RESUMEN
Patients with diabetes tend to have an increased incidence of osteoporosis, which may be associated with hyperglycemia; however, the pathogenic mechanisms governing this interaction remain unknown. The present study sought to investigate whether elevated extracellular glucose levels of bone mesenchymal stem cells (BMSCs) could influence osteoblastic differentiation and whether the intracellular Sonic hedgehog (Shh) pathway could adjust the effects. Furthermore, to verify the results in vivo, a rat tooth extraction model was constructed. BMSCs were incubated in eight types of culture medium, including low glucose (LG), LG + lentivirus (Lenti), LG + Lentismall interfering RNA (LentisiRNA), LG + LentiShh, high glucose (HG), HG + Lenti, HG + LentisiRNA and HG + LentiShh. The lentiviral transfection efficiency was observed using a fluorescence microscope; protein and mRNA expression was detected by western blotting and reverse transcriptionquantitative polymerase chain reaction (RTqPCR). The matrix mineralization and alkaline phosphatase (ALP) activity of BMSCs were examined by Alizarin red staining and ALP activity assays, respectively. The expression of osteogenesisrelated genes in BMSCs were quantified by RTqPCR. The alveolar ridge reduction was measured and histological sections were used to evaluate new bone formation in the tooth socket. With high concentrations of glucose, Shh expression, matrix mineralization nodules formation, ALP activity and the levels of bone morphogenic protein 4 (BMP4), bone sialoprotein (BSP) and osteopontin (OPN) expression were greatly reduced compared with LG and corresponding control groups. Whereas activated Shh signaling via LentiShh could increase the number of matrix mineralization nodules, ALP activity, and the expression levels of BMP4, BSP and OPN in BMSCs. Additionally, in vivo assays demonstrated that LentiShh induced additional bone formation. Collectively, the results of the present study indicated that HG inhibited the Shh pathway in osteoblasts and resulted in patterning defects during osteoblastic differentiation and bone formation, while the activation of Shh signaling could suppress these deleterious effects.
Asunto(s)
Glucosa/farmacología , Proteínas Hedgehog/biosíntesis , Lentivirus , Osteoblastos/metabolismo , Osteogénesis/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Animales , Proteínas Hedgehog/genética , Masculino , Osteoblastos/patología , Osteogénesis/genética , Ratas , Ratas Sprague-Dawley , Transducción GenéticaRESUMEN
Cirrhosis is the terminal stage of hepatic diseases and is prone to develop into hepatocyte carcinoma. Increasing evidence suggests that the transplantation of dental pulp stem cells (DPSCs) may promote recovery from cirrhosis, but the key regulatory mechanisms involved remain to be determined. In this study, we overexpressed human hepatocyte growth factor (hHGF) in primary rat DPSCs and evaluated the effects of HGF overexpression on the biological behaviors and therapeutic efficacy of grafted DPSCs in cirrhosis. Liver cirrhosis was induced via the intraperitoneal injection of CCl4 twice weekly for 12 weeks and was verified through histopathological and serological assays. HGF was overexpressed in DPSCs via transduction with a hHGF-lentiviral vector and confirmed based on the elevated expression and secretion of HGF. The HGF-overexpressing DPSCs were transplanted into rats intravenously. The HGF-overexpressing DPSCs showed increased survival and hepatogenic differentiation in host liver tissue at 6 weeks after grafting. They also exhibited a significantly greater repair potential in relation to cirrhosis pathology and impaired liver function than did DPSCs expressing HGF at physiological levels. Our study may provide an experimental basis for the development of novel methods for the treatment of liver cirrhosis in clinical practice.