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Objective:To prepare nanoprobes by using polydopamine (PDA) as a carrier which is modified with the sonosensitizer protoporphyrin Ⅸ (PpⅨ) and labeled with 131I, 99Tc m or 177Lu, and to explore the value of these new nanoprobes in diagnosis and combination therapy of breast cancer. Methods:PDA particles were synthesized by aqueous oxidation, and a layer of polyethylene glycol (PEG) and PpⅨ were modified on the surface to product PDA-PEG-PpⅨ. Then the nuclides 131I, 99Tc m and 177Lu were labeled on PDA, respectively, and the labeling yield and stability were determined. The cytotoxicity test was conducted by comparing the viabilities of 4T1 tumor cells in free 131I group and 131I-PDA-PEG-PpⅨ group. The 4T1 cells were divided into 7 groups according to different treatment methods: PDA-PEG-PpⅨ group, PDA-PEG-PpⅨ+ photothermal therapy (PTT) group, PDA-PEG-PpⅨ+ sonodynamic therapy (SDT) group, 131I-PDA-PEG-PpⅨ+ PTT group, 131I-PDA-PEG-PpⅨ+ SDT group, 131I-PDA-PEG-PpⅨ+ PTT+ SDT group (100 μg/ml PDA-PEG-PpⅨ, 925 kBq/ml 131I), and the control group (DMEM culture medium). The cell viabilities of those groups were compared to evaluate the therapeutic effect. 4T1 tumor bearing mouse models were established, then 99Tc m-PDA-PEG-PpⅨ was injected through the tail vein (29.6 MBq) or intratumorally (14.8 MBq) to perform gamma imaging. The independent-sample t test and one-way analysis of variance were used for data analysis. Results:The PDA particles were uniform in size, with a particle size of (160.0±1.5) nm. They had a good photothermal conversion effect. A characteristic peak consistent with PpⅨ (400 nm) appeared in the UV-Vis absorption spectrum of PDA-PEG-PpIX. In the cytotoxicity test, when the radioactivity was 1.850 or 3.700 or 7.400 MBq/ml, the cell viabilities of free 131I group and 131I-PDA-PEG-PpⅨ group were significantly different ((72.18±6.57)% vs (86.07±5.17)%, (59.31±9.06)% vs (80.85±4.21)%, (42.90±1.30)% vs (72.99±5.73)%; t values: 3.71, 4.82, 11.46, P values: 0.006, 0.001, <0.001). The 131I-PDA-PEG-PpⅨ+ PTT+ SDT combination therapy had a better killing effect on 4T1 tumor cells than the combination of 131I-PDA-PEG-PpⅨ+ PTT and 131I-PDA-PEG-PpⅨ+ SDT (cell viabilities: (10.09±2.50)% vs (16.04±2.63)%, (28.65±4.72)%; F=351.66, P<0.001). In vivo imaging showed that 99Tc m-PDA-PEG-PpⅨ was stable in mouse models and could be effectively enriched in tumors. Conclusions:A multifunctional nanoprobe based on PDA is successfully prepared. The radionuclide labeling method is simple and effective, with a good stability. 131I-PDA-PEG-PpⅨ can kill 4T1 cells efficiently. 99Tc m-PDA-PEG-PpⅨ has an obvious tumor concentration effect in mouse models.
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Objective:To develop the anti-CD30 monoclonal antibody 64Cu-1, 4, 7-trizacyclononane-1, 4, 7-triacetic acid (NOTA)-CD30 and visualize CD30 expression in lymphoma non-invasively. Methods:The CD30 expression levels of 5 cell lines (Karpas299, Raji, Daudi, Ramos, and U266) were assessed by Western blot. Cell lines with high and low CD30 expression were selected for flow cytometry to evaluate the specific binding affinity of anti-CD30 monoclonal antibody. Thirteen NSG mice were used to established CD30 positive and negative subcutaneous xenograft models. 64Cu-NOTA-CD30 was obtained and 64Cu-NOTA-immunoglobulin (Ig)G was used as the control. ImmunoPET imaging was performed 2, 24, and 48 h after the injection of 64Cu-NOTA-CD30 or 64Cu-NOTA-IgG. Finally, the biodistribution studies were conducted. Repeated-measures analysis of variance and Bonferroni test were conducted for comparison. Results:Karpas299 showed the highest CD30 expression, while Raji showed the lowest. Flow cytometry showed specific binding affinity of the anti-CD30 monoclonal antibody to the Karpas299 cell line. The radiochemical purities of the probes were both higher than 95%. In microPET, the 64Cu-NOTA-CD30 uptake of Karpas299 xenograft tumors increased over time, with (11.46±0.58), (17.60±1.16) and (19.46±0.99) percentage activity of injection dose per gram of tissue (%ID/g) at 2, 24 and 48 h respectively. The contrast to normal tissue was good at 48 h, with the tumor/heart (blood) ratio of 2.20±0.22. The uptake of 64Cu-NOTA-CD30 in Karpas299 tumor at 48 h after injection was significantly higher than that in Raji tumor ((6.10±1.03) %ID/g) and 64Cu-NOTA-IgG in Karpas299 tumor ((5.12±0.89) %ID/g; F=290.99, t values: 19.65 and 22.25, all P<0.001). The uptake of 64Cu-NOTA-CD30 and the control probe in the heart and liver decreased over time in all groups. Ex vivo biodistribution at 48 h was mainly consistent with the results of microPET in vivo. Conclusions:64Cu-NOTA-CD30 is able to visualize the expression level and distribution of CD30 non-invasively. It is promising to be applied for screening the beneficial groups and evaluating efficacy for CD30-targeted immunotherapy.
