本文選題：成骨細(xì)胞 + 力生長(zhǎng)因子　； 參考：《重慶大學(xué)》2011年碩士論文

【摘要】：最近力生長(zhǎng)因子(Mechano growth factor, MGF)是生物力學(xué)及生長(zhǎng)因子研究方面的新熱點(diǎn)。MGF是Igf-1基因的選擇性剪接變異體,在拉伸刺激的骨骼肌和損傷的神經(jīng)、肌肉等組織中高表達(dá)。進(jìn)一步研究發(fā)現(xiàn),MGF及其E肽(MGF-Ct24E)能夠促進(jìn)肌肉肥大,損傷修復(fù),并且具有神經(jīng)保護(hù)和心肌保護(hù)等功能。本實(shí)驗(yàn)室的研究發(fā)現(xiàn)在拉伸刺激的成骨細(xì)胞中也有MGF mRNA的高表達(dá),提示MGF也可能是骨組織中響應(yīng)力信號(hào)的效應(yīng)因子,還發(fā)現(xiàn)MGF能夠調(diào)控成骨細(xì)胞的增殖分化過(guò)程,促進(jìn)骨缺損修復(fù)作用。應(yīng)力在骨組織的生長(zhǎng)、重建過(guò)程中發(fā)揮了重要的調(diào)控作用,骨折損傷部位往往因?yàn)槿狈?yīng)力而導(dǎo)致愈合后的骨質(zhì)疏松甚至再骨折現(xiàn)象。因此,本課題比較分析應(yīng)力與MGF-Ct24E作用后成骨細(xì)胞的基因表達(dá)譜,尋找兩者在骨代謝調(diào)控方面的異同,設(shè)想以MGF代替應(yīng)力解決骨損傷修復(fù)過(guò)程因固定醫(yī)療措施使得應(yīng)力嚴(yán)重降低的問(wèn)題。主要內(nèi)容和結(jié)論如下: (1)運(yùn)用組織塊培養(yǎng)法培養(yǎng)出新生大鼠顱骨成骨細(xì)胞,利用差時(shí)黏附法對(duì)培養(yǎng)出的細(xì)胞進(jìn)行純化。通過(guò)形態(tài)學(xué)觀察、堿性磷酸酶(ALP)染色、基質(zhì)前體染色、礦化結(jié)節(jié)染色等鑒定方法證實(shí)該組織塊法培養(yǎng)的細(xì)胞具有典型的成骨細(xì)胞生物學(xué)特征,符合后續(xù)實(shí)驗(yàn)要求。將傳代培養(yǎng)至第三代的成骨細(xì)胞用于后續(xù)實(shí)驗(yàn)研究。 (2)對(duì)細(xì)胞施加12%的周期性拉伸刺激和50ng/mL MGF-Ct24E直接作用8h,以靜態(tài)培養(yǎng)的細(xì)胞作為對(duì)照組,提取細(xì)胞總RNA,進(jìn)行基因芯片雜交實(shí)驗(yàn)分析各組細(xì)胞的差異表達(dá)基因。以對(duì)照組為參照,在力學(xué)加載組的細(xì)胞中共發(fā)現(xiàn)差異表達(dá)基因1866條,其中上調(diào)1113條,下調(diào)753條;在MGF-Ct24E處理組的細(xì)胞中共發(fā)現(xiàn)差異表達(dá)基因1178條,其中上調(diào)基因796條,下調(diào)基因382條。以力學(xué)加載組為參照,分析MGF-Ct24E處理組的細(xì)胞發(fā)現(xiàn)差異表達(dá)基因共602條,其中上調(diào)基因290條,下調(diào)基因312條。 (3)應(yīng)用Gene Ontology(GO)數(shù)據(jù)庫(kù)對(duì)篩選出的差異表達(dá)基因進(jìn)行基因本體論分析,分類分析各差異表達(dá)基因的功能類型。應(yīng)力和MGF-Ct24E作用成骨細(xì)胞后,得到的差異表達(dá)基因所參與調(diào)控的GO類型有眾多相似之處。這些共同之處主要是關(guān)于細(xì)胞增殖和分化、細(xì)胞周期、骨發(fā)育和骨化的調(diào)節(jié),細(xì)胞遷移及對(duì)應(yīng)力刺激的響應(yīng)等。除了兩者共同調(diào)節(jié)的表達(dá)類型外,應(yīng)力作用后的差異表達(dá)基因還參與調(diào)控了肌動(dòng)蛋白細(xì)胞骨架組織、細(xì)胞生長(zhǎng)、蛋白區(qū)域特異性結(jié)合和蛋白質(zhì)二聚化活性等;MGF-Ct24E作用后的差異表達(dá)基因還參與調(diào)控了軟骨發(fā)育、細(xì)胞骨架、生長(zhǎng)因子結(jié)合、蛋白激酶結(jié)合和受體結(jié)合等。以力學(xué)加載組為參照,分析MGF-Ct24E處理組的基因表達(dá)譜,發(fā)現(xiàn)MGF-Ct24E處理組中的差異表達(dá)基因主要與胚胎和組織發(fā)育、轉(zhuǎn)錄與基因表達(dá)的正調(diào)節(jié)等有關(guān)。MGF-Ct24E處理組與力學(xué)加載組的差異表達(dá)基因在調(diào)控骨代謝方面有很高的相似性,其差異基因表達(dá)的變化趨勢(shì)與差異表達(dá)倍數(shù)也基本一致。 (4)通過(guò)細(xì)胞增殖、細(xì)胞周期分析和細(xì)胞分化檢測(cè)等實(shí)驗(yàn)進(jìn)一步分析了MGF-Ct24E對(duì)成骨細(xì)胞生物學(xué)活性的影響。結(jié)果顯示:MGF-Ct24E在成骨細(xì)胞生長(zhǎng)早期促進(jìn)了細(xì)胞的增殖,抑制了ALP活性;細(xì)胞周期分析發(fā)現(xiàn)MGF-Ct24E作用組在第一天和第三天S期的細(xì)胞所占比例顯著高于對(duì)照組,細(xì)胞增殖指數(shù)顯著提高,隨著培養(yǎng)時(shí)間的延長(zhǎng)增殖速度逐漸減緩,促增殖效率逐漸降低;在細(xì)胞分化晚期,MGF-Ct24E促進(jìn)了膠原的表達(dá)和礦化結(jié)節(jié)的形成,促進(jìn)了成骨細(xì)胞的分化。說(shuō)明MGF-Ct24E并非是抑制成骨細(xì)胞的分化,而是促進(jìn)成骨細(xì)胞早期的增殖活性,延遲了其分化活性。細(xì)胞實(shí)驗(yàn)所得結(jié)果與基因芯片分析結(jié)果一致。 綜上所述,MGF-Ct24E和應(yīng)力作用在骨代謝基因表達(dá)的調(diào)控方面有著極為類似的效應(yīng),細(xì)胞實(shí)驗(yàn)分析再一次證實(shí)了MGF-Ct24E促進(jìn)成骨細(xì)胞的增殖,延遲其分化,最終促進(jìn)骨形成作用。因此,MGF-Ct24E有望能代替應(yīng)力刺激用于骨損傷修復(fù)的臨床治療。
