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Liver transplantation is one of the best approaches for the treatment of hepatocellular carcinoma. The concept of transplant oncology could shed light on the whole process management of hepatocellular carcinoma patients. The success rate of operation and perioperative safety were the major concerns in the past, whereas the focus of treatment is gradually shifting to cancer treatment and improving patient survival and quality of life, with the promotion of neoadjuvant and postoperative adjuvant therapy for hepatocellular carcinoma. The prognosis of different group of patients might be heterogeneous. Therefore, refined stratification should be carried out for heterogeneous patients before and after liver transplantation to achieve the best prognosis. The present study classified patients for three clusters: primary hepatocellular carcinoma patients within the transplant criteria, hepatocellular carcinoma recurrence after liver resection, and patients after down-staging or neoadjuvant therapy. Fine stratified management are essential for the whole process management in the new era of transplant oncology.
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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 effects of lncRNA associated with microvascular invasion in hepatocellular carcinoma (lncRNA MVIH) on proliferation, invasion and tumorigenic ability of colorectal cancer SW620 cells.Methods:SW620 cells cultured in vitro were divided into empty vector group (transfected with pcDNA3.1 empty vector plasmid), MVIH overexpression group (transfected with pcDNA3.1-MVIH overexpression vector plasmid), NC siRNA group (transfected with interference sequence negative control NC siRNA) and MVIH siRNA group (transfected with MVIH siRNA interference sequence). The mRNA expression level of MVIH in SW620 cells in each group was detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). Cell counting kit-8 (CCK-8), clone formation test and transwell chamber were used to detect the proliferation activity, clone formation ability and invasion ability of SW620 cells; The subcutaneous transplanted tumor of nude mice was constructed by subcutaneous injection of SW620 cells in each group 48 hours after transfection. The transplanted tumor volume was measured every 5 days to evaluate the tumor growth. On the 25th day, the nude mice in each group were killed and the tumor weight was measured. Results:The mRNA MVIH expression level, cell proliferation, clone formation rate and number of invasive cells of SW620 cells in the MVIH overexpression group were significantly higher than those in the empty vector group ( P<0.05); the MVIH mRNA expression level, cell proliferation , clone formation rate and number of invasive cells of SW620 cells in the MVIH-siRNA group were significantly lower than those in NC-siRNA group ( P<0.05). The results of subcutaneous transplantation tumor in nude mice showed that the growth rate of tumor in MVIH overexpression group was faster and the tumor weight was higher than those in the empty vector group ( P<0.05); the growth rate of tumor in MVIH overexpression group was slower and the tumor weight was lower than those in NC-siRNA group ( P<0.05). Conclusions:LncRNA MVIH can promote the proliferation, invasion and tumorigenesis of SW620 cells in colorectal cancer.
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Objective To assess the imaging characteristics of 18F-Alfalide II in different tumorbearing mice and pharmacokinetics in Beagle dogs.Methods BALB/c nude mice(n-24)were used for subcutaneous tumor models(A549 and U87MG),orthotopic lung cancer models(A549)and orthotopic breast cancer models(MDA-MB-231)(n=6 in each group).18F-Alfatide II and 18F-fluorodeoxyglucose(FDG)microPET/CT images were compared in the 4 types of tumor-bearing nude mice models.18F-Alfatide II blocking experiment,biodistribution experiment and imaging studies in tumors of different growth cycles were performed in A549 subcutaneous tumor-bearing nude mice models.Pharmacokinetic experiments were carried out in Beagle dogs(n = 6)and CD-1 mice(n = 9).Two-sample t test was used to analyze the data.Results Compared with 18F-FDG,18F-Alfatide II microPET/CT images showed better imaging quality and contrast in subcutaneous A549,U87MG tumors and orthotopic A549(tumor/heart:4.50±1.17 vs 0.95±0.31;t = 4.125,P<0.01),orthotopic MDA-MB-231(tumor/muscle:6.60±1.53 vs 0.92±0.43;t = 3.984,P<0.01)transplantation nude mice models.18F-Alfatide II could specifically target A549 tumors,and the tumor uptake of 18F-Alfatide II was reduced by about 75% after pre-injection with cyclo(Arg-Gly-Asp-D-Tyr-Lys)(c(RGDyk)).18F-Alfatide II was rapidly cleared from the blood of Beagle dogs(T1/2 was(57.34±11.69)min).It was cleared in the form of prototype drug and(69.24±6.82)% of cumulative dose was excreted through the urine within 4 h after administration.Conclusions 18F-Alfatide II shows a higher target/non-target ratio than,18F-FDG in the imaging of A549,MDA-MB-231 and U87MG tumor-bearing nude mice models,which is more conducive to the diagnosis of tumor.18F-Alfatide II has excellent pharmacokinetic properties.