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Objective:To evaluate the role of has_circ_0008039 and miR-484 in oxygen-glucose deprivation/reoxygenation (OGD/R) injury in SK-N-SH cells and the relationship with Fis1.Methods:SK-N-SH cells were cultured in vitro to logarithmic growth stage and divided into 5 groups ( n=25 each) according to the random number table method: control group (group C), OGD/R group, has_circ_0008039 siRNA group (group S), hsa_circ_0008039 over-expression group (group E) and has_circ_0008039 siRNA plus miR-484 inhibitor group (group S+ I). Cells were cultured in normal condition in group C. In S, E and S+ I groups, after the cells were transfected with hsa_circ_0008039 siRNA, has_circ_0008039 over-expression vector, hsa_circ_0008039 siRNA and miR-484 inhibitor, the cells were subjected to oxygen-glucose deprivation for 12 h followed by 24 h restoration of O 2-glucose supply to develop the OGD/R model.At 24 h of restoration of O 2-glucose supply, the cell viability and amount of lactic dehydrogenase (LDH) released were measured using CCK-8 assay, the expression of hsa_circ_0008039, miR-484 and Fis1 mRNA was detected using real-time polymerase chain reaction, and the expression of Fis1 was detected by Western blot.A dual-fluorescein experimental report was used to verify the targeting relationship between hsa_circ_0008039 and miR-484. Results:Compared with group C, the cell viability was significantly decreased, and the amount of LDH released was increased in the other 4 groups, the expression of hsa_circ_0008039 and Fis1 was significantly up-regulated, and the expression of miR-484 was down-regulated in OGD/R and E groups, the expression of hsa_circ_0008039 and Fis1 was significantly down-regulated, and miR-484 was up-regulated in group S, and the expression of hsa_circ_0008039 and miR-484 was significantly down-regulated, and the expression of Fis1 was up-regulated in group S+ I ( P<0.05). Compared with group OGD/R, the cell viability was significantly decreased, and the amount of LDH released was increased in E and S+ I groups, the cell viability was significantly increased, and the amount of LDH released was decreased in group S, the expression of hsa_circ_0008039 and Fis1 was significantly up-regulated, and the expression of miR-484 was down-regulated in group E, the expression of hsa_circ_0008039 and Fis1 was significantly down-regulated, and the expression of miR-484 was up-regulated in group S, and the expression of hsa_circ_0008039 and miR-484 was significantly down-regulated, and the expression of Fis1 was up-regulated in group S+ I ( P<0.05). Compared with group S, the cell viability was significantly decreased, the amount of LDH released was increased, the expression of miR-484 was down-regulated, and the expression of Fis1 was up-regulated in group S+ I ( P<0.01). The dual-fluorescein experimental report verified that miR-484 was the target of hsa_circ_0008039 which binded to miR-484 specifically. Conclusions:has_circ_0008039 is involved in OGD/R injury in SK-N-SH cells by targetedly binding to miR-484, which is associated with up-regulation of Fis1 expression.
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Objective:To explore the isolation and culture methods of mouse parietal epithelial cells (PECs) of Bowman′s capsule, so as to provide a cell tool for further study.Methods:Mouse renal corpuscles were isolated by cell sieving combined with magnetic separation. After primary culture, identified parietal epithelial cells were induced to differentiate into podocytes. Immunofluorescence staining, real-time quantitative PCR and Western blotting were used to detect specific markers of parietal epithelial cells and podocytes.Results:Primary cultured PECs grew like paving stone and expressed Claudin-1 (PECs specific marker), CD133 (stem cell marker) and CD24 (stem cell marker), without the expression of tubular epithelial cell proteins, mesangial cell and podocyte specific proteins. Cultured to 6 generations in vitro, the PECs still expressed Claudin-1, CD133 and CD24. After incubated with differentiation medium, PECs were able to express podocyte markers WT-1 and Synaptopodin. Conclusion:The renal corpuscles are extracted by cell sieving combined with magnetic separation, and the mouse PECs successfully cultured in vitro can be induced to express podocytes′ markers.
