不同发育阶段绒山羊皮肤中FGF5基因mRNA表达的RT-PCR检测

为深入研究成纤维细胞生长因子5对毛囊生长发育的生物学功能提供理论依据。在10月左右和1月左右,即皮肤毛囊处于生长旺期和退行期时采集了12只内蒙古阿尔巴斯白绒山羊皮样,利用TRIZOL试剂盒提取皮肤总RNA(一步法),利用RT-PCR方法检测FGF5基因在绒山羊绒毛周期性生长不同阶段皮肤中的表达分布情况,试验结果表明,FGF5基因mRNA在绒山羊毛囊生长旺期和退行期均有表达,却只得到了一种剪切形式的表达,经测序是长片段,而缺失外显子2的短片段形式没有检测到。

TALENs编辑绵羊成纤维细胞FGF 5基因

类转录激活因子效应物(Transcription activator-like effector,TALE)已经成为基因组编辑和基因转录调控的有效工具,在多个物种的基因组中实现特定位点的删除、插入和突变。针对绵羊成纤维细胞生长因子5(Fibroblast growth factor 5,FGF5)基因起始密码子ATG位点,设计构建TALENs(Transcription activator-like effector nucleases,TALENs)。通过比较分析正常培养和基因组编辑处理的绵羊成纤维细胞,电转TALENs组合,Surveyor突变检测,筛选获得1对有效的TALENs。有限稀释细胞传代培养,PCR扩增FGF5基因片段,经PAGE检测筛选发生突变的细胞,测序确认FGF5基因ATG位点产生缺失突变细胞。获得具有编辑活性的TALENs,为该基因的定点编辑奠定基础。Surveyor检测和测序结果表明,在绵羊FGF5基因ATG起始密码子上游104碱基位点,存在1个G/C单核苷酸多态。

Novel nano-microspheres containing chitosan, hyaluronic acid, and chondroitin sulfate deliver growth and differentiation factor-5 plasmid for osteoarthritis gene therapy

Objective:To construct a novel non-viral vector loaded with growth and differentiation factor-5(GDF-5) plasmid using chitosan,hyaluronic acid,and chondroitin sulfate for osteoarthritis (OA)gene therapy.Methods: Nano-microspheres (NMPs)were prepared by mixing chitosan,hyaluronic acid,and chondreitin sulfate.GDF-5 plasmid was encapsulated in the NMPs through electrostatic adsorption.The basic characteristics of the NMPs were observed,and then they were co-cultured with chondrocytes to observe their effects on extracellular matrix (ECM) protein expression.Finally,NMPs loaded with GDF-5were inje.cted into the articular cavities of rabbits to observe their therapeutic effects on OA in vivo.Results:NMPs exhibited good physicochemical properties and low cytotoxicity.Their average diameter was (0.61±0.20)μm,and encapsulation efficiency was (38.19±0.36)%.According to Cell Counting Kit-8(CCK-8)assay,relative cell viability was 75%-99%when the total weight of NMPs was less than 560μg. Transfection efficiency was (62.0±2.1)% in a liposome group,and (60.0±1.8)% in the NMP group.There was no sig- nificant difference between the two groups (P>0.05).Immunohistochemical staining results suggested that NMPs can successfully transfect chondrocytes and stimulate ECM protein expression in vitro.Compared with the control groups, the NMP group significantly promoted the expression of chondrocyte ECM in vivo (P<0.05),as shown by analysis of the biochemical composition of chondrocyte ECM.When NMPs were injected into OA model rabbits,the expression of ECM proteins in chondrocytes was significantly promoted and the progression of OA was slowed down.Conclusions: Based on these data,we think that these NMPs with excellent physicochemical and biological properties could be promising non-viral vectors for OA gene therapy.

Adenovirus-mediated GDF-5 promotes the extracellular matrix expression in degenerative nucleus pulposus cells

Objective： To construct a recombinant adenovirus vector-carrying human growth and differentiation factor-5 （GDF-5） gene, investigate the biological effects of adenovirus-mediated GDF-5 （Ad-GDF-5） on extracellular matrix （ECM） expression in human degenerative disc nucleus pulposus （NP） cells, and explore a candidate gene therapy method for intervertebral disc degeneration （IDD）. Methods： Human NP cells of a degenerative disc were isolated, cultured, and infected with Ad-GDF-5 using the AdEasy-1 adenovirus vector system. On Days 3, 7, 14, and 21, the contents of the sulfated glycosaminoglycan （sGAG）, deoxyribonucleic acid （DNA） and hydroxyproline （Hyp）, synthesis of proteoglycan and collagen II, gene expression of collagen II and aggrecan, and NP cell proliferation were assessed. Results： The adenovirus was an effective vehicle for gene delivery with prolonged expression of GDF-5. Biochemical analysis revealed increased sGAG and Hyp contents in human NP cells infected by Ad-GDF-5 whereas there was no conspicuous change in basal medium （BM） or Ad-green fluorescent protein （GFP） groups. Only cells in the Ad-GDF-5 group promoted the production of ECM, as demonstrated by the secretion of proteoglycan and up-regulation of collagen II and aggrecan at both protein and mRNA levels. The NP cell proliferation was significantly promoted. Conclusions： The data suggest that Ad-GDF-5 gene therapy is a potential treatment for IDD, which restores the functions of degenerative intervertebral disc through enhancing the ECM production of human NP ceils.

Relationship between FGF12 expression in high-grade gliomas and clinical features

Objective:gliomas are the most common intracranial tumors.Fibroblast growth factor-12(FGF12),which belongs to the fibroblast growth factor(FGFs)family,plays an important role in cell mitosis,as well as in other life functions,such as embryo development,tissue repair,cell proliferation,and tumor growth and invasion.The purpose of this study was to explore the potential value of FGF12 in high-grade gliomas and to predict its drug sensitivity.To provide a possible therapeutic target for glioma.Methods:high-grade glioma gene expression data and clinical information were downloaded from the gene expression omnibus(GEO)database,using the R language“impute”and“survival”survival analysis package.The FGF12 genes closely related to survival were screened,a survival curve was drawn,and clinical correlation analysis was conducted.The differentially expressed genes(DEGs)were defined as |logFC|>1,adj.PVal<0.05 as the standard.We used the David for Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis,and constructed the protein-protein interactions(PPI)network.Then we used the Connectivity Map(CMAP)database for drug location,and the validation group was verified by the Chinese Glioma Genome Atlas(CGGA)database in the same way.Results:we found that high FGF12 expression was associated with a higher survival rate.The same validation was performed in the validation group through the CGGA database,and the survival curve showed the same trend.The expression level of FGF12 is an independent factor that affects the life time and status of the samples,and it is a low risk factor.GO enrichment analysis showed that differential genes were enriched in matrix transmembrane transporter activity,ion channels and calcium ion active channels.KEGG showed that DEGs were enriched in the phosphatidylinositol 3-kinase(PI3K)-protein kinase B(Akt)signaling pathway,dopaminergic synapse and cyclic adenosine monophosphate(cAMP)signaling pathway.Four seed genes,GRIA2,COLLA2,GRIA4 and HES6,were obtained by PPI network analysis.The cAMP was used to analyze and obtained 7 small molecule drugs,such as merbromin,naloxone,AH-2384&ticarcillin,vincamine,amoxicillin,azacyclonol,which may be helpful in the prognosis of high-grade gliomas.Conclusion:FGF12 and its pathway may serve as a biomarker or therapeutic target for high-grade gliomas.