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Objective To develop the automated preparation of 18F-Alfatide II using newly-designed 18F-minireactor and perform 18F-Alfatide D microPET/CT imaging in tumor.Methods The automated preparation of 18F-Alfatide H was developed by using 18F-microreactor and water phase Al18F-chelating method,and the radiochemical yield and quality analysis were measured.The nude mice bearing breast tumor ZR-75-1 and nasopharyngeal tumor CNE1 were established(n = 3 respectively).MicroPET/CT imaging was performed at 0.5,1.0 and 2.0 h after the injection of 18F-Alfatide II.The region of interest(ROI)was depicted and the tumor/muscle(T/M)ratio was calculated.Results 18F-Alfatide II was automatically prepared with the total synthesis time of 40 min,the radiochemical yield of(28±6)%(no decay corrected,n=11),and the radiochemical purity >97%.All quality analysis indexes accorded with the radiopharmaceutical requirements.18F-Alfatide II microPET/CT images of ZR-75-1 and CNE1 tumors were clear due to the high radioactivity uptake of tumor lesions(T/M ratio was greater than 4.0 at 1.0 h after injection).Conclusion Based on the 18F-minireactor,the,8F-Alfatide II can be prepared successfully with short synthesis time and high radiochemical yield,which can help the application studies in 18F-Alfatide II microPET/CT imaging.
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Objective To elucidate the inhibitory effect of 131I-fulvestrant on the growth of human breast cancer cells and the effect on the important organs.Methods MTT assay was used to clarify the difference in killing effects of the 131I-fulvestranton on MCF-7 cells and MDA-MB-231 cells.Breast cancer MCF-7 cell xenografts in nude mice was establishied,and two different administration methods of the 131I-fulvestrant in the MCF-7 cell to nude mice were given respectively.Organs and tumours of nude mice were observed.Results MTT assay demonstrated that 131I-fulvestrant had similar cytotoxicity against MCF-7 cells and MDA-MB-231 cells,and the former was slightly stronger.Transient contact experiments showed that 131I-fulvestrant could play a tumor suppressor effect on MCF-7 cells continually,but MDA-MB-231 cells wasn't.After the injection of 131I-fulvestrant via caudal vein,the radioactivity concentration on tumor site accounted for (4.33 ± 0.28)％ of the total injection,and the volume of the tumor reduced before gradually increasing again.Radioactivity in the blood accounted for (20.76 ± 2.54)％ of the total injection.Qrgans like liver and kidney also showed radioaction distribution.Its distribution was accorded with the distribution of estrogen receptor.Local injection of 131I-fulvestrant got powerful killing effect on the tumor,and the distribution of the radioaction was mainly confined within the tumor.Conclusion 131I-fulvestrant has a good inhibitory effect on MCF-7 breast cancer cells,which is a superposition of radiotherapy and endocrine therapy,and it is controllable on the general condition and important organs of nude mice.
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Objective To establish neoplasm transplantation models of breast cancer cells in BALB /c nude mice and to isolate tumor infiltrating myeloid cells.Methods pHAGE-EF-ZsG-DEST plasmid,pMD2.G plasmid and psPAX2 were transfected into BT474 using the method of calcium phosphate transfection .The positive cells were selected by flow cytometry and implanted in the fat pad of nude mice .A tumor model of breast cancer cells implanted in nude mice was constructed, and the tumor infiltrating myeloid cells were isolated .Conclusion Tumor infiltrating myeloid cells are successfully isolated, which will contribute to the study of the functions of tumor infiltrating myeloid cells .
