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Objective To investigate the inhibitory effects of a recombinant adenovirus vector ex-pressing human IFN-λ1 ( r-Ad-hIFN-λ1) on the growth of orthotopic gastric cancer and to analyze its possi-ble mechanism in a nude mice model of orthotopic human gastric carcinoma .Methods Orthotopic trans-plantation was performed to establish the nude mice model of orthotopic gastric cancer .Thirty mice were ran-domly divided into three groups including PBS control group , Ad-Lac Z empty vector group and r-Ad-hIFN-λ1 experiment group .The mice in each group were given the corresponding interventions once a week for three times.The sizes of tumor were detected by using B-mode ultrasound at different time points to draw growth curves .The expression of IFN-λ1 at mRNA level in skeletal muscle tissues was analyzed by RT-PCR.Western blot assay and immunohistochemical staining were used to detect IFN-λ1 protein in gastric tumor samples .The apoptosis of cells in paraffin-embedded tumor tissues was detected by TUNEL .The per-centages of natural killer ( NK) cells in spleen tissues were analyzed by flow cytometry .Results Compared with control group and empty vector group , the mice in r-Ad-hIFN-λ1 experiment group showed the smallest tumor size [(331.25±6.00) mm3, (322.92±5.92) mm3 vs (248.39±7.60) mm3; P<0.05].hIFN-λ1 was transfected into skeletal muscle successfully and expressed in gastric cancer tissue .Highly expressed hIFN-λ1 was observed in cytoplasm of tumor cells from experiment group by immunohistochemical staining . The apoptotic index of tumor tissues for experiment group was 0.700±0.059 which was significant different from that of control group (0.271±0.026, P<0.05) and empty vector group (0.333±0.028, P<0.05). The percentage of NK cells in spleens from mice in experiment group [(26.49±1.89)%] was significantly higher than that of control group [(13.94 ±1.31)%, P<0.05)] and empty vector group [(19.12 ±1.69)%, P<0.05)].Conclusion Transfection of r-Ad-hIFN-λ1 and expression of hIFN-λin skeletal muscle could significantly inhibit the growth of gastric cancer by inducing tumor cells ′apoptosis and enhan-cing NK cells.
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Objective To explore the effects of Ad-hIFN-λ1 recombinant adenovirus on prolifera-tion of gastric adenocarcinoma cell line SGC-7901 and its mechanism .Methods Ad-hIFN-λ1 recombinant adenovirus and empty plasmid Ad-LacZ were respeetively transfated to human gastric adenocarcinoma SGC-7901 cells.The proliferation of transfected cells was detected by MTT assay .IFN-λ1 gene expression at mR-NA and protein levels in the cells were measured by RT-PCR analysis and immunofluorescence assay , re-spectively .Tunel assay and flow cytometry were used to analyze the rate of cell apoptosis .Results The proliferation of gastric adenocarcinoma SGC-7901 cells were significantly inhibited with Ad-hIFN-λ1 inter-vention in accordance with highly expressed IFN-λ1 at mRNA and protein levels , respectively .The apoptosis rate of Ad-hIFN-λ1 transfected cells was markedly higher than that of the empty plasmid Ad-LacZ group and PBS blank control group .Conclusion The expression of hIFN-λ1 could be detected after transfection of Ad-hIFN-λ1 recombinant adenovirus into gastric adenocarcinoma SGC-7901 cells.Ad-hIFN-λ1 could signifi-cantly inhibit the proliferation of gastric adenocarcinoma SGC-7901 cells by promoting the apoptosis of cancer cells.
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Objective To investigate the effects of integrin β1 gene expression inhibited by short hairpin RNA (shRNA) on invasion of pancreatic carcinoma PANC1 cells in vitro,and investigate the mechanism.Methods The eukaryotic expression plasmid of shRNA targeting integrin β1 gene ( integrin β1 shRNA) and control eukaryotic expression plasmid shRNA (c-shRNA) was constructed and was transfected into PANC1 cells.The cells without plasmid transfection were used as control.The expressions of integrinβ1,MMP 2,MMP 9 mRNA and protein were detected by real-time PCR and Western blotting.The invasive ability of PANC1 cells was observed with Transwell cell culture chamber.Results Integrinβ1 mRNA expressions in integrinβ1 shRNA group,c-shRNA group and control group were 0.0029 ± 0.0004,0.0131 ± 0.0009,0.0138 ± 0.0005 ; the expressions of integrinβ1 protein were 0.0159 ± 0.0062,0.3215 ± 0.0126,0.3107 ±0.0094; the inhibitory rate of integrinβ1 mRNA and protein expression in integrinβ1 shRNA group was (78.6 ±7.2 ) ％ and (92.9 ± 3.2) ％ ( P ＜ 0.01 ).But there was no difference between the c-shRNA group and control group (P =0.2999).Number of penetrating cells in integrinβ1 shRNA group decreased from 52 ±5 to 21 ±4( P ＜ 0.01 ) ; the expression of MMP 2 and MMP 9 mRNA decreased from 0.592 ± 0.073,0.847 ± 0.069 to 0.102 ± 0.034,0.273 ± 0.071 ; the expression of M MP2 and MMP 9 protein decreased from 0.225 ± 0.046,0.416 ±0.081 to 0.059 ±0.013,0.106 ±0.022(P ＜0.05).Conclusions Recombinant integrinβ1 shRNA expression plasmid can effectively inhibit the expression of integrinβ1 gene and suppress the invasion of PANC1 cells in vitro by down-regulating MMP 2 and MMP 9 gene expression.
