BMP2和VEGF基因共修饰的骨髓基质干细胞在低氧条件下成骨能力研究

发布时间：2018-07-04 16:53

本文选题：骨髓基质干细胞 + 骨形态发生蛋白-2　；参考：《山东大学》2010年硕士论文

【摘要】： 目的：骨形态发生蛋白(Bone Morphogenetic Protein 2,BMP2)是目前已知的具有最强促进异位成骨能力细胞因子,血管内皮生长因子(Vascular endothelial growth factor, VEGF)能显著促进血管再生有利于骨缺损的愈合。BMP2和VEGF两种蛋白的分泌存在相互促进的正反馈作用。骨缺损的愈合,尤其是在愈合过程的初期,是在低氧环境下进行的。骨髓基质干细胞(Bone Marrow Stromal Cells,BMSC)具有向骨形成细胞和血管形成细胞等多种细胞分化的潜能。本课题研究模拟低氧环境下同时转染hBMP2和VEGF基因的BMSC在成骨方面的相关特性。方法： 1.大鼠骨髓基质干细胞(BMSC)的分离、培养利用Percoll分离液(相对密度1.077),用密度梯度离心法从大鼠骨髓中分离骨髓基质干细胞,进行体外培养及扩增。 2.通过形态学观察和生长曲线的测定及Elisa,观察BMSC转染hBMP-2的腺病毒载体(Ad-hBMP-2)和VEGF的腺病毒载体(Ad-VEGF)后生物学行为的变化和目的基因的表达。 BMSC转染绿色荧光蛋白(Green Fluorescent Protein,GFP)的腺病毒载体(Ad-GFP)后,使用荧光显微镜来测定腺病毒的转染效率,并观测转染Ad-GFP的细胞传代后绿色荧光蛋白的表达情况。 3.MTT法检测低氧控制系统下,转染Ad-hBMP-2和VEGF的BMSC细胞增值及活力取第二代骨髓基质干细胞进行实验,实验分为四组：(1)BMSC培养组；(2)Ad-hBMP-2+BMSC培养组；(3)VEGF+BMSC培养组；(4)Ad-hBMP-2+VEGF+BMSC培养组。体外连续培养7天,分别在此期间内用MTT法检测各组细胞的增值能力,酶标仪测定光密度值(OD值),每个样本设六个复孔,求其平均值并绘制细胞生长曲线。 4.低氧控制系统下,检测转染Ad-hBMP-2和VEGF的BMSC成骨能力取第二代骨髓基质干细胞进行实验,实验分为四组：(1)BMSC培养组；(2)Ad-hBMP-2+BMSC培养组；(3)VEGF+BMSC培养组；(4)Ad-hBMP-2+VEGF+BMSC培养组。96孔板内于低氧控制系统下,连续诱导培养6天,分别在此6天内用PNPP法检测各组细胞的碱性磷酸酶(Alkaline Phosphatase,ALP)的活性的表达；酶标仪测定光密度值(OD值),每个样本设五个复孔,求其平均值,并绘制ALP活性增长曲线；6孔板内四组样本体外连续诱导培养7天,在第7天行茜素红染色,观察矿化结节形成情况。结果： 1.密度梯度离心法,可获取高纯度的BMSC,并且细胞活性较好。贴壁3天后细胞成长为梭形及多角形,5、6天后增殖迅速,形成细胞集落。传代后细胞形态更加单一,5、6天后即可铺满瓶底。 2.体外实验转染骨髓基质干细胞。Ad-GFP转染12小时后即可检测到绿色荧光蛋白表达,转染2天后平均转染效率为90%,传至5代后依然可检测到绿色荧光蛋白表达。 3.MTT法检测低氧控制系统下,转染Ad-hBMP-2和Ad-VEGF的BMSC细胞增值及活力显示：第1、2天各组细胞数量没有明显增加,各组间细胞增殖能力统计学上没有显著性差异(P0.05)；第3天后,各组细胞数量明显增加,转染Ad-hBMP-2的细胞增殖能力在统计学上显著高于其他培养组(P0.01),转染Ad-VEGF细胞增殖能力与空白组无显著性差别(P0.05)。 4.低氧控制系统下,检测转染Ad-hBMP-2和VEGF的BMSC成骨能力 1)PNPP法检测各组ALP活性表达,结果显示：Ad-hBMP-2+VEGF+BMSC培养组细胞成骨诱导培养后,ALP活性表达统计学上显著高于其他三组(P0.05);Ad-hBMP-2+BMSC成骨诱导培养组高于BMSC组和VEGF+BMSC组(P0.05)；BMSC组和VEGF+BMSC组无显著差别(P0.05)。 2)低氧控制系统下,4组样本培养7天后,茜素红染色结果显示：Ad-hBMP-2+VEGF+BMSC成骨诱导培养组细胞密集生长区有多量矿化结节形成,茜素红染色成橘红色。Ad-hBMP-2+BMSC和VEGF+BMSC成骨诱导培养组细胞有少量矿化结节。BMSC成骨诱导培养组只有零星几个矿化结节。结论：在一周的培养时间内,骨形态形成蛋白(Bone Morphogenetic Protein2,BMP2)可以增强骨髓基质干细胞(BMSC)的成骨活性；血管内皮生长因子(vascular endothelial growth factor, VEGF)短期内对BMSC成骨活性影响和对照组无统计学差异；BMP-2和VEGF联合作用比单独转染BMP-2更能显著提高BMSC的成骨活性。
[Abstract]:Objective:
Bone morphogenetic protein (Bone Morphogenetic Protein 2, BMP2) is known to be the strongest promoter of ectopic osteogenesis, and vascular endothelial growth factor (Vascular endothelial growth factor, VEGF) can significantly promote vascular regeneration and promote the secretion of healing.BMP2 and VEGF two proteins, which are beneficial to bone defects. The healing of bone defects, especially at the early stage of the healing process, is carried out in the hypoxic environment. Bone Marrow Stromal Cells (BMSC) has the potential of multiple cell differentiation to bone morphogenetic cells and angiogenic cells. This topic is to simulate the simultaneous transfection of B to hBMP2 and VEGF genes in hypoxic environment. MSC related characteristics in osteogenesis.
Method:
1. rat bone marrow stromal cells (BMSC) were isolated and cultured with Percoll separation solution (relative density 1.077). The bone marrow stromal stem cells were isolated from rat bone marrow by density gradient centrifugation, and were cultured and amplified in vitro.
2. the changes in the biological behavior and the expression of the target genes after BMSC transfection of hBMP-2 adenovirus vector (Ad-hBMP-2) and VEGF adenovirus vector (Ad-VEGF) were observed by morphological observation and determination of growth curve and Elisa.
After BMSC transfected to the adenovirus vector (Ad-GFP) of Green Fluorescent Protein (GFP), the transfection efficiency of adenovirus was measured by fluorescence microscope, and the expression of green fluorescent protein after transfection of Ad-GFP was observed.
3.MTT assay was used to detect the proliferation and viability of BMSC cells transfected with Ad-hBMP-2 and VEGF under hypoxia control system.
