Background Mostly because of the limited number and proliferative ability of the transplanted hepatocytes, hepatocyte transplantation offers only temporary support to the hepatic function with rather poor functional r...Background Mostly because of the limited number and proliferative ability of the transplanted hepatocytes, hepatocyte transplantation offers only temporary support to the hepatic function with rather poor functional replacement of the damaged liver parenchyma. This study aimed to observe the therapeutic effect of human thioredoxin （hTrx） gene-modified hepatocytes on experimental acute liver failure in rats. Methods hTrx cDNA was obtained by reverse transcription-polymerase chain reaction （RT-PCR） from human osteosarcoma 143 （TK-） cells to construct the recombinant retrovirus vector pLEGFP/hTrx, which was packaged into PA317 cells to collect the recombinant retrovirus containing hTrx gene. After titration and characterization, the recombinant retrovirus was applied to primary cultured rat hepatocyte for infection to generate hTrx gene-modified rat hepatocytes, whose viability and antioxidative capacity were examined by immunohistochemistry and MTT assay, respectively. In a Sprague-Dawley （SD） rat model of acute liver failure, the modified hepatocytes were injected into the spleen, and the hepatic function and survival rate of the recipient rats were evaluated at different time points after the transplantation. Results NIH3T3 cells infected by the recombinant retrovirus were capable of expressing bioactive hTrx in the form of fusion proteins. Immunohistochemistry demonstrated normal function of the hTrx gene-modified hepatocytes, which possessed strong antioxidative capacity as shown by MTT assay. Transplantation of the modified hepatocytes in rats with acute liver failure resulted in significantly lowered serum alanine aminotransferase （ALT） and total bilirubin （TBIL） levels （P 〈0.05）. The hepatocytes exhibited long-term survival and efficient proliferation after transplantation. Fourteen days after the operation, the rat models receiving hTrx gene-modified hepatocytes had significantly higher survival rate than those without the transplantation. Conclusion hTrx gene-modified hepatocyte transplantation can effectively alleviate acute liver failure in rats.展开更多
Objective To clone and determine the sequence and expression of a cDNA segment for human thioredoxin. Methods The cDNA segment of thioredoxin was obtained through amplification by RT PCR cloning from 143 (TK -) hu...Objective To clone and determine the sequence and expression of a cDNA segment for human thioredoxin. Methods The cDNA segment of thioredoxin was obtained through amplification by RT PCR cloning from 143 (TK -) human osteosarcoma cell. The amplified products were cloned into pGEM T Easy vector and sequenced. Then the expressed vector pBV220 hTRX was constructed and transformed into E.coli strain DH5α for hTRX expression. The hTRX was purified by DEAE Sephadex A 50 column and the activity of recombinant hTRX was determined by the insulin disulfide reduction assay. Results Comparison of cDNA sequence of the cloned fragments with that of the reported hTRX (GenBank J04026) demonstrated that there were two differences compared to the reported cDNA sequence for hTRX at bp180 and bp284, and the amino acids enceoded altered respectively, but motif of the sequence was identical to that of the reported hTRX. The recombinant hTRX can catalyze insulin reduction by DTT. Conclusion The successful cloning and expression of hTRX cDNA formed a basis for further study on biological functions and utilization of hTRX.展开更多
Human thioredoxin and antibacterial peptide, PR39, have been shown to have potent antioxidant effects that may prolong survival of cells during hypoxia. The pSSCMV/human thioredoxin-PR39 vector was successfully constr...Human thioredoxin and antibacterial peptide, PR39, have been shown to have potent antioxidant effects that may prolong survival of cells during hypoxia. The pSSCMV/human thioredoxin-PR39 vector was successfully constructed in this study and used to infect ECV304 cells. Transfected ECV304 cells were incubated at 1%, 5% hypoxic, and normal oxygen conditions. We found that the number of apoptotic cells after transfection with recombinant adeno-associated virus-human thioredoxin -PR39 was significantly lower than controls, suggesting a protective effect of the recombinant human thioredoxin-PR39 protein on hypoxic cells.展开更多
基金The study was supported by grants from the Major State Basic Research Development Program （973 Program） of China （No. 2009CB522404） and the National Natural Science Foundation of China （No. 30772135）.
