Fluorescent labeling application of graphene oxide quantum dots in living human periodontal ligament stem cells / 医学研究生学报

ObjectiveNano-graphene oxide quantum dots (GOQDs) can be used to target fluorescent markers. The stem cell labeling is an important method in studying stem cell treatments. Our study aims to explore the possibility of using GOQDs as living cell fluorescent marker materials for human periodontal ligament stem cells (hPDLSCs), and to evaluate the biosecurity and effect as live cell fluorescence markers of GOQDs.Methods GOQDs were testified by TEM, DLS, UV-vis, and PL spectra. hPDLSCs were obtained by tissue cultivation and separated by single cell-derived colony selection. Then the source of the cells was carried out by immunocytochemical staining of anti-vimentin, anti-cytokeratin, and multipotent differentiation was used in the identification of stem cells. hPDLSCs were incubated with different concentrations of GOODs (0, 10, 25, and 50 μg/mL) for 24h and 72 h. Cytotoxicity and proliferation effects were determined using CCK-8, and cell cycles were detected using flow cytometry after the co-culture of GOQDs and hPDLSCs. The fluorescent labeling effect of GOQDs was tested using laser scanning confocal microscopy.ResultsThe characterization of GOQDs showed that the nanoparticles were evenly dispersed in water and showing blue light at 365 nm. TEM and DLS showed GOQDs had good dispersion, and the particle size was (6.36±1.41) nm. Immunocytochemical staining of anti-vimentin was positive while anti-cytokeratin was negative. The results of cytotoxicity showed there were no significant differences in cell activity after incubated with different concentrations of GOODs (0, 5, 10, 25, 50, 100, 200, and 400 μg/mL) (P>0.05), and there was no significant decrease in cell activity between 24h and 72h (P>0.05). There was no significant difference in the proportional distribution of G1, G2, and S phases between the two concentrations of GOQDs (0 μg/mL and 50 μg/mL) (P>0.05). Fluorescent images showed that GOQDs could enter the cell membrane and increase the fluorescence intensity at the concertation of 50 μg/mL.ConclusionGOQDs were confirmed to have good biocompatibility and could be used for live cell labeling of hPDLSCs.

Fluorescent labeling application of graphene oxide quantum dots in living human periodontal ligament stem cells / 医学研究生学报

ObjectiveNano-graphene oxide quantum dots (GOQDs) can be used to target fluorescent markers. The stem cell labeling is an important method in studying stem cell treatments. Our study aims to explore the possibility of using GOQDs as living cell fluorescent marker materials for human periodontal ligament stem cells (hPDLSCs), and to evaluate the biosecurity and effect as live cell fluorescence markers of GOQDs.Methods GOQDs were testified by TEM, DLS, UV-vis, and PL spectra. hPDLSCs were obtained by tissue cultivation and separated by single cell-derived colony selection. Then the source of the cells was carried out by immunocytochemical staining of anti-vimentin, anti-cytokeratin, and multipotent differentiation was used in the identification of stem cells. hPDLSCs were incubated with different concentrations of GOODs (0, 10, 25, and 50 μg/mL) for 24h and 72 h. Cytotoxicity and proliferation effects were determined using CCK-8, and cell cycles were detected using flow cytometry after the co-culture of GOQDs and hPDLSCs. The fluorescent labeling effect of GOQDs was tested using laser scanning confocal microscopy.ResultsThe characterization of GOQDs showed that the nanoparticles were evenly dispersed in water and showing blue light at 365 nm. TEM and DLS showed GOQDs had good dispersion, and the particle size was (6.36±1.41) nm. Immunocytochemical staining of anti-vimentin was positive while anti-cytokeratin was negative. The results of cytotoxicity showed there were no significant differences in cell activity after incubated with different concentrations of GOODs (0, 5, 10, 25, 50, 100, 200, and 400 μg/mL) (P>0.05), and there was no significant decrease in cell activity between 24h and 72h (P>0.05). There was no significant difference in the proportional distribution of G1, G2, and S phases between the two concentrations of GOQDs (0 μg/mL and 50 μg/mL) (P>0.05). Fluorescent images showed that GOQDs could enter the cell membrane and increase the fluorescence intensity at the concertation of 50 μg/mL.ConclusionGOQDs were confirmed to have good biocompatibility and could be used for live cell labeling of hPDLSCs.

