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Objective@#To investigate the effect of overexpression of Notch intracellular domain (NICD) on proliferation and osteogenic differentiation of human periodontal ligament stem cells (hPDLSC).@*Methods@#The third generation hPDLSC with stable overexpressing of NICD were assigned as experimental group, normal hPDLSC were as negative control group and hPDLSC transfected with empty vector were as blank control group. The effect of overexpressing NICD on proliferation ability of hPDLSC was detected by using cell counting kit-8 (CCK-8). Alizarin Red staining and real-time quantitative PCR (qPCR) were used to detect the effects of NICD on cementum attachment proteins (CAP), osteocalcin (OCN), Runt-related transcription factor 2 (RUNX2) and Notch signal pathway receptor Notch1. The effect of overexpressing NICD on hPDLSC osteogenic protein RUNX2 and flag marker protein (used to label NICD) were detected by using Western blotting.@*Results@#CCK-8 results showed that there were no significant differences in A values amongst the three groups for 1-2 days (P>0.05). The number of cells in the experimental group was significantly increase than that of the two control groups from the third to seventh days (A values were 0.203±0.016, 0.364±0.014, 0.449±0.020, 0.549±0.020 and 0.570±0.020, respectively) (P<0.05). Alizarin red staining showed that compared with the blank control group and negative control group, the mineralized nodules in the experimental group had smaller formation range and lighter color, and the differences were statistically significant (P<0.05). The expressions of CAP gene (0.751±0.058, 0.887±0.025), osteocalcin gene (0.592±0.051, 0.670±0.045) and RUNX2 gene (0.319±0.038, 0.684±0.055) at 14 and 21 days in the experimental group were significantly lower than those in the negative control group respectively (P<0.05). However, the expression levels of Notch1 gene at 14 and 21 days (2.507±0.047, 4.041±0.219) were significantly higher than those of negative and blank control groups (P<0.05). The results of Western blotting showed that the expressions of flag marker protein (0.167±0.007, 0.204±0.010) at 14 and 21 days in the experimental group were significantly higher than those in the negative and blank control groups (P<0.05). However, the expressions of RUNX2 protein (0.075±0.006, 0.074±0.013) at 14 and 21 days were significantly lower than that in the negative control group (0.092±0.003, 0.118±0.008) and blank control group (0.174±0.006, 0.212±0.008) (P<0.05).@*Conclusions@#Overexpression of NICD can promote the proliferation capacity of hPDLSC and inhibit its osteogenic differentiation.
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OBJECTIVE： To study the effects of Capparis spinosa total alkaloid on Notch pathways related protein Notch2， Delta-like 3 （DLL3）， Jagged1 and Notch intracellular domain 1 （NICD1） in mice with systemic sclerosis （SSc）. METHODS： BALB/c mice were randomly divided into blank control group， model group， positive control group （penicillamine 125 mg/kg）， C. spinosa total alkaloid low-dose， medium-dose and high-dose groups （225， 450， 900 mg/kg）， with 16 mice in each group. Except for blank control group， other groups were given bleomycin subcutaneously for 4 weeks to induce SSc model. C. spinosa total alkaloid groups were given relevant dose of C. spinosa total alkaloid cream for external use. Positive control group was given relevant dose of penicillamine intragastrically. Blank control group and model groups were given cream matrix without drug， once a day， for consecutive 8 weeks. 4 h after last administration， the skin of the administration area of each group of mice was collected. mRNA expression of Notch2 and NICD1 was detected by real-time PCR； the content of DLL3 was measured by ELISA； the protein expression of Jagged1 in skin tissue was detected by immunohistochemstry. RESULTS： Compared with blank control group， mRNA expression of Notch2 and NICD1， DLL3 content， protein expression of Jagged1 were markedly increased， with statistical significance （P＜0.01）. Compared with model group， mRNA expression of NICD1， DLL3 content and protein expression of Jagged1 were decreased significantly in C. spinosa total alkaloid medium-dose and high-dose groups， positive control group， mRNA expression of Notch2 in skin tissue were decreased significantly in C. spinosa total alkaloid high-dose group and positive control group， with statistical significance （P＜0.05 or P＜0.01）. CONCLUSIONS： C. spinosa total alkaloid can inhibit the abnormal expression of Notch2， NICD1， DLL3 and Jagged1 in skin tissue of SSc model mice， and inhibit over activation of Notch pathway in SSc model mice.
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ObjectiveNotch signaling is highly conservative in evolution and plays an important role in cell's proliferation and differentiation.Construction of tentiviral vector containing Notch intracellular domain (NICD) would lay the foundation for the study of Notch signaling.MethodsTotal RNA was extracted from the myeloid tissue of C57BL/6 mice.cDNA was composed via reverse transcription.NICD sequence was obtained by PCR and recombined into lentiviral vector.Lentiviral vector with NICD was infected into target HEK293T cell.Real-time PCR and Western blot were used to examine NICD expression in HEK293T cell.ResultsNICD expression increased in HEK293T cells.ConclusionThe successful construction of lentiviral vector involving mice NICD expression provides the foundation for the future study.