Delayed DNA double-strand break in S-phase H1299 cells after thermal damage / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the pattern of DNA double-strand break (DSB) formation in S-phase cells after thermal damage and explore the mechanisms behind heat sensitivity of S-phase cells and delayed DSBs.</p><p><b>METHODS</b>Flow cytometry was used to analyze the cell cycle arrest in H1299 cells exposed to thermal damage, and EdU incorporation assay was employed to evaluate the DNA replication capacity of the cells. The cells synchronized in S phase were obtained by serum starvation and DSBs were observed dynamically using neutral comet assay. Trypan blue dye exclusion technique was used to analyze the cell viability after thermal damage. Western blotting (WB) was used to detect the phosphorylation of ATM and DNA binding RAD18.</p><p><b>RESULTS</b>The percentage of S-phase cells increased significantly after exposure of the cells to 45 degrees celsius; for 1 h (P<0.01). The time-dependent variation pattern of EdU incorporation was similar to that of S-phase cell fraction. The comet tail began to appear only after incubation of the cells at 37 degrees celsius; for some time and the Olive tail moment (OTM) increased with prolonged incubation. Cell death remained low until 7.5 h after heat exposure of the S-phase cells and then increased rapidly. The phosphorylation of ATM first increased but then decreased drastically. In cells with heat exposure, DNA binding RAD18 was attenuated obviously compared that in non-exposed cells.</p><p><b>CONCLUSION</b>Thermal damage causes cell cycle arrest in S phase, and delayed fatal DSBs occur in the arrested cells due to persistent replication and DNA damage repair suppression, which are the possible cause of heat sensitivity of S-phase cells.</p>

Therapeutic and immunological effects of microwave ablation combined with CpG ODN on transplanted colon carcinoma in mice / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the therapeutic and immunological effects of microwave ablation (MA) combined with CpG ODN in mice bearing transplanted colon carcinoma.</p><p><b>METHODS</b>A mouse model bearing colon carcinoma was established by subcutaneously inoculating CT26 cells into the right flank of Balb/c mice. The tumor-bearing mice were randomized into control group with PBS injection in the peritumoral area, MA group, MA combinated with CpG ODN group, and CpG ODN group with CpG ODN injection in the peritumoral area. The tumor volume changes were observed, and serum CD3(+)CD4(+) and CD3(+)CD8(+) T lmyphocytes were analyzed by flow cytometry after the treatments. Serum levels of interleukin (IL)-2, IL-12 and IFN-gamma were detected by ELISA. The mice in the MA group and the combined treatment group showing tumor regression were rechallenged with CT26 cells.</p><p><b>RESULTS</b>No significant difference was found in the number of serum CD3(+), CD3(+)CD4(+), or CD3(+)CD8(+) T lymphocytes between the 4 groups. The ratio of CD3(+)CD4(+)/CD3(+)CD8(+) T lymphocytes in the combined treatment group and MA group were 1.58-/+0.10 and 1.53-/+0.13, respectively, significantly higher than that in PBS group and CpG ODN group (P<0.05). The serum concentration of IL-2, IL-12 and IFN-gamma in the combined treatment group were 64.6-/+7.4 pg/ml, 314.1-/+26.9 pg/ml and 61.9-/+7.3 pg/ml, respectively, significantly higher than those in the other 3 groups (P<0.05). The tumor formation rate in the combined treatment group was significantly lower than that in MA group (25.0% vs 75.0%, P<0.05).</p><p><b>CONCLUSION</b>CpG ODN can enhance the immunity and decrease the tumor formation rate following a rechallenge with CT26 cells in mice treated with MA.</p>