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Objective To synthesize 18F labeled N-(2-18 F-fluoropropionyl)-L-glutamine (18 F-FPGLN) for tumor PET imaging,and to perform its biodistribution study on normal mice and PC-3 tumorbearing nude mice.Methods 4-nitrophenyl-2-18F-fluoropropionate (18F-NFP) was synthesized on the MF2V-IT-I synthesizer and was purified by semi-preparative HPLC.Anhydrous 18F-NFP was added to a solution of L-glutanine t-butyl ester to synthesize 18F-FPGLN t-butyl ester,which was hydrolyzed by HCl (3 mol/L) and neutralized with NaOH (2 mol/L) solution.18F-FPGLN product was collected for further study.Biodistribution study was performed on normal Kunming mice and PC-3 prostate cancer tumor-bearing nude mice,respectively.Results 18F-FPGLN was synthesized with 10％-15％ (decay uncorrected) overall radiochemical yield after 130 min of radiosynthesis.The radiochemical purity was higher than 96％.Rapid and high uptake of radiotracer was observed within the kidneys,and was quickly excreted through the urinary bladder.The uptake in kidney reached (35.0±1.2) ％ID/g at 5 min post-injection,and descended to (1.5±0.3) ％ID/g at 120 min.The liver,lung,heart and small intestine showed relatively moderate uptake of radioactivity.The uptake in the pancreas,muscle,spleen,stomach and brain was low,and the lowest uptake of (1.5±0.3) ％ID/g was found in the brain at 30 min post injection.High accumulation of 18F-FPGLN in PC-3 xenograft was observed,and the tumor/muscle ratio reached 2.07 at 60 min post injection.Conclusion A novel N-position 18F-labeled glutamine analogs 18F-FPGLN,with favorable pharmacokinetic characteristics,is synthesized successfully,which makes it possible to perform tumor PET imaging using 18F-FPGLN subsequently.
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OBJECTIVE To study the antitumor activity and underlying mechanisms of Xiaoaiping injection(Xap)combined with gefitinib(Gef)in nude mice bearing resistant non-small cell lung cancer (NSCLC) cells H460 or H975. METHODS BALB/c nude mice were inoculated with human NSLCL cells H460 or H1975 and the drug treatment did not start until the tumor volume reached 50-100 mm3. The tumor bearing mice were divided into four groups:control group,Xap group(5 g · kg-1,ip),Gef group (50 mg · kg-1,ig),and Xap plus Gef group. All the administration lasted for 21 d continuous. Tumor volumes were measured two or three times per week,and the body weight of animals was re-corded. At the end of the test,tumors were weighed after the sacrifice of mice. Tumor inhibition rate and relative tumor proliferation rate were calculated based on tumor weight and tumor volume. The related biomarkers and proteins in tumor tissues were detected by immunohistochemistry and Western blot assay, respectively. RESULTS Compared with the control group,no significant effect was observed on the growth of H460 and H1975 xenografts in groups of Xap or Gef alone in nude mice. However,the two-drug combination significantly suppressed tumor volume,with (1103 ± 340) versus (3020 ± 450) mm3 for H460 and(487 ± 153)versus(1269 ± 161)mm3 for H1975,respectively(P<0.05). The combined Xap and Gef application also significantly reduced the tumor weight,with(1.20±0.52)versus(2.78± 0.93)g for H460 and(0.52 ± 0.32)versus(0.92 ± 0.42)g for H1975,respectively(P<0.05). The relative tumor proliferation rate and inhibition rate in the combination group was 42.1%and 43.5%for H460(P<0.01),43.0%and 52.5% for H1975(P<0.01). Compared with Xap and Gef drug alone,their combination showed significant difference in reducing tumor weight,suppressing tumor proliferation rate and increasing tumor inhibition rate(P<0.05). Immunohistochemistry results showed that each drug alone had no effect on tumor angiogenesis markers of vascular endothelial growth factor(VEGF)and CD105 expression,or on drug resistance related proteins of p-ERK,p-Akt and p-mTOR,whereas the combination of Xap and Gef obviously reduced the expression of these biomarkers in H460 and H1975 tumor tissues. The decreased drug resistance related proteins of p-PI3K and its downstream molecules p-Akt,p-ERK and p-mTOR by the two-drug combination were also confirmed by Western blot results(P<0.01,compared with control), and showed significant difference compared with each single treatment(P<0.05). CONCLUSION The addition of Xap significantly improves the antitumor activity of Gef in H460 and H1975 xenografts,and this synergistic effect may be ascribed to the inhibition of tumor angiogenesis,the down-regulation of PI3K and its downstream signaling molecules which are associated with drug resistance.
