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Objective To investigate the effect and mechanism of miR-20a-5p on human nephroblastoma cell line WiT49 transplanted tumor in nude mice.Methods The gene expression chip was downloaded from GEO database,and the differential gene miR-20a-5p was obtained by GEO2R.The NF-κB gene was positively correlated with the expression of miR-20a-5p through cBioPortal database.The target gene of miR-20a-5p was predicted to be NFKBIB of the NF-κB transcription factor suppressor protein family by targetscan database,and was verified by dual luciferase assay.Nephroblastoma cell line WiT49 was cultured in vitro and transfected into WiT49 cells with lentiviral vectors constructed with miR-20a-5p mimics and its suppressor gene.Twelve nude mice were randomly divided into three groups:WiT49 model group,WIT49-miR-20a-5p overexpression group and WIT49-miR-20a-5p knockdown group.The tumor mass and volume of each group were detected by tumor formation experiment in nude mice.real time fluorescent quantitative polymerase chain reaction(qRT-PCR)was used to detect the expression of miR-20a-5p,NFKBIB and NF-κB in each group;CCK-8 cell proliferation assay was used to verify the proliferation of tumor cells in each group.Results miR-20a-5p is highly expressed in nephroblastoma and is positively correlated with the expression of NF-κB.miR-20a-5p and NFKBIB have mutual binding sites and binding effects.In the tumor formation experiment of nude mice,the tumor volume and mass of WIT49-miR-20a-5P overexpression group were significantly increased compared with WiT49 model group,and the difference was statistically significant(P<0.05).In the qRT-PCR test,the expressions of miR-20a-5p and NF-κB in the WIT49-miR-20a-5p overexpression group were higher than those in the WiT49 model group,and NFKBIB expression in the WIT49-miR-20a-5p overexpression group was lower than that in the WiT49 model group,with statistical significance(P<0.05).CCK-8 cell proliferation assay showed that the absorbance of WIT49-miR-20a-5p overexpression group at 24 and 48 hours was higher than that of WiT49 model group,and the absorbance of WIT49-miR-20a-5p knockdown group at 24,48 and 72 hours was lower than that of WiT49 model group,and the difference was statistically significant(P<0.05).Conclusion miR-20a-5p may promote the growth of human nephroblastoma cell WiT49 transplanted tumor in nude mice by regulating NFKBIB activation of NF-κB pathway.
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Objective:To prepare a fluorescent probe Cetuximab-IRDye800CW targeting epidermal growth factor receptor (EGFR) and investigate its application value in surgical navigation of glioblastoma (GBM).Methods:The fluorescence properties of Cetuximab-IRDye800CW were determined by fluorescence spectrophotometer. The specificity of Cetuximab-IRDye800CW bound to GBM cells was verified by Western blot. The competitive binding method of enzyme-linked immunosorbent assay (ELISA) was used to prove whether the probe could achieve tumor targeting by binding to EGFR. Subcutaneous models of 6 nude mice of GBM were divided into experimental group ( n=3; injected with Cetuximab-IRDye800CW) and control group ( n=3; injected with IRDye800CW), and images were obtained at 5 min, 24 h, 48 h and 72 h after injection. Differences of mean fluorescence intensity (MFI) and tumor to background ratio (TBR) between experimental group and control group were compared. In situ models of GBM nude mice were established ( n=6), and MRI and intraoperative navigation were conducted, which were compared with pathological distribution. Independent-sample t test was used to analyze the data. Results:The maximum emission wavelength of Cetuximab-IRDye800CW was 820 nm, which could be received by near infrared fluorescence imaging equipment. Western blot showed that Cetuximab-IRDye800CW was only bound to GBM cells. The competitive binding of ELISA showed that Cetuximab-IRdye800CW could achieve tumor targeting by binding with EGFR. At 5 min, 24 h, 48 h and 72 h after injection of fluorescent materials, the MFI values of experimental group were 109.00±3.81, 73.36±9.93, 55.24±8.82, 37.71±6.11, which were higher than those of control group (91.32±4.17, 42.91±5.39, 25.08±6.05, 8.33±1.00; t values: 4.36-9.40, P values: 0.011-0.049). The TBR of experimental group was higher than that of control group at 24 h and 48 h after injection (24 h: 2.40±0.28 vs 1.57±0.07, t=4.94, P=0.039; 48 h: 2.07±0.12 vs 1.22±0.08, t=9.85, P=0.010). GBM in situ model was successfully constructed and verified by MRI, and the tumor was visualized under the fluorescence device navigation. Pathological distribution of the tumor with HE staining was consistent with fluorescence imaging. Conclusion:Cetuximab-IRDye800CW has fluorescence imaging capability and can identify tumor boundaries in intraoperative navigation of GBM, which has potential clinical application value.