[Abstract]:Recently, Mechano growth factor (MGF) is a new hot spot in the study of biomechanics and growth factors.MGF is a selective splicing variant of the Igf-1 gene, which is highly expressed in the skeletal muscles and injured nerves and muscles. Further studies have found that MGF and its E peptide (MGF-Ct24E) can promote muscle hypertrophy and loss of muscle. This laboratory study found that there is a high expression of MGF mRNA in the osteoblasts stimulated by tension, suggesting that MGF may also be an effector factor of response signal in bone tissue. It is also found that MGF can regulate the proliferation and differentiation process of osteoblasts and promote the repair of bone defects. Force plays an important regulatory role in the process of bone tissue growth and reconstruction. The fracture site often causes osteoporosis and even refracture after the lack of stress. Therefore, we compare and analyze the gene table of the osteoblasts after the stress and MGF-Ct24E action, and find the similarities and differences in the regulation of bone metabolism. It is envisaged that MGF should be used instead of stress to solve the problem of serious stress reduction due to fixed medical measures in the process of bone repair.
(1) the cultured rat calvarial osteoblasts were cultured by tissue mass culture, and the cultured cells were purified by differential time adhesion method. By morphological observation, alkaline phosphatase (ALP) staining, matrix precursor staining, mineralized nodule staining and other identification methods confirmed that the cells cultured in this tissue block have typical osteoblast biological characteristics. The results were consistent with the requirements of follow-up experiment. The osteoblasts from third generations were used for subsequent experimental studies.
(2) 12% periodic stretching stimulation and direct action of 50ng/mL MGF-Ct24E on 8h were applied to the cells. The static cultured cells were used as the control group, and the total RNA was extracted. The differential expression genes of each cell were analyzed by gene chip hybridization experiment. In the control group, 1866 differentially expressed genes were found in the cells of the mechanical loading group. There were 1113 up-regulated and 753 down regulation. In the MGF-Ct24E treatment group, there were 1178 differentially expressed genes, among which 796 were up-regulated, and 382 down regulated genes. A total of 602 differentially expressed genes were found in the MGF-Ct24E treatment group. Among them, 290 and 312 down regulated genes were up-regulated.
(3) using the Gene Ontology (GO) database to carry out the gene ontology analysis of the selected differentially expressed genes and classify the functional types of the differentially expressed genes. After the action of stress and MGF-Ct24E into osteoblasts, there are many similarities in the GO types involved in the regulation of differentially expressed genes. These similarities are mainly about the cells. Proliferation and differentiation, cell cycle, regulation of bone development and