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Objective:To synthesize a novel site-specifically labelled probe 68Ga-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid (DOTA)-Cys-Asp-Val (CDV)-Nb109 and explore its potential for detection of the programmed cell death ligand 1 (PD-L1) expression level in different tumors. Methods:Firstly, CDV was inserted into the tail of the sequence of Nb109 by genetic engineering. Then the precursor DOTA-CDV-Nb109 was prepared by mixing the maleimide-DOTA and the single-domain antibody CDV-Nb109 (amount of substance ratio 1∶1) via the maleimide-cysteine site-specific coupling strategy. Subsequently, the DOTA-CDV-Nb109 was labeled with 68Ga and purified by PD-10 column. Human melanoma A375, human PD-L1 transfected melanoma A375-hPD-L1 and human glioma U87 tumor-bearing mice models were established, and the diagnostic value of 68Ga-DOTA-CDV-Nb109 was evaluated by stability assay, cellular uptake, and microPET imaging. One-way analysis of variance and the least significant difference t test were used to analyze the data. Results:The probe 68Ga-DOTA-CDV-Nb109 was obtained with the radiochemical yield of (69.79±4.69)%, radiochemical purity more than 97%, and molar activity of (12.85±1.51) GBq/μmol. 68Ga-DOTA-CDV-Nb109 had strong binding affinity for A375-hPD-L1 with the dissociation constant ( Kd) of (66.43±17.89) nmol/L. The uptake of 68Ga-DOTA-CDV-Nb109 in A375-hPD-L1 and U87 cells were (3.17±0.15) percentage of the added radioactivity dose (%AD) and (2.08±0.03) %AD respectively, which were significantly higher than that in A375 cells ((1.21±0.14) %AD; F=82.87, t values: 15.23, 9.98, P values: <0.001, 0.003). The tumor uptake of the probe in A375-hPD-L1 ((5.21±0.35) percentage of injected dose per ml (%ID/ml)) and U87 tumor-bearing mice ((3.44±0.69) %ID/ml) were significantly higher than that in A375 tumor-bearing mice ((2.17±0.36) %ID/ml; F=249.72, t values: 35.70, 3.43, both P<0.001). Conclusion:The site-specifically labelled probe 68Ga-DOTA-CDV-Nb109, which can non-invasively and dynamically monitor the change of PD-L1 expression level in different tumors and help screen patients who can benefit from PD-L1 immune checkpoint blocking therapy, is successfully synthesized with high radiochemical purity.
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Objective:To prepare specific molecular probe 18F-AlF-1, 4, 7-triazacylononane-1, 4, 7-triacetic acid-(polyethylene glycol) 4-ZD2 ( 18F-AlF-NOTA-PEG 4-ZD2) for targeting extradomain-B fibronectin (EDB-FN), and evaluate its properties in vitro and in vivo. Methods:18F-AlF-NOTA-PEG 4-ZD2 was prepared by one-step chelation labeling with Al 18F. The radiochemical purity and in vitro stability were determined by high performance liquid chromatography (HPLC). The partition coefficient (logP) of 18F-AlF-NOTA-PEG 4-ZD2 was evaluated, and the cell uptake experiment was carried out (triple-negative breast cancer (MDA-MB-231) cells (1×10 6/tube) were divided into 3 groups ( n=3 per group); positive group, inhibition group, control group). MicroPET imaging was performed on MDA-MB-231 bearing nude mice ( n=3) after 18F-AlF-NOTA-PEG 4-ZD2 injection (30, 60, 90, 120 min) and compared with blocking group ( n=3, NOTA-PEG 4-ZQ 2 was preinjected at 0.5 h before 18F-AIF-NOTA-PEG a-ZD2 injection). Independent-sample t test was used to analyze the data. Results:18F-AlF-NOTA-PEG 4-ZD2 was successfully prepared. The optimized radiochemical yield was (33.8±2.1)% (undecay corrected, n=8) and the radiochemical purity was >96%. After incubating 120 min at 37 ℃, the radiochemical purity of 18F-AlF-NOTA-PEG 4-ZD2 in human serum and PBS was >93%, indicating its good stability in vitro. The specific activity was (11.1±3.2) GBq/μmol, and logP was -1.43±0.05. The uptake value of tumor cells was (1.77±0.28) percentage applied activity (%AR)/10 6 cells at 120 min post-injection in positive group, and the total uptake value of the inhibition group was (0.76±0.07) %AR/10 6 cells ( t=4.30, P=0.032). MicroPET imaging in tumor bearing nude mice showed that 18F-AlF-NOTA-PEG 4-ZD2 was mainly metabolized by the liver and kidneys. The tumor uptake value was (1.94±0.21) percentage activity of injection dose per gram of tissue (%ID/g) at 60 min post-injection and the tumor/muscle ratio was 3.80±0.25 at 90 min post-injection in the experimental group, while the tumor uptake value of tumor bearing nude mice in the blocking group was (0.43±0.09) %ID/g at 60 min post-injection ( t=3.18, P=0.006). Conclusions:18F-AlF-NOTA-PEG 4-ZD2 can be prepared simply with high labeling rate and good stability in vitro, with high tumor uptake and tumor/muscle ratio in microPET imaging, and good specificity and long tumor residence time. The probe has good application prospect in breast cancer with high expression of fibronectin subtype EDB-FN.