Second generations of bone marrow stromal stem cells were taken and divided into four groups: (1) BMSC culture group; (2) Ad-hBMP-2+BMSC culture group; (3) VEGF+BMSC culture group; (4) Ad-hBMP-2+VEGF+BMSC culture group. Continuous culture for 7 days in vitro, MTT method was used to detect the increment of cells in each group, and the enzyme meter measured light density value (o value), each The sample has six holes, and the average value is drawn and the cell growth curve is drawn.
4. osteogenic ability of BMSC transfected with Ad-hBMP-2 and VEGF under hypoxia control system
Second generations of bone marrow stromal cells were taken and divided into four groups: (1) BMSC culture group; (2) Ad-hBMP-2+BMSC culture group; (3) VEGF+BMSC culture group; (4) Ad-hBMP-2+VEGF+BMSC culture group in.96 orifice plate under hypoxia control system for 6 days for 6 days, and the PNPP method was used to detect alkaline phosphatase in each group of cells (Alk) within this 6 day (Alk The expression of the activity of aline Phosphatase, ALP); the light density value (o value) was measured by the enzyme labeled instrument. Five compound holes were set in each sample, and the average value of each sample was set, and the growth curve of ALP activity was drawn. The four groups of samples in the 6 hole plate were continuously induced and cultured for 7 days in vitro, and the seventh days were stained with alizarin red, and the formation of mineralized nodules was observed.
Result:
The 1. density gradient centrifugation method can obtain high purity BMSC, and the cell activity is good. Cells grow into spindle and polygon after 3 days. After 5,6 days, the cells proliferate rapidly and form cell colonies. After the passage, the cell morphology is more single and can be covered with the bottom of the bottle after 5,6 days.
2. the expression of green fluorescent protein could be detected after transfection of bone marrow stromal stem cells.Ad-GFP in vitro for 12 hours. The average transfection efficiency was 90% after 2 days of transfection, and the expression of green fluorescent protein could still be detected after 5 generations.
The proliferation and vitality of BMSC cells transfected with 3.MTT and Ad-VEGF showed that the number of BMSC cells transfected with Ad-hBMP-2 and Ad-VEGF showed no significant increase in number of cells on day 1,2, and there was no significant difference in cell proliferation between each group (P0.05). After third days, the number of cells in each group increased significantly, and the proliferation ability of Ad-hBMP-2 transfected to Ad-hBMP-2 Compared with other culture groups (P0.01), the proliferation of Ad-VEGF cells was not significantly different from that of the blank group (P0.05).
4. osteogenic ability of BMSC transfected with Ad-hBMP-2 and VEGF under hypoxia control system
1) the expression of ALP activity in each group was detected by PNPP method. The results showed that the expression of ALP activity was significantly higher than that of the other three groups (P0.05) after the induction of osteogenesis in the Ad-hBMP-2+VEGF+BMSC culture group, and the Ad-hBMP-2+BMSC osteogenic induction group was higher than the BMSC group and VEGF+BMSC group (P0.05), and there was no significant difference between the BMSC group and the VEGF+BMSC group (P0.05).
2) under the hypoxic control system, 4 groups of samples were cultured for 7 days. The results of alizarin red staining showed that there were many mineralized nodules in the cell intensive growth area of Ad-hBMP-2+VEGF+BMSC osteogenesis induced culture group. Alizarin red was stained into orange red.Ad-hBMP-2+BMSC and VEGF+BMSC induction culture group with a small amount of mineralized nodules in.BMSC osteogenic induction culture group. There are sporadic mineralized nodules.
Conclusion:
Bone morphogenetic protein (Bone Morphogenetic Protein2, BMP2) can enhance the osteogenic activity of bone marrow stromal cells (BMSC) in a week of culture, and there is no statistical difference between the vascular endothelial growth factor (VEGF) and the control group in the short term. Compared with the transfection of BMP-2 alone, the osteogenic activity of BMSC could be significantly improved.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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