ABSTRACT
Objective@#To study the effects of nerve growth factor (NGF) on the proliferation, osteogenic differentiation and mineralization of type 2 diabetic mice bone marrow stromal cell (BMSC), providing basis for clinical application of NGF.@*Methods@#Three 8-week-old male db/db mice and two 8-week-old male C57BL/6J mice were used in the study. BMSC derived from femur were cultured though adherence method. BMSC of C57BL/6J mice and db/db mice was divided into normal group and diabetic group to conduct the osteogenic potential experiment, named experiment one. In experiment two, diabetic BMSC was divided into 3 groups: diabetic control group, NGF group, and K252a+NGF group [K252a was the inhibitor of tyrosine kinase A (TrkA), which was the high affinity receptor of NGF], to investigate effect of NGF on osteogenic potential of diabetic mice BMSC. After seeding BMSC, K252a was added into K252a+NGF group, then NGF was added 30 min later. NGF was added into NGF group and K252a+NGF group, but not diabetic control group. The proliferation of BMSC at 1, 3, 5 and 7 d in experiment one and the proliferation of BMSC at 1, 2 and 3 d in experiment two were evaluated through methyl thiazolyl tetrazolium, and the level of alkaline phosphatase (ALP) at 3, 5 and 7 d in both experiments were measured. After being osteogenic induced for 14 d, mineralized nodules in both experiments were quantitated by alizarin red calcium stain. Five holes were set in every group, and all experiments were repeated 3 times.@*Results@#The BMSC proliferation of diabetic group was significantly higher than that of the normal group at 3, 5 and 7 d (P<0.05). After being osteogenic inducted for 3, 5 and 7 d, ALP level of diabetic group were significantly lower than that of normal group (P<0.05). After being osteogenic inducted for 14 d, calcium nodule count of diabetic group [(23.1±6.4) nodule/field] were significantly lower than that of normal group [(36.9±7.9) nodule/field](P<0.05). At 1, 2 and 3 d, BMSC proliferations of diabetic control group, NGF group and K252a+NGF group were not statistically different (P>0.05). After being osteogenic inducted for 3 and 5 d, ALP level of NGF group was significantly higher than that of diabetic control group (P<0.05). After being osteogenic inducted for 3, 5, and 7 d, ALP level of K252a+NGF group was significantly lower than that of NGF group (P<0.05) and diabetic control group (P<0.05). After being osteogenic induced for 14 d, calcium nodule count of NGF group [(45.2±6.8) nodule/field] was significantly more than that of diabetic control group [(23.1±6.4) nodule/field](P<0.05); while calcium nodule count of K252a+NGF group [(18.0±4.5) nodule/field] was significantly less than that of NGF group (P<0.05) and diabetic control group (P<0.05).@*Conclusions@#The differentiation and mineralization of type 2 diabetic mice BMSC was significantly reduced. NGF promoted the osteoblastic differentiation and mineralization of diabetic mice BMSC in viro though combining with TrkA.
ABSTRACT
BACKGROUND: Bone morphogenetic protein-2(BMP-2) production of targeted cells is promoted by transfection of adenoviral vectors containing gene, but there are some immune responses. Transfection with plasmid as vector holds promise.OBJECTIVE: To explore the feasibility to construct human bone morphogenetic protein-2 eukaryotic expression vector labeled with green fluorescent protein (GFP).DESIGN: Single sample observation.SETTING: Tianjin Hospital.MATERIALS: The experiment was performed at the Key Laboratory of Hormone and Development, Ministry of Health, Tianjin Medical University from March 2006 to March 2007. pcDNA3.1/CT-hBMP2 plasmid containing full-length hBMP2 gene fragment was provided by Dr. Li; bicistronic eukaryotic expression vector pSELECT-GFPzeo-MCS and Zeo was provided by Invivogen; pTA2(R)-T Easy by Dingguo, China; restriction enzymes BamHI and NheI, T4 DNA ligase by Jingmei Biotech; PCR upstream and downstream primer synthesis and sequencing by Augct, Beijing.METHODS: With pcDNA3.1/CT-hBMP2 as template, hBMP2 target fragment was subcloned by PCR binding with designed specific primers. The fragment was bound with pTA2-T-easy and pSELECT-GFPzeo-MCS, separately, and transfected into DH5 α cells. pSELECT-GFPzeo-hBMP2 containing GFP was obtained after screening.MAIN OUTCOME MEASURES: hBMP2 sequence was identified by PCR; whether hBMP2 was cloned into pTA2-hBMP2 and pSELECT-GFPzeo-MCS was identified by digestion and sequencing.RESULTS: A target fragment of 1 216 bp was obtained by PCR amplification, and cloned into pTA2-T-easy and pSELECT-GFPZeo-MCS. The screening and sequencing results showed that the target fragment was 100% matched with BMP2cDNA sequence (NM-001200) from GenBank.CONCLUSION: hBMP2 eukaryotic expression vector labeled with green fluorescent protein is successfully constructed.