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ObjectiveTo observe the primary tumor of tree shrews and to provide a basis for studying the pathogenesis and prevention of trichoepithelioma. MethodsA tumor was discovered in the chest and abdomen of a tree shrew during natural cultivation. The tree shrew was anesthetized, and the tumor was surgically removed. Hematoxylin and eosin (HE) staining and immunohistochemical staining were performed on the tumor tissue after paraffin section, and the tumor cells were isolated and cultured by passage. The isolated tumor cells were subcutaneously injected into healthy tree shrews and nude mice. The tumorigenesis of tumor cells in vivo was observed once a day, with nude mice continuously observed for 2 months and tree shrews observed for more than 6 months. ResultsHE staining showed that the basal cells in the dermis were arranged as a whole, like a string of petals, forming nests and stripe-like structures with clear boundaries. The observation results after magnification revealed that the tumor cells were arranged in a pallisade-like and basal pattern, with deep nuclear staining and minimal cytoplasmic. Immunohistochemical staining showed the high expression of CK protein and low proportion expression of ki-67 protein in tumor cells, as well as the high expression of vimentin and low expressions of Bcl2 and CD10 in tumor cell mesenchyme. The isolated tumor cells grew well in DMEM medium containing 10% fetal bovine serum and could be cultured by passage, but no tumor formation was observed in healthy tree shrews and nude mice inoculated with tumor cells. ConclusionCombined with the location of the tumor, overall morphology, HE staining, and immunohistochemical results, the thoracoabdominal mass of the tree shrew was diagnosed as a trichoepithelioma.
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Objective:To evaluate the effects of high mobility group protein box 1 (HMGB1) on clinical prognosis of esophagus squamous cell carcinoma (ESCC) patients treated with chemoradiotherapy and the radiosensitivity of xenograft in nude mice.Methods:A total of 90 endoscopic biopsy specimens were obtained from ESCC patients treated with chemoradiotherapy. The expression level of HMGB1 was determined by immunohistochemical staining. High expression level was defined when staining was observed on ≥50% of the tumor cells. All patients were divided into the high expression group ( n=48) and low expression group ( n=42), and their survival information was retrospectively analyzed. Cell transfection was performed with the plasmid carrying human HMGB1-shRNA to knockdown HMGB1 expression in ECA109 cells and xenograft mouse models were established. The tumor volume and mass were calculated after irradiation with a dose of 15 Gy. The cell apoptosis in xenograft tissues were detected. Survival analysis was performed using Kaplan-Meier method. Univariate prognostic analysis was conducted by log-rank test. Intergroup comparison was performed by analysis of variance (ANOVA). Results:The expression level of HMGB1 was significantly associated with gross tumor volume, longest diameter of tumor, T staging and distant metastasis ( χ2=9.663, 5.625, 4.068, 7.146, all P<0.05). In the low expression group, the overall survival (OS) ( χ2=4.826, P=0.028), progression-free survival (PFS) ( χ2=4.390, P=0.036) were longer compared with that in the high expression group. Further analysis of HMGB1-high expression patients showed that the radiation dose and the combination of chemoradiotherapy did not significantly affect the OS or PFS of ESCC patients. We observed that knockdown of HMGB1 slowed the growth rate of xenograft, decreased the tumor volume and increased the apoptosis rate after irradiation. Conclusions:ESCC patients with high expression level of HMGB1 obtain poor prognosis after chemoradiotherapy, which can be enhanced by increasing the sensitivity to radiotherapy and chemotherapy. HMGB1 knockdown can effectively increase the radiosensitivity of xenograft in ESCC nude mice.
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Objective:To explore the application potential of 18F-Asp-Glu-val-Asp (DEVD)-Cys(StBu)-PPG(CBT)-AmBF 3 ( 18F-1; PPG: propargyl-glycine; CBT: 2-cyanobenzothiazole; AmBF 3: ammoniomethyl-trifluoroborate) PET imaging in early monitoring of triple-negative breast cancer (TNBC) radiotherapy response. Methods:Ten MDA-MB-231 tumor bearing nude mice models were constructed and divided into radiotherapy group ( n=5) and non-radiotherapy group ( n=5) by random sampling method. The radiotherapy group was treated with single irradiation at a dose of 8 Gy. 18F-1 microPET imaging was performed in the radiotherapy and non-radiotherapy groups, and the tumor uptake and muscle uptake in 2 groups at different time points (2.5, 7.5, 12.5, 17.5, 22.5, 27.5, 32.5, 37.5, 42.5, 47.5, 52.5, 57.5 min after injection) were analyzed. The specific uptake of the probe in apoptotic cells was verified by radioautography, HE staining and immunofluorescent staining. Repeated measures analysis of variance and one-way analysis of variance were used to analyze data. Results:18F-1 microPET imaging showed that there was significant difference between tumor uptake and muscle uptake in radiotherapy group ( F=20.27, P=0.011). The uptake of radiotherapy group was the highest at 7.5 min after injection ((4.64±0.35) percentage activity of injection dose per gram of tissue(%ID/g)). There was no significant difference between tumor uptake and muscle uptake in the non-radiotherapy group ( F=1.81, P=0.215). The tumor/muscle (T/M) ratio of radiotherapy group was higher than that of non-radiotherapy group ( F=31.95, P=0.005), with the highest at 47.5 min after injection (2.49±0.46). Radioautography showed that the tumor radioactivity in radiotherapy group was higher than that of muscle in radiotherapy group, and was also higher than tumor and muscle radioactivies in non-radiotherapy group ( F=116.79, P<0.001). HE staining and immunofluorescent staining verified that 18F-1 could specifically detect the activity of caspase-3 activated in tumor cells after radiotherapy. Conclusion:18F-1 can specifically recognize the activated caspase-3 after TNBC radiotherapy, and monitor radiotherapy response at the molecular level by apoptosis PET imaging.
