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Objective@#To observe the effects of basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), and vascular endothelial growth factor C (VEGF-C) on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into lymphatic endothelial cells (LECs).@*Methods@#The third to the fifth passage of BMSCs of rats were collected for the following experiments. (1) BMSCs of rats were collected and divided into negative control group, CD90 group, CD44 group, and CD34 group according to the random number table (the same grouping method below), with 3 samples in each group. Phosphate buffer of 5 μL was added to cells in negative control group, and cells in the other 3 groups were added with 5 μL corresponding antibodies respectively. The positive expression of cell surface antigen was detected by flow cytometer. (2) BMSCs of rats in 3 batches were collected and divided into blank control group, VEGF-C group, HGF group, bFGF group, VEGF-C+ HGF group, VEGF-C+ bFGF group, HGF+ bFGF group, and VEGF-C+ HGF+ bFGF group, with 3 samples in each group. Cells in blank control group were added with 2 mL complete medium, cells in VEGF-C group were added with 2 mL complete medium and 10 μL VEGF-C of 10 μg/mL, cells in HGF group were added with 2 mL complete medium and 16 μL HGF of 10 μg/mL, and cells in bFGF group were added with 2 mL complete medium and 20 μL bFGF of 1 μg/mL. Cells in VEGF-C+ HGF group, VEGF-C+ bFGF group, HGF+ bFGF group, and VEGF-C+ HGF+ bFGF group were added with 2 mL complete medium and induction factors with corresponding concentration and volume as above. On 10 d of culture, the morphology of the cells was observed by the inverted phase contrast microscope, and the protein and mRNA expressions of lymphatic vessel endothelial hyaluronic acid receptor 1 (LYVE-1), VEGF receptor 3 (VEGFR3), and integrin α9 were detected by Western blotting and real-time fluorescent quantitative reverse transcription polymerase chain reaction respectively. (3) BMSCs of rats were collected and divided into blank control group, HGF+ VEGF-C+ bFGF group, bFGF+ VEGF-C+ HGF group, and VEGF-C+ HGF+ bFGF group, with 3 samples in each group. Cells in blank control group were added with 2 mL complete medium. Cells in HGF+ VEGF-C+ bFGF group were added with 2 mL complete medium, 16 μL HGF of 10 μg/mL, and 10 μL VEGF-C of 10 μg/mL, after 6 hours, 20 μL bFGF of 1 μg/mL was added. Cells in bFGF+ VEGF-C+ HGF group were added with 2 mL complete medium, 20 μL bFGF of 1 μg/mL, and 10 μL VEGF-C of 10 μg/mL, after 6 hours, 16 μL HGF of 10 μg/mL was added. Cells in VEGF-C+ HGF+ bFGF group were simultaneously added with 2 mL complete medium and the same concentration and volume of three inducing factors as above. In addition, BMSCs of rats in another 2 batches were collected and grouped, and they were dealt with the same methods as above except that the interval time of 6 hours in HGF+ VEGF-C+ bFGF group and bFGF+ VEGF-C+ HGF group was adjusted to 12 and 24 hours. On 10 d of culture, protein expressions of LYVE-1, VEGFR3, and integrin α9 were detected by Western blotting. Data were processed with analysis of variance of factorial design, one-way analysis of variance, and least significant difference t test, and Bonferroni correction.@*Results@#(1) The positive expression rates of surface antigen of cells in negative control group, CD90 group, CD44 group, and CD34 group were 0.39%, 99.84%, 99.90%, and 0.57%, respectively. (2) On 10 d of culture, cells in blank control group, HGF group, bFGF group, and HGF+ bFGF group presented long fusiform, while cells in the other groups presented polygonal shape. (3) On 10 d of culture, there were no protein expressions of LYVE-1, VEGFR3, and integrin α9 in cells of blank control group, HGF group, bFGF group, and HGF+ bFGF group. On 10 d of culture, protein expressions of LYVE-1, VEGFR3, and integrin α9 in cells of VEGF-C+ HGF+ bFGF group were significantly higher than those in VEGF-C group (t=24.21, 11.04, 15.43, P<0.01), VEGF-C+ HGF group (t=10.81, 9.93, 10.20, P<0.01), and VEGF-C+ bFGF group (t=11.67, 6.32, 19.00, P<0.01). Protein expressions of LYVE-1 in cells of VEGF-C+ HGF group and VEGF-C+ bFGF group were significantly higher than the protein expression in VEGF-C group (t=8.69, 15.20, P<0.01). Protein expression of VEGFR3 in cells of VEGF-C+ bFGF group was obviously higher than the protein expressions in VEGF-C group and VEGF-C+ HGF group (t=8.67, 7.21, P<0.01). Protein expression of integrin α9 in cells of VEGF-C+ HGF group was obviously higher than the protein expressions in VEGF-C group and VEGF-C+ bFGF group (t=8.80, 8.83, P<0.01). (4) On 10 d of culture, there were no mRNA expressions of LYVE-1, VEGFR3, and integrin α9 in cells of blank control group, HGF group, bFGF group, and HGF+ bFGF group. On 10 d of culture, mRNA expressions of LYVE-1 and VEGFR3 in cells of VEGF-C group were significantly lower than those in VEGF-C+ bFGF group and