腺病毒介导BMP-2基因转染诱导性多能间充质干细胞成骨方向的研究

发布时间：2018-06-21 15:15

本文选题：骨形态蛋白-2 + 腺病毒　；参考：《青岛大学》2017年硕士论文

【摘要】：背景和目的:各种原因造成的骨组织缺损是临床常见疾患,随着骨组织工程技术的兴起,给传统治疗难点的骨缺损带来了一些曙光,组织工程利用自体细胞的生物活性,给予外在因素特定的干扰下将其诱导分化为所需的细胞,从而达到修复组织的能力。传统的间充质干细胞、胚胎干细胞已在体外成功被诱导定向分化,但涉及到一些伦理、取材限制等问题,因此其在组织学上作为种子方面的研究受到一定的限制;而随着体内体细胞的研究,通过使用几个特定的转录因子诱导体细胞的重编程而获得的可不断自我更新且具有多向分化潜能的诱导性多能干细胞(induced pluripotent stem cells,i PSCs)的出现,可作为组织学中理想的种子细胞,本课题在前期已成功将人、山羊、大鼠成纤维细胞诱导为i PSCs的基础上,运用诱导因子将山羊i PSCs定向分化为i PSCs-MSC,作为骨组织工程的优化种子细胞,然后利用体外构建腺病毒搭载生物因子BMP-2,从而去转染诱导i PSCs-MSC,探讨载体介导下种子细胞转染的可行性,同时观察研究i PSCs-MSC在生物因子BMP-2的作用下,对其体外增殖和分化成骨能力的影响。方法:1.体外扩增预先构建好的i PSCs-MSC,取传代至第三代的i PSCs-MSC,按照普通培养基、含BMP-2的培养基、腺病毒-BMP-2的培养基对其进行体外繁殖,而分别设定为空白对照组、阴性对照组和实验组,然后分别往各组加入成骨诱导液,在体外进行培养,分别在培养1、2、3、4、5、6、7d用CCK-8试剂检测i PSCs-MSC的存活及增殖活性;2.三组处理好的细胞,转染后,分别于3、7、14天后终止培养,利用碱性磷酸酶试剂盒检测其碱性磷酸酶表达情况;3.然后在三组细胞培养增殖至20天后,使用茜素红染液,然后分别于染色后1周、2周后用光学显微镜下观察矿化结节的形成;4.使用Western Blotting法测定三组细胞,检测转染后BMP-2的表达情况。结果:1.CCK-8检测后,实验组细胞的可见光吸收度OD高于另外两项对照组,说明实验组中细胞增殖能力活性明显强于另外两组;2.腺病毒转染后的三组细胞ALP测定呈阳性反应,且细胞内ALP活性随着时间的延长而增加,但是实验组中ALP含量明显高于另外两组,同时随着时间延长,阴性对照组较空白对照组也表现出强ALP表达能力;3.实验组红色矿化结节含量明显,阴性对照组矿化结节表达稍高于空白对照组,空白对照组未见明显红色结节;4.各组细胞转染后,实验组细胞BMP-2表达明显高于阴性对照组,阴性对照组表达不明显,空白对照组未见表达。结论:1.证实了i PSC-MSCs在体外成骨的可行性;2.说明了体外生物因子BMP-2具有能显著促进i PSC-MSCs向成骨细胞的分化和功能;3.腺病毒能明显加快体外BMP-2对i PSC-MSCs向成骨细胞的分化速率;4.BMP-2基因转染诱导性多能间充质干细胞后在体外可明显促进其向成骨细胞分化,并为其作为骨组织工程优化种子细胞奠定了实验基础。
[Abstract]:Background and objective: bone tissue defect caused by various causes is a common clinical disease. With the rise of bone tissue engineering technology, it brings some light to the traditional treatment of bone defect. Tissue engineering makes use of the biological activity of autosomal cells. The ability of tissue repair can be achieved by inducing the cells to differentiate into the required cells under the specific interference of external factors. Traditional mesenchymal stem cells, embryonic stem cells have been successfully induced directional differentiation in vitro, but related to a number of ethical, material constraints, so the study of histology as a seed is limited to a certain extent; With the study of somatic cells in vivo, induced pluripotent stem cells induced by inducing pluripotent stem cells, which can be self-renewing and have multi-differentiation potential, were obtained by reprogramming several specific transcription factor inducers. It can be used as an ideal seed cell in histology. We have successfully induced human, goat and rat fibroblasts into I PSCs in the early stage. Goat I-PSCs were differentiated into I-PSCs-MSCs by inducing factors, which were used as seed cells for bone tissue engineering. Then, the adenovirus was constructed to carry the biological factor BMP-2 in vitro, which was used to transfect and induce iPSCs-MSC.To explore the feasibility of vector mediated seed cell transfection, and to observe the effect of iPSCs-MSC on the biological factor BMP-2. Effects on proliferation and osteogenic differentiation in vitro. Method 1: 1. The pre-constructed I PSCs-MSCs were amplified in vitro, and then passed to the third generation I PSCs-MSC.According to the normal medium, the medium containing BMP-2 and the medium of adenovirus BMP-2, they were propagated in vitro, and were set as blank control group, negative control group and experimental group, respectively. Then the osteoblasts were added to each group and cultured in vitro. The survival and proliferative activity of iPSCs-MSC were detected by CCK-8 reagent on the 7th day of culture. The three groups of treated cells were transfected, and the culture was terminated after 14 days after transfection. The alkaline phosphatase expression was detected by alkaline phosphatase kit. The cells in the three groups were cultured for 20 days and then stained with alizarin red. The formation of mineralized nodules was observed under optical microscope at 1 week and 2 weeks after staining. The expression of BMP-2 was detected by Western blotting. Results 1. The OD of the cells in the experimental group was higher than that in the other two control groups after CCK-8 detection, which indicated that the proliferative activity of the cells in the experimental group was significantly higher than that in the other two groups. The ALP activity of the three groups after adenovirus transfection increased with time, but the ALP content in the experimental group was significantly higher than that in the other two groups, and the ALP activity increased with time. The negative control group also showed stronger ALP expression ability than the blank control group. The content of red mineralized nodules in the experimental group was obvious, the expression of the mineralized nodules in the negative control group was slightly higher than that in the blank control group, and no obvious red nodule was found in the blank control group. After transfection, the expression of BMP-2 in the experimental group was significantly higher than that in the negative control group, but not in the blank control group. Conclusion 1. The feasibility of I PSC-MSCs osteogenesis in vitro was confirmed. The results showed that BMP-2 could significantly promote the differentiation and function of I PSC-MSCs into osteoblasts in vitro. Adenovirus could significantly accelerate the differentiation rate of I PSC-MSCs into osteoblasts in vitro. 4. BMP-2 gene transfection into induced pluripotent mesenchymal stem cells could obviously promote the differentiation of I PSC-MSCs into osteoblasts in vitro. It also lays the experimental foundation for the optimization of seed cells in bone tissue engineering.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R68

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