The integral mature peptide gene of human growth differentiation factor-5 (GDF-5) was cloned to provide the essential foundation for study on the biological characteristics of GDF-5 at gene and protein levels. Two primers were chemosynthesized according to the hGDF-5 sequence reported in Genbank. The hGDF-5 gene was gained by RT-PCR methods from the total RNA extracted from human fetus cartilage tissue, and was cloned into vector pMD18-T. The sequence of recombinant plasmid pMD18-T-hGDF-5 was analyzed by sequence analysis. DNA agarose gel electrophoresis showed that the product of RT-PCR was about 380bp, and double enzyme digestion of the recombinant plasmid corresponded with it. The result of sequence assay was in agreement with the reported hGDF-5 sequence in Genbank. Our results showed that the integral mature peptide gene of human GDF-5 was cloned successfully from human fetal cartilage tissue, and totally identified with the sequence of human GDF-5 in Genbank.
Bone Morphogenetic Proteins ; biosynthesis ; genetics ; Cartilage ; chemistry ; Cloning, Molecular ; Fetus ; Genetic Vectors ; Growth Differentiation Factor 5 ; Humans ; Transforming Growth Factor beta ; biosynthesis ; genetics

This experimental study was aimed to construct the recombinant adenovirus vector containing human GDF-5 gene, and to use it for infecting human MSCs and detecting the expression of the gene GDF-5. The core sequence of human GDF-5 was amplified by PCR from pCMV-SPORT6, and then was cloned to pAdtrack-CMV. The linearized shuttle plasmid pAdtrack-CMV-GDF-5 was homogenously recombined with pAdeasy-1 in BJ5183. The potential clone was analyzed by restriction endonuclease digestion. The correct clone was linearized and transfected into QBI-293 cells for packing and amplifying so as to obtain adenovirus pAd-GDF-5 and identify it, while the titer was also determined by TCID50. MSCs were infected by the harvested virus, and the expression of GDF-5 was detected by RT-PCR. The recombinant adenovirus vector containing human GDF-5 gene was constructed successfully, its titer was 1 x 10(9) PFU/ml, and it could infect MSCs efficiently. The human MSCs infected by constructed adenovirus vector could continue expressing GDF-5 in a certain time, and the transgenic MSCs would be much potential on tissue regeneration.
Adenoviridae ; genetics ; metabolism ; Genetic Vectors ; genetics ; Growth Differentiation Factor 5 ; biosynthesis ; genetics ; Humans ; Mesenchymal Stromal Cells ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Transfection

Brachydactyly type C (BDC) is characterized by shortening of the middle phalanges of the index, middle, and little fingers. Hyperphalangy of the index and middle finger and shortening of the first metacarpal can also be observed. BDC is a rare genetic condition associated with the GDF5 gene, and this condition has not been confirmed by genetic analysis so far in the Korean population. Herein, we present a case of a 6-yr-old girl diagnosed with BDC confirmed by molecular genetic analysis. The patient presented with shortening of the second and third digits of both hands. Sequence analysis of the GDF5 gene was performed and the pathogenic mutation, c.1312C>T (p.Arg438Cys), was identified. Interestingly, this mutation was previously described in a patient who presented with the absence of the middle phalanges in the second through fifth toes. However, our patient showed no involvement of the feet. Considering intrafamilial and interfamilial variability, molecular analysis of isolated brachydactyly is warranted to elucidate the genetic origin and establish a diagnosis.
Asian Continental Ancestry Group/*genetics ; Brachydactyly/diagnosis/*genetics ; Child ; DNA Mutational Analysis ; Female ; Fingers/anatomy & histology ; Growth Differentiation Factor 5/*genetics ; Humans ; Mutation ; Republic of Korea

Adult ; Carrier Proteins ; genetics ; Female ; Finger Joint ; physiopathology ; Growth Differentiation Factor 5 ; genetics ; Humans ; Joint Diseases ; congenital ; genetics ; physiopathology ; Male ; Middle Aged ; Pedigree ; Young Adult

Osteoarthritis is a common cause of functional deterioration in older adults and is an immense burden on the aging population. Altered chondrogenesis is the most important pathophysiological process involved in the development of osteoarthritis. However, the molecular mechanism underlying the regulation of chondrogenesis in patients with osteoarthritis requires further elucidation, particularly with respect to the role of microRNAs. MiR-21 expression in cartilage specimens was examined in 10 patients with knee osteoarthritis and 10 traumatic amputees. The effect of miR-21 on chondrogenesis was also investigated in a chondrocyte cell line. The effect of miR-21 on the expression of growth differentiation factor 5 (GDF-5) was further assessed by luciferase reporter assay and western blot. We found that endogenous miR-21 is upregulated in osteoarthritis patients, and overexpression of miR-21 could attenuate the process of chondrogenesis. Furthermore, we identified GDF-5 as the direct target of miR-21 during the regulation of chondrogenesis. Our data suggest that miR-21 has an important role in the pathogenesis of osteoarthritis and is a potential therapeutic target.
Cartilage/metabolism/pathology ; Case-Control Studies ; Cell Line ; Chondrocytes/*metabolism/pathology ; Growth Differentiation Factor 5/genetics/*metabolism ; Humans ; MicroRNAs/genetics/*metabolism ; Osteoarthritis/*metabolism/pathology ; Up-Regulation

OBJECTIVETo analyze the relationship of genetic and mechanical factors with age of onset of knee osteoarthritis (OA).

METHODSCross-sectional and longitudinal studies were conducted in 863 patients with knee OA and 999 age-matched controls from Chinese Han population with complete clinical data. Single nucleotide polymorphisms of + 104T/C in rs143383 of the growth differentiation factor 5 (GDF5) gene and the aspartic acid repeat polymorphism of the asporin gene ASPN were genotyped by taqman probe and polyacrylamide gel electrophoresis respectively.