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Objective To transfect the recombinant mIL-28A adenovirus vector into lung adenocarcinoma cell line LA795 and research its anticancer activity. Methods Transfected the constructed mouse IL-28(mIL-28) recombinant adenovirus vector into LA795 cell line, detected with PCR, immunocytal fluorescence, Tunel, Annexin V and MTT. Results Transfected with rAd-mlL-28A into the LA795 cells, mIL-28A gene expression products mRNA increased obviously, IL-28 expression was detected in cells obviously,apoptosis cell number increased, and the growth of LA795 cells transfected with rAd-mIL-28A were inhibited obviously. Conclusion The recombinant miL-28A adenovirus vector we have constructed, which expresses IL-28 when transfected to lung adenocarcinoma cell line LA795, inhibits growth of carcinoma cell to some extent, and may work by promoting the apoptosis of cancer cells.
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Objective To express mouse IFN-λ2 stably and study its biological activity. Methods Full-length of mIFN-λ2 cDNA was obtained by using RT-PCR from cells of mouse spleen stimulated by ve-sicular stomatitis virus(VSV) and then subcloned to eukaryotic expressing vector PCAGG-EGFP. The recom-binant was transfected into CHO cells. VSV * GFP-B16 system was used to measure the antivirus activity. The constructed cell line MDBK-Mxp-Luc was used to study the character of Mx1 protein induced by the mIFN-λ2. Results The recombinant pMD18-T-mIFN-λ2 was digested by two kinds of enzyme, Sac I and Xho I, to produce the fragment was of 582 bp, and of which the sequence analysis of sequence shows it was entirely consistent with the nucleotide sequences reported in GenBank. PCAGG-EGFP-mIFN-λ2 eukaryotic expressing vector was constructed successfully and expressed stably in CHO cells, and the mRNA of mIFN-λ2 was verified expressing in CHO-PCAGG-EGFP-mIFN-λ2 cell line by RT-PCR. The antivirus activity of in the supernatant secreted by the CHO-PCAGG-EGFP-mIFN-λ2 cell line was 10~4 AU/ml. The mIFN-λ2 pro-tein can could induce the expression of the antivirus protein Mx1, and the expression of Mx1 protein induced by mIFN-λ2 enhanced with time going, 9 to 12 hours achieved the peak, 24 hours vanished. Conclusion Gene cloning of mIFN-λ2 was successful. The eukaryotic expressing vector of mIFN-λ2 was constructed suc-cessfully and expressed stably in CHO cells, and its product has obvious antivirus activity in vitro. And the antivirus activity of the product was closely correlated with inducing expression of antivirus protein Mx1.
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Objective To explore the characteristics of endothelial progenitor cells (EPCs) on different scaffolds and to find a new bio-engineered synthetic hybrid scaffold for artificial bio-engineered blood vessels. Methods EPCs were induced from mesenchymal stem cells isolated from rat bone marrow and seeded on ECM scaffold. The surface structure of the scaffold and growth status of EPCs on the scaffold were observed and analysed by electron microscopy. The characteristics and number of those EPCs on different kinds of scaffolds were studied with EPC-specific VWF by immunofluorescence, Western blotting and real-time PCR technique at different time points. Results The cell adhesion rate at 1,3,5 h after seeded on pressed scaffold were higher than that on unpressed scaffolds( P < 0. 01 ). Pressed scaffolds has got a larger cell number( P < 0. 05 )at DIV1, DIV3, DIV7, but there was no significant difference after DIV10. Furthermore, cell shapes of EPCs on pressed scaffolds were more mature and more similar to endothelial cells. A level cell surface on pressed scaffolds was achieved. Western blotting assays revealed EPCs on pressed scaffolds expressed more protein VWF at DIV3, DIV7, DIV10. Real-time PCR results showed EPCs on the two different groups of scaffolds all expressed VWF gene, The quantity of their expression in the two groups were all enhanced after DIV7 (P < 0. 05 ). The quantity of VWF gene expression in the pressed group was much higher than that in the unpressed group at DIV3 ,DIV7,DIV10 (P <0, 01), but there was no significant difference after DIV14. Conclusions Pressed ECM scaffolds can promote adhesion, proliferation and differentiation on EPCs. Pressed scaffolds can be used as the matrix for EPC and fabricated into a novel synthetic tissue bio-engineered vascular scaffold.
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Objective To isolate and identify smooth muscle progenitor cells(SPCs) from rat bone marrow and to observe specific expression of smooth muscle progenitor cells during proliferation and differentiation in vitro.Methods MNCs were isolated by density gradient centrifugation from rat marrow and cultured in conditioned nutrient medium,identification was performed by immunofluorescent staining(?-SMA,CD14).Smooth muscle cells specific markers(?-SMA) were checked with Western blotting and Real-time PCR at different time.Results During culturing,cells adhered and became spindle shaped with outgrowth at 4 d and 7 d,and showed typical "peak" "valley" at 14 d.Both ?-SMA and CD14 were positive after 4 d.Expression of ?-SMA was not found at 1 d with Western blotting,but it gradually enhanced at 4 d and reached the peak from 10 d to 14 d,then maintaned high-level at 21 d.The results with Real-time PCR indicated that no expression of ?-SMA mRNA within non-induced cells was found,but after being induced it gradually enhanced at 4 d and got to peak at 14 d,then kept the high-level at 21 d,low-expression at 1 d was significantly different from the other ones(P