成骨生长肽及其诱导大鼠骨髓间充质干细胞成骨分化机制的研究

发布时间：2018-06-12 02:06

  本文选题：成骨生长肽 + 骨髓间充质干细胞　； 参考：《山东大学》2008年硕士论文

【摘要】： 成骨生长肽(Osteogenic Growth Peptide,OGP)是组蛋白H4基因编码的一种多肽。研究表明,OGP具有广泛的成骨和造血活性。体内OGP可以刺激骨形成,增加小梁骨密度,促进骨折的愈合;体外OGP可以调节多种成骨细胞、骨髓间充质干细胞的增殖,提高碱性磷酸酶活性,促进基质矿化。OGP还可以刺激造血细胞的生成,提高骨髓移植的成活率,OGP的造血活性与OGP改善了造血微环境有关。推测OGP的成骨和造血活性可能与骨髓间充质干细胞有关。 骨髓间充质干细胞(BMSCs)是骨髓中一类具有多向分化潜能的干细胞,是骨髓造血微环境的重要组成部分,在适当的条件下可以分化为成骨细胞、软骨细胞、脂肪细胞等。骨髓间充质干细胞构成造血细胞生长、分化必不可少的微环境,同时也是成骨祖细胞的前体细胞,对骨髓造血和成骨均具有十分重要的作用。 为了研究OGP的生物学活性与骨髓间充质干细胞的关系及可能的应用前景,本论文从以下几个方面作了研究: (1)大鼠骨髓间充质干细胞(rBMSCs)的分离培养及OGP对大鼠骨髓间充质干细胞增殖和成骨分化的作用。结果表明,OGP在低浓度(10~(-10)mol/L、10~(-11)mol/L)时对大鼠骨髓间充质干细胞具有有丝分裂原活性,高浓度(10~(-8)mol/L、10~(-9)mol/L)时促进大鼠骨髓间充质干细胞成骨分化。RT-PCR结果及基因芯片结果均表明,OGP促进大鼠骨髓间充质干细胞成骨分化的早期可能与成骨特异性转录因子Cbfα1无关;OGP诱导大鼠骨髓间充质干细胞成骨分化的早期,成骨细胞特异性表达的标志分子骨钙素(Osteocalcin)表达阴性,表明大鼠骨髓间充质干细胞还没有完成向成骨细胞的分化。 (2)成骨生长肽诱导大鼠骨髓间充质干细胞基因表达谱变化的研究。通过博奥27K Rat Genome Array芯片,研究了OGP诱导大鼠骨髓间充质干细胞早期基因表达谱的变化。发现共有145个差异表达的基因,其中91个上调表达的基因,54个下调表达的基因。差异表达的基因中可能与成骨及造血活性相关的基因共有35个:包括4个血管发生相关的基因、9个骨代谢相关的基因、14个炎症与免疫相关的基因、8个细胞增殖与分化相关的基因。基因表达谱芯片结果为进一步研究OGP促进大鼠骨髓间充质干细胞成骨和造血活性的分子机制建立了很好的基础。 (3)重组载体转染大鼠骨髓间充质干细胞及其成骨分化活性与成骨生长肽诱导的大鼠骨髓间充质干细胞的成骨分化活性的比较。以pEGFP-N1质粒为基础,构建了OGP正义载体和Pre-OGP反义载体,并成功的转染了大鼠骨髓间充质干细胞。荧光观察表明,重组载体可以成功地转染大鼠骨髓间充质干细胞。碱性磷酸酶(ALP)活性测定表明,Pre-OGP反义载体转染的大鼠骨髓间充质干细胞成骨分化活性受到抑制,而OGP正义载体转染的大鼠骨髓间充质干细胞更易于成骨分化。 (4)成骨生长肽免疫学检测方法的建立。为了研究OGP与骨质疏松等骨代谢疾病、骨折延迟愈合和骨折不愈合等临床疾病的关系,本论文建立了一种OGP的免疫学检测方法。通过改进的免疫学方法建立了一种可用于临床检测血清OGP浓度的方法,与传统方法相比,灵敏度大大提高。
[Abstract]:Osteogenic Growth Peptide (OGP) is a peptide encoded by the histone H4 gene. Studies have shown that OGP has extensive osteogenesis and hematopoiesis. In vivo OGP can stimulate bone formation, increase bone density of trabecular bone and promote fracture healing; in vitro OGP can regulate the proliferation of multiple osteoblasts, bone marrow mesenchymal stem cells and improve alkali. The activity of sexual phosphatase, promoting matrix mineralization.OGP can also stimulate the formation of hematopoietic cells and increase the survival rate of bone marrow transplantation. The hematopoietic activity of OGP is related to the improvement of the hematopoietic microenvironment of OGP. It is presumed that the osteogenesis and hematopoiesis of OGP may be related to bone marrow mesenchymal stem cells.
Bone marrow mesenchymal stem cell (BMSCs) is a kind of stem cells with multiple differentiation potential in bone marrow. It is an important part of bone marrow hematopoietic microenvironment. It can be differentiated into osteoblasts, chondrocytes and adipocytes under appropriate conditions. Bone marrow mesenchymal stem cells constitute the microenvironment which is essential for the growth of blood cells, and also the differentiation of hematopoietic cells. It is a precursor cell of osteoblast progenitor cells and plays a very important role in bone marrow hematopoiesis and osteogenesis.
In order to study the relationship between biological activity of OGP and bone marrow mesenchymal stem cells and its potential application, the following aspects were studied in this paper.
