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@#This study was to evaluate the cellular inhibition effects induced by anti-AGR2 monoclonal antibody 18A4 in two AGR2-negative melanoma cells, A375 and B16-F10, treated with external oncoprotein AGR2. MTT assay and colony formation assay were performed to detect the cell proliferation rate. Flow cytometry analysis was performed to evaluate cell cycle transition. Cell migration rate was analyzed by wound healing assay. Cell morphological changes were detected by phalloidin staining. The expression of p53 was detected by Western blotting and immunofluorescence. Results showed that 18A4 inhibited the AGR2-dependent tumor properties including enhanced proliferation, accelerated cell cycle, increased cell migration and morphological changes of cells. In addition, by Western blot analysis and immunofluorescence, AGR2 blocking antibody 18A4 also restored p53 expression that was repressed by external AGR2 treated by chemotherapeutic drug doxorubicin. These findings suggest that 18A4 is able to inhibit the cellular tumorigenic properties induced by external AGR2 and is a potential drug against fumor.
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Objective To investigate the effects of Buyang Huanwu decoction and its decomposed recipes on neurological function and angiogenesis after focal cerebral ischemia in rats and its mechanism. Methods Fifty-two clean grade SD male rats were randomly divided into sham-operation,model,whole prescription,invigorating qi,and promoting blood circulation groups (n = 8 in each group)according to the random number table. In addition to the sham-operation group,the middle cerebral artery occlusion models of the rats in other groups were induced by the suture method. The patients with the first Longa nerve function scores 1 to 3 were used as successful modeling. The whole Buyang Huanwu decoction included dried root of Astragalus membranaceus 120. 0 g,dried root of angelica sinensis 6. 0 g,dried root of Paeonia lactiflora 4. 5 g, dried rhizome Ligusticum chuanxiong 3. 0 g,dried body of Pheretima aspergillum 3. 0 g,dried flowers of Carthamus tinctorius 3. 0 g,and seed of Prunus persica 3. 0 g;the invigorating qi prescription included dried root of Astragalus membranaceus 120. 0 g;the promoting blood circulation prescription included dried root of angelica sinensis 6. 0 g,dried root of Paeonia lactiflora 4. 5 g,dried rhizome Ligusticum chuanxiong 3. 0 g, dried body of Pheretima aspergillum 3. 0 g,dried flowers of Carthamus tinctorius 3. 0 g,and seed of Prunus persica 3.0 g. On the first day after procedure,the rats began to be administered intragastrically. The intragastric doses of the whole prescription group,invigorating qi group,and promoting blood circulation group were 13. 1,10. 8,and 2. 2 g/ kg,respectively. The sham-operation group and the model group were given equal volume of isotonic saline,once a day for 14 days. 5-bromodeoxyuridine (BrdU,50 mg/ kg)were injected intraperitoneally,once a day for 14 days. The modified neurological severity score (mNSS)and the corner test were used to evaluate sensorimotor function at day 1,7 and 14 after procedure. BrdU and rat von Willebrand factor (vWF)double immunofluorescent staining were used to detect the angiogenesis in ischemic peripheral region;Western Blot was used to detect the protein expression of vascular endothelial growth factor (VEGF). Results (1)Compared with the model group,the mNSS score in rats in the whole prescription group was lower at day 7 and 14 after procedure (6. 8 ±1. 0 vs. 8. 5 ±1. 1,6. 1 ± 0. 8 vs. 8. 0 ± 1. 4;all P < 0. 01). The number of turning right in the whole prescription group was reduced (7. 1 ±0. 6 vs. 8. 6 ±1. 2 and 6. 1 ± 0. 8 vs. 7. 9 ±1. 1;all P < 0. 01). The number of turning right in the invigorating qi group was reduced (7. 5 ± 0. 5 vs. 8. 6 ± 1. 2 and 6. 2 ± 1. 0 vs. 7. 9 ± 1. 1;all P < 0. 01). At day 14 after procedure,the number of BrdU / vWF co-labeled immunopositive cells in ischemic peripheral zone of the whole prescription group was increased significantly. There was significant difference between the groups (30 ± 8 / mm2 vs. 24 ± 7 / mm2;P < 0. 01). The VEGF protein expression was increased (0. 