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Objective:To explore the effect of different glucose concentrations on the uptake of 18F-FDG and the expression of glucose transport protein(Glut)-1 and Glut-3 in non-small cell lung cancer (NSCLC). Methods:NSCLC cell line A549 cells were cultured in DMEM medium with glucose concentrations of 3.9, 5.0, 6.1, 8.3 and 11.1 mmol/L respectively for 24 h. Then 3.7×10 4 Bq 18F-FDG was added into each group and γ counter was used to measure the radioactivity count 1 h later. Western blot was used to examine the expression of Glut-1 and Glut-3. One-way analysis of variance and Bonferroni test were used for data analysis. The correlation was analyzed by Pearson correlation analysis. Results:The 18F-FDG uptake rates in 3.9, 5.0, 6.1, 8.3 and 11.1 mmol/L groups were (4.89±0.83)%, (4.07±0.23)%, (3.66±0.29)%, (3.34±0.16)% and (3.29±0.24)%, respectively ( F=7.05, P=0.006). Compared with 3.9 mmol/L group, the 18F-FDG uptake rates in 8.3 and 11.1 mmol/L groups were reduced and differences were statistically significant ( P values: 0.013, 0.010), while there were no statistical differences between the other groups ( P values: 0.057-0.999). The relative expressions of Glut-1 and Glut-3 in each group were 1.17±0.10, 1.00±0.00, 0.84±0.07, 0.70±0.18, 0.61±0.16, and 1.14±0.05, 1.00±0.00, 0.86±0.12, 0.71±0.05, 0.40±0.06, respectively ( F values: 10.26 and 51.94, P values: 0.001, <0.001). Moreover, the 18F-FDG uptake rates were positively correlated with the expression of Glut-1 and Glut-3 ( r values: 0.775 and 0.744, both P=0.001). Conclusions:When the glucose concentration fluctuates within 3.9-11.1 mmol/L, the change of glucose will affect the 18F-FDG uptake rate and the expression of Glut-1 and Glut-3 in A549 cells. Moreover, the 18F-FDG uptake rate is related to the expressions of Glut-1 and Glut-3.
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Objective:To investigate the effects of miR-5011-5p on apoptosis and migration of bladder cancer cell line J82 and the underlying mechanism.Methods:J82 cells were transfected with random sequence molecules (NC group) and miR-5011-5p sequence molecules (miR-5011-5p group). Flow cytometry and scratch experiment were performed to analyze the effects of miR-5011-5p on apoptosis and migration of J82 cells. The target gene of miR-5011-5p was predicted by bioinformatics. Real-time fluorescent quantitative polymerase chain reaction and western blot assay were performed to investigate the effects of miR-5011-5p on target gene expression.Results:The relative expression of miR-5011-5p in J82 cells in the miR-5011-5p group was significantly higher than that in the NC group (10.73 ± 1.67 vs. 1.04 ± 0.16, t = 5.81, P < 0.01). There was significant difference in the apoptosis rate of J82 cells between NC and miR-5011-5p groups [(8.83 ± 1.67)% vs. (34.96 ± 3.80)%, t = 6.30, P < 0.01]. The migration rate of J82 cells differed significantly between NC and miR-5011-5p groups [(71.31 ± 7.69)% vs. (37.43 ± 5.01)%, t = 3.69, P < 0.05]. The target gene of miR-5011-5p may be Yes-related protein 1 (YAP1). Compared with the NC group, miR-5011-5p exhibited an obvious inhibitory effect on the YAP1 expression in J82 cells ( P < 0.01). Conclusion:miR-5011-5p may promote the apoptosis of J82 cells and inhibit their migration in bladder cancer through targeted inhibition of YAP1 gene expression.
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Objective:To prepare 68Ga-2-(4, 7-bis(carboxymethyl)-1, 4, 7-triazonan-1-yl)pentanedioic acid (NODAGA)-YHWYGYTPQNVI (GE11) and evaluate its feasibility of PET imaging for pancreatic cancer. Methods:GE11 peptide was conjugated with NODAGA and then labeled with 68Ga. The labeling yield, radiochemical purity, hydrophilicity, stability and specificity in vitro were determined. Human pancreatic cancer BxPC3 nude mice models ( n=9) were established. MicroPET imaging was then obtained after 30 and 90 min, and mice were sacrificed at 90 min to acquire the radioactivity distribution of main organs and tumors. Pair t test was used to analyze the data. Results:The labeling yield was (73.5±5.4)% and radiochemical purity was more than 98%. After incubation 120 min in mouse serum at 37 ℃, radiochemical purity was more than 92%. The uptake was specific in BxPC3 cell lines. MicroPET images showed that 68Ga-NODAGA-GE11 could accumulate quickly in tumor. Value of tumor uptake was significantly higher than that of normal pancreas at 90 min ((1.38±0.25) vs (0.49±0.07) %ID/g; t=12.67, P<0.05), and the radio-uptake of blood, muscle and bone was lower than that of tumor. Conclusions:68Ga-NODAGA-GE11 is easy to be prepared with high radiochemical purity and good stability, and can specifically target BxPC3 xenograft tumor. However, due to the high uptake in the kidneys and liver, the value of 68Ga-NODAGA-GE11 in PET imaging for pancreatic tumor needs further study.
