primary condition for the study of tissue engineering bone. This experiment will start with the establishment of co culture system and then study the effect of co culture system on the expression of insulin growth factor 1 (insulin-like growthfactor-1, IGF-1). And the effect on biological characteristics of vascular endothelial cells. Methods: this experiment was divided into three parts. The first part was Human osteosarcoma MG-63cell (MG63) and vascular endothelial cell line (Vascularendothelial cells, VECs). The cells were cultured to sixth generations and were established by 5 different proportions. MG63-VECs co culture system, the optimum co culture ratio of two kinds of cells was determined by the observation of cell morphology under inverted microscope. The second part, using the best co culture ratio of the first part to establish the MG63-VECs co culture system as the experimental group. MG63 and VECs were cultured separately as the group, and respectively in 12h, 24h, 36h and 48h, respectively. The ELISA test method was used to detect the IGF-1 expression value at each time point of the experimental group and the control group. Then the Transwell chamber was used to direct the indirect co culture of VECs and MG63 according to the optimum proportion. The experiment was divided into 4 groups. The experiment was divided into groups of A groups, and a total of B, MG63, VECs groups were cultured in the B, MG63, and VECs groups. The expression of IGF-1mRNA was detected by quantitative PCR. The third part observed the effect of co culture system on the biological characteristics of vascular endothelial cells. On the one hand, the VECs was stained by DIi staining, and MG63 was directly co cultured with 1:1 as the experimental group. VECs was stained by DIi to be the control group, and 96h after the fluorescence microscope. On the other hand, the tube type of the two groups was observed. On the other hand, the rat tail collagen was observed. The three-dimensional culture environment was prepared by the rat tail collagen. The experimental group was co cultured with MG63 and VECs according to 1:1. The control group observed the tube type under the inverted microscope after the single culture of VECs by the inverted microscope. All the above original experimental data were divided into SPSS13.0 software. Analysis, the data was expressed with mean mean standard deviation (x + s). The average number of two samples was compared with independent sample t test; 22 samples were compared with variance analysis (ANOVA) first, and then compared with SNK in multi sample 22 comparison. P0.05 and P0.01 showed significant and significant difference. The first part, MG63 was cultivated to fifth generations. Under the inverted microscope, the morphology of the cells was irregular, and the.VECs was round or oval in the shape of triangle and polygon. The growth of the two cells was the best in the direct co culture of MG63 and VECs cells, and the cell growth was nodular, and the osteoblasts grew in the blood vessels. The second part of the endothelial cell nodules. The results of ELISA showed that the expression of IGF-1 in the co culture group was significantly higher than that in the control group (P0.05). The results of fluorescence quantitative PCR showed that the IGF-1mRNA expression value of the 12h-48h co culture A group and the MG63 group increased with the time, and the co culture A group was significantly higher than the MG63 group IGF-1mRNA expression value (P0.05). The expression value of IGF-1mRNA in group VECs and VECs showed a trend of unstable change along with time, which showed an upward trend in 12h and 36h, and decreased in 24h and 48h. The IGF-1mRNA expression value at each time point in the co culture A group was obviously higher than that in co culture B group (P0.01). The third part, in vitro tube type experiment, DIi tracing method under 96h fluorescence microscope, observed the occurrence class of experimental group. In the control group, there was no obvious tube like structure in the control group. The rat tail gum was observed under 72h inverted microscope and the control group formed a blood vessel like structure. In the experimental group, a small number of cells were necrotic and no obvious vascular cavity like structure was formed. Conclusion: 1, the growth of the two cells in the direct co culture of MG63 and VECs in 1:1 was the most important. Well, cell growth, VECs nodular growth, MG63 growth in VECs nodules.2, IGF-1 expression value in MG63-VECs co culture system is superior to two individual culture; under the indirect co culture condition, the expression of IGF-1mRNA in co culture A group is significantly higher than that in co culture B group.3, through the tube type experiment of co culture system, it is proved to be bone fine. The vascular endothelial cells are more likely to form a tube type with the participation of the cells. The osteoblasts in the vascularization of bone tissue not only construct the basic structure of the bone tissue, but also as a type of tubular induced functional cells, and participate in the migration and formation of VECs.
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R782

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相关期刊论文 前1条

1 马红梅;邹进;王跃中;艾红军;杨向红;;人成骨细胞与脐静脉血管内皮细胞直接混合培养的实验研究[J];口腔医学;2007年06期

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