【摘要】：目的：建立從人臍帶中有效分離培養(yǎng)間充質(zhì)干細(xì)胞(MSCs, mesenchymal stem cells),并能進(jìn)行體外擴(kuò)增傳代的方法；同時構(gòu)建并表達(dá)、純化人角質(zhì)細(xì)胞生長因子2 (keratinocyte growth factor 2, KGF-2)以及穿膜型KGF-2,研究其對人臍帶MSCs(hUC-MSCs, human umbilical cord MSCs)增殖的影響；并研究KGF-2對細(xì)胞成脂成骨成脂分化的影響,同時對其作用機(jī)制進(jìn)行初步探討。 方法：采用組織塊貼壁法,體外分離獲得hUC-MSCs;構(gòu)建pET28a-KGF-2及TAT-pET28a-KGF-2重組質(zhì)粒,將其轉(zhuǎn)化大腸桿菌Rosetta(DE3),通過表達(dá)條件優(yōu)化獲得目的蛋白并進(jìn)行可溶性純化；在對所純化蛋白進(jìn)行鑒定及活性檢測后,MTT法檢測其對細(xì)胞增殖的作用；利用Western blot及試劑盒檢測KGF-2蛋白對細(xì)胞堿性磷酸酶ALP及鈣沉積量等成骨標(biāo)記的影響,通過實(shí)時定量PCR檢測KGF-2蛋白對PPARγ2及GPDH等成脂標(biāo)記的影響;利用Western blot檢測MAPK通路中ERK1/2及p38途徑的改變。 結(jié)果：1、組織塊貼壁法可在10-15天內(nèi)獲得可體外擴(kuò)增傳代的,且具有MSCs表面抗原標(biāo)記的hUC-MSCs,體外誘導(dǎo)可分化為脂肪細(xì)胞和成骨細(xì)胞。 2、成功構(gòu)建pET28a-KGF-2及TAT-pET28a-KGF-2重組質(zhì)粒,通過表達(dá)條件的優(yōu)化獲得高效、可溶性表達(dá)的KGF-2及TAT-KGF-2重組蛋白。 3、高濃度的KGF-2及TAT-KGF-2重組蛋白均可對hUC-MSCs產(chǎn)生促增殖作用,且TAT-KGF-2達(dá)到最大細(xì)胞增殖速率所需蛋白濃度高于KGF-2重組蛋白。 4、一定濃度的KGF-2重組蛋白可有效促進(jìn)細(xì)胞成骨分化,但對成脂分化無明顯影響。在對其機(jī)制的研究中發(fā)現(xiàn),KGF-2可激活ERK1/2通路,在該通路被抑制時,KGF-2的促成骨分化作用也受到抑制。結(jié)論：成功分離得到可體外傳代的hUC-MSCs;獲得高效、可溶性表達(dá)、且具有活性的KGF-2及TAT-KGF-2重組蛋白,其能夠促進(jìn)hUC-MSCs的增殖；其中KGF-2重組蛋白對細(xì)胞成骨分化也具有促進(jìn)作用,該作用很可能是通過激活ERK1/2途徑實(shí)現(xiàn)。
[Abstract]:Objective: to establish an effective method to isolate and culture mesenchymal stem cells (MSCs, mesenchymal stem cells),) from human umbilical cord and to amplify and subculture them in vitro. The expression and expression of human keratinocyte growth factor 2 (KGF-2) and transmembrane KGF-2, were used to study the effect of human keratinocyte growth factor 2 (KGF-2) on the proliferation of MSCs (hUC-MSCs, human umbilical cord MSCs) in human umbilical cord. The effects of KGF-2 on adipogenic differentiation of osteoblasts were also studied. Methods: hUC-MSCs; was isolated by tissue mass adherence in vitro. The recombinant plasmids of pET28a-KGF-2 and TAT-pET28a-KGF-2 were constructed and transformed into Escherichia coli Rosetta (DE3). After the purified protein was identified and its activity was detected, the effect of the purified protein on cell proliferation was detected by MTT assay. The effects of KGF-2 protein on osteogenic markers such as alkaline phosphatase ALP and calcium deposition were detected by Western blot and kit. The effects of KGF-2 protein on PPAR 緯 2 and GPDH were detected by real-time quantitative PCR. The changes of ERK1/2 and p38 pathway in MAPK pathway were detected by Western blot. Results: 1. HUC-MSCs, with MSCs surface antigen could be induced to differentiate into adipocytes and osteoblasts in vitro. 2. The recombinant plasmids of pET28a-KGF-2 and TAT-pET28a-KGF-2 were successfully constructed, and the recombinant proteins of KGF-2 and TAT-KGF-2 were obtained by optimizing the expression conditions. 3High concentration of KGF-2 and TAT-KGF-2 could promote the proliferation of hUC-MSCs, and the protein concentration of TAT-KGF-2 to reach the maximum cell proliferation rate was higher than that of KGF-2 recombinant protein. 4. KGF-2 recombinant protein at a certain concentration could effectively promote osteogenic differentiation, but had no effect on adipogenic differentiation. It was found that KGF-2 could activate the ERK1/2 pathway, and when the pathway was inhibited, the effect of KGF-2 on bone differentiation was also inhibited. Conclusion: the recombinant protein of KGF-2 and TAT-KGF-2, which can be subcultured in vitro, is highly efficient, soluble and active, which can promote the proliferation of hUC-MSCs. Among them, KGF-2 recombinant protein can also promote osteogenic differentiation, which may be achieved by activating the ERK1/2 pathway.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 Tyler ZARUBIN;Activation and signaling of the p38 MAP kinase pathway[J];Cell Research;2005年01期

2 廖清船,肖洲生,秦艷芳,趙彥,潘瑋,劉霆,周宏灝;p44/42和p38 MAPKs在骨髓間充質(zhì)干細(xì)胞向成骨細(xì)胞分化中發(fā)揮不同的功能[J];中國骨質(zhì)疏松雜志;2004年03期

，

本文編號：2365736