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Objective:To screen 89Zr-labeled anti-epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) monoclonal antibody molecular probes suitable for monitoring the gastric mucinous adenocarcinoma bearing mouse models with low glucose metabolism. Methods:The expression of EGFR and HER2 in the MGC803 gastric cancer cell line was verified by analyzing cell slides and xenograft tumor sections. 89Zr-Deferoxamine (DFO)-Cetuximab and 89Zr-DFO-Pertuzumab were prepared and the radiochemical purity was detected. Cell binding experiments and blocking experiments were performed to verify the binding ability and specificity of the probes. Twelve gastric mucinous adenocarcinoma bearing mouse models were divided into 3 groups ( n=4 in each group): 89Zr-DFO-Cetuximab group (7.4 MBq/mouse, 74 μg/mouse), 89Zr-DFO-Pertuzumab group (7.4 MBq/mouse, 70 μg/mouse) and 18F-fluorodeoxyglucose (FDG) group (7.4 MBq/mouse). MicroPET imaging was performed at 4, 24 and 48 h ( 18F-FDG group underwent imaging at 1 h only) post-injection. The biodistribution study of 89Zr-DFO-Cetuximab and 89Zr-DFO-Pertuzumab was conducted in 2 groups ( n=4 in each group) 48 h after the injection. The independent sample t test was used for data analysis. Results:The immunofluorescent staining demonstrated EGFR expression was significantly higher than HER2 expression in MGC803 gastric cancer cell line. The radiochemical purity of 89Zr-DFO-Cetuximab and 89Zr-DFO-Pertuzumab were both more than 95%, and the specific activities were 100 and 95 MBq/mg, respectively. The two probes had good stability in normal saline and fetal bovine serum, with the radiochemical purity higher than 80% at 72 h. MicroPET imaging showed that the uptake of 89Zr-DFO-Cetuximab in the MGC803 tumor was significantly higher than that of 18F-FDG and 89Zr-DFO-Pertuzumab. The biodistribution study demonstrated the 89Zr-DFO-Cetuximab uptake (percentage activity of injection dose per gram of tissue, %ID/g) of tumors at 48 h was significantly higher than that of 89Zr-DFO-Pertuzumab (56.3±12.0 vs 22.0±3.6; t=4.31, P<0.05). Conclusion:Compared with 89Zr-DFO-Pertuzumab, 89Zr-DFO-Cetuximab has a better potential for non-invasive monitoring of gastric mucinous adenocarcinoma with low glucose metabolism.
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Objective:To synthesize a 68Ga-labeled oxalyldiaminopropionic acid (ODAP)-urea based prostate specific membrane antigen (PSMA) targeting probe, and evaluate its properties in vitro and in vivo. Methods:Ligand P151 was synthesized by solid-phase synthesis and its Ki value was determined. The ligand P151 was added into the mixture of 68GaCl 3 and NaOAc solution and was reacted at 95 ℃ for 10 min. The labeling yield and in vitro stability of 68Ga-P151 were determined by high performance liquid chromatography (HPLC). The lipid-water partition coefficient (log P) and cell binding ability were determined. The biodistribution of 68Ga-P151 in normal KM mice was determined. MicroPET imaging of 68Ga-P151 was carried out in prostate cancer 22Rv1 tumor-bearing mice and compared with 68Ga-PSMA 617. Independent sample t test was used to analyze the data. Results:P151 was successfully synthesized with the Ki of 0.58 nmol/L, the labeling yield more than 95% and the radiochemical purity more than 95%. After placement in saline or human serum albumin (HSA) solution at 37 ℃ for 2 h, the radiochemical purity of 68Ga-P151 was still more than 95%, indicating a good stability in vitro. The lipid-water partition coefficient (log P) of 68Ga-P151 was -2.65±0.17, indicating a good hydrophilicity. 68Ga-P151 specifically bound to PSMA in prostate cancer LNCaP cells with the uptake value of (0.83±0.04) percentage injection activity (%IA)/10 5 cells. Biodistribution of normal mice showed that 68Ga-P151 was mainly excreted through kidneys and other organs showed low uptake. MicroPET imaging of tumor-bearing mice showed the maximum standardized uptake value (SUV max: 0.79±0.23 vs 0.54±0.05; t=2.12), tumor/kidney ratio (2.04±0.65 vs 1.88±0.33; t=0.44) and tumor/muscle ratio (12.83±5.18 vs 6.95±1.63; t=2.17) between 68Ga-P151 and 68Ga-PSMA 617 were not significantly different (all P>0.05). Conclusions:68Ga-P151 can be prepared simply and labeled in high yield and show improved pharmacokinetic properties in vivo. The imaging of 68Ga-P151 on PSMA positive tumor is comparable to that of 68Ga-PSMA 617, making it a potential radiopharmaceutical for the diagnosis of prostate cancer.
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ABSTRACT Objective: To evaluate genotoxicity of zinc oxide, P. A. calcium hydroxide, mineral trioxide aggregate and an iodoform paste using comet assay on human lymphocytes. Material and Methods: Two positive controls were used: methyl-methanesulfonate for the P.A. calcium hydroxide and mineral trioxide aggregate; and doxorubicin for the iodoform paste and zinc oxide. There were also two negative controls: distilled water for the P.A. calcium hydroxide and mineral trioxide aggregate; and DMSO for the iodoform paste and zinc oxide. Comets were identified using fluorescence microscopy and 100 of them were counted on each of the three slides analyzed per drug test. A damage index was established, taking into consideration the score pattern that had previously been determined from the size and intensity of the comet tail. Analysis of variance, followed by Tukey's test, was used to compare the means of the DNA damage indices. Results: The DNA damage index observed for mineral trioxide aggregate (7.08 to 8.58) and P.A. calcium hydroxide (6.50 to 8.33), which were similar to negative control index. On the other hand, damage index for zinc oxide (104.7 to 218.50) and iodoform paste (115.7 to 210.7) were similar to positive control index. Conclusion: Iodoform paste and zinc oxide showed genotoxicity at all concentrations used.
