KGF-2和穿膜型KGF-2的构建、纯化及其对人脐带间充质干细胞增殖、分化的影响
发布时间：2018-12-06 08:18
【摘要】：目的：建立从人脐带中有效分离培养间充质干细胞(MSCs, mesenchymal stem cells),并能进行体外扩增传代的方法；同时构建并表达、纯化人角质细胞生长因子2 (keratinocyte growth factor 2, KGF-2)以及穿膜型KGF-2,研究其对人脐带MSCs(hUC-MSCs, human umbilical cord MSCs)增殖的影响；并研究KGF-2对细胞成脂成骨成脂分化的影响,同时对其作用机制进行初步探讨。 方法：采用组织块贴壁法,体外分离获得hUC-MSCs;构建pET28a-KGF-2及TAT-pET28a-KGF-2重组质粒,将其转化大肠杆菌Rosetta(DE3),通过表达条件优化获得目的蛋白并进行可溶性纯化；在对所纯化蛋白进行鉴定及活性检测后,MTT法检测其对细胞增殖的作用；利用Western blot及试剂盒检测KGF-2蛋白对细胞碱性磷酸酶ALP及钙沉积量等成骨标记的影响,通过实时定量PCR检测KGF-2蛋白对PPARγ2及GPDH等成脂标记的影响;利用Western blot检测MAPK通路中ERK1/2及p38途径的改变。 结果：1、组织块贴壁法可在10-15天内获得可体外扩增传代的,且具有MSCs表面抗原标记的hUC-MSCs,体外诱导可分化为脂肪细胞和成骨细胞。 2、成功构建pET28a-KGF-2及TAT-pET28a-KGF-2重组质粒,通过表达条件的优化获得高效、可溶性表达的KGF-2及TAT-KGF-2重组蛋白。 3、高浓度的KGF-2及TAT-KGF-2重组蛋白均可对hUC-MSCs产生促增殖作用,且TAT-KGF-2达到最大细胞增殖速率所需蛋白浓度高于KGF-2重组蛋白。 4、一定浓度的KGF-2重组蛋白可有效促进细胞成骨分化,但对成脂分化无明显影响。在对其机制的研究中发现,KGF-2可激活ERK1/2通路,在该通路被抑制时,KGF-2的促成骨分化作用也受到抑制。结论：成功分离得到可体外传代的hUC-MSCs;获得高效、可溶性表达、且具有活性的KGF-2及TAT-KGF-2重组蛋白,其能够促进hUC-MSCs的增殖；其中KGF-2重组蛋白对细胞成骨分化也具有促进作用,该作用很可能是通过激活ERK1/2途径实现。
[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 纬 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.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329
本文编号：2365736
[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 纬 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.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329
【参考文献】
相关期刊论文 前2条
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,秦艳芳,赵彦,潘玮,刘霆,周宏灏;p44/42和p38 MAPKs在骨髓间充质干细胞向成骨细胞分化中发挥不同的功能[J];中国骨质疏松杂志;2004年03期
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