ossification, cell migration and response to stress stimulation are also involved in the regulation of actin cytoskeleton, cell growth, protein region specific binding and protein dimerization activity, in addition to the expression types that are co regulated by both. The differentially expressed genes after the action of MGF-Ct24E also regulate the development of cartilage, cytoskeleton, growth factor binding, protein kinase binding and receptor binding. Based on the mechanical loading group, the gene expression profiles in the MGF-Ct24E treatment group are analyzed, and the differential expression genes in the MGF-Ct24E treatment group are mainly developed with the embryo and tissue, and the transcriptional and transcriptional genes are found. The positive regulation of gene expression related to the differential expression genes of the.MGF-Ct24E treatment group and the mechanical loading group have high similarity in the regulation of bone metabolism, and the variation trend of the differential gene expression is also basically consistent with the differential expression multiplier.
(4) the effects of MGF-Ct24E on the biological activity of osteoblasts were further analyzed by cell proliferation, cell cycle analysis and cell differentiation test. The results showed that MGF-Ct24E promoted cell proliferation and inhibited ALP activity in the early stage of osteoblast growth, and the cell cycle analysis found that the MGF-Ct24E action group was in the first and third days S. The proportion of cells in the stage was significantly higher than that in the control group, and the proliferation index increased significantly. The proliferation rate gradually slowed down with the incubation time, and the proliferation efficiency gradually decreased. In the late differentiation of the cells, MGF-Ct24E promoted the expression of collagen and the formation of mineralized nodules, and promoted the differentiation of osteoblasts. It showed that MGF-Ct24E was not a inhibition. The differentiation of osteoblasts, but the early proliferation activity of osteoblasts, delayed the differentiation activity. The results of cell experiments were in accordance with the results of gene chip analysis.
In summary, MGF-Ct24E and stress play a very similar effect on the regulation of gene expression in bone metabolism. Cell experimental analysis again confirms that MGF-Ct24E promotes the proliferation of osteoblasts, delays its differentiation, and ultimately promotes bone formation. Therefore, MGF-Ct24E is expected to replace stress stimulation in the clinical treatment of bone damage repair. Therapy.
【學(xué)位授予單位】：重慶大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R329

【引證文獻(xiàn)】

相關(guān)博士學(xué)位論文 前3條

1 傅亞;骨修復(fù)因子功能化聚乳酸的制備及其生物相容性研究[D];重慶大學(xué);2012年

2 孫姣霞;基于新型PEG衍生物共聚改性聚乳酸的骨再生材料的研究[D];重慶大學(xué);2012年

3 李玉筱;力生長(zhǎng)因子24肽修飾的聚乳酸仿生骨基質(zhì)材料的研究[D];重慶大學(xué);2012年

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本文編號(hào)：2078256