Subject(s)
Humans , Tooth, Deciduous , Zinc Oxide , Comet Assay , Genotoxicity , Mutagenicity Tests/instrumentation , Zinc Oxide , Brazil , Calcium Hydroxide , Analysis of Variance , Microscopy, FluorescenceABSTRACT
Objective:To investigate the expression of microRNA (miRNA, miR)-4699-3p in ovarian cancer cell lines, and observe its ability to regulate the expression of mitochondrial ribosomal protein S23 (MRPS23) and its effect on the migration and proliferation of ovarian cancer cells.Methods:Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-4699-3p in ovarian cancer cell lines (OVCAR-3, SKOV-3, HO-8910, OC3, A2780) and normal ovarian epithelial cells (IOSE80). The liposome transfection method was used to transfect miR-4699-3p mimic or negative control miR-NC to the cell line with the lowest miR-4699-3p expression, which was defined as the experimental group and the control group. qRT-PCR was used to verify transfection efficiency. Bioinformatics technology predicted the candidate target gene of miR-4699-3p, and the dual luciferase reporter gene experiment identified its ability to regulate the target gene. qRT-PCR and Western blot were used to detect the expression of target genes at the mRNA and protein levels. Cell counting method (CCK-8) and transwell migration experiment were used to detect the effect of miR-4699-3p on the proliferation and migration of ovarian cancer cells.Results:The expression of miR-4699-3p in ovarian cancer cell lines was significantly lower than that of normal ovarian epithelial cells ( P<0.05), and the lowest expression in OVCAR-3 cells ( P<0.01). After transfection of miR-4699-3p, the expression of miR-4699-3p in OVCAR-3 cells in the experimental group was significantly increased ( P<0.01), which proved that the transfection was successful. Bioinformatics technology predicted that the candidate target gene of miR-4699-3p may be MRPS23. The dual luciferase reporter gene experiment confirmed that miR-4699-3p can directly target the 3′-UTR of MRPS23 gene mRNA ( P<0.01). Compared with the OVCAR-3 cells in the control group, the mRNA and protein expression levels of the MRPS23 gene were significantly reduced, while the expressions of Twist, Slug, CDK6 and Cyclin D3 were significantly decreased ( P<0.01) in the experimental group after up-regulating miR-4699-3p expression ( P<0.01). After up-regulating miR-4699-3p expression, the proliferation ability of OVCAR-3 cells decreased ( P<0.05) and the migration ability of OVCAR-3 cells was reduced ( P<0.01). Conclusions:miR-4699-3p is under-expressed in ovarian cancer cell lines. Up-regulation of miR-4699-3p expression in OVCAR-3 cells can inhibit ovarian cancer cell proliferation and migration by interfering with MRPS23 gene expression.
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Objective:The purpose of this study was to investigate the expression and role of N-myc downstream regulatory gene 2 (NDRG2) in radiation resistance of bladder cancer cells.Methods:T24 cells were cultured in vitro and irradiated with different doses of X-ray (0, 2, 4, 8, 10 and 20 Gy). The best dose of X-ray was selected for subsequent treatment. The radioresistant BCa cell line T24/R was established. The cytotoxicity of T24/R cells was detected by counting kit-8 (CCK-8) method. The proliferation and invasion ability of T24/R cells and T24 cells were detected by flow cytometry and transwell, respectively. Western blot was used to detect the expression of epithelial mesenchymal transition (EMT) related proteins. The survival rate of T24/R group (control group) and T24/R-NDRG 2 group was detected, and the migration ability of T24/R-NDRG 2 cells was detected after 2 Gy treatment. Results:The cell viability was inhibited significantly when the dose of X-ray was ≥2 Gy X-ray, so 2 Gy X-ray irradiation was chosen as the best condition for BCa cytotoxicity and T24/R radiation resistance cell line was successfully established; Apoptosis test showed that the number of S-phase cells was increased in T24/R group, and the proportion of S-phase cells in T24/R vs T24 was (26.49±4.5)% vs (14±2.6)% ( P<0.05); Transwell test showed that T24/R cells showed stronger migration ability than control group ( P<0.05), but there was no significant difference in EMT related protein expression between the two groups ( P>0.05). Overexpression of NDRG2 can significantly decreased the activity and migration ability of radiation-resistant T24/R cells ( P<0.05) when the radiation dose was gradually increasing in both groups. Conclusions:The radiation resistance of BCa cells is one of the causes of local tumor recurrence. Up-regulation of NDRG2 expression can inhibit the radiation resistance of T24 cells, so it can be used as a candidate for treatment of radiation-resistant BCa patients.
