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Objective@#To assess the effect of viral macrophage inflammatory protein (vMIP) -Ⅱ on the expression of apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G) , and to explore the mechanisms.@*Methods@#A recombinant plasmid pEGFP-N3-K4 (vMIP-Ⅱ plasmid group) and an empty plasmid pEGFP-N3 (empty plasmid group) were separately transfected into 293T cells, and quantitative PCR and Western blot analysis were performed to evaluate the effect of transfection with vMIP-Ⅱ gene on the APOBEC3G expression in 293T cells. Some 293T cells in the empty plasmid group and vMIP-Ⅱ plasmid group were treated with 1 000 IU/ml interferon (IFN) -α for 36 hours, and then Western blot analysis was conducted to determine the APOBEC3G expression in the empty plasmid group and vMIP-Ⅱ plasmid group with or without IFN-α treatment. Some 293T cells transfected with vMIP-Ⅱ plasmids were treated with 75 μmol/L AG490 (a JAK/STAT signaling pathway inhibitor) and 20 μmol/L U0126 (an ERK signaling pathway inhibitor) separately; after 24 hours, total protein was extracted from 293T cells, and Western blot analysis was conducted to determine the expression of APOBEC3G. A recombinant plasmid containing APOBEC3G promoter was constructed by using a luciferase reporter gene, and the promoter fragment included the full-length promoter sequence (POS) of APOBEC3G, sequences with the lengths of 1 560, 960, 720, 480, 420, 360, 330 and 240 bp, and the regulatory element-free region (NEG) of APOBEC3G, separately. Some 293T cells were co-transfected with the recombinant plasmid carrying luciferase reporter gene and vMIP-Ⅱ plasmid (experimental group), or the recombinant plasmid and empty plasmid (control group). Subsequently, the activity of the APOBEC3G promoter was evaluated, and the key promoter region through which the transcriptional activity of APOBEC3G was regulated by vMIP-Ⅱ was analyzed. Statistical analysis was carried out by using t test, one-way analysis of variance and least significant difference (LSD) -t test.@*Results@#The mRNA and protein expression of APOBEC3G was significantly higher in the vMIP-Ⅱ plasmid group (2.500 ± 0.013, 1.472 ± 0.013 respectively) than in the control group (1, 0.364 ± 0.030 respectively; t = 6.22, 6.54 respectively, both P < 0.05) . The APOBEC3G expression significantly differed among the empty plasmid group, vMIP-Ⅱ plasmid group, empty plasmid + IFN-α group and vMIP-Ⅱ plasmid + IFN-α group (1, 2.030 ± 0.108, 2.700 ± 0.081 and 2.600 ± 0.099 respectively; F = 67.026, P < 0.001) , but there was no significant difference between the vMIP-Ⅱ plasmid group and empty plasmid + IFN-α group (t = 3.46, P > 0.05) . The APOBEC3G expression also significantly differed among the vMIP-Ⅱ plasmid group, vMIP-Ⅱ plasmid + AG490 group and vMIP-Ⅱ plasmid + U0126 group (0.617 ± 0.025, 0.179 ± 0.061, 0.359 ± 0.012 respectively; F = 70.019, P < 0.001) , and was significantly lower in the vMIP-Ⅱ plasmid + AG490 group and vMIP-Ⅱ plasmid + U0126 group than in the vMIP-Ⅱ plasmid group (t = 9.66, 11.836 respectively, both P < 0.01) . Luciferase activity assay showed that the promoter activity significantly differed among the vMIP-Ⅱ plasmid groups transfected with POS, 1 560-, 960-, 720-, 480-, 420-, 360-, 330-, 240-bp or NEG sequences (F = 81.092, P < 0.001) , and the APOBEC3G promoter activity decreased greatly from the 720-bp group to 480-bp group.@*Conclusion@#vMIP-Ⅱ upregulates the expression of APOBEC3G, likely through the JAK/STAT signaling pathway or the key promoter region regulating the transcriptional activity of APOBEC3G.
