BACKGROUND: In vivo experiments have confirmed that fibroblast growth factor can effectively protect gentamicin-induced renal tubular epithelial cell injury, but the effect on the in vitro cultured cells is still rare. OBJECTIVE: To explore the mechanisms of basic fibroblast growth factor (bFGF) at different concentrations on preventing nephrotoxity mediated by genamicin on the primarily cultivated renal tubular epithelial cell models. METHODS: By use of enzyme and mesh screening, renal tubular epithelial cells were isolated from Kunming mice and purified, adjusting the cell concentration of 1×10~8/L, then cell suspension was moved to a 96-well culture plate and divided into different groups for culture: blank control group: normal culture; gentamicin group: 10, 30, 50 μL/hola (ie, 400, 1 200, 2 000 U/holes)arerecorded as G1, G2, G3; bFGF group: 20, 50, 80 μL/hole (ie, 90,225, 360 ng/hole) are recorded as B1, B2, B3; gentamicin plus bFGF intervention group: after adding bFGF 12 hours, then added gentamicin 12 hours, assigned into 9 dose subgroups, namely, G1B1, G1B2, G1B3, G2B1, G2B2, G2B3, G3B1, G3B2, G3B3, each subgroup contained four-hole complex. Cell morphology and quantity was observed. RESULTS AND CONCLUSION: Gentamicin showed a dose-dependent effect on the renal tubular epithelial cell injury, epithelial cells in the medium and high concentration groups exhibited shrinkage, rounded, swelling, poor adhesion, severely damaged cytoplasm and structural disorder. In the low concentration group, the number change of cells was not obvious, and fibroblasts began to appear; In the bFGF groups, cells were full, exhibited strong refraction, the cell number increased significantly, these manifestations were significant in 50 μL/hole concentration, and there was no significant difference compared with 80 μL/hore concentration; in case of gentamicin plus bFGF intervention, cells with low concentrations of gentamicin had no obvious damage to cells, which increased, the damaged cells collapse was reduced in the group of low concentration of gentamicin, cell shrinkage and poor adhesion were slightly relieved, high concentrations of bFGF intervention could yield to good cell morphology, but high concentrations of gentamicin caused cell swelling and necrosis of injury, which could not be improved by any concentrations of bFGF intervention. 50 μL/hole bFGF has antagonistic effect on the nephrotoxicity mediated by medium and low concentrations of gentamicin, but has no protection on high concentration of gentamicin-induced nephrotoxicity.

OBJECTIVETo generate a mouse model of chronic hepatitis B (CHB) infection by performing in vivo transduction of hepatitis B virus (HBV) covalently closed circular (ccc)DNA.

METHODSNude mice were injected with HBV cccDNA at doses of 1.5, 1.0 or 0.5 mug/ml. A control group was generated by giving equal injection volumes of physiological saline. The serum levels of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) on post-injection days 1 and 3, weeks 1-6, 8 and 10 were assayed by reflection immunoassay. At post-injection week 10, all animals were sacrificed and liver tissues were collected. Copies of HBV DNA in serum and liver tissue were detected by real-time PCR. HBV antigens in liver tissue were detected of by immunohistochemistry. Pathological analysis of liver tissue carried out with hematoxylin-eosin staining. Linear correlation of data was determined by statistical analysis.

RESULTSHBsAg and HBeAg were detected in sera from all three groups of cccDNA-injected mice staring at post-injection day 1 and lasting through week 10. The levels of HBsAg over the 10-week period showed two patterns of increase-decrease;the lowest level was detected at week 4 and the highest level was detected at week 8. In contrast, the levels of HBeAg over the 10-week period showed three patterns of increase-decrease; the lower levels were detected at weeks 2 and 4 and the higher levels at weeks 3 and 6. HBV DNA copies in liver tissues showed a cccDNA dose-dependent descending trend over the 10-week study period (1.5 mug/ml:1.14E+07 ± 6.51E+06 copies/g, 1.0 mug/ml:9.81E+06 ± 9.32E+06 copies/g, and 0.5 mug/ml:3.72E+06 ± 2.35E+06 copies/g; Pearson's r =0.979). HBV DNA copies in sera showed the pattern of 1.0 mug/ml cccDNA more than 1.5 mug/ml cccDNA more than 0.5 mug/ml cccDNA, and in general were higher than those detected in the liver tissues. Liver tissues from all cccDNA-injected mice showed positive immunohistochemistry staining for both HBsAg and HBeAg. HE staining showed that the liver tissues of all cccDNA-injected mice had severe fatty and vacuolar degeneration and less obvious structure of liver lobules (compared to the liver tissues from control mice).

CONCLUSIONThe CHB mouse model successfully established in this study by in vivo transduction of HBV cccDNA may represent a useful tool to study the pathogenic mechanisms and potential antiviral treatments of human CHB.

Animals ; DNA, Circular ; administration & dosage ; DNA, Viral ; administration & dosage ; Disease Models, Animal ; Hepatitis B Surface Antigens ; blood ; Hepatitis B e Antigens ; blood ; Hepatitis B virus ; genetics ; physiology ; Hepatitis B, Chronic ; virology ; Male ; Mice ; Mice, Nude ; Transduction, Genetic ; Virus Replication