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Objective:To develop a tetramer probe targeting fibroblast activation protein (FAP), named 1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid (DOTA)-4P(FAP inhibitor (FAPI)) 4, evaluate its biodistribution and PET image in FAP-positive-tumor bearing nude mice, and explore its feasibility as a novel radio-regent for treatment of FAP-positive tumor. Methods:FAP tetramer probe was constructed on the FAPI-46 motif with four mini-polyethylene glycol (PEG)(PEG 3) spacers between the four FAPI motifs, denoted as 4P(FAPI) 4. DOTA was used as the chelator for radiolabeling with 68Ga and 177Lu. The FAP binding characteristics were test by in vitro cell competitive binding experiment. Small-animal PET, in vivo biodistribution, and radionuclide targeting therapy were performed in HT-1080-FAP tumor bearing nude mice ( n=39). Independent-sample t test was performed to analyze tumor uptake data, and two-factor repeated measures analysis of variance was utilized to compare tumor volume data in radioactive isotope therapy. Results:Cell experiment showed that FAPI-tetramer and FAPI-monomer had similar half maximal inhibitory concentration values (3.29 and 2.15 nmol/L). 68Ga/ 177Lu radiolabeled FAPI-tetramer had better tumor uptake and retention than FAPI-monomer in small-animal PET and in vivo biodistribution experiment, with the tumor uptake for 177Lu-DOTA-4P(FAPI) 4 and 177Lu-FAPI-46 at 48 h of (18.72±1.32) vs (2.72±1.20) percentage activity of injection dose per gram of tissue (%ID/g) ( t=15.55, P<0.001). 177Lu-DOTA-4P(FAPI) 4 group showed best anti-tumor efficacy compared with 177Lu-FAPI-46 and control group in radionuclide targeting therapy. On the 2nd day after the start of treatment, the tumor volume in the tetramer treatment group was significantly smaller than that in the control group (mean difference 67.19 mm 3, P=0.049); on the 14th day after the start of treatment, the tumor volume in the tetramer treatment group was significantly smaller than that in the monomer treatment group (mean difference 414.33 mm 3, P=0.005). Conclusion:FAPI-tetramer can improve tumor uptake and retention ability compared with FAPI-46, and 177Lu-DOTA-4P(FAPI) 4 can be a promising radio-agent for FAP-positive tumor therapy.
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AIM:To investigate the inhibitory effect of the"decoction for soothing liver and removing stasis and toxicity(SGQYJDF)"on hepatocellular carcinoma(HCC)proliferation in nude mice by inducing ferroptosis via the tumor protein 53(p53)/solute carrier family 7 member 11(SLC7A11/xCT)/glutathione peroxidase 4(GPX4)pathway.METHODS:An ectopic subcutaneous tumor model was established by injecting SK-Hep-1 cells subcutaneously into the right axilla of nude mice.Upon formation of tumor,the mice were randomly divided into five groups(i.e.,control group,low-,medium-and high-dose SGQYJDF groups and medium-dose SGQYJDF plus Sorafenib group).Each group of mice was orally administered with the corresponding therapy for 14 consecutive days,during which the tumor size was observed regularly.At the end of treatment,the tumor growth inhibition rate was calculated based on tumor mass,and histopatho-logical changes were observed by HE staining.Then,the levels of malondialdehyde(MDA),glutathione(GSH)and fer-rous ions(Fe2+)were detected by colorimetric assays.The expression of the proliferation markers Ki-67 and GPX4 was de-tected by immunohistochemistry(IHC).The expression of p53 and xCT was detected by Immunofluorescence(IF).And the expression of p53,xCT and GPX4 was determined by Western blot.RESULTS:(1)SGQYJDF was found to dose-de-pendently decrease tumor volume(P＜0.01)and inhibit tumor mass growth(P＜0.01),and meanwhile,reduce the per-centage of Ki-67-positive cells(P＜0.01)and their proliferation ability in tumor tissues,as compared to the control group.(2)In terms of Ferroptosis-related indicators,SGQYJDF was found to dose-dependently increase the levels of Fe2+ and MDA but decrease the level of GSH in tumor tissues(P＜0.01),as compared to the control group.(3)In terms of protein expression,SGQYJDF was found to dose-dependently upregulate the expression of p53(P＜0.05)but inhibit the expres-sion of xCT(P＜0.05)and GPX4(P＜0.01).Notably,medium-dose SGQYJDF plus sorafenib showed a stronger effect than high-dose SGQYJDF.CONCLUSION:Our findings suggest that SGQYJDF can induce Ferroptosis to inhibit the proliferation of subcutaneously transplanted tumor tissues in nude mice by upregulating the expression of p53,and inhibit-ing the expression of xCT and GPX4.
