Objective To investigate the survival profile of the intradermally injected mouse autologous skin fibroblasts and the changes of the collagen fibers by using green fluorescent protein labeling and two-photon fluorescence microscopy. Methods The cultured cells were transfected by EGFP lentivirus, and then the cells were injected into the corresponding mouse skin. Biopsy was taken from the animals after 1 and 2 months. The specimens made serial frozen sections, the survival profile of the injected cells and the changes of the collagen fibers were observed by two-photon fluorescence microscopy. The collagenic area and dermal thickness were measured with image analysis software, and statistical analysis was also carried out. Results Two-photon fluorescence microscopy showed clear images of the injected cells and collagen fibers. Both the area of collagen fibers and the dermal thickness were significantly increased in injected cells after 2 months (P<0.05), however, there were no difference between injected cells and control after 1 mouth (P>0.05). Conclusions Autologous cultured fibroblasts could survive in a long time after transplantating into the skin, and collagen could be newly produced, the depth of dermis increases, which provides a possibility to treat mini-defects of the tissue.

ObjectiveTo investigate the effect of basic fibroblast growth factor-polyactide release nanospheres on proliferation and adipogenic induction of adipose-derived stem cells in vitro.MethodsAdipose-derived stem cells were isolated and induced for three-line differentiation in vitro.The culture medium and inductive medium of stem cells were prepared containing 0,1,2,3,4 and 5 mg/ml basic fibroblast growth factor-polyactide release nanospheres,respectively.Adipose-derived stem cells were cultured ina 96-well plate and replaced the culture medium containing release nanospheres the second day.The cells proliferation was detected by the method of MTT every day and quanti fication of oil red O every other day.The data obtained were analyzed with SPSS13.0 statistical software.ResultsThe basic fibroblast growth factor polyactide release nanospheres had the ability promoting proliferation and adipogenic induction of adipose-derived stem cells.The best concentration of nanospheres was 3 mg/ml and 4 mg/ml,respectively.ConclusionsThe basic fibroblast growth factor-polyactide release nanospheres could promote proliferation and adipogenic induetionof adipose-derived stem cells significantly.It could be used as an ideal cytokine release nanospheres in adipose tissue engineering.

Aim To study the function of VR1 in chronic pain, to construct VR1 siRNA expression vectors and to study their silencing effect in the DRG neurons of rats were detected.Methods The hairpin sequences of siRNAs targeting VR1 gene of rat were designed, and two pairs of oligonucleotide sequence were synthesized. The annealed oligonucleotide fragments were cloned into linearized pRNAT-U6.2/Lenti expression vector and identified by PCR and DNA sequencing.Then, they were co-transfected by lipofectamine into 293T cells.The silencing effects of the lentivector-mediated VR1 siRNAs on the expression of VR1 mRNA were determined by RT-PCR after intrathecal injection in rats.Results DNA sequencing showed that the oligonucleotide fragments were correctly cloned into linearized pRNAT-U6.2/Lenti expression vector and the expression of VR1 mRNA in L4-L6 DRG neurons was inhibited significantly by pRNAT-U6.2/Lenti-siVR1 after intrathecal injection in rats.Conclusion The lentivector-mediated siRNAs are successfully constructed and they inhibit the expression of VR1 mRNA in the DRG neurons of rats, which may provide a potential tool for the further study and treatment of chronic pain.