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Objective To determine the effect of ostecytic TGF-β/Smad4 signaling on osteoblastic and osteoclastic differentiation in bone marrow stromal cells (BMSCs).Methods Mice with osteocytic TGF-β/Smad4 conditional knock down (Smad4ot CKD) were generated as previously by crossing DMP1-8kb-Cre mice with Smad4lox(ex8)/lox(ex8) mice.The osteocytes were isolated from tibial and femoral diaphysis and co-cultured with wild-type BMSCs.ALP staining, Alizarin red staining and TRAP staining were performed to show osteoblastic and osteoclastic differentiation.Then, their marker genes were detected by qPCR and proteins measured by Western blot.ResultsThe expression of Runx2 and Osterix were reduced in smad4 CKDot co-cultured with BMSCs compared with controls(P<0.01).Similarly, the specific markers of osteoblastic differentiation were decreased (P<0.01).Additionally, the expression of RANKL was not significantly changed in with BMSCs.However, OPG was highly expressed incontrol group compared with smad4 CKD in co-cultured group (P<0.05).Thus, the radio of RANKL/OPG was significantly reduced (P<0.05).Furthermore, the expression of RANK was inhibited.Conclusions The terminally-differentiated osteocytes are the cells regulating bone metabolism, while down-regulation of osteocytic-TGF-β/Smad4 inhibits BMSC osteoblastic and osteoclastic differentiation.
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Objective To investigate the clinical efficacy of domestic porous tantalum rod in treatment of early avascular necrosis of femoral head (ANFH).Methods A prospective study was made on 18 cases (19 hips) diagnosed as early ANFH treated by domestic porous tantalum rod from July 2014 to December 2015.There were 14 males and four females, with a mean age of 44.2 years (range, 30-62 years).According to the modified Ficat staging, four cases were identified as stage Ⅱa and 14 stageⅡb.Efficacy of the treatment was evaluated by Harris score, Womac score, radiological images of the hip, complications and bone growth.Results All cases were followed up for 8-24 months (mean, 16 months).No complications such as infection, wound healing problems, immunological rejection, tantalum rod breakage, loosening or displacement were observed at last follow-up.Harris score was (82.7±9.0)points, (84.5±10.8)points and (87.2±10.0)points at postoperative 3, 6 and 12 months respectively, significantly higher than that pre-operation [(75.5±11.9)points] (P<0.05).Harris score was rated excellent in 10 cases, good in three, fair in five and poor in one at the last follow-up, with the excellent and good rate of 68%.Womac score was (17.4±9.4)points, (12.4±7.3)points, and (11.1±8.4)points at postoperative 3, 6 and 12 months respectively, significantly lower than that pre-operation [(28.3±13.1)points] (P<0.05).Seventeen cases (18 hips) showed no obvious deterioration in femoral head necrosis, with femoral head survival rate of 95%.One case underwent total hip arthroplasty after operation because of progressive hip pain and collapsed femoral head.Bone ingrowth was detected in the porous tantalum biomaterial after operation.Conclusion Domestic porous tantalum rod can effectively promote bone ingrowth, relieve pain, prevent the collapse of the femoral head, delay total hip arthroplasty time and finally improve hip function in treatment of early avascular necrosis of femoral head.
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Objective:To investigate the feasibility and its action of the therapeutic DNA vaccine for treatment of HBV infection.Methods:By genetic recombinant technique,have constructed 2 eukaryotic expressing plasmids namely pS2.S and pFP,encoding HBV envelope middle protein(preS2+HBsAg) and human leukocyte cytokines fusion protein(IL 2/IFN ?) genes respectively and evaluated its efficacy for inducing cellular immunity in healthy BALB/c mice and HBsAg serum conversion in HBV transgenic(Tg) mice after immunization of the plasmids by intramuscular administration.Results:1.The T cell proliferation by in vitro HBsAg stimulation closely correlated with HBsAg concentration,with its stimulation index(SI=5.6?0.9) in pS2.S immunized group,being significantly higher than that (2.0?0.5) of the control.The IL 2 and IFN ? secretion level in supernatant of the DNA vaccine immunized spleen cell culture were significantly higher than that of the control,while the IL 4 secretion level was not affected.2.The dendritic cells(DCs) extracted from the local draining lymph node(LN) after DNA vaccination induced a HBsAg specific T cell proliferation with its SI(4.20) being higher than that(2.55) of the control.3.In each group of high dose pS2.S and the middle dose combined with pFP inoculation,the HBsAg serum conversion occurred in one HBV Tg mouse,with the serum anti HBs level increasing as the time prolonged,while the serum HBsAg level of the rest mice were significantly lower than that of the control.Conclusion:The results suggest that HBV DNA vaccine can effectively induced cellular immunity in healthy mice;and the preliminary results of the immunized HBV Tg mice may provide an experimental evidence for further investigation of DNA vaccine for its use in treatment of HBV infection.
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To develop an effective therapeutic HBV DNA vaccine, two eukaryotic expressing plasmids, namely pcDNAS 2 ?S and pcDNAIIF , which respectively encoding HBV middle envelope protein preS 2 ?S and human IL 2/IFN ? fusion protein, were constructed by DNA recombinant technique. After identification by restriction analysis and DNA sequencing, the constructed recombinant plasmids were transfected into COS 7 cells in vitro with transfection reagent Lipofectamine. The expressed product in supernatant was quantified by ELISA , and the biologic activities of IL 2 and IFN ? were assayed by CTLL 2 cell line proliferation and cytopathogenic inhibition, respectively. The results showed that secretive expression of preS 2 ?S and hIL 2/hIFN ? fusion protein peaked at 48h after transfection, and the expression levels were HBsAg(P/N)=7 63, IL 2=10 35ng/ml, and IFN ?=7 90ng/ml. The IL 2 and IFN ? activities in the culture supernatant of COS 7 cells transfected with pcDNAIIF were 998U/ml and 249U/ml, respectively. These results suggested that the recombinant plasmids pcDNAS 2 ?S and pcDNAIIF were correctly constructed and the transfected cells could express secretive active protein.