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C17 is an orally available anti-tumor compound inhibiting cancer stem cell (CSC). In this study, a stable, sensitive and simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was established and validated, and was further applied to a pharmacokinetic study in nude mice receiving C17 by gavage. Using propranolol as the internal standard, the plasma samples were pre-treated by precipitation with methanol and analyzed on an Intersil C8-3 column (100 mm × 2.1 mm, 3 μm), and gradient elution was performed with a mobile phase consisting of 0.1% formic acid aqueous and solution mixed up by 90% isopropanol and 10% acetonitrile. The analyte was detected by a triple quadrupole tandem mass spectrometer, and multiple reaction monitoring was employed to select C17 at m/z 439.3/247.1 and propranolol at m/z 260.2/116.2 in the positive ion mode. The calibration curves were linear (r > 0.995) over the range of 5-800 ng·mL-1. The intra- and inter-day precisions and accuracies were 7.42%-13.22% and -8.99%-8.81% respectively. The method was successfully applied to a PK study in nude mice administered with a single oral dose of 50 mg·kg-1 C17, and the PK data were analyzed with non-linear mixed effect model (NONMEM). Two separated absorption peaks were found in the PK curve of C17, and a two-compartment model with two sequential first-order absorption rate was utilized to describe the PK properties of C17, and the model could provide insights into the physiological process and exposure of C17 in nude mice. All animal experiments were in strict accordance with the regulations of the Biomedical Ethics Committee of Peking University.
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Objective:To observe the inhibitory effects of Huangqi Jiedu Decoction on lung metastasis of breast cancer in nude mice; To explore the mechanism of intervening epithelial mesenchymal transformation (EMT) induced by Wnt/β-catenin signaling pathway.Methods:Totally 30 nude mice were divided into model group, adriamycin group and Huangqi Jiedu Decoction low-, medium-, and high-dosage groups according to random number table method. Each group was injected subcutaneously with mouse breast cancer 4T1 cells to construct tumor - bearing nude mice model. Huangqi Jiedu Decoction low-, medium- and high-dosage groups were intragastrically administrated with Huangqi Jiedu Decoction 17.82, 35.64 and 71.28 g/kg; adriamycin group was injected intraperitoneally adriamycin 0.05 g/kg; model group was intragastrically administrated with normal saline of the same volume for 21 d. Tumor volume was measured at 9, 15, and 21 days after modeling. After the end of administration, the tumor tissue was separated, the tumor weight was measured, and the tumor inhibition rate was calculated. The lung tissue was Isolated,, the number of lung metastatic nodules and the inhibition rate of lung metastasis was counted. HE staining was used to observe the tissue morphology and evaluate the effectiveness of the model. The protein expressions of β-catenin, E-Cadherin and Vimentin in lung tissue were detected by Western Blot. The mRNA levels of β-catenin, E-Cadherin and Vimentin in lung tissue were detected by real-time fluorescent quantitative PCR.Results:Compared with the model group, the tumor volume and mass of Huangqi Jiedu Decoction low-, medium- and high-dosage groups decreased ( P<0.01); the number of pulmonary metastasis nodules in Huangqi Jiedu Decoction high-dosage group significantly decreased ( P<0.01); the mRNA and protein expressions of β-catenin and Vimentinm decreased in the Huangqi Jiedu Decoction low-, medium- and high-dosage groups ( P<0.01), and the protein and mRNA expressions of E-Cadherin increased in the Huangqi Jiedu Decoction high-dosage group ( P<0.01). Conclusion:Huangqi Jiedu Decoction can effectively inhibit the growth and lung metastasis of breast cancer transplanted tumor, and the mechanism may be to down-regulate the expression of key molecules in the Wnt/β-catanin signaling pathway, thereby inhibiting the EMT process, so as to inhibit the lung metastasis of breast cancer.
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Objective:To evaluate the effect of compound UC2288 on the radiosensitivity of CNE-2R cell line and nude mouse transplanted tumor.Methods:The UC2288 concentration was referenced to previous experimental results (IC 50=12.20 μmol/L). The effect of UC2288 combined with 2, 4, 6, 8 Gy X-ray irradiation on the radiosensitivity of CNE-2R cell line was detected by clone formation experiment. The effect of UC2288 combined with 2, 4, 6, 8 Gy X-ray irradiation on the proliferation of CNE-2R cell line was determined by CCK8 assay. The nude mouse model of transplanted tumor was constructed with CNE-2R cell line. The radiosensitivity of transplanted tumor of UC2288 combined with 2 Gy/fraction X-ray irradiation for three consecutive days was evaluated. Results:The experimental concentration of UC2288 was 8 μmol/L. The clonality of CNE-2R cell line was reduced under UC2288 combined with X-ray 2, 4, 6, and 8 Gy irradiation, andthe radiosensitizationratio was 1.60. The proliferation of CNE-2R cell line was significantly decreased under UC2288 combined with X-ray 2, 4, 6, and 8 Gy irradiation. UC2288 inhibited the growth of transplanted tumor in nude mice, and the inhibitory effect was strengthened with the extension of observation time, and the most obvious effect was observed at 16 d. ( P<0.01). Theradiosensitizationratio was 4.33. The proliferation of CNE-2R cell line was decreased under UC2288 combined with X-ray irradiation. Conclusion:UC2288 can increase the radiosensitivity of nasopharyngeal carcinoma radioresistant cell line CNE-2R.