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Objective To study the significance of circadian gene Period2 expression in epithelial ovarian cancer tissues and the effect of gene overexpression on the growth of ovarian cancer xenografts in nude mice. Methods Twenty-two cases of ovarian cancer paraffin specimens in the First Hospital of Shanxi Medical University (ovarian cancer group) were chosed during Jau. 2010 to Dec. 2013, including 8 cases of stageⅠ, 8 cases of stageⅡ, and 6 cases of stageⅢ, while 6 cases of benign ovarian epithelial tumor paraffin specimens were selected as control (benign tumor group). Period2 gene were detected by real-time quantitative PCR and western blot methods in different stages of ovarian cancer tumor tissues. Established theovarian cancer xenografts in nude mice with ovarian cancer cell line SKOV3, and they weredivided into 3 groups (n=8), including the recombinant plasmid group, empty plasmid group and control group. Using gene transfection technique to transfer Period2 gene into tumor tissues, tested the expression of Period2 mRNA in tumor tissues by real-time quantitative PCR after transfection into all nude mice, monitoredthetransplant tumor growth and calculating the tumor inhibition rate,detected the antiapoptotic gene BRE, apoptosis related tumor necrosis factor receptor (TNFR1) andtumor suppressor gene NIX in tumor tissues by real-time PCR and western blot in different groups. Results (1)The expression level of Period2 mRNA in tumor tissues amongovarian cancer group stageⅠ,ⅡandⅢwere respectively 2.59±0.50, 0.47± 0.08 and 0.42 ± 0.08, but benign tumor group was 6.59 ± 1.05. The expression level of Period2 protein in ovarian cancer group stage Ⅰ,Ⅱ and Ⅲ were respectively 0.835 ± 0.087, 0.412 ± 0.035 and 0.199 ± 0.031, while benign tumor group was 0.874 ± 0.094. The expression level of Period2 mRNA and protein in benign tumor group was higher than those in ovarian cancer group stageⅠ,ⅡorⅢ(P<0.01). With ovarian cancer stage increased, the expression of Period2 mRNA and protein were decreased or absent (P<0.05).(2)Two weeks after transfection, the expression level of Period2 mRNA in recombinant plasmid group tumor tissue was significantly higher than those in the empty plasmid group or the control group (6.11±0.56 vs 0.50±0.09 vs 0.44 ± 0.08, respectively;P<0.01), the transplanted tumor volume of recombinant plasmid group was significantly less than those in empty plasmid group or the control group[ (486±70) mm3 vs (835±106) mm3 vs (846 ± 110) mm3, respectively;P<0.01], the tumor inhibition rate of the recombinantin plasmid group was as high as 42.9%, that was significantly higher than those in the empty plasmid group and the control group (3.8% and 0, respectively;P<0.05).(3)The expression level of BRE mRNA and protein in transplanted tumor tissues in the recombinant plasmid group were significantly lower than those in empty plasmid group and the control group;the expression level of TNFR1 and NIX were significantly higher than those in the empty plasmid group and the control group (all P<0.05). Conclusions Period2 mRNA and protein expression are absent in ovarian cancer of advanced stage. Transfection and stable expression of Period2 gene could slow down the growth of ovarian cancer, and the tumor inhibition rate could be significantly increased. Period2 gene may promote ovarian cancer cells apoptosis through inhibition of BRE gene expression and promoting TNFR1, NIX gene expressionto exert anti-tumor effect.