VEGF-C+ HGF+ bFGF group (tLYVE-1=6.22, 18.01, tVEGFR3=8.49, 15.34, P<0.01), and mRNA expression of integrin α9 were significantly lower than that in VEGF-C+ HGF group and VEGF-C+ HGF+ bFGF group (t=13.24, 9.65, P<0.01). The mRNA expressions of LYVE-1, VEGFR3, and integrin α9 in cells of VEGF-C+ HGF+ bFGF group were obviously higher than those in VEGF-C+ HGF group and VEGF-C+ bFGF group (t=13.92, 11.95, 13.72, 5.27, 5.64, 9.10, P<0.01). Compared with those of VEGF-C+ bFGF group, the mRNA expression of VEGFR3 of cells in VEGF-C+ HGF group was significantly lower (t=6.91, P<0.01), while the mRNA expression of integrin α9 of cells in VEGF-C+ HGF group was significantly higher (t=11.69, P<0.01). (5) On 10 d of culture at interval time of 6, 12, 24 h, there were no protein expressions of LYVE-1, VEGFR3, or integrin α9 in cells of blank control group. On 10 d of culture at interval time of 6, 12, 24 h, the protein expressions of LYVE-1, VEGFR3, and integrin α9 in cells of HGF+ VEGF-C+ bFGF group, bFGF+ VEGF-C+ HGF group, and VEGF-C+ HGF+ bFGF group were close (F6 h=2.25, 2.47, 2.19, F12 h=2.93, 1.47, 3.25, F24 h=0.28, 0.20, 1.01, P>0.05).@*Conclusions@#VEGF-C is a necessary factor for inducing BMSCs to differentiate into LECs. HGF and bFGF may promote the differentiation by up-regulating the expressions of integrin α9 and VEGFR3 respectively. But the induction effects of the two factors may be independent. The combination of VEGF-C, HGF, and bFGF have the best effects of promoting differentiation.
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Aim To investigate the effects of classic calcium antagonists verapamil(Ver),nifedipine(Nif),diltiazem(Dil)and the novel calcium antagonist N-n-butyl haloperidol iodide(F2)which was synthesized by our lab by regulating Ca2+-independent phospholipase A2(iPLA2)on hypoxia/reoxygenation(H/R)injury of cardiac microvascular endothelial cells(CMECs)and the mechanisms.Methods The CMECs were isolated from SD neonatal rats.The H/R model was established,then cells were treated with different concentrations of calcium antagonists and F2.The content of LDH in the cell supernatant was measured by colorimetric method.The levels of IL-6 and AA in cell supernatant were measured by ELISA;and late-stage apoptosis was measured by TUNEL.The mRNA and protein expression levels of iPLA2 in CMECs were examined by real time-PCR and Western blot analysis.Results Calcium antagonists except Dil decreased the generation of LDH,IL-6 and AA in a dose-dependent manner(P<0.05),and reduced the apoptosis(P<0.05).F2 and Ver decreased the mRNA and protein expression of iPLA2 in a dose-dependent manner,while there were no such effects for Nif and Dil.Conclusions Calcium antagonists except Dil have protective effects against H/R injury.F2 and Ver protect CMECs against H/R injury partly through iPLA2.
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Aim To investigate the effect of N-n-butyl haloperidol iodide(F2) on mitochondria-dependent apoptotic pathway of H9c2 cardiac myocytes during hypoxia/reoxygenation(H/R) injury.Methods The H/R models of H9c2 cardiac myocytes were established.The H9c2 cardiac myocytes were randomly divided into five groups: control group(C group), hypoxia/reoxygenation group(H/R group), F2 low concentration group(L), F2 medium concentration group(M), F2 high concentration group(H).Apoptotic rate was evaluated by flow cytometry(FCM).The levels of Cyto C, Bcl-2, Bax were observed by Western blot.Caspase-3 activity was measured with colorimetry.Results Compared with H/R group, F2 low, medium and high concentrations group could significantly decrease apoptosis rate and increase the ratio of Bcl-2 to Bax proteins and inhibit the release of Cyto C into the cytosolic fraction, and decrease caspase-3 activity.Conclusion F2 can protect H9c2 cardiac myocytes against H/R-induced injury through interfering in mitochondria-dependent pathway.
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Objective To investigate the effect of PNS on malignant melanoma and the expression of connexin32 in melanoma. Methods The spontaneous and experimental lung metastasis models of B16 melanoma were used to investigate the inhibitory effect of PNS on tumor growth and metastasis. The expression of connexin in melanoma were detected by immunohistoehemistry. Results (1) PNS can obviously inhibit the growth of B16 melanoma. The inhibition rate of the 480mg/kg PNS group was 50. 85%. (2) PNS can obviously inhibit the lung metastasis of B16 melanoma. The number of tumor colonies in lung of the 240 mg/kg PNS group and 480 mg/kg PNS group were lower than that in the negative control group. ( 3 ) The expression of connexin32 in melanoma was detected by immunohistochemistry:PNS could up-regulate the expression of connexin32 in membrane of melanoma. Conclusion PNS can inhibit malignant melanoma growth and metastasis and could also up-regulate the expression of connexin32 in membrane of melanoma.