Generation of the regulatory protein rtTA transgenic mice

AIM: To translate Tet-on system into a conditional mouse model, in which hepatitis B or C virus (HBV or HCV) gene could be spatiotemporally expressed to overcome 'immune tolerance' formed during the embryonic development and 'immune escape' against hepatitis virus antigen(s), an effector mouse, carrying the reverse tetracycline-responsive transcriptional activator (rtTA) gene under the tight control of liver-specific human apoE promoter, is required to be generated. METHODS: To address this end, rtTA fragment amplified by PCR was effectively inserted into the vector of pLiv.7 containing apoE promoter to create the rtTA expressing vector, I.e., pApoE-rtTA. ApoE-rtTA transgenic fragment (-6.9 kb) released from pApoE-rtTA was transferred into mice by pronucleus injection, followed by obtaining one transgene (+) founder animal from microinjection through PCR and Southern blot analysis.RESULTS: rtTA transgene which could be transmitted to subsequent generation (F1) derived from founder was expressed in a liver-specific fashion. CONCLUSION: Taken together, these findings demonstrate that rtTA transgenic mice, in which rtTA expression is appropriately targeted to the murine liver, are successfully produced, which lays a solid foundation to 'off-on-off' regulate expression of target gene (s) (e.g., HBV and/or HCV) in transgenic mice mediated by Tet-on system.

Inhibitory effect of HuangqiZhechong decoction on liver fibrosis in rat

AIM. To assess the inhibitory effect of HuangqiZhechong decoction on hepatic fibrosis in rats induced by CCl4 plus alcohol and high fat low protein diet.METHODS: Male SD rats were randomly divided into hepati cfibrosis model group, control group and 3 treatment groups consisting of 12 rats in each group. Except for the normal control group, all the rats were subcutaneously injected with CCI4 at a dosage of 3 mL/kg. In 3 treated groups, eithe rhigh-dose group (9 mL/kg), or medium-dose group (6 mL/kg),or low-dose group (3 mL/kg) was daily garaged with Huangqi Zhechong decoction, and saline vehicle was given to model and normal control rats. Enzyme-linked immunosorbent assay (ELISA) and biochemical examinations were used to determine the changes of alanine aminotransferase (ALT),aspartate aminotransferase (AST), hyaluronic acid (HA),laminin (LN), type-III-procollagen-N-peptide (PIIIP), and type IV collagen content in serum, and hydroxyproline (Hyp) content in liver after sacrificing the rats. Pathologic changes,particularly fibrosis were examined by hematoxylin and eosin (HE) and Van Gieson staining.RESULTS: Compared with the model control group; serum ALT, AST, HA, LN, PIIIP and type IV collagen levels dropped markedly in Huangqi Zhechong decoction groups, especially in the medium-dose Huangqi Zhechong decoction group (1 954±576 U/L vs 759±380 U/L, 2 735±786 U/L vs 1259±829 U/L, 42.74±7.04 ng/mL vs20.68±5.85 ng/mL,31.62±5.84 ng/mL vs 14.87±1.45 ng/mL, 3.26±0.69 ng/mL vs 1.47±0.46 ng/mL, 77.68±20.23 ng/mL vs 25.64±4.68 ng/mL, respectively) (P<0.05). The Hyp content in liver tissue was also markedly decreased (26.47+11.24 mg/mgprot vs 9.89±3.74 mg/mgprot) (P<0.01). Moreover, the stage ofthe rat liver fibrosis in Huangqi Zhechong decoction groups was lower than that in model group, and more dramatic drop was observed in medium-dose Huangqi Zhechong decoction group (P<O,01),CONCLUSION: Huangqi Zhechong decoction can inhibit hepatic fibrosis resulted from chronic liver injure, retard the development of cirrhosis, and notably ameliorate the liver function. It may be a safe and effective therapeutic drug for patients with fibrosis.