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Objective:Apt-A10-3.2 (aptamer of prostate specific membrane antigen (PSMA)) can be used as a specific ligand for early diagnosis and targeted treatment of prostate cancer. Mouse double minute 2 homolog (MDM2) is closely related to the malignancy of prostate cancer, and MDM2 small interfering RNA (siRNA) can silence MDM2 gene through RNA interference. To design a novel chimera of PSMA Apt-MDM2 siRNA and combine it with docetaxel (DTX) to explore a new diagnosis and treatment model combining targeted therapy of PSMA-positive prostate cancer with 99Tc m-chimera imaging monitoring. Methods:Apt-siRNA were obtained by covalent connection of PSMA Apt-A10-3.2 and MDM2 siRNA, which was combined with DTX to treat PSMA-positive prostate cancer cell lines (22RV1 and LNCaP). Cell lines were treated with Apt-siRNA alone or in combination with DTX. The levels of MDM2 and apoptosis-related proteins (B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X (Bax), poly ADP-ribose polymerase (PARP), caspase-3) were detected by Western blot, which were used to evaluate the therapeutic effect. Fifteen BALB/c mice bearing 22RV1 xenografts were treated with PBS, DTX+ Apt-siRNA (200 pmol) and DTX+ Apt-siRNA (400 pmol), respectively. Tumor volume and MDM2 level were observed, and 99Tc m-Apt-siRNA SPECT imaging was performed to obtain the tumor/muscle (T/M) ratio. One-way analysis of variance, Tukey′s test and linear regression analysis were used for data analysis. Results:The levels of MDM2 protein were significantly decreased by Apt-siRNA (0.25±0.02, F=183.40, P<0.001; 0.56±0.03, F=37.15, P<0.001) in 22RV1 and LNCaP cells. After the treatment of Apt-siRNA+ DTX, the levels of Bcl-2 were significantly decreased, and the levels of Bax, PARP and caspase-3 were significantly increased. MDM2 protein level (400 pmol: 0.59±0.12; F=49.99, P=0.023) and tumor volume (400 pmol: (0.22±0.07) cm 3;F=71.30, P=0.039) were significantly inhibited by Apt-siRNA+ DTX in mice bearing 22RV1 xenografts. As for 99Tc m-Apt-siRNA SPECT imaging in vivo, T/M ratio of treatment group was significantly decreased (400 pmol: 2.07±0.22; F=34.99, P=0.022), and there was a linear regression relationship between T/M ratio and the expression level of MDM2 ( R2=0.875, P<0.001). Conclusion:Apt-siRNA combined with DTX can effectively inhibit the progression of prostate cancer, and realize visual targeted diagnosis and treatment of PSMA-positive prostate cancer by coupling radionuclide technetium.
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Objective:To investigate the value of 18F-FDG total-body PET/CT dynamic imaging in evaluating the efficacy of early radiotherapy for MDA-MB-231 breast cancer bearing nude mice. Methods:MDA-MB-231 breast cancer bearing nude mice were established and divided into control group and radiotherapy group based on the random number table method ( n=10 for each group). 18F-FDG total-body PET/CT dynamic imaging was performed before and after the radiotherapy. The SUV max and the maximum tracer uptake net influx constant ( Kimax) of tumors, and the target-to-background ratio (TBR) were calculated. The change of tumor volume was recorded. The value of 18F-FDG total-body PET/CT dynamic imaging in evaluating the efficacy of radiotherapy was accessed using pathological findings as the reference. Paired t test, independent-sample t test and Pearson correlation analysis were used for data analysis. Results:After radiotherapy, SUV max and Kimax(4.66±0.46 and 0.14±0.03) were reduced in the radiotherapy group compared with those before radiotherapy (5.30±0.52 and 0.19±0.03; t values: 4.61, 8.31, P values: 0.001, <0.001), while the SUV max (5.94±0.74 vs 5.24±0.50) and Kimax (0.23±0.03 vs 0.19±0.02) were increased compared with baseline in the control group ( t values: 4.77, 6.87, P values: 0.001, <0.001). TBR Ki was significantly higher than TBR suv based on all images of the 2 groups (14.11±5.58 vs 5.91±1.60; t=8.92, P<0.001). The tumor volume in the radiotherapy group decreased compared with that before radiotherapy, but the difference was not statistically significant ((0.74±0.12) vs (0.81±0.08) cm 3; t=2.24, P=0.052). The results of immunohistochemistry showed that glucose transport protein (Glut)1 was highly expressed in tumors, and the Glut1 positive cell percentage of the radiotherapy group was significantly lower than that of the control group ((38.30±6.18)% vs (69.78±5.37)%; t=12.17, P<0.001). The expression of Glut1 was significantly positively correlated with SUV max and Kimax(the control group: rsuv=0.75, P=0.012; rKi=0.77, P=0.010; the radiotherapy group: rsuv=0.67, P=0.035; rKi=0.77, P=0.010). The apoptosis index (AI) of tumor cells in the radiotherapy group was higher than that in the control group ((24.15±4.00)% vs (10.15±3.05)%; t=8.85, P<0.001). Conclusion:18F-FDG total-body PET/CT dynamic imaging can sensitively monitor the effect of early radiotherapy in MDA-MB-231 breast cancer bearing nude mice.
