【摘要】： 隨著骨組織工程研究的不斷發(fā)展,種子細(xì)胞的來源、培養(yǎng)、誘導(dǎo)、增殖、分化等研究成為近年來骨組織工程進(jìn)行研究的熱點(diǎn)。間充質(zhì)干細(xì)胞具有能大量增殖、分化為多種組織細(xì)胞和表達(dá)外源目的基因等優(yōu)點(diǎn),并且可以在特定的條件下定向向成骨細(xì)胞分化,因其可能成為用于臨床的組織工程化骨種子細(xì)胞而受到越來越多的關(guān)注。 近年來體外沖擊波療法正被廣泛的用于治療骨不連、骨折延遲愈合和股骨頭壞死以及運(yùn)動系統(tǒng)慢性勞損性疾病,并取得了很好的療效。人們發(fā)現(xiàn),機(jī)械物理刺激促使間充質(zhì)干細(xì)胞分化。沖擊波作為一種機(jī)械物理刺激能夠促使間充質(zhì)干細(xì)胞向成骨細(xì)胞分化,但這一過程不是自發(fā)完成的,其中可能涉及到多條信號通路的調(diào)控,其中MAPK通路可能起著極其重要的調(diào)節(jié)作用,但具體通過MAPK傳導(dǎo)路途徑的ERK、JNK以及p38MAPK通路中哪一條通路參與以及各通路在誘導(dǎo)成骨分化過程中的作用尚不十分清楚。本試驗(yàn)通過前期獲得最佳沖擊波(工作電壓為8.5kv,作用頻次為120次)作用于分離培養(yǎng)后的人骨髓間充質(zhì)干細(xì)胞,設(shè)立空白對照組,并分別加入ERK、JNK以及p38MAPK通路抑制劑,通過western blot檢測ERK、JNK以及p38MAPK的磷酸化以及MAPK通路作用底物AP-1(c-fos和c-jun mRNA)的表達(dá)。用MTT法檢測細(xì)胞增殖,用成骨特性鑒定的方法(鈣-鈷法染色檢測堿性磷酸酶的表達(dá)及其活性、Von Kossa染色檢測鈣結(jié)節(jié)和鈣沉積量的檢測)判斷沖擊波是否誘導(dǎo)人骨髓間充質(zhì)干細(xì)胞向成骨細(xì)胞分化,研究ERK、JNK、p38 MAPK通路在體外沖擊波誘導(dǎo)下人骨髓間充質(zhì)干細(xì)胞向成骨細(xì)胞分化過程中的作用。 本研究發(fā)現(xiàn)在沖擊波誘導(dǎo)人骨髓間充質(zhì)干細(xì)胞誘導(dǎo)向成骨細(xì)胞分化過程中,通過Western blot方法證實(shí)ERK、JNK以及p38MAPK通路均早期磷酸化激活,提示ERK、JNK以及p38MAPK通路均參與了成骨細(xì)胞增殖、分化。應(yīng)用ERK、JNK以及p38MAPK通路相應(yīng)的阻斷劑研究結(jié)果表明:ERK通路促進(jìn)人骨髓間充質(zhì)干細(xì)胞增殖,抑制ERK通路后細(xì)胞增殖作用明顯降低。抑制JNK通路后向成骨細(xì)胞分化作用雖有降低,但不具有統(tǒng)計(jì)學(xué)意義;對細(xì)胞增殖未見有明顯作用。抑制p38MAPK通路后MAPK作用底物AP-1 (c-fos和c-jun mRNA)表達(dá)明顯受抑制,成骨鑒定證實(shí)向成骨細(xì)胞分化作用明顯降低,并呈劑量依賴性;低濃度的p38MAPK抑制劑反而促進(jìn)細(xì)胞增殖。應(yīng)用阻斷劑后行同樣條件進(jìn)行誘導(dǎo),提示p38MAPK通路是沖擊波誘導(dǎo)人骨髓間充質(zhì)干細(xì)胞誘導(dǎo)向成骨細(xì)胞分化的主要途徑。
[Abstract]:With the development of bone tissue engineering, the research on the origin, culture, induction, proliferation and differentiation of seed cells has become the focus of bone tissue engineering research in recent years. Mesenchymal stem cells (MSCs) have the advantages of proliferation, differentiation into various histocytes and expression of exogenous target genes. Mesenchymal stem cells can differentiate into osteoblasts under specific conditions. More and more attention has been paid to its potential as tissue engineered bone seed cells for clinical use. In recent years, extracorporeal shock wave therapy has been widely used in the treatment of nonunion, delayed union of fracture, necrosis of femoral head and chronic strain disease of motor system. Mechanical physical stimulation has been found to induce differentiation of mesenchymal stem cells. Shock wave, as a mechanical physical stimulation, can induce mesenchymal stem cells to differentiate into osteoblasts, but this process is not spontaneous and may involve the regulation of multiple signaling pathways. The MAPK pathway may play an extremely important role in regulating osteogenesis, but it is not clear which one of the ERKN JNK pathways and which p38MAPK pathway is involved in the process of osteogenic differentiation through the MAPK pathway, and the role of each pathway in the process of osteogenic differentiation is not well understood. In this experiment, the optimal shock wave (working voltage 8.5 kv, action frequency 120 times) was obtained for isolation and culture of human bone marrow mesenchymal stem cells, blank control group was established, and ERKN JNK and p38MAPK pathway inhibitor were added respectively. The phosphorylation of p38MAPK and the expression of AP-1 (c-fos and c-jun mRNA) in MAPK pathway were detected by western blot. Cell proliferation was detected by MTT assay. The osteogenic characteristics of human bone marrow mesenchymal stem cells (BMSCs) were determined by the method of identification of osteogenic characteristics (detection of alkaline phosphatase expression by calcium cobalt staining and detection of calcium nodules and calcium deposition by Von Kossa staining) in order to determine whether shock wave induced the differentiation of human bone marrow mesenchymal stem cells into osteoblasts. To study the role of ERKN JNKN p38 MAPK pathway in the differentiation of human bone marrow mesenchymal stem cells into osteoblasts induced by extracorporeal shock wave. In the course of inducing osteoblast differentiation from human bone marrow mesenchymal stem cells (BMSCs) induced by shock wave, it was confirmed by Western blot method that early phosphorylation activation of ERKN JNK and p38MAPK pathway, both ERKN JNK and p38MAPK pathway were involved in osteoblast proliferation. To divide. The results showed that p38MAPK pathway promoted the proliferation of human bone marrow mesenchymal stem cells and inhibited the proliferation of human bone marrow mesenchymal stem cells after inhibition of ERK pathway. The inhibitory effect of JNK pathway on osteoblast differentiation was decreased, but there was no significant difference between the two groups, and there was no obvious effect on the proliferation of osteoblasts. The expression of AP-1 (c-fos and c-jun mRNA) in the substrate of MAPK was obviously inhibited after inhibiting the p38MAPK pathway. Osteogenic identification showed that the differentiation into osteoblasts was obviously decreased in a dose-dependent manner, and the low concentration of p38MAPK inhibitor could promote the proliferation of cells. The same condition was used to induce human bone marrow mesenchymal stem cells (BMSCs) to differentiate into osteoblasts under the same conditions. The results indicated that p38MAPK pathway was the main pathway of inducing human bone marrow mesenchymal stem cells to differentiate into osteoblasts by shock wave.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R329

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 王芳;奶山羊骨髓間充質(zhì)干細(xì)胞的分離培養(yǎng)及向雄性生殖細(xì)胞誘導(dǎo)分化[D];西北農(nóng)林科技大學(xué);2011年

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