(1) the isolation and culture of rat bone marrow mesenchymal stem cells (rBMSCs) and the effect of OGP on the proliferation and osteogenesis of bone marrow mesenchymal stem cells in rats. The results show that OGP has mitogen activity at low concentration (10~ (-10) mol/L, 10~ (-11) mol/L) in rat bone marrow mesenchymal stem cells, and the high concentration (10~ (-8) mol/L) promotes rats The results of osteogenic differentiation of bone marrow mesenchymal stem cells (.RT-PCR) and gene chip results showed that OGP promoted bone differentiation of bone marrow mesenchymal stem cells in the early stages of bone marrow mesenchymal stem cells may not be related to bone specific transcription factor Cbf alpha 1; OGP induced bone marrow mesenchymal stem cells in the early stage of osteogenesis and osteoblast specific expression of osteoblast bone calcium. Negative expression of Osteocalcin showed that bone marrow mesenchymal stem cells did not differentiate into osteoblasts in vitro.
(2) a study of gene expression profiles in rat bone marrow mesenchymal stem cells induced by osteogenic growth peptide. The changes in gene expression profiles of bone marrow mesenchymal stem cells (MSCs) induced by OGP were studied by boo 27K Rat Genome Array chip. There were a total of 145 differentially expressed genes, 91 up-regulated genes and 54 down-regulated genes. There are 35 genes associated with osteogenesis and hematopoiesis in differentially expressed genes, including 4 angiogenesis related genes, 9 bone metabolism related genes, 14 inflammatory and immune related genes, and 8 cell proliferation and differentiation related genes. The result of gene expression spectrum core is to further study OGP promoting rat bone marrow The molecular mechanism of osteogenic and hematopoietic activities of mesenchymal stem cells has laid a good foundation.
(3) a comparison of recombinant vector transfected rat bone marrow mesenchymal stem cells and their osteogenic differentiation activity and osteogenic differentiation activity of rat bone marrow mesenchymal stem cells induced by osteogenic growth peptide. Based on pEGFP-N1 plasmid, a OGP justice carrier and Pre-OGP Antisense Vector were constructed, and the rat bone marrow mesenchymal stem cells were transfected successfully. The results showed that the recombinant vector could be successfully transfected into rat bone marrow mesenchymal stem cells. The activity of alkaline phosphatase (ALP) activity showed that the osteogenic differentiation activity of rat bone marrow mesenchymal stem cells transfected by Pre-OGP antisense vector was inhibited, and the rat bone marrow mesenchymal stem cells transfected by OGP just carrier were more prone to osteogenic differentiation.
(4) establishment of an immunological detection method for osteogenic growth peptide. In order to study the relationship between OGP and bone metabolic diseases such as osteoporosis, fracture delayed union and fracture nonunion, a OGP immunoassay method was established in this paper. An improved immunological method was used to establish a prescription for clinical detection of serum OGP concentration. Compared with the traditional method, the sensitivity of the method is greatly improved.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329
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本文编号：2007769