Efficacy of cryoablation combined with CpG oligonucleotides in the treatment of murine transplanted colon carcinoma / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To investigate the efficacy of cryoablation combined with CpG ODN in the treatment of murine transplanted colon carcinoma.</p><p><b>METHODS</b>Colon carcinoma model was established by subcutaneously inoculating CT26 cells into the right flank in BALB/c mice. The tumor-bearing mice were randomly divided into 4 groups:the group of PBS injected in peritumoral area, the group of cryoablation, the group of cryoablation combined with CpG ODN, the group of CpG ODN injected in peritumoral area. The tumor size changes were measured. Serum levels of interleukin (IL)-12 and interferon-gamma(IFN-gamma) were assayed by ELISA. The rates of CD3(+)CD4(+)T, CD3(+)CD8(+)T lymphocytes in serum were counted with flow cytometry. Mice in the cryoablation group and the combined group with tumor regression were re-challenged with CT26 cells.</p><p><b>RESULTS</b>The survival time of cryoablation group and combined therapy group were (80.3 + or - 5.4) days and (83.8 + or - 5.5) days, respectively, longer than (53.7 + or - 3.7) days in PBS group and (51.5 + or - 6.8) days in CpG ODN group(all P<0.05). The suppress rates of tumor cells in cryoablation group and combined therapy group were 83.8% and 86.2% respectively. After 20 days following treatment, CD3(+)CD4(+)T/CD3(+)CD8(+)T ratio and the concentrations of IL-12 and IFN-gamma in mice serum of cryoablation group and combined therapy group were higher than those in PBS group and CpG ODN group(all P<0.05). No significant difference was found in CD3(+)CD4(+)T/CD3(+)CD8(+)T ratio between cryoablation group and combined therapy group(P>0.05). However, the concentrations of IL-12 and IFN-gamma in combined therapy group were higher than those of cryoablation group(all P<0.05). After re-challenging, tumor formation rate in the cryoablation combined with CpG ODN group was 16.7%, significantly lower than that in the cryoablation group(83.8%)(P<0.05).</p><p><b>CONCLUSION</b>Cryoablation combined with CpG ODN can increase antitumor immune response in mice, and therefore can decrease the tumor formation when re-challenged with CT26 cells.</p>

Transferrin receptor imaging for tracing mesenchymal stem cells implanted in the spinal cord / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the feasibility of tracing mesenchymal stem cells in vivo with scintigraphy.</p><p><b>METHODS</b>Transferrin receptor expression of cultured mesenchymal stem cells (hMSCs) was quantified with radioligand-receptor binding assay before the cells were transplanted into the spinal cord of rabbits. (131)I-labeled transferrin was then administered into the subarachnoid space of the rabbits, and scintigraphic images were acquired with a gamma camera at different time points after the administration. In the control experiments, (131)I-labeled human serum albumin was used in stead of (131)I-transferrin as the tracer, or only PBS was injected without stem cell transplantation. The images were semi-quantitatively analyzed with region of interest (ROI) techniques, and the phosphor imaging on the spinal sections were performed.</p><p><b>RESULTS</b>Radioligand-receptor binding assay showed 10 770 binding sites with high affinity (KD=0.982 nmol/L) for Fe saturated transferrin on each human mesenchymal cell. Visible accumulation of radioactivity at the cell transplantation sites was observed 16 h and 24 h after intrathecal injection of (131)I-transferrin tracer, but not in two control groups. ROI analysis showed that the difference between (131)I-transferrin and the control groups was statistically significant (P<0.05). Phosphor imaging further verified that it was the specific coupling of transferrin to the implanted cells that resulted in radioactivity accumulation at the transplantation sites.</p><p><b>CONCLUSIONS</b>Transferrin receptor imaging is capable of in vivo tracing of the implanted stem cells, and has the potential for use in non-invasive monitoring for stem cell transplantation therapy after further technical improvements.</p>

D2 receptor expression on immortalized human neural progenitor cell line hNPC-TERT in vitro and in vivo / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe D(2) receptor expression on human neural progenitor cell line hNPC-TERT before and after transplantation into rabbit central nervous system.</p><p><b>METHODS</b>D(2) receptor expression on cultured hNPC-TERT cells was verified and quantitatively analyzed with immunofluorescence assay and receptor radio ligand binding assay, respectively. 3 x 10(6) hNPC-TERT cells were implanted in the spinal cord of New Zealand rabbit with HeLa cells as the control. Two days after implantation, positron-emission tomography (PET) scan with (11)C-raclopride as the radiotracer was performed in the living animals or for the isolated spinal cords, and cryosections of the spinal cord containing the implanted cells were prepared for immunofluorescence assay.</p><p><b>RESULTS</b>Cultured hNPC-TERT cells showed high expression of D(2) receptor (Bmax=8 x 10(4)). PET scans of the rabbits identified visible radioactive accumulations at the site where hNPC-TERT cells were implanted but not at the site of HeLa cell implantation. Region of interest analysis showed a significant difference between the two cells in the maximal standard uptake value at the cell implantation sites. The results were further confirmed with ex vivo PET imaging of the spinal cord and tissue immunofluorescence assay.</p><p><b>CONCLUSION</b>Human neural progenitor cells hNPC-TERT highly express dopamine D(2) receptors and retain this capacity after implantation into the spinal cord, suggesting their potential for treatment of such nerve system disease as Parkinson syndrome.</p>