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Objective:To evaluate the effects of adipose-derived stem cells (ADSCs) and ADM microparticle on diabetic wound healing.Methods:ADSCs was co-cultured with ADM microparticle in vitro. The models of diabetic nude mice were established by intraperitoneal injection of STZ and the full-thickness skin defects were designed on the back. All 24 diabetic mice were randomly divided into 4 group: experimental groups were transplanted with ADSCs and ADM microparticle and the other groups were transplanted with ADSCs, ADM microparticle and blank control group was set up. On the 7th and 14 th days, the wound healing rate of 3 mice randomly selected from each group was calculated, and the thickness between dermis and epidermis was measured by hematoxylin and eosin staining. The density of neovascularization was measured by immunohistochemical staining. The differences were compared between the groups.Results:Compared to the ADSCs groups, the mice of the experimental groups showed higher cell survival rate. The wound healing rate in the experimental groups was (86.0±2.7)% (7 days) and (98.5±1.1)% (14 days), thicker dermis-epidermis distance was (99.1±1.8) μm (7 days) and (124.3±4.3) μm (14 days) ( P<0.05), and higher density of neovascularization was noted. Conclusions:The transplantation with active ADM microparticle can significantly promote neovascularization and wound healing of diabetic wound.
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Objective:To explore the mechanism of fat formation induced by PLA-adipose derived stem cells (ADSC) and LAF-ADSC cells, and to provide a new direction and idea for cell-assisted granular fat transplantation.Methods:The liposuction operation of normal human body was performed in the plastic surgery operating room of Chengdu Badachu Medical Cosmetic Hospital. The liposuction obtained through the operation was placed still, and the upper granular adipose tissue was digested, isolated, cultured and identified by PLA-ADSC; the obtained lower liquid tissue was centrifuged, and the precipitate was digested, isolated, cultured and identified by LAF-ADSC; the differentiation characteristics and growth ability of PLA-ADSC and LAF-ADSC were compared. Then animal experiments were carried out: PLA-ADSC and LAF-ADSC were mixed with matrix glue Matrigel and transplanted into nude mice as experimental group 1 and experimental group 2. Matrix glue Matrigel was transplanted into nude mice as control group. The lipogenic ability of the three groups in nude mice was compared.Results:The cell experiment showed that the cells extracted from the static granular fat and the lower filtrate sediment could adhere to the wall and grew and subcultured smoothly. The cells from the above two different sources expressed CD44, CD73 and CD105, with positive rates of 99.5%, 99.99% and 99.7% respectively, while CD19, CD31 and CD45 were negative. The results of lipogenic induction and differentiation showed that there were lipid droplets in the cytoplasm, and the lipid droplets were orange red after cell oil red O staining. The results of animal experiment showed that three months after transplantation, the average graft volume of experimental group 1 was (0.070±0.009) cm 3, that of experimental group 2 was (0.067±0.007) cm 3, and that of the control group was (0.009±0.005) cm 3. The difference between the graft volume of experimental group 1 and experimental group 2 and the control group was statistically significant ( t=26.522 and t=37.183, all P<0.01), There was no significant difference in graft volume ( t=1.250, P>0.05). The wet weight of grafts in experimental group 1 was (0.200±0.021) g, that in experimental group 2 was (0.175±0.019) g, and that in the control group was (0.129±0.012) g, there was significant difference between experimental groups 1 and 2 and the control group ( t=11.601 and t=9.978, P<0.05), but there was no significant difference between experimental group 1 and experimental group 2 ( t=3.650, P>0.05). After oil red O staining, the grafts in experimental groups 1 and 2 were generally orange yellow, and the control group was scattered light yellow. The expression of CD31 in the experimental groups 1 and 2 was positive, and the expression of CD31 in control group was negative. Conclusions:Active ADSCs can be extracted from the granular fat layer and the lower filtrate of the static fat aspirate, and the ADSCs from both sources have good lipogenic ability in nude mice.
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Objective:Enriching and isolating breast cancer stem cells from breast cancer transplantation tumors in nude mice.Methods:Human breast cancer MDA-MB-231 cells were injected into the right axilla subcutaneous of 20 nude mice, and the tumor growth was observed .After 30 days, tumors were isolated and stained with hematoxylin and eosin, and then tumor cells from tissues were isolated. DMEM medium containing serum was used to cultivate isolated transplantation tumor cells, cell morphology and growth were also observed. Flow cytometry was used to detect the proportion of stem cells (CD44 +/CD24 -/low cells) in transplantation tumor cells. Serum-free DMEM medium containing epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and B27 cell supplement were used to cultivate transplantation tumor cells and to obtain cell microspheres. The proportion of stem cells on the 10th day in cell microspheres was detected by using flow cell sorter and stem cells were isolated according to the markers of cell surface. Results:After subcutaneously injecting MDA-MB-231 cells into 20 nude mice for 9 days, 17 nude mice had subcutaneous tumors with more parenchymal cells, little interstitial cells, arranged cords tumor cells, large volume of the cell and abundant cytoplasm, the nuclei in different sizes and hyperchromatic state, mitotic more common, the nucleoli clear and obvious pleomorphy. After cultivating transplantation tumor cells with DMEM medium containing serum, the cells began to grow adherent after 24 h, and the adherent proportion rose to 60% after 3 days; after 7 days, the cell proliferation was accelerated; and the cell morphology was more consistent, most of which were spindle shaped and were not significantly different from MDA-MB-231 cells; the proportion of stem cells in transplantation tumor cells was (0.10±0.02)%. After cultivating transplantation tumor cells with serum-free DMEM medium containing cell cultured supplement, the cells grow in spherical patterns, the proportion of stem cells in cell microspheres got up to (70.47±2.03)% on the 10th day.Conclusions:Subcutaneously injecting MDA-MB-231 cells in nude mice can build breast cancer nude mice ectopic transplantation tumor model. Breast cancer stem cells in the transplantation tumors can be enriched from isolated transplantation tumor cells through serum-free medium, and more stem cells can be isolated to provide the research basis for the biological characteristics of breast cancer stem cells.
