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Objective To study the effect of the recombinant Lentivirus containing calcitonin gene related peptide (CGRP) gene on cells biological activity and differentiation of rat bone marrow stem cells(MSCs).Methods Rat MSCs were isolated and cultured by granulocytes adherent.MSCs were transfected with Lenti-EGFP CGRP(MSCsCGRP+/+ group),While MSCs were transfected with Lenti-EGFP as control group.Cell transfection rate was detected by flow cytometry,protein secretion in the above-mentioned MSCsCGRP+ + supernatant was detected using ELISA method.Cells surface markers weare detected by flow cytometry and immunohistochemistry.Trypan blue was used to examin the survive rate,β galactosidase staining was used to examin aging of MSCs transfection,and MTT was used to examine cell vitality.Results At first day after transfecting with Lenti-EGFP-CGRP,fluorescence was not observed by fluorescence microscope,but a small amount of CGRP protein was detected by ELISA in MSCsCGRP+/+ group,at 3 days and 4 days after transfecting with MSCs,strong fluorescence was observed by fluorescence microscope (the cell transfection rates were 77.87％ and 79.58％).The CGRP expression was significantly higher in MSCsCGRP+ + group than in control group [(19.53±0.50) pg/ml vs.(3.12±0.00) pg/ml,t=48.964,P＜0.01].At three days after transfection with MSCs,CD29 and CD90 expression were significantly higher,as compared with control group,CD31 expression was increased in MSCsCGRP+ /+ group.Seven days after transfection with MSCs,CD31 expression was significantly increased in MSCsCGRP+ + group,vWF expression was significantly increased in MSCsCGRP+ + group after MSCs were transfected with LentiEGFP CGRP for 14 days,but a SMA expression was decreased in MSCsCGRP+ +group.At 3 days and 7 days after transfection with Lenti-EGFP-CGRP,the proliferation,survive and aging showed no difference in MSCsCGRP+/+group and in control group (the proliferation of cell:t=0.253,0.290the survive of cell t=-0.307,0.690,all P＞0.05).At 14 days after transfection with Lenti-EGFP-CGRP,aging of cell were decreased in MSCsCGRP+ + group as compared with control group (t=2.446,P＜ 0.05).Conclusions After MSCs are transfected with Lenti EGFP-CGRP,biological characteristics of MSCs has no significant effects,there is still proliferation and differentiation activity.Cell secretion of CGRP can promote the endothelial cell differentiation,and inhibit the differentiation to smooth muscle cells.The CGRP modification of MSCs may play a role in the regulation of angiogenesis.
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<p><b>OBJECTIVE</b>To investigate the impact of calcitonin gene-related peptide (CGRP) modified bone marrow mesenchymal stem cell (MSC) on the migration of vascular smooth muscle cell (VSMC) and related mechanisms.</p><p><b>METHODS</b>The MSC and VSMC were isolated from rats and cultured, CGRP was transfected to MSC with the high expression lentivirus vector, VSMC was transfected with high expression lentivirus vector of receptor activity modifying protein 1 (RAMP1) and the silence expression lentivirus vector of RAMP1. Then MSC was co-cultured with VSMC. Experimental groups were as follows: (1) Ang II group (MSC + VSMC + Ang II); (2) MSC(CGRP+) group (MSC(CGRP+) + VSMC + Ang II); (3) MSC(CGRP+) RAMP1(-) group (MSC(CGRP+) + VSMC(RAMP1-) + Ang II); (4) MSC(CGRP+) RAMP1(+) group (MSC(CGRP+) + VSMC(RAMP1+) + Ang II); (5) RAMP1(+) group (MSC + VSMC(RAMP1+) + Ang II). Transwell assay was applied to detect the migration of smooth muscle cells, Western blot was applied to detect the protein expression of cells in various groups.