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Objective:To investigate the effects of triptolide on radiosensitization of lung cancer A549 cells and the underlying mechanism.Methods:During June-September 2019, lung cancer A549 cells were treated with different concentrations of triptolide for 24 and 48 hours in Animal Experiment Center, Zhejiang Chinese Medical University, China. The inhibitory effects of triptolide on the proliferation of lung cancer A549 cells were determined using MTT method. Appropriate concentrations of triptolide and double distilled water were added to the experimental and control groups, respectively. The effects of triptolide on radiosensitization of lung cancer A549 cells was determined by colony formation assay. Radiosensitization ratio was calculated. Lung cancer A549 cells were divided into blank control, triptolide, radiotherapy, and radiotherapy + triptolide groups. The effects of triptolide on apoptosis and cell cycle of lung cancer A549 cells were determined by flow cytometry.Results:The 10% inhibitory concentration (IC 10) and half maximal inhibitory concentration (IC 50) of triptolide for treating lung cancer A549 cells were 36.61 nmol/L and 259.38 nmol/L, respectively at 24 hours, and they were 9.05 nmol/L and 61.49 nmol/L, respectively at 48 hours. Triptolide had an radiosensitization effect on lung cancer A549 cells, with the radiosensitization ratio of 1.135. The apoptosis rate in the radiotherapy + triptolide group was significantly higher than that in the radiotherapy [(45.47 ± 8.29)% vs. (5.25 ± 0.59)%, t = 6.847, P = 0.002]. The proportion of lung cancer A549 cells at the G2/M phase in the radiotherapy group was significantly higher than that in the radiotherapy + triptolide group [(27.82 ± 0.96)% vs. (11.98 ± 0.55)%, t = 20.176, P < 0.05]. The proportion of lung cancer A549 cells at the G2/M phase in the black group was significantly higher than that in the triptolide group [(17.31 ± 3.42)% vs. (8.05 ± 0.71)%, t = 3.749, P = 0.02]. Conclusion:Triptolide has a radiosensitization effect on lung cancer A549 cells, and the underlying mechanism may be related to its participation in cell apoptosis and cycle regulation.
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Objective To explore the effects of graphene oxide (GO)-polyethylene glycol (PEG)-folic acid (FA)-pyrenemethylamine hydrochloride (PyNH2)-mediated RNA interference (RNAi) of hypoxia-inducible factor-1α (HIF-1α) on the biological behaviors of human pancreatic cancer Patu8988 cells.Methods GO-PEG-FA-PyNH2 and RNAi targeting HIF-1α gene (GO-PEG-FA-PyNH2-HIF-1α-RNAi)was constructed.The expressions of HIF-1α and glucose transporter 1 (Glut-l) in Patu8988 cells were determined after knockdown of HIF-1α by RNAi.The invasive ability,the proliferation and the cell cycle of Patu8988 cells were investigated.The effect of HIF-1α knockdown on the uptake of 18F-fluorodeoxyglucose (FDG) in Patu8988 cells was also detected.Comparison of data was conducted by one-way analysis of variance and least significant difference t test.Results The GO-PEG-FA-PyNH2 was successfully constructed,and no cytotoxicity was found.Under the hypoxia or normoxia state,the mRNA and protein levels of HIF-1α and mRNA level of Glut-1 in cells transfected with GO-PEG-FA-PyNH2-HIF-1α-RNAi (study group) were lower than those in cells transfected with GO-PEG-FA-PyNH2 (negative group) and cells transfected with Opti-minimal essential medium (Opti-MEM,control group;F=30.25-32.58,t=3.66-5.81,all P＜0.05);the numbers of migrated cells in the study group were much lower than those in the negative group and the control group (F=38.63 and 41.35,t=20.51-35.25,all P＜0.01);the cell proliferation in the study group was significantly lower than that in the negative group and the control group (F=35.19 and 38.11,t =15.11-22.19,all P＜0.05).The proportions of G0/G1 cells in the study group were higher than those in the negative group and the control group (F=34.60 and 34.83,t=11.55-34.56,all P＜0.05);the 18 F-FDG uptake in the study group was lower than that in the negative group and control group (F=29.85 and 31.69,t =3.35-4.35,all P＜0.05).Conclusion GO-PEG-FA-PyNH2-mediated HIF-1α RNAi inhibits the expression of HIF-1α in pancreatic cancer cells,leading to changes in related biological behaviors.
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Objective To fabricate manganese-doped carbon quantum dots(Mn-CDs)@anti-human epididymis protein 4(HE4)monoclonal antibody(Mn-CDs@Anti-HE4 mAb)dual-modal fluorescent-magnetic nanoprobe for ovarian cancer cells targeting imaging,and evaluate its potential on fluorescent imaging and MRL Methods Mn-CDs were synthesized at 150 ℃ with solvothermal method.The average diameter,fluorescent capability and MRI efficiency were determined.The cytotoxicity of Mn-CDs in vitro was evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS)assay with HO-8910 ovarian cancer stem cells and EA.hy926 human umbilical vein endothelial cells.Mn-CDs@Anti-HE4 mAb was fabricated with condensation reaction and characterized by ultraviolet(UV)absorption spectra.Fluorescence imaging and MRI in vitro was performed for cancer cell-targeting study.One-way analysis of variance and the least significant difference t test were used to analyze the data.Results The Mn-CDs with diameter of(4.64±0.85)nm showed a well-defined spherical morphology.The fluorescent spectra of Mn-CDs exhibited a typical excitation-dependent behavior with an excitation maximum at 360 nm and emission maximum at 440 nm.The T1 relaxation rate was(3.26±0.04)mmol ? L-1 ? s-1.The cytotoxicity tests in vitro showed that the survival rates of HO-8910 cells and EA.hy926 cells were both significantly different after treated with different concentrations of Mn-CDs(F= 1 947.509,260.174,both P<0.05),and there was no cytotoxicity in both HO-8910 cells and EA.hy926 cells at concentrations of MnCDs within 0-2.5 mg/ml(all P>0.05),while the survival rates of the two kinds of cells were descended with the increasing of concentration within 3.0-4.5 mg/ml(P<0.05).Mn-CDs@Anti-HE4 mAb could target HO-8910 cells on fluorescence imaging and MRI.Conclusions Mn-CDs@Anti-HE4 mAb,with good potential on fluorescence imaging,MRI and targeting ability,is successfully synthesized.It may provide a new method for early diagnosis of ovarian cancer.
