發(fā)布時間：2018-06-12 02:06

  本文選題：成骨生長肽 + 骨髓間充質干細胞��； 參考：《山東大學》2008年碩士論文

【摘要】： 成骨生長肽(Osteogenic Growth Peptide,OGP)是組蛋白H4基因編碼的一種多肽。研究表明,OGP具有廣泛的成骨和造血活性。體內(nèi)OGP可以刺激骨形成,增加小梁骨密度,促進骨折的愈合;體外OGP可以調(diào)節(jié)多種成骨細胞、骨髓間充質干細胞的增殖,提高堿性磷酸酶活性,促進基質礦化。OGP還可以刺激造血細胞的生成,提高骨髓移植的成活率,OGP的造血活性與OGP改善了造血微環(huán)境有關。推測OGP的成骨和造血活性可能與骨髓間充質干細胞有關。 骨髓間充質干細胞(BMSCs)是骨髓中一類具有多向分化潛能的干細胞,是骨髓造血微環(huán)境的重要組成部分,在適當?shù)臈l件下可以分化為成骨細胞、軟骨細胞、脂肪細胞等。骨髓間充質干細胞構成造血細胞生長、分化必不可少的微環(huán)境,同時也是成骨祖細胞的前體細胞,對骨髓造血和成骨均具有十分重要的作用。 為了研究OGP的生物學活性與骨髓間充質干細胞的關系及可能的應用前景,本論文從以下幾個方面作了研究: (1)大鼠骨髓間充質干細胞(rBMSCs)的分離培養(yǎng)及OGP對大鼠骨髓間充質干細胞增殖和成骨分化的作用。結果表明,OGP在低濃度(10~(-10)mol/L、10~(-11)mol/L)時對大鼠骨髓間充質干細胞具有有絲分裂原活性,高濃度(10~(-8)mol/L、10~(-9)mol/L)時促進大鼠骨髓間充質干細胞成骨分化。RT-PCR結果及基因芯片結果均表明,OGP促進大鼠骨髓間充質干細胞成骨分化的早期可能與成骨特異性轉錄因子Cbfα1無關;OGP誘導大鼠骨髓間充質干細胞成骨分化的早期,成骨細胞特異性表達的標志分子骨鈣素(Osteocalcin)表達陰性,表明大鼠骨髓間充質干細胞還沒有完成向成骨細胞的分化。 (2)成骨生長肽誘導大鼠骨髓間充質干細胞基因表達譜變化的研究。通過博奧27K Rat Genome Array芯片,研究了OGP誘導大鼠骨髓間充質干細胞早期基因表達譜的變化。發(fā)現(xiàn)共有145個差異表達的基因,其中91個上調(diào)表達的基因,54個下調(diào)表達的基因。差異表達的基因中可能與成骨及造血活性相關的基因共有35個:包括4個血管發(fā)生相關的基因、9個骨代謝相關的基因、14個炎癥與免疫相關的基因、8個細胞增殖與分化相關的基因�；虮磉_譜芯片結果為進一步研究OGP促進大鼠骨髓間充質干細胞成骨和造血活性的分子機制建立了很好的基礎。 (3)重組載體轉染大鼠骨髓間充質干細胞及其成骨分化活性與成骨生長肽誘導的大鼠骨髓間充質干細胞的成骨分化活性的比較。以pEGFP-N1質粒為基礎,構建了OGP正義載體和Pre-OGP反義載體,并成功的轉染了大鼠骨髓間充質干細胞。熒光觀察表明,重組載體可以成功地轉染大鼠骨髓間充質干細胞。堿性磷酸酶(ALP)活性測定表明,Pre-OGP反義載體轉染的大鼠骨髓間充質干細胞成骨分化活性受到抑制,而OGP正義載體轉染的大鼠骨髓間充質干細胞更易于成骨分化。 (4)成骨生長肽免疫學檢測方法的建立。為了研究OGP與骨質疏松等骨代謝疾病、骨折延遲愈合和骨折不愈合等臨床疾病的關系,本論文建立了一種OGP的免疫學檢測方法。通過改進的免疫學方法建立了一種可用于臨床檢測血清OGP濃度的方法,與傳統(tǒng)方法相比,靈敏度大大提高。
[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.
【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R329
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本文編號：2007769