RESULTSBody mass index (BMI) in subjects with knee OA was significantly higher than that in controls (P< 0.01). Obesity (BMI≥ 25) was a high risk factor for knee OA (P< 0.01). Regression analysis showed that there was a certain correlation between the BMI and age of onset of the knee OA (Pearson= 0.15, P< 0.01). To analyze the BMI distribution, the two groups were subgrouped by age with 5-year interval. The BMI in early-onset patients was lower than that in the late-onset patients (F= 2.497, P= 0.011). However, there was no significant difference in the control subgroups. Frequencies of the GDF5 and ASPN alleles distributed differently between the early- and late-onset patients (P< 0.05).

CONCLUSIONGenetic factors play an important role in knee osteoarthritis of early-onset patients, whereas mechanical factor play an important part in knee osteoarthritis of late-onset patients.

Adult ; Age of Onset ; Aged ; Aging ; genetics ; Biomechanical Phenomena ; Body Weight ; Case-Control Studies ; Extracellular Matrix Proteins ; genetics ; Female ; Gene Frequency ; Growth Differentiation Factor 5 ; genetics ; Humans ; Male ; Mechanical Phenomena ; Middle Aged ; Osteoarthritis, Knee ; epidemiology ; etiology ; genetics ; physiopathology

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 chondroitin 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-5 were injected 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 560 μg. Transfection efficiency was (62.0±2.1)% in a liposome group, and (60.0±1.8)% in the NMP group. There was no significant 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.
Animals ; Cell Differentiation ; Cell Survival/drug effects* ; Chitosan/chemistry* ; Chondrocytes/cytology* ; Chondroitin Sulfates/chemistry* ; Drug Carriers ; Extracellular Matrix/metabolism* ; Genetic Therapy/methods* ; Growth Differentiation Factor 5/genetics* ; Hyaluronic Acid/chemistry* ; Microspheres ; Nanomedicine ; Osteoarthritis/therapy* ; Plasmids/metabolism* ; Rabbits

Insulin-like growth factor-binding proteins(IGFBPs) are critical regulators of the mitogenic activity of insulin-like growth factors (IGFs). IGFBP5, one of these IGFBPs, has special structural features, including a nuclear transport domain, heparin-binding motif, and IGF/extracellular matrix/acid-labile subunit-binding sites. Furthermore, IGFBP5 has several functional effects on carcinogenesis and even normal cell processes, such as cell growth, death, motility, and tissue remodeling. These biological effects are sometimes related with IGF (IGF-dependent effects) and sometimes not (IGF-independent effects). The functional role of IGFBP5 is most likely determined in a cell-type and tissue-type specific manner but also depends on cell context, especially in terms of the diversity of interacting proteins and the potential for nuclear localization. Clinical findings show that IGFBP5 has the potential to be a useful clinical biomarker for predicting response to therapy and clinical outcome of cancer patients. In this review, we summarize the functional diversity and clinical importance of IGFBP5 in different types of cancers.
Animals ; Apoptosis ; Cell Differentiation ; Cell Movement ; Humans ; Insulin-Like Growth Factor Binding Protein 5 ; genetics ; metabolism ; physiology ; Neoplasm Metastasis ; Neoplasms ; metabolism ; pathology ; Protein Binding ; RNA, Messenger ; metabolism ; Signal Transduction ; Somatomedins ; metabolism

It is widely known that hypoxia can promote chondrogenesis of human bone marrow derived mesenchymal stem cells (hMSCs) in monolayer cultures. However, the direct impact of oxygen tension on hMSC differentiation in three-dimensional cultures is still unknown. This research was designed to observe the direct impact of oxygen tension on the ability of hMSCs to "self assemble" into tissue-engineered cartilage constructs. hMSCs were cultured in chondrogenic medium (CM) containing 100 ng/mL growth differentiation factor 5 (GDF-5) at 5% (hypoxia) and 21% (normoxia) O2 levels in monolayer cultures for 3 weeks. After differentiation, the cells were digested and employed in a self-assembly process to produce tissue-engineered constructs under hypoxic and normoxic conditions in vitro. The aggrecan and type II collagen expression, and type X collagen in the self-assembled constructs were assessed by using immunofluorescent and immunochemical staining respectively. The methods of dimethylmethylene blue (DMMB), hydroxyproline and PicoGreen were used to measure the total collagen content, glycosaminoglycan (GAG) content and the number of viable cells in each construct, respectively. The expression of type II collagen and aggrecan under hypoxic conditions was increased significantly as compared with that under normoxic conditions. In contrast, type X collagen expression was down-regulated in the hypoxic group. Moreover, the constructs in hypoxic group showed more significantly increased total collagen and GAG than in normoxic group, which were more close to those of the natural cartilage. These findings demonstrated that hypoxia enhanced chondrogenesis of in vitro, scaffold-free, tissue-engineered constructs generated using hMSCs induced by GDF-5. In hypoxic environments, the self-assembled constructs have a Thistological appearance and biochemical parameters similar to those of the natural cartilage.
Aggrecans ; genetics ; metabolism ; Bone Marrow Cells ; drug effects ; metabolism ; Cartilage ; cytology ; metabolism ; Cell Differentiation ; drug effects ; genetics ; Cell Hypoxia ; Cells, Cultured ; Chondrogenesis ; drug effects ; genetics ; Collagen Type II ; genetics ; metabolism ; Collagen Type X ; metabolism ; Female ; Gene Expression ; drug effects ; Glycosaminoglycans ; metabolism ; Growth Differentiation Factor 5 ; pharmacology ; Humans ; Immunohistochemistry ; Male ; Mesenchymal Stromal Cells ; drug effects ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Tissue Engineering ; methods