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Objective To assess the effect of viral macrophage inflammatory protein (vMIP)-Ⅱ on the expression of apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G),and to explore the mechanisms.Methods A recombinant plasmid pEGFP-N3-K4 (vMIP-Ⅱ plasmid group) and an empty plasmid pEGFP-N3 (empty plasmid group) were separately transfected into 293T cells,and quantitative PCR and Western blot analysis were performed to evaluate the effect of transfection with vMIP-]Ⅱ gene on the APOBEC3G expression in 293T cells.Some 293T cells in the empty plasmid group and vMIP-Ⅱ plasmid group were treated with 1 000 IU/ml interferon (IFN)-α for 36 hours,and then Western blot analysis was conducted to determine the APOBEC3G expression in the empty plasmid group and vMIP-Ⅱ plasmid group with or without IFN-α treatment.Some 293T cells transfected with vMIP-Ⅱplasmids were treated with 75 μ mol/L AG490 (a JAK/STAT signaling pathway inhibitor) and 20 μ mol/L U0126 (an ERK signaling pathway inhibitor) separately;after 24 hours,total protein was extracted from 293T cells,and Western blot analysis was conducted to determine the expression of APOBEC3G.A recombinant plasmid containing APOBEC3G promoter was constructed by using a luciferase reporter gene,and the promoter fragment included the full-length promoter sequence (POS) of APOBEC3G,sequences with the lengths of 1 560,960,720,480,420,360,330 and 240 bp,and the regulatory element-free region (NEG) of APOBEC3G,separately.Some 293T cells were co-transfected with the recombinant plasmid carrying luciferase reporter gene and vMIP-Ⅱ plasmid (experimental group),or the recombinant plasmid and empty plasmid (control group).Subsequently,the activity of the APOBEC3G promoter was evaluated,and the key promoter region through which the transcriptional activity of APOBEC3G was regulated by vMIP -Ⅱ was analyzed.Statistical analysis was carried out by using t test,one-way analysis of variance and least significant difference (LSD)-t test.Results The mRNA and protein expression of APOBEC3G was significantly higher in the vMIP-Ⅱ plasmid group (2.500 ± 0.013,1.472 ± 0.013 respectively) than in the control group (1,0.364 ± 0.030 respectively;t =6.22,6.54 respectively,both P ＜ 0.05).The APOBEC3G expression significantly differed among the empty plasmid group,vMIP-Ⅱ plasmid group,empty plasmid + IFN-oα group and vMIP-Ⅱ plasmid + IFN-α group (1,2.030 ± 0.108,2.700 ± 0.081 and 2.600 ± 0.099 respectively;F =67.026,P ＜ 0.001),but there was no significant difference between the vMIP-Ⅱ plasmid group and empty plasmid + IFN-α group (t =3.46,P ＞ 0.05).The APOBEC3G expression also significantly differed among the vMIP-Ⅱ plasmid group,vMIP-Ⅱ plasmid + AG490 group and vMIP-Ⅱplasmid + U0126 group (0.617 ± 0.025,0.179 ± 0.061,0.359 ± 0.012 respectively;F =70.019,P ＜ 0.001),and was significantly lower in the vMIP-Ⅱ plasmid + AG490 group and vMIP-Ⅱ plasmid + U0126 group than in the vMIP-Ⅱ plasmid group (t =9.66,11.836 respectively,both P ＜ 0.01).Luciferase activity assay showed that the promoter activity significantly differed among the vMIP-Ⅱ plasmid groups transfected with POS,1 560-,960-,720-,480-,420-,360-,330-,240-bp or NEG sequences (F =81.092,P ＜ 0.001),and the APOBEC3G promoter activity decreased greatly from the 720-bp group to 480-bp group.Conclusion vMIP-Ⅱ upregulates the expression of APOBEC3G,likely through the JAK/STAT signaling pathway or the key promoter region regulating the transcriptional activity of APOBEC3G.
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Objective To investigate the effect of exogenous vascular endothelial growth factor (VEGF) on bone activity of rabbit heterotopic allograft decalcified bone.Methods 140 adult healthy China white rabbits were selected,no limitation with sex,20 rabbits as the donor preparation of allogenic decalcified bone,according to the random number table,the rest was divided into the experimental group (allograft decalcified bone ± VEGF) and the control group (Allograft decalcified bone),each group contained 60 rabbits.For the experimental group,the prepared 1.5 cm long homologous decalcified tibia was placed in rabbit right thigh of rectus femoris and vastus medialis muscle gap near by saphenous artery,and fixed on the femur with two 0.8 mm Kirschner wire.In the vicinity of the skin,implanted an osmotic pump which contain the VEGF solution 200 μl with concentration was 0.5 μg/ml.In the control group,implanted the isometric allograft decalcified bone in rabbit right thigh corresponding parts with the same method.Each group respectively at 0,2,4,6,8,10 weeks to death 10 white rabbits,By specimen observation,HE dyeing observation and detection of type Ⅰ glue protein fluorescence intensity,Analysis the bone activation degree of two groups of bone allograft decalcified.Results Experimental allograft decalcified bone gradually wrapped by connective tissue membrane,its surface appear different size of the pits and gradually increased and become deep,while the control group pits relatively little and shallow.In the experimental group and control group,the fluorescence intensity of type Ⅰ collagen reached its peak respectively at 8 weeks (47.57 ±3.50) and 10 weeks (45.07±6.02),with no statistically significant (P ＞ 0.05).Conclusion Rabbit allograft decalcified bone implanted in the muscle clearance with abundant blood supply can be transformed into activated bone after 10 weeks,and after applying exogenous VEGF,allograft decalcified bone can be transformed into activated bone after 8 weeks,the bone activation process obviously speed up.The reaults confirmed the exogenous VEGF can obviously promote the ectopic rabbit bone allograft decalcified bone activation process.
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Objective To understand and identify the molecules related to the natural resistance to Schistosoma japonicum infection in Mirotus fortis. Methods Sera from Mirotus fortis without schistosome infection were collected. The S.japonicum adult worm cDNA library was immunologically screened with the sera. The positive recombinants were identified, cloned, sequenced and analysed with software and internet. Results Seven genes encoding antigens relevant to sera antibodies in Mirotus fortis were cloned and sequenced. These antigens included glyceraldehyde 3-phosphate dehydrogenase (GAPDH), serine protease inhibitors(SERPIN), 70 kDa heat shock protein(HSP70), 22^6 kDa membrane-associated antigen, paramyosin (Sj97), cytochrome C and cathepsin B. Conclusion Many protein molecules might have been involved in natural resistance to \{S.japonicum\} infection in Mirotus fortis. The above 7 kinds of molecules may be identified as new candidates of vaccine against \{S.japonicum\} infection.