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Objective To evaluate the value of 18F-FDG PET/CT in early in vivo monitoring of tumor response to cisplatin,and analyze the relationship between 18F-FDG uptake in tumor and the corresponding pathological changes.Methods Thirty VX2 rabbits were divided into 5 groups by random number table with 6 in each group,including 4 treatment groups and 1 control group.18F-FDG PET/CT were performed before and after (6,12,24 and 36 h post-injection respectively) intravenous administration of cisplatin (7 mg/kg) in the treatment groups,respectively.The control group was injected with physiological saline followed by 18F-FDG PET/CT.The ROI was drawn and the SUVmax and T/NT ratio were calculated.The tumor necrosis rate and apoptosis index were observed by histopathologic examination.Paired t test,GamesHowell test and arcuation correlation analysis were used to analyze the data.Results Significant differences were found in SUVmax and T/NT of the control group before and after injection of physiological saline (6.58±1.67 vs 9.77±2.45,52.93±3.90 vs 29.34±3.31;t=-5.480,17.593,both P＜0.05).18F-FDG uptake decreased after 6 h post-injection of cisplatin,with the mean SUVmax decrease rate of (11.83±8.89) ％ and the mean T/NT decrease rate of (59.00±8.22)％.In the 24 h treatment group,18F-FDG uptake decreased most,and the mean SUVmax decrease rate was (42.33±33.80)％,the mean T/NT decrease rate was (83.50± 7.69) ％.The SUVmax and T/NT of those 2 groups were significantly different from those of the control group,and no difference was found between the 2 treatment groups(all P＜0.05).The changes of SUVmax and T/NT were positively correlated with apoptosis index and tumor necrosis rate (r=0.750,0.794,0.804,0.874,all P＜0.05).Conclusion 18F-FDG PET/CT is a sensitive method for monitoring early response to tumor chemotherapy in VX2 tumor-bearing rabbits at 24 h after treatment.
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Objective To explore the inhibitory effects of lobaplatin and cisplatin and their regulation of apoptosis-related genes in ovarian cancer cells in nude mice.Methods SKOV3 cells were implanted into nude mice.In monotherapy treatment study,the nude mice bearing human SKOV3 cells were randomly divided into control,lobaplatin,and cisplatin groups,with 7 mice in each group.The mice in each group were received corresponding treatment.The volume of tumor and the weight of nude mice were measured three times per week,respectively.Tumor inhibitory rate was calculated.The protein expressions of bax and bcl-2 were detected by flow cytometry.Results The growth inhibitory rate was 47.2％ in lobaplatin group and 42.8％ in cisplatin group,without significant difference between two groups (P ＞ 0.05).The expression of bcl-2 was decreased but the bax was increased in lobaplatin and ciaplatin groups compared to the control group.Conclusions Lobaplatin can significantly inhibit the growth of ovarian cancer cells,induce apoptosis by down-regulation of bcl-2 and up-regulation of bax.
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AIM:To investigate the effect of recombinant lentiviral vector for RNA interference (RNAi) on the expression of fatty acid-binding protein 5 (FABP5) gene in hepatocellular carcinoma HepG2 cells and tumor formation in nude mice.METHODS:RNAi lentiviral vector was used in the experiment.Human hepatocellular carcinoma HepG2 cells were divided into 3 groups:the HepG2 cells in experimental group were transfected with the recombinant lentivirirus vector LV-shRNA-FABP5, the cells in negative control group were transfected with a control lentiviral vector LV-shRNA-NC, and the cells in normal control group were without any treatment.The nude mice were randomly divided into 3 groups.The growth of the transplanted tumor cells in the nude mice was observed.The tumor growth curve, volume and weight were de-termined 4 weeks after the cell inoculation.The expression of FABP5 was detected by real-time PCR, Western blot and im-munohistochemical staining.RESULTS:Transfection of the lentiviral vector FABP5-shRNA obviously reduced FABP5 ex-pression in the HepG2 cells.Tumor formation was all positive in the 3 groups of the nude mice inoculated with the tumor cells.Compared with normal control group and negative control group, the tumor growth slowed significantly in experimental group with smaller volume and weight.FABP5 expression in the transplanted tumor tissues was significantly down-regulated at mRNA and protein levels in experimental group as compared with normal control group and negative control group. CONCLUSION:RNAi-induced down-regulation of FABP5 effectively inhibits the growth of transplanted hepatocellular carcinoma, suggesting that FABP5 gene may be an effective target for gene therapy in treating liver cancer.
