[1]陈方舟 谭俊峰 孙凯 刘洋△.生长分化因子5基因转染大鼠脂肪干细胞在RAD16-Ⅱ自组装肽纳米凝胶中的培养[J].中国中医骨伤科杂志,2018,26(08):11-13，17.
　CHEN Fangzhou TAN Junfeng SUN Kai LIU Yang.Transfecting Growth and Differentiation Factor 5 Gene of Adipose-derived Stem Cells Cultured in Self-assembling Peptide Hydrogel of RAD16-Ⅱ[J].Chinese Journal of Traditional Medical Traumatology & Orthopedics,2018,26(08):11-13，17.
点击复制

生长分化因子5基因转染大鼠脂肪干细胞在RAD16-Ⅱ自组装肽纳米凝胶中的培养()
分享到：

《中国中医骨伤科杂志》[ISSN:1005-0205/CN:42-1340/R]

卷:
第26卷
期数:
2018年08期
页码:
11-13，17
栏目:
实验研究
出版日期:
2018-08-02

文章信息/Info

Title:
Transfecting Growth and Differentiation Factor 5 Gene of Adipose-derived Stem Cells Cultured in Self-assembling Peptide Hydrogel of RAD16-Ⅱ
文章编号:
1005-0205(2018)08-0011-03
作者:
陈方舟1 谭俊峰1 孙凯2 刘洋1△
1武汉市第五医院(江汉大学第二附属医院)骨科(武汉,430050) 2武汉大学人民医院
Author(s):
CHEN Fangzhou1 TAN Junfeng1 SUN Kai2 LIU Yang1△
1Fifth Hospital of Wuhan(Second Hospital Affiliated of Jianghan University), Wuhan 430050, China; 2Renmin Hospital of Wuhan University, Wuhan 430060, China.
关键词:
生长分化因子5;  脂肪干细胞;  自组装肽纳米凝胶
Keywords:
Keywords: growth and differentiation factor-5;  adipose-derived stem cells;  self-assembling peptide hydrogel
分类号:
R-33
文献标志码:
A
摘要:
目的:将生长分化因子5(GDF-5)基因转染于大鼠脂肪干细胞并将其培养于RAD16-Ⅱ自组装肽纳米凝胶,并比较其与二维培养大鼠脂肪干细胞成软骨细胞分化效率。方法:选取雄性SD大鼠采用酶消化-密度梯度离心法提取脂肪干细胞行体外培养,并通过流式细胞术鉴定脂肪干细胞。采用LipofectamineTM2000进行脂质体pcDNA3.1(+)/GDF-5重组质粒瞬间转染大鼠脂肪干细胞。将转染大鼠脂肪干细胞分别置于三维RAD16-Ⅱ自组装肽纳米凝胶(实验组)及普通二维培养基培养(对照组)。通过RT-PCR、免疫组化及免疫荧光等方法检测培养1和2周后特异性细胞外基质Ⅱ型胶原蛋白(Collagen Ⅱ)和蛋白聚糖(Aggrecan)表达水平。结果:RT-PCR、免疫组化及免疫荧光检测结果表明RAD16-Ⅱ自组装肽纳米凝胶组细胞在诱导7 d后有Collagen Ⅱ和Aggrecan表达,并且RT-PCR检测结果表明RAD16-Ⅱ自组装肽纳米凝胶组细胞的Collagen Ⅱ和Aggrecan mRNA表达水平较对照组高,差异有统计学意义(P<0.05)。结论:三维RAD16-Ⅱ自组装肽纳米凝胶有利于生长分化因子5转染脂肪干细胞Ⅱ型胶原蛋白和蛋白聚糖的表达,促进生长分化因子5转染脂肪干细胞向成软骨细胞方向分化,RAD16-Ⅱ自组装肽纳米凝胶是较好的软骨组织工程细胞支架。
Abstract:
Abstract Objective:To transfect the GDF-5 gene into ADSCs cultured in three-dimensional self-assembling peptide hydrogel of RAD16-Ⅱ, and to compare the transfection efficiency with ADSCs cultured in two-dimensional medium.Methods: Male SD rats were selected for in vitro culture of adipose stem cells by enzyme digestion and density gradient centrifugation, and adipose stem cells were identified by flow cytometry.The liposome pcDNA3.1(+)/GDF-5 recombinant plasmid was transiently transfected into rat adipose stem cells using LipofectamineTM2000.Adipose-derived stem cells were transfected into three-dimensional RAD16-Ⅱ self-assembled peptide nanogels(experimental group)and normal two-dimensional culture medium(control group).The level of specific extracellular matrix Collagen Ⅱ and Aggrecan expression were measured by RT-PCR, immunohistochemistry, and immunofluorescence 1 and 2 weeks after culture.Results: The results of RT-PCR, immunohistochemistry and immunofluorescence showed that the RAD16-Ⅱ self-assembled peptide nanosels were expressed in Collagen Ⅱ and Aggrecan after 7 d of induction, and the RT-PCR detection results showed that the mRNA expression level of Collagen Ⅱ and Aggrecan in the RAD16-Ⅱ self-assembled peptide nanosels was higher than that of the control group, and the difference was statistically significant(P<0.05).Conclusion: Three-dimensional RAD16-Ⅱ self-assembling peptide nanogels are beneficial to the expression of type Ⅱ Collagen and proteoglycan in adipose stem cells transfected with growth factor 5, and promote the differentiation of adipose stem cells transfected with growth factor 5 into chondrocytes.RAD16-Ⅱ self-assembling peptide nanogels are a preferred scaffold for cartilage tissue engineering.