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【Objective】 To investigate the effect of polymerized human cord hemoglobin (PolyCHb) on the chemosensitivity of human breast cancer MCF-7 cell subcutaneous xenografts in nude mice and its mechanism. 【Methods】 The MCF-7 cells in exponential growth phase were collected and made into suspension cells at a density of 5×107 cells/mL.Subsequently, the cells were inoculated subcutaneously in the right limb of 18 BALB/c-nu nude mice with 0.2 mL cells per mouse to establish subcutaneous xenograft.When the tumor volume reached about 100 mm3, they were randomly divided into chemotherapy group: doxorubicin 5 mg·kg-1, once/week; chemotherapy + PolyCHb group: in addition to doxorubicin (chemotherapy group), PolyCHb 600 mg·kg-1, 3 times/week; the control group: normal saline 90 mg·kg-1, once/week; all were injected through tail vein continuously for 4 weeks.From the day of injection (d 0), the tumor volume of each group of nude mice was measured every 3 days, and the tumor growth curves were drawn accordingly.After 38 days, the tumor growth observation was completed.The tumor was removed and weighed to calculate the tumor inhibition rate.HE staining, immunohistochemistry and TUNEL method were used to observe the pathological changes of tumor tissue, detect the expression of HIF-1α, and detect tumor cell apoptosis respectively.The content of reactive oxygen species (ROS) of each group was determined by fluorescence staining. 【Results】 The tumor volume (mm3) of chemotherapy + PolyCHb group, chemotherapy group and the control group at day 38 were 196.35±103.45 vs 316.29±62.88 vs 519.42±177.33 (P<0.05), and the tumor inhibition rate (%) of chemotherapy + PolyCHb treatment group and chemotherapy group was 62.20 vs 39.11, respectively.HE staining and TUNEL detection showed that cell necrosis and apoptosis in the growth area of tumor tissue increased in chemotherapy + PolyCHb group.Immunohistochemistry and fluorescence staining showed that HIF-1α expression in chemotherapy + PolyCHb group decreased and reactive oxygen species (ROS) content increased. 【Conclusion】 PolyCHb increases the chemosensitivity of subcutaneous xenograft in nude mice with breast cancer, and its mechanism may be related to the increase of ROS in tumor tissue and the promotion of tumor cell apoptosis.
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AIM: To optimize an orthopedic non-muscle invasive bladder cancer (NMIBC) model in nude mouse by comparing four different ways of cellular transplantation, and to evaluate the efficacy of drug by bladder instillation, so as to provide a stable and efficient animal model for the treatment of bladder cancer. METHODS: After disruption of bladder mucosa by dilute acid-alkali or silver nitrate, T24 cells were instilled into the nude mouse bladder. T24 cells were injected directly into the bladder with mechanical injury of bladder mucosa. T24 cells were injected into the bladder wall. On the 14th day after making models, the nude mice were sacrificed. And the bladder mass and histopathological changes of tumor (including bladder) was observe to confirm the formation of orthopedic bladder cancer. The dynamic changes of orthopedic bladder cancer were observed after injecting T24 cells into the bladder wall. Gemcitabine was used to verify the applicability of the model of injecting T24 cells into the bladder wall in vivo. RESULTS: No tumor was found in the bladder after intravesical instillation of T24 cells with dilute acid-alkali or silver nitrate treatment. With mechanical injury of bladder mucosa, all nude mice had tumors after injection T24 cells. But the number of tumors varied and often occurred at multiple sites. The tumor was found in the bladder of all nude mice by injecting T24 cells into bladder wall, and there was only one tumor. The tumor showed slow linear growth within 15 days and rapid linear growth from day 18 to 31. In vivo efficacy evaluation, gemcitabine 150 mg/kg intravesical perfusion could significantly inhibit the growth of NMIBC in nude mice replicated by direct injection of T24 cells into the bladder wall, and the tumor inhibition rate was 97.1%. CONCLUSION: The orthotopic NMIBC model can not be established with the bladder mucosa injuried by dilute acid-alkali or silver nitrate treatment. The number and size of orthotopic bladder cancer are different by mechanical injury of bladder mucosa. Injection of T24 cells into the bladder wall of nude mouse can successfully establish the orthotopic NMIBC model, which can be used for the evaluation of NMIBC therapeutic drugs.