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BACKGROUND:After glucose regulated protein 94 (GRP94) was knockout in model mice of transplanted tumor, cel ular adhesion is terminated, thus stimulating the proliferation of liver-derived cel s and promoting the development of liver cancer. We speculate that GRP94 plays a protective role against liver cancer. OBJECTIVE:To investigate the expression of endoplasmic reticulum molecular chaperone GRP94 mRNA and protein with smal interfering RNA technique in nude mice model of transplantable human ovarian carcinoma, and to explore the effect of GRP94 mRNA and protein expression on the growth of transplanted tumor. METHODS:The gene sequences of human GRP94 were obtained from Gene Bank. psiSTRIKETM/GRP94 was constructed, which is eukaryotic expression vector control ed by the U6 promoter of human RNA polymerase Ⅲ. The transplantable model of human ovarian carcinoma in nude mice was established using human ovarian cancer HO-8910 cel line. The eukaryotic expression vector was transfected into the transplanted tumors in nude mice, and the growth of the tumor was observed. The nude mice models were divided into three groups, specific smal interfering RNA group, non-specific smal interfering RNA group and saline control group. The volumes of the subcutaneous tumor were determined. RT-PCR and immunohistochemistry were used to detect the mRNA and protein expression of GRP94 respectively. RESULTS AND CONCLUSION:The recombinant plasmid of RNA interference specific for GRP94 was successful y constructed. The subcutaneous tumors appeared in al the nude mice 5 days after transplantation. The diameter of subcutaneous tumors was 7-10 mm 14 days after transplantation. The growth of subcutaneous tumors in nude mice with interference specific for GRP94 treatment was significantly decreased as compared with non-specific smal interfering RNA group and control group (P<0.05). The proliferation activity was inhibited by 65.1%. The expression of GRP94 mRNA and protein was significantly down-regulated after treatment of psiSTRIKETM/GRP94 (P<0.01). The transfection of psiSTRIKETM/GRP94 could significantly induce inhibitory effects on the growth of ovarian carcinoma in nude mice, and the underlying mechanism is associated with the down-regulated expression of GRP94 mRNA and protein.
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Objective: To investigate the suppression effect of mouse marrow-derived endothelial progenitor cells (EPCs) modified by cytosine deaminase (CD) gene and enzyme prodrug 5-fluorocytosine (5-Fc) on the growth of hepatocellar carcinoma in orthotopic H22 cell-transplanted mice. Methods: The EPCs were transfected by lentiviral vector carrying CD gene (plenti6.3-EGFP-CD) using Polybrene technique. The mouse hepatoma H22 cells were treated by the supernatant of EPCs carrying CD gene and 5-Fc, and then the number and morphology of the cells were observed under an inverted microscope. C57BL/6 mice bearing orthotopic transplanted H22 hepatocellar carcinoma were treated by EPCs carrying CD gene and 5-Fc. The volume of tumor in mice was monitored by magnetic resonance imaging, and the apoptosis in tumor was tested by terminal deoxynucleotidyl transferase-mediated dUTP nick and labeling (TUNEL) assay. The expression of CD protein in the transplanted tumor was identified by Western blotting. Results: The proliferation of H22 cells treated by the supernatant of EPCs carrying CD gene combined with 5-Fc was significantly reduced. Compared with the control, the tumor growth in the hepatocellular carcinoma orthotopic-transplantation mouse models treated by EPCs carrying CD gene and 5-Fc was inhibited to (47.29±5.81)% (P < 0.05), and the apoptosis index of tumor cells was markedly increased [(39.98±5.13)%]. Conclusion: The EPCs modified by CD gene combined with 5-Fc administration can effectively inhibit the proliferation of the mouse orthotopic transplanted hepatocellular carcinoma, and induce the apoptosis of tumor cells. Copyright © 2014 by TUMOR.
