Published online Jun 1, 2004. doi: 10.3748/wjg.v10.i11.1634
Revised: October 4, 2003
Accepted: October 29, 2003
Published online: June 1, 2004
AIM: To test the hypothesis that introduction of antisense TβR I and TβR II eukaryotic expressing plasmids into a rat model of immunologically induced liver fibrosis might block the action of TGF-β1 and halt the progression of liver fibrosis.
METHODS: RT-Nest-PCR and gene recombination techniques were used to construct rat antisense TβR I and TβR II recombinant plasmids which could be expressed in eukaryotic cells. The recombinant plasmids and empty vector (pcDNA3) were encapsulated by glycosyl-poly-L-lysine and then transducted into rats of pig serum-induced liver fibrosis model. Expression of exogenously transfected gene was assessed by Northern blot, and hepatic expressions of TβR I and TβR II were evaluated by RT-PCR and Western blot.We also performed ELISA for serum TGF-β1, hydroxyproline of hepatic tissues, immunohistoche-mistry for collagen types I and III, and VG staining for pathological study of the liver tissues.
RESULTS: The exogenous antisense TβR I and TβR II plasmids could be well expressed in vivo, and block mRNA and protein expression of TβR I and TβR II in the fibrotic liver at the level of mRNA respectively. These exogenous plasmid expressions reduced the level of TGF-β1 (antisense TβR I group 23.998 ± 3.045 ng/mL, antisense TβR II group 23.156 ± 3.131 ng/mL, disease control group 32.960 ± 3.789 ng/mL; F = 38.19, 36.73, P < 0.01). Compared with disease control group, the contents of hepatic hydroxyproline (antisense TβR I group 0.169 ± 0.015 mg/g liver, antisense TβR II group 0.167 ± 0.009 mg/g liver, disease control group 0.296 ± 0.026 mg/g liver; F = 14.39, 15.48, P < 0.01) and the deposition of collagen types I and III decreased in the two antisense treatment groups (antisense TβR I group, collagen type I 669.90 ± 50.67, collagen type III 657.29 ± 49.48; antisense TβR II group, collagen type I 650.26 ± 51.51, collagen type III 661.58 ± 55.28; disease control group, collagen type I 1209.44 ± 116.60, collagen type III 1175.14 ± 121.44; F = 15.48 to 74.89, P < 0.01). Their expression also improved the pathologic classification of liver fibrosis models (compared with disease control group, χ2 = 17.14, 17.24, P < 0.01). No difference was found in the level of TGF-β1, the contents of hepatic hydroxyproline and collagen types I and III and pathologic grade between pcDNA3 control group and disease control group or between the two antisense treatment groups (F = 0.11 to 1.06, χ2 = 0.13 to 0.16, P > 0.05).
CONCLUSION: Antisense TβR I and TβR II recombinant plasmids have certain reverse effects on liver fibrosis and can be used as possible candidates for gene therapy.