33 ±0. 01 vs. 0. 30 ±0. 01;P <0. 01). (2)Compared with the invigorating qi group,the rat mNSS scores of the whole prescription group were lower at day 7 and 14 after procedure (the invigorating qi group 8.2 ±1.3 and 7.5 ±0.9 respectively;all P <0. 05). The number of BrdU/ vWF immunopositive cells in the whole prescription group was increased at day 14 after procedure (26 ±5/ mm2 in the invigorating qi group;P < 0. 05). The VEGF protein expression was increased (0.31 ±0.01 in the invigorating qi group;P <0.01). (3)Compared with the promoting blood circulation group, the mNSS scores of the whole prescription group were lower at day 7 and 14 after procedure (the promoting blood circulation group 8.5 ±0.9 and 7.6 ±0.7 respectively;all P <0. 05). The number of turning right was reduced (8.5 ±0. 8 and 7. 6 ± 0. 9 respectively in the promoting blood circulation group;all P < 0. 05). The number of BrdU/ vWF immunopositive cells in ischemic peripheral zone of the whole prescription group at day 14 after procedure was increased (26 ± 6 / mm2 ,P < 0. 05). The relative expression level of VEGF was increased (0. 31 ±0. 01 in the promoting blood circulation group,P <0. 05). Conclusion Buyang Huanwu decoction can promote angiogenesis and recovery of neurological function after cerebral ischemia. Its mechanism may be associated with the up-regulation of the VEGF protein. The traditional Chinese medicines for invigorating qi and invigorating the circulation of blood in the prescription have synergistic effect.
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Objective To analyze the expression of hsa-miR-10b in three cervical cancer cell lines , and to find the target genes of hsa-miR-10b.Methods PCR was applied to measure expression levels of hsa-miR-10b in C-33A, HeLa and CaSki .The relative studies on hsa-miR-10b were retrieved from PubMed .The sequence and genome char-acteristics of hsa-miR-10b were analyzed on line by miRbase and NCBI .The target genes of hsa-miR-10b were searched by TargetScan , PicTar and miRanda , and then demonstrated by Gene Ontology and Pathway Enrichment analysis.Results Compared with C-33A, the expression of hsa-miR-10b significantly reduced in HeLa and Caski (P<0.01).Current studies showed that hsa-miR-10b was related with multiple tumorigenesis .hsa-miR-10b was lo-cated in human chromosome 2q31.1 and highly conserved among different species .The target genes were enriched in the biological processes of transcription , gene expression regulation , cell proliferation and etc .(P<0.05).Pathway analysis showed that these target genes were responsible for biochemical erents mediated by to the signaling pathways of cancer, cell cycle, ARF and etc.(P<0.05).Conclusions hsa-miR-10b may have extensive functions , and closely related with the occurrence and development in cervical cancer .Prediction of target genes provides a theoreti-cal basis for the further study .
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Objective To investigate the effect of Buyanghuanwu decoction(BYHWD) on the expression of growth-associated protein 43(GAP-43) and synaptophysin(SYP) after focal cerebral ischemia in mice.Methods Focal cerebral ischemia was induced by 30 min of middle cerebral artery occlusion followed by reperfusion.BYHWD (20 g/kg) was administered orally 24 h after ischemia and once a day.The neurological score and the corner test were used to evaluate sensorimotor function on 1,3,7,and 14 days after ischemia.The expression of GAP-43 and SYP in the ischemic boundary zone was determined by immunohistochemistry 14 days after ischemia.Results Compared with the model group,BYHWD significantly ameliorated neurological dysfunction at 3,7 and 14 days and reduced the number of right turn at 7 and 14 days after ischemia(P ＜ 0.05).The average optical density of GAP-43 and SYP was 0.217 ±0.012 and 0.278 ±0.019 at the boundary zone in model group 14 days after ischemia,respectively.BYHWD also significantly increased the immunoreactivity of GAP-43 (0.250 ± 0.013)and SYP(0.323 ± 0.017) (P＜ 0.01).Conclusion BYHWD promotes axonal regeneration and synaptic plasticity in the ischemic boundary zone,which may contribute to functional recovery after focal cerebral ischemia.