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Objective To construct human canstatin gene eukaryotic expression vector and investigate the therapeutic effect of intramuscular canstatin gene delivered by electroporation on tumor growth.Methods Canstatin cDNA was amplified from total RNA extracted from fresh fetal liver by reversing transcription polymerase chain reaction (RT-PCR).The canstatin cDNA fragment was in serted into pEGFP-N1 eukaryotic expression vector.The recombination plasmid was delivered to the quadriceps of the mice with Lewis lung carcinomas by electroporation intramuscular.Fluorescence intension measured by fluorescence microscope,reverse-PCR assay,and immunohistochemistry assay were performed to detect the expression of canstatin gene in the muscle and in circulation.The tumor weight and volume were used to detect the biological effects of canstatin gene delivery.Results Recombinant eukaryotic expression vector of recombinant human canstatin was successfully constructed.The canstatin mRNA was significantly increased in the skeletal muscle and intramuscular delivery of canatatin gene by electroporation acquired the expression of enhanced green fluorescent protein (EGFP)/canstatin protein in the circulation and significantly inhibited tumor growth.The percent of the inhibition of tumor weight was 57.7 ％.Conclusions Electroporation mediated gene transfer efficiency in skeletal muscle was compared to simple plasmid injection and lasted for a long time.It was an efficient and safe,convenient and economic,gene transfer methods and might have certain clinical application value.Electroporation mediated canstatin gene transfer in skeletal muscle had obvious inhibitory effect on Lewis lung cancer in mice subcutaneous xenograft tumor growth.
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Objective To detect apoptosis of transplanted lung adenocarcinoma cells in nude mice after 125I brachytherapy by 99Tcm-Annexin V combined with diffusion weighted MRI (MR-DWI).Methods Twenty-five BALB/c-nu nude mice models subcutaneously transplanted with A549 cells were divided into experimental group (EG,n=13) and control group (CG,n=12) by random number table method.One 125I seed with apparent activity of (24.8±6.3) MBq was implanted into each mouse in EG,while CG underwent cold seed implantation.Both of 99Tcm-Annexin V imaging and MR-DWI were performed within 7-10 d after brachytherapy,then all mice were sacrificed and tumor cell apoptosis was detected by terminal oxynucleotidyl transferase mediated dUTP biotin nick end labeling (TUNEL) immunofluorescence,Survivin expression was assayed by SP.Two-sample t test,x2 test and Pearson correlation analysis were used for data analysis.Results Positive rate of cell apoptosis by 99Tcm-Annexin V imaging was 69.2％(9/13) and 8.3％(1/12) respectively in EG and CG (x2 =12.73,P＜0.01).The uptake ratio of 99Tcm-Annexin V,apparent diffusion coefficient(ADC) value and apoptosis index(M) of the tumor in EG were 2.91±0.85,(2.03±0.44)×10-3 mm2/s and (49± 18) ％,respectively,which were significantly higher than those of CG (1.26 ± 0.37,(1.29 ± 0.21) ×10-3 mm2/s and (11±4)％ respectively,t=5.930,5.452,7.606,all P＜0.05).Survivin expression in EG and CG was (46± 13) ％ and (15±7) ％ respectively (t =5.158,P＜0.05).The value of ADC was correlated with AI and uptake ratio(r=0.756,0.788,both P＜0.05).Uptake ratio was correlated with AI (r=0.754,P＜0.05),while Survivin expression was negatively correlated with AI (r =-0.772,P＜0.05).Conclusions Down-regulation of Survivin expression may play an important role in apoptosis induced by 125I brachytherapy.99Tcm-Annexin V combined with MR-DWI could effectively evaluate apoptosis of lung adenocarcinoma cells in a non-invasive way,thus it might be helpful in evaluation of early efficacy of 125I brachytherapy.
