The Protective Effect Of Fusion Protein TAT-HO-1-pET32a On Rat Liver Partial Reperfusion Injury

Objective1 To construct a prokaryotic expression vector of TAT-HO-1-pET32a containing TAT protein transduction domain (PTD) and HO-1(heme oxygenase-1, HSP-32), express and purify it, and determine its enzyme activity and cytotoxity and its transductive ability into cells in vivo and in vitro.2 To set up the protein transduction system of transducing fusion protein TAT-HO-1-pET32a into PLC cells in vitro and the liver cells of SD rats.3 To set up the model of liver partial reperfusion injury of adult SD rats.4 To evaluate the protective effect of fusion protein TAT-HO-1-pET32a on apoptosis of L-02 cells and on the liver partial reperfusion injury of rat in order to set a basis for the clinical use of fusion protein TAT-HO-1-pET32a to relieve the liver partial reperfusion injury.Methods1 HO-1 and TAT-PTD were cloned to the fusion expression vector pET32a by PCR and cloning techniques and the fusion expression vector TAT-HO-1-pET32a was testified by enzyme cutting and genomic sequencing. E.coli BL-21 was transformed by the correct plasmid TAT-HO-1-pET32a and under the induction of IPTG E.coli BL-21 expressed the soluble fusion protein TAT-HO-1-pET32a. The fusion protein was purified by Ni-NTA-agarose and was identified by SDS-PAGE and Western-Blot.2 The enzyme activity of the fusion protein was determined and its cytotoxity was testified by CCK-8 kit. Its transductive ability into cells in vitro was testified by adding it into the culture of PLC cells and its transductive ability into cells in vivo was testified by injecting it into SD rat from the tail vein .3 The first classification of L-02 cells in vitro:(I)control group(n = 4);(II)L-02 cells were treated with THF-α(5000u/mL)+CHX(1.5mg/mL) for 24h (n = 4);(III)fusion protein TAT-HO-1-pET32a(10μg/mL) was added to the L-02 cells 4h before they were treated with THF-α(5000u/mL)+CHX(1.5mg/mL) for 24h(n = 4);(IV)fusion protein TAT-HO-1-pET32a(100μg/mL) was added to the L-02 cells 4h before they were treated with THF-α(5000u/mL)+CHX(1.5mg/mL) for 24h (n=4). And the protective effect of fusion protein TAT-HO-1-pET32a on cells was testified by CCK-8 kit.4 The second classification of L-02 cells in vitro:(I)control group(n = 4);(II)L-02 cells were treated with THF-α(5000u/mL)+CHX(1.5mg/mL) for 24h (n = 4);(III)fusion protein TAT-HO-1-pET32a(100μg/mL) was added to the L-02 cells 4h before they were treated with THF-α(5000u/mL)+CHX(1.5mg/mL) for 24h(n = 4). And the protective effect of fusion protein TAT-HO-1-pET32a on cells was testified by AO-PI.5 Set up the model of liver partial reperfusion injury of adult SD rat according to Nauta. Twenty four male SD rats, weighing 200-250g, were randomly divided into the following four groups: (I)control group (n = 6 ) , just sham operation, exposed the pedicle of the left and middle lobes without blood blocking;(II)ischemia and reperfusion group (n = 6 ) , the left and middle lobes were ischemiaed for 60 min and reperfused for 6 h; (III)TAT-HO-1-pET32a group(n = 6 ) , fusion protein TAT-HO-1-pET32a (10mg/kg) was injected into SD rats from the tail vein for 4 h and then the left and middle lobes were ischemiaed for 60 min and reperfused for 6 h; (IV)HO-1-pET32a group (n = 6 ), fusion protein HO-1-pET32a (10mg/kg) was injected into SD rats from the tail vein for 4 h and then the left and middle lobes were ischemiaed for 60 min and reperfused for 6 h. 2mL blood was taken from the infrahepatic vena cava and some hepatic tissue was taken and frozen at -80℃for assays, the animals were sacrificed.6 The assay of animal specimen: serum ALT and AST levels and SOD, MDA and MPO levels were assayed, apoptotic cells were assayed by in situ nick end labeling(TUNEL) method and the light microscopy assay. Results1 Genomic sequencing verified the prokaryotic expression vector of TAT-HO-1-pET32a was constructed correctly. TAT-HO-1-pET32a fusion protein was highly expressed in E.coli BL-21 under the induction of IPTG and successfully purified by Ni-NTA-agarose and was identified by SDS-PAGE and Western-Blot. CCK-8 kit assay proved that the fusion protein had no cytotoxity. Enzyme activity assay proved that the fusion protein had stable enzyme activity. And the fusion protein can be transduced into L-02 cells in vitro and into hepatic cells of SD rats.2 The addition of TAT-HO-1-pET32a(10μg/mL)into the cultures of L-02 cells for 4h before they were treated by THF-α(5000u/mL)+CHX(1.5mg/mL) for 24h can raise its survival rate from 25.52%±0.47% to 41.76%±5.49%, and the addition of TAT-HO-1-pET32a(100μg/mL)can raise its survival rate from 25.52%±0.47% to 52.98%±6.29%.3 Compared with the control group, the serum ALT and AST levels of reperfusion injury group, TAT-HO-1-pET32a group, HO-1-pET32a group were significantly higher (P<0.05); the SOD levels were significantly lower(P<0.05); and the MDA and MPO levels were significantly higher (P<0.05); the injury of hepatic tissue was more severe. Compared with reperfusion injury group, the serum ALT and AST levels of TAT-HO-1-pET32a group were significantly lower (P<0.05); the SOD levels were significantly higher(P<0.05); and the MDA and MPO levels were significantly lower(P<0.05); the injury of hepatic tissue was significantly lessened; and the apoptotic index was 34.28%±3.00% which was significantly lower than 56.12%±1.71% of reperfusion injury group(P<0.05). Compared with TAT-HO-1-pET32a group , the serum ALT and AST levels of HO-1-pET32a group were significantly higher ( P<0.05 ) ; the SOD levels were significantly lower(P<0.05); the MDA and MPO levels were significantly higher (P<0.05); the injury of hepatic tissue was more severe; and the apoptotic index was 56.96%±2.63% which was significantly higher than 34.28%±3.00% of TAT-HO-1-pET32 group ( P<0.05 ) . Compared with the reperfusion injury group , the serum ALT and AST levels, the SOD, MDA and MPO levels of HO-1-pET32a group had no significant difference (P >0.05); the injury of hepatic tissue was similar; and the apoptotic index was 56.96%±2.63% which had no significant difference with 56.12%±1.71% of ischemia and reperfusion group(P >0.05).Conclusion1 Prokaryotic expression vector of TAT-HO-1-pET32a was successfully constructed. TAT-HO-1-pET32a fusion protein with full activity was successfully achieved. And these set a basis for the further research of TAT-HO-1-pET32a fusion protein.2 The partial liver reperfusion injury model of adult SD rat was successfully established. 3 TAT-HO-1-pET32a fusion protein had some protective effect on apoptosis of L-02 cells induced by THF-α+CHX.4 Fusion protein TAT-HO-1-pET32a can significantly improve the liver partial reperfusion injury of SD rats.