Study of 5-bromodeoxyuridine labeling of endothelial progenitor cells from the circulating blood from tooth movement rat / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the optimal dosage and timing for 5-bromodeoxyuridine (BrdU) labeling of endothelial progenitor cells (EPCs) from rat circulating blood.</p><p><b>METHODS</b>The animal model for rat tooth movement was established. EPCs were obtained by density gradient centrifugation. The expressions of specific antigens on cell surface were analysed by immunocytochemistry and fluorescenceochemistry. EPCs were incubated with BrdU at different concentrations (5, 10, 15 micromol/L) for different incubating time (12, 24, 48, 72, 96 h) to identify the optimal BrdU concentration and incubating time for cell labeling. Immunohistochemistry was performed to calculate the labeling index (LI).</p><p><b>RESULTS</b>The culture cell positively expressed CD34, CD133 and could be shown to endocytose DiI-ac-LDL, FITC-UEA-1. Incubation of the EPCs with BrdU at 10 micromol/L and for an optimal length of 72 h appeared to achieve the highest LI (66.8+/-2.9)%, which was significantly higher than group of 5 micromol/L (P<0.05), while there was no significant difference between the group of 15 micromol/L and 10 micromol/L (P>0.05).</p><p><b>CONCLUSION</b>EPCs can be isolated from tooth movement rat circulating blood and cultured. Incubation of the EPCs with BrdU at 10 micromol/L and for an optimal length of 72 h appeared to achieve the optimal LI. This provides a foundation for us to investigate the mechanism of chemiotaxis and differentiation for EPCs.</p>

Effect of TRAIL gene in human squamous cell carcinoma cell line induced by adenovirus / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To study the apoptotic effect on the squamous cell carcinoma cell line TCa83 induced by recombined adenovirus vector containing TRAIL gene and CMV promoter.</p><p><b>METHODS</b>The TCa83 cell line was firstly infected with different titre of AdCMV-EGFP containing enhanced green fluorescence protein gene (EGFP) as control, and investigated the transducing rate through fluorescence to obtain the definite titre. Then TCa83 cell line was infected with AdCMV-TRAIL in proper titre, and TRAIL gene was detected by means of RT-PCR. After TCa83 cell line was infected with AdCMV-TRAIL and AdCMV-EGFP at day 1, 3, 5, 7, the activity of TCa83 cell line were evaluated by MIT and the apoptosis were detected by flow cytometer.</p><p><b>RESULTS</b>Proper titre was of 1,000 particles/cell, and TCa83 cell line could be infected 100% in this titre. TRAIL gene was detected by RT-PCR after infected with AdCMV-TRAIL. The activity of TCa83 decreased in both groups, but the AdCMV-TRAIL group decreased more sharply than AdCMV-EGFP group (P < 0.001). Both AdCMV-TRAIL and AdCMV-EGFP could lead to apoptosis of TCa83 cells, but the AdCMV-TRAIL, function stronger than AdCMV-EGFP. Especially there was remarkable statistic difference between two groups (P < 0.0001).</p><p><b>CONCLUSION</b>AdCMV-TRAIL could effectively decrease the activity of TCa83 cell line and induce apoptosis.</p>

Study of methods of decalcification for making united slices of tooth and affiliated periodontic tissues / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To study the methods of decalcification for making united slices of tooth and affiliated periodontic tissues.</p><p><b>METHODS</b>Twenty-one samples containing dog molars and affiliated periodontic tissues were divided into seven mean groups. The pH value of solution, time of decalcification, weight and volume of samples, and content of decalcified calcium were detected. The slices were observed by HE, specific, and immunohistochemical stain.</p><p><b>RESULTS</b>The velocity of decalcification increased with decrease of solution pH. The weight of samples lightened by 37.61%, the volume reduced by 25.97% on average, and calcium decalcified was 174.49 mg per gram humid samples. The EDTA decalcification was slowest, but it was best. Decalcification was fast in Plank-Rycho solution while the section was worst, and faster in the formyl solution containing aluminium chloride than in EDTA, and the section was better.</p><p><b>CONCLUSIONS</b>The 50% formyl solution containing aluminium chloride is an ideal decalcifying solution.</p>