文摘Background Mostly because of the limited number and proliferative ability of the transplanted hepatocytes, hepatocyte transplantation offers only temporary support to the hepatic function with rather poor functional replacement of the damaged liver parenchyma. This study aimed to observe the therapeutic effect of human thioredoxin （hTrx） gene-modified hepatocytes on experimental acute liver failure in rats. Methods hTrx cDNA was obtained by reverse transcription-polymerase chain reaction （RT-PCR） from human osteosarcoma 143 （TK-） cells to construct the recombinant retrovirus vector pLEGFP/hTrx, which was packaged into PA317 cells to collect the recombinant retrovirus containing hTrx gene. After titration and characterization, the recombinant retrovirus was applied to primary cultured rat hepatocyte for infection to generate hTrx gene-modified rat hepatocytes, whose viability and antioxidative capacity were examined by immunohistochemistry and MTT assay, respectively. In a Sprague-Dawley （SD） rat model of acute liver failure, the modified hepatocytes were injected into the spleen, and the hepatic function and survival rate of the recipient rats were evaluated at different time points after the transplantation. Results NIH3T3 cells infected by the recombinant retrovirus were capable of expressing bioactive hTrx in the form of fusion proteins. Immunohistochemistry demonstrated normal function of the hTrx gene-modified hepatocytes, which possessed strong antioxidative capacity as shown by MTT assay. Transplantation of the modified hepatocytes in rats with acute liver failure resulted in significantly lowered serum alanine aminotransferase （ALT） and total bilirubin （TBIL） levels （P 〈0.05）. The hepatocytes exhibited long-term survival and efficient proliferation after transplantation. Fourteen days after the operation, the rat models receiving hTrx gene-modified hepatocytes had significantly higher survival rate than those without the transplantation. Conclusion hTrx gene-modified hepatocyte transplantation can effectively alleviate acute liver failure in rats.
文摘Objective To clone and determine the sequence and expression of a cDNA segment for human thioredoxin. Methods The cDNA segment of thioredoxin was obtained through amplification by RT PCR cloning from 143 (TK -) human osteosarcoma cell. The amplified products were cloned into pGEM T Easy vector and sequenced. Then the expressed vector pBV220 hTRX was constructed and transformed into E.coli strain DH5α for hTRX expression. The hTRX was purified by DEAE Sephadex A 50 column and the activity of recombinant hTRX was determined by the insulin disulfide reduction assay. Results Comparison of cDNA sequence of the cloned fragments with that of the reported hTRX (GenBank J04026) demonstrated that there were two differences compared to the reported cDNA sequence for hTRX at bp180 and bp284, and the amino acids enceoded altered respectively, but motif of the sequence was identical to that of the reported hTRX. The recombinant hTRX can catalyze insulin reduction by DTT. Conclusion The successful cloning and expression of hTRX cDNA formed a basis for further study on biological functions and utilization of hTRX.
基金sponsored by the National Natural Science Foundation of China,No.30970992
文摘Human thioredoxin and antibacterial peptide, PR39, have been shown to have potent antioxidant effects that may prolong survival of cells during hypoxia. The pSSCMV/human thioredoxin-PR39 vector was successfully constructed in this study and used to infect ECV304 cells. Transfected ECV304 cells were incubated at 1%, 5% hypoxic, and normal oxygen conditions. We found that the number of apoptotic cells after transfection with recombinant adeno-associated virus-human thioredoxin -PR39 was significantly lower than controls, suggesting a protective effect of the recombinant human thioredoxin-PR39 protein on hypoxic cells.