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Objective To investigate the effect of HIF-2a silencing by transfection of siRNA into MG-63 cells un-der hypoxia. Methods HIF-2αexpression level in MG-63 cells under hypoxia was determined by Western Blot. Small in-terfering RNA (siRNA) was used to construct MG-63/siHIF-2α(siHIF-2α)cells and control MG-63/scramble (NC) cells. The expression levels of HIF-2α, Vascular endothelial growth factor (VEGF), p-Erk/ErK and Mcl-1 in MG-63, NC and si-HIF-2αcells was determined by Western Blot. NC and siHIF-2αcells were cultured under hypoxia. Cell viability was as-sessed by MTT assay. Migration was identified by scratch migration assay. Tumor formation was identified by clone formation assay. Nude mouse subcutaneous xenograft model was used to investigate tumor development in vivo. Results Hypoxia im-proved HIF-2αexpression in MG-63 cells in a time-dependent manner (F=2 037.412,P<0.001). HIF-2αexpression un-der hypoxia in siHIF-2αcells was lower than that in NC cells (P<0.01). Cell viability of siHIF-2αcells under hypoxia for 12 h and 24 h were lower than that in NC cells (P<0.05 or P<0.01). The relative width of scratch in siHIF-2αgroup under hypoxia for 12 h and 24 h were larger than that in NC group (P<0.01 or P<0.01). When cell counts reach 1 000-5 000, the clone formation rates of siHIF-2αcells were lower than that in NC cells (P<0.05 or P<0.01). The expression of VEGF, p-Erk/Erk and Mcl-1 protein under hypoxia in siHIF-2αcells was lower than that in NC cells(P<0.01). Tumor sizes, weights and density of siHIF-2α group in nude mice were suppressed compared with those in NC group (P<0.01). Conclusion Blocking HIF-2αsignal pathway warrants its investigation as a potential strategy in osteosarcoma treatment.
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Objective To investigate the conventional ultrasound characteristics of VX2 tumors in fatty liver and normal liver of rabbits,furthermore,to analysis the blood flow characteristics of them with contrast-enhanced ultrasound (CEUS).Methods Rabbit model of fatty liver and normal liver with implanted VX2 tumors were established,and examined using conventional and CEUS.QontraXt quantitative software of CEUS was used.Results The fatty liver models with 20 VX2 tumors and normal liver models with 23 VX2 tumors were successfully developed in rabbits.In conventional ultrasonography,hyperechoic VX2 tumor lesions in fatty liver were far less than them in normal liver (P =0.008).No significant difference was found in iso-echoic and hypoechoic VX2 tumor lesions between two groups (P ＞ 0.05).Compared with normal liver parenchyma,arrival time of fatty liver parenchyma was later,peak intensity was lower (P ＜0.05).There was no significant difference in arrival time,peak intensity,sharpness,area under curve between the VX2 tumors of normal liver and fatty liver (P ＞ 0.05).Conclusions There is a significant difference characteristic in conventional ultrasonography between VX2 tumors in fatty liver and normal liver.Fatty liver blood perfusion effects are lower than normal liver in CEUS.Quantitative parameters of CEUS are more objective and accurate for hemodynamic of VX2 tumors and fatty liver.
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Objective To explore the diagnostic value of the dual-energy technique with dual-source in the evaluation of therapeutic effect of interventional therapy for hepatocarcinoma.Methods 8 rabbits with tumor growing well were treated with intervention operation,paralleled with double energy CT scan and focal area image,CT and color order change were measured by dual energy liver OVERLAY workstation.Meanwhile,The necrosis and survival of tumor were evaluated by dual energy CT after hepatocarcinoma intervention therapy.Results Taking pathologic diagnosis as gold standard,compared with imaging and pathological results in the order of black,red,orange and red,accuracies were 87.2％,72.3％,71.5％ and 83.6％ respectively.Sensitivities were 95.1％,37.4％,54.7％ and 63.8％ respectively.Specificities were 85.4％,86.3％,81.5％ and 85.8％ respectively.Positive predictives value were 61.4％,54.2％,62.4％ and 55.6％ respectively.Negative predictive values were 96.3％,76.2％,79.7％ and 91.3 ％ respectively.According to the Spearman relevance analysis,there was strong positive correlation between survival of tumor cells and image color scale (r =12.35,P ＜ 0.05).Conclusion The necrosis and survival of tumor cells in rabbit VX2 liver tumor model can be preliminarily reflected by dual source CT dual energy enhancement scanning.The results provide good reference to the application of dual-source and dual-energy in clinical diagnosis and treatment.