</p><p><b>RESULTS</b>VSMC migration number was significantly lower in MSC(CGRP+) group compared with Ang II group (50.8 ± 2.6 vs. 71.4 ± 2.3, P < 0.05), but higher than in MSC(CGRP+) RAMP1(+) group (50.8 ± 2.6 vs. 30.4 ± 3.0, P < 0.05). When RAMP1 expression reduced in VSMC, compared with MSC(CGRP+) RAMP1(+) group, VSMC migration increased in the MSC(CGRP+) RAMP1(-) group compared to MSC(CGRP+)RAMP1(+) (69.0 ± 5.6 vs. 30.4 ± 3.0, P < 0.05) and was similar to Ang II group (69.0 ± 5.6 vs. 71.4 ± 2.3, P > 0.05) and RAMP1(+) group (71.6 ± 3.4). According to the result of Western blot, P-P65 protein expression in MSC(CGRP+) group was lower than that in Ang II group (0.475 ± 0.022 vs.0.642 ± 0.035, P < 0.05). P-P65 protein expression in MSC(CGRP+)RAMP1(-) group was higher than that in MSC(CGRP+) RAMP1(+) group (0.670 ± 0.030 vs. 0.373 ± 0.041, P < 0.05), and there was no difference between MSC(CGRP+)RAMP1(-) group and Ang II group (P > 0.05). P-P65 protein expression was similar between RAMP1(+) group (0.643 ± 0.039) and Ang II group (P > 0.05).</p><p><b>CONCLUSIONS</b>CGRP inhibits VSMC migration through RAMP1. NF-κB and RAMP1 play crucial role in the inhibiting effects of CGRP on VSMC migration. Thus, RAMP1-CGRP signaling inhibits VSMC migration through NF-κB signal pathways.</p>
Subject(s)
Animals , Rats , Bone Marrow Cells , Calcitonin Gene-Related Peptide , Cell Movement , Coculture Techniques , Hematopoietic Stem Cells , In Vitro Techniques , Muscle, Smooth, Vascular , Myocytes, Smooth Muscle , NF-kappa B , Receptor Activity-Modifying Protein 1 , Signal Transduction , TransfectionABSTRACT
<p><b>OBJECTIVE</b>To investigate the effects of arsenic trioxide (As(2)O(3)) on the apoptosis and p-glycoprotein (P-gp) expression of multidrug-resistant human leukemia cells.</p><p><b>METHODS</b>Human multidrug-resistant leukemia cell line K562/ADM overexpressing the MDR1 gene, was used as the target cells. The cell proliferating activity was assessed using the MTT colorimetric assay. Cytomorphology was investigated under light, confocal and electron microscopes. DNA fragmentation was examined using agarose gel electrophoresis, while p-gp expression, cell cycle status and sub-G1 cells were determined using flow cytometry.</p><p><b>RESULTS</b>Zero point five to 20 micromol/L As(2)O(3) inhibited the proliferation of K562/ADM cells, and K562/ADM cells were more sensitive to As(2)O(3) than the parental K562 cells. As(2)O(3)-induced apoptosis of K562/ADM cells was determined by the observance of typical morphological changes and the appearance of DNA ladder and sub-G1 cell populations. As(2)O(3) significantly inhibited the P-gp expression of K562/ADM cells, and synergistically enhanced the sensitivity of the drug-resistant cells to adriamycin.</p><p><b>CONCLUSIONS</b>As(2)O(3) induces growth-inhibition and apoptosis, down-regulates P-gp expression and exerts a synergistic effect in combination with adriamycin in multidrug-resistant leukemia cells.</p>
Subject(s)
Humans , ATP Binding Cassette Transporter, Subfamily B, Member 1 , Apoptosis , Arsenicals , Pharmacology , Drug Resistance, Multiple , Gene Expression , Genes, MDR , Leukemia , Genetics , Metabolism , Oxides , PharmacologyABSTRACT
Object To study the augmentation of the T activated killer (T AK) cell proliferating and tumor killing activities of the polysaccharide fraction from Rhodiola kirilowii (Regel) Regel, Lycium barbarum L., Hedysarum polybotrys Hand. Mazz., Glehnia littoralis F. Schmidt ex Miq., Rehmannia glutinosa (Gaert.) Libosch. ex Fisch. et Mey. and Aloe barbadensis Mill. in vitro. Methods The T AK cells were induced by anti CD 3 antibody (CD 3McAb) and rIL 2 from human peripheral blood mononuclear cells (PBMC). The effects of the above six plant polysaccharides (1～100 ?g/mL) on the proliferation of T AK cell, the cytotoxicity to Raji cells and L 1210 cells, and the IL 2 receptor (IL 2R) expression in T AK cells were determined. Results The six polysaccharides alone had no obvious effect on the proliferation of T AK cells. In the presence of rIL 2 and CD 3McAb, they could reinforce the proliferation of T AK cells and its tumor killing activities against Raji cells and L 1210 cells at a different extent, and a 21%～68% increase of IL 2R expression in T AK cells was observed. Conclusion The plant polysaccharides significantly enhance the proliferation and the tumoricidal activities of T AK cells and the enhancing actions related to the increase of IL 2R expression.