参考文献/References:

[1] Chen YL,Sun CK,Tsai TH,et al.Adipose-derived mesenchymal stem cells embedded in platelet-rich fibrin scaffolds promote angiogenesis,preserve heart function,and reduce left ventricular remodeling in rat acute myocardial infarction[J].Am J Transl Res,2015,7(5):781-803. [2] Feng C,Liu H,Yang Y,et al.Growth and differentiation factor-5 contributes to the structural and functional maintenance of the intervertebral disc[J].Cell Physiol Biochem,2015,35(1):1-16. [3] Suzuki E,Fujita D,Takahashi M,et al.Adipose tissue-derived stem cells as a therapeutic tool for cardiovascular disease[J].World J Cardiol,2015,7(8):454-465. [4] 吴磊,刘洋,谭俊峰.生长分化因子5诱导脂肪干细胞向成软骨细胞方向分化的影响[J].生物医学工程与临床,2017,21(4):429-434. [5] Murphy MK,Huey DJ,Hu JC,et al.TGF-β1,GDF-5,and BMP-2 stimulation induces chondrogenesis in expanded human articular chondrocytes and marrow-derived stromal cells[J].Stem Cells,2015,33(3):762-773. [6] Zuk P.Adipose-derived stem cells in tissue regeneration:a review[J].ISRN Stem Cell,2013(5). [7] 赵明璨,刘畅.脂肪干细胞在软骨组织工程中的研究进展[J].中国生物医学工程学报,2014,33(4):475-481. [8] Fan J,Park H,Tan S,et al.Enhanced osteogenesis of adipose derived stem cells with noggin suppression and delivery of BMP-2[J].PLOS One,2013,8(8):e72474. [9] Bigot N,Mouche A,Preti M,et al.Hypoxia differentially modulates the genomic stability of clinical-grade ADSCs and BM-MSCs in long-term culture[J].Stem Cells.2015,33(12):3608-3620. [10] Daans M,Luyten FP,Lories RJ.GDF-5 deficiency in mice is associate with instability-driven joint damage,gait and subchondral bone changes[J].Ann Rheum Dis,2011,70(1):208-213. [11] Chhabra A,Zijerdi D,Zhang J,et al.BMP-14 deficiency inhibits long bone fracuure healing:a biochemical,histologic,and radiographic assessment[J].J Orthop Trauma,2005,19(9):629-634. [12] Toulouse A,Collins GC,Sullivan AM.Neurotrophic effects of growth/differentiation factor 5 in a neuronal cell line[J].Neutrotox Res,2012,21(3):256-265. [13] Zaidi SH,Huang Q,Momen A,et al.Growth differentiation factor 5 regulates cardiac repair after myocardial infarction[J].J Am Coll Cardiol,2010,55(2):135-143. [14] Gao S,Zheng Y,Cai Q,et al.Different methods for inducing adipose-derived stem cells to differentiate into Schwann-like cells[J].Arch Med Sci,2015,11(4):886-892. [15] Zhu P,Liu J,Shi J,et al.Melatonin protects ADSCs from ROS and enhances their therapeutic potency in a rat model of myocardial infarction[J].J Cell Mol Med,2015,19(9):2232-2243. [16] Alexander TH,Sage AB,Chen AC,et al.Insulin-like growth factor Ⅰ and growth differentiation factor-5 promote the formation of tissue-engineered human nasal septal cartlage[J].Tissue Eng Part C Methods,2010,16(5):1213-1221. [17] 李明辉,刘洋,孙凯,等.生长分化因子5诱导脂肪干细胞向软骨细胞的转化[J].中组织工程研究,2016,20(51):7628-7633. [18] Holmes T,De Lacalle S,Su X,et al.Extensive neurite out-growth and active synapse formation on self-assembling peptide scaffolds[J].Proc Natl Acad Sci USA,2000,97(12):6728-6733. [19] Daniunaite K,Serenaite I,Misgirdaite R,et al.Epigenetic regulation of human adipose-derived stem cells differentiation[J].Mol Cell Biochem,2015,410(1-2):111-120.

备注/Memo

备注/Memo:
基金项目:武汉市科学技术局基金资助项目(2013060602010265) △通信作者 E-mail:liuyang19642000@163.com
更新日期/Last Update: 2018-08-02