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Objective@#To investigate the Rhizoma Pleionis mediating vascular endothelial growth factor A (VEGF-A)-related angiogenesis in breast cancer cells to inhibit epithelial-mesenchymal transition (EMT) in breast cancer.@*Methods@#Firstly, the proliferation activity of MDA-MB-231 and 4T1 of breast cancer cells was detected by methyl thiazolyl tetrazolium (MTT) method at different concentrations (0.2 g/ml, 0.4 g/ml, 0.6 g/ml, 0.8 g/ml, 1.0 g/ml); secondly, the specific concentration of Rhizoma Pleionis was selected and tested by Transwell test for its effect on breast cancer cell metastasis and invasion. The formation of human umbilical vein endothelial cells (HUVEC) capillary wall was measured to detect the effect of Rhizoma Pleionis on cell angiogenesis. The EMT-related protein and angiogenesis-related molecules were detected by Western blot.@*Results@#MTT results showed that Rhizoma Pleionis of 0.8 g/ml and 1.0 g/ml can inhibit the MDA-MB-231 and 4T1 cells proliferation in a dose-dependent manner, with statistically significant difference (P<0.05). Rhizoma Pleionis can inhibit the invasion of breast cancer cells and inhibit the formation of blood vessel walls in HUVECs. Western blot confirmed that the expression level of EMT-related proteins: E-cadherin was significantly increased and the expression level of N-cadherin, Vimentin and Snail was significantly decreased after treatment with Rhizoma Pleionis (P<0.05); and the level of the related molecule hypoxia inducible factor-1α (HIF-1α), VEGF-A and vascular endothelial growth factor receptor-2 (VEGFR-2) was significantly reduced (P<0.05).@*Conclusions@#Rhizoma Pleionis inhibits the epithelial-mesenchymal transition of breast cancer by influencing the angiogenesis of VEGF-A, and inhibits the invasion and metastasis of breast cancer.
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Objective@#To synthesis 177Lu-prostate specific membrane antigen (PSMA)-I&T with automated module, evaluate the biodistribution and pharmacokinetics in mice and study the targeting property in human prostate cancer cell line LNCaP Clone FGC.@*Methods@#The iQS-TS automated module was applied in labeling 177Lu-PSMA-I&T. Radiochemical purity and stability were determined with high performance liquid chromatography (HPLC). The biodistribution was observed in normal ICR mice and U-SPECT/CT imaging was performed in LNCaP Clone FGC tumor-bearing mice. Independent-sample t test was used to analyze the data.@*Results@#177Lu-PSMA-I&T was stable in vitro and in vivo, with the radiolabeled yield of (91.5±4.9)% and radiochemical purity >99%. The half maximal inhibitory concentration (IC50) of 177Lu-PSMA-I&T binding to LNCaP Clone FGC cells was (26.74±3.53) nmol/L. The uptake of 177Lu-PSMA-I&T by LNCaP Clone FGC cells increased with time and significantly decreased after the inhibitor addition (t values: 4.301-27.483, all P<0.05). 177Lu-PSMA-I&T was cleared from blood rapidly and predominantly excreted by kidneys. Significant radioactive uptake was observed in tumors with a long retention time.@*Conclusion@#177Lu-PSMA-I&T can be produced in a convenient and efficient procedure using iQS-TS automated module, with good biological properties and excellent affinity and targeting property towards prostate cancer cells, which making it a potential radiopharmaceutical for prostate cancer therapy.
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Objective@#To prepare manganese-doped carbon quantum dots (Mn-CDs) dual-modal nanoprobe for fluorescent-magnetic imaging, and evaluate its characteristics and potential on fluorescence imaging and MRI.@*Methods@#Mn-CDs were synthesized at 150 ℃. The form, diameter, component, fluorescent capability, T1 relaxation rate, stability and cytotoxicity of Mn-CDs in vivo were verified. The fluorescence imaging of HO-8910 tumor-bearing mice was performed on small animal imager, and the whole-body enhanced imaging was performed on 3.0 T MRI scanner. One-way analysis of variance was used to analyze the data.@*Results@#The Mn-CDs with the diameter of (4.64±0.85) nm showed a well-defined spherical morphology. The fluorescent spectra of Mn-CDs exhibited that the excitation maximum was at 360 nm and the emission maximum was at 440 nm. The T1 relaxation rate was (3.26±0.04) mmol·L-1·s-1. The Mn-CDs had good stability of fluorescent and magnetic imaging capability at 0, 0.25, 0.5, 0.75, 1.0 and 2 months at room temperature with no significant differences of fluorescent and magnetic signals (F=1.566 and 0.987, both P>0.05). After injection of 200 μl Mn-CDs (15 g/L), mice were all alive and had no viscera damage. The tumor could be observed obviously on fluorescence imaging at 5 min. Enhanced MRI showed that the tumor was unevenly enhanced and Mn-CDs were mainly cleared away through urinary system.@*Conclusion@#Mn-CDs are stable and have good potential on fluorescence imaging and MRI, which provides a promising multimodal imaging method for tumor detection and monitoring.