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Objective To explore the relationship between miR-26a and metadherin (MTDH), and to verify the relationship between miR-26a and MTDH in vivo in nude mice. Methods Immunohistochemical SP staining method was used to detect the expression of MTDH and in situ hybridization was used to detect the expression of miR-26a in 86 cases of esophageal cancer, and the correlation between the expressions was analyzed. The bioinformatics prediction Targetscan Human 7. 2 software could predicte the binding fragment of MTDH on the miR-26a sequence, and the luciferase report experiment was used to verify the targeted regulatory relationship between MTDH and miR-26a. Nude mice were injected esophageal cancer cell lines subcutaneously which were lentiviral interferenced and overexpressed miR-26a to observe the fonnation of tumors. The tumors from nude mice were made into paraffin, and each was detected. The expression of MTDH in miR-26a in the tumor groups was detected by immunohistochemical staining and in situ hybridization and the relationship was analyzed. Results The expression of miR-26a in esophageal cancer tissues was significantly lower than that in paired nonnal esophageal tissues, and the expression of MTDH in esophageal cancer tissues was significantly higher than that in paired normal esophageal tissues. The expression of miR-26a was related to the patient' s pathological grade (P<0. 05), N stage(P<0. 05), and tumor volume (P<0. 01). The expression of MTDH in esophageal cancer was related to the N stage (P<0. 05) and the degree of differentiation (P<0. 01). Targetscan Human7. 2 bioinformatics software predicted that the MTDH gene contained a target sequence of hsa-miR-26a.The luciferase reporter gene experiment also verified the targeted regulation relationship between miR-26a and MTDH. The expression of miR-26a was the highest in KYSE-450 cells and the lowest in Ecal09 cells. The mRNA expression of MTDH in the lv-miR-26a group was significantly lower than that in the lv-NC group, and the mRNA expression in the lv-miR-26a-inhibitor group was significantly higher than that in the lv-NC group. Alter tumor formation, miR-26a expression increased and MTDH expression decreased in miR-26a group. Alter tumor formation, the expression oi miR-26a decreased and the expression oi MTDH increased in miR-26a inhibitor group. Conclusion MiR-26a can inhibit the expression oi MTDH in esophageal cancer cells. Both in vitro and in vivo experiments can verily the targeted regulatory relationship between miR-26a and MTDH. MiR-26a ma)' play a role in the occurrence and development oi esophageal cancer through the MTDH.
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@#[Abstract] Objective: To explore the application value of human IL-15 transgenic NCG mice (NCG-hIL-15 mice) in preclinical evaluation of chimeric antigen receptor modified NK (CAR-NK) cell therapy for tumor treatment. Methods: qPCR and WB were performed to detect the expression of human IL-15 in the bone marrow and main organs (spleen, liver, lung, kidney and pancreas) of transgenic mice. After being transfused with human PBMC-derived NK (PB-NK) cells, the NCG-hIL-15 mice and control NCG mice were continuously monitored for the in vivo amplification of NK cells and the changes in body weight and survival time. Flow cytometry was used to detect the differential expressions of activated receptors and inhibitory receptors in amplified NK cells. WB was used to detect the expressions of perforin and granzyme-B. NCG-hIL-15 mice or NCG mice bearing MIAPaca-2 cell transplanted tumor were treated with anti-MUC1-CAR-NK cell reinfusion; then, the CAR-NK cell survival in different groups of mice was detected by Flow cytometry, and the survival time of tumor bearing mice was recorded and tumor growth was detected by in vivo imaging. Results: The results indicated that PB-NK cells could proliferate stably within 10 weeks in NCG-hIL-15 mice without obvious graft versus host diseases (GVHD) during the observation period. The in vivo-expanded human NK cells maintained the original expression patterns of various surface molecules, including KARs and KIRs. Compared with the NK cells in NCG mice, the NK cells in NCG-hIL-15 mice contained significantly higher amounts of granzyme-B and perforin (all P<0.05). CAR-NK cells showed significantly increased survival rate and stronger tumor-inhibitory effect in NCG-hIL-15 mice as compared with those in control NCG mice, resulting in significantly prolonged survival in NCG-hIL-15 mice (all P<0.01). Conclusion: NCG-hIL-15 mouse model has potential application value in preclinical trial and biological evaluation of NK cell-based immunotherapy.
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Sempervirine, a yohimbane-type alkaloid isolated from Gelsemium elegans, was found to significantly inhibit the cellular proliferation of U251 cells in vitro and in vivo in a dose-dependent manner. U251 cells were treated with 0-16 μmol·L-1 of sempervirine for 24, 48 or 72 h. An MTT assay and clone formation assay were used to investigate cell survival and clone formation. Hoechst staining and Annexin V-FITC/PI staining were used to measure cell apoptosis. The expression of PI3K, AKT, p-AKT, Bax, Bcl-2, caspase-3 and cleaved caspase-3 was determined by Western blot analysis. The antitumor effect of sempervirine in vivo was investigated by inoculating nude mice with U251 cells. All animal experiments were in strict accordance with the regulations of the Biomedical Ethics Committee of Fujian Medical University (Fujian, China). The results show that sempervirine significantly inhibits the proliferation and induces the apoptosis of U251 cells, promotes cleavage of caspase-3, down-regulates the protein expression of PI3K and Bcl-2/Bax, and inhibits phosphorylation of AKT in vitro. Intraperitoneal injection of 4 or 8 mg·kg-1·day-1 of sempervirine inhibits U251 cells tumor growth in the xenograft nude mice, and tumor weight decreased by 44.76% and 61.26%, respectively. Our study shows that sempervirine significantly inhibits the proliferation of U251 cells in vitro and in vivo, laying a foundation for further research and development of its anti-glioma effect.