[Secretable eukaryotic expression of recombinant chicken Iglambda and preparation of monoclonal antibodies against chicken Iglambda].

AIM: To construct the eukaryotic expression vector for chicken Iglambda light chain, to express it on COS7 cells and to prepare the monoclonal antibodies against chicken Iglambda. METHODS: The cDNA of chicken Iglambda light chain with signal peptide sequence was amplified and then inserted into eukaryotic expression plasmid pcDNA3 after double enzyme cutting. The constructed recombinant vector was transfected into COS7 cells by lipofectamin and the secretable eukaryotic expression of chicken Iglambda light chain was verified by Western blot. The monoclonal antibodies (mAbs) against chicken Iglambda light chain were prepared by immunizing BALB/c mice with 2 x 10(6) chicken B cells and by cell fusion technology. RESULTS: The eukaryotic expression vector was successfully constructed. Western blot demonstrated that chicken Iglambda light chain existed in the cultural supernatant. The hybridoma lines secreting anti-Iglambda mAbs were screened by indirect ELISA. The specific reactivity between anti-Iglambda mAbs and recombinant chicken Iglambda light chain was detected by Western blot. CONCLUSION: The secreted recombinant chicken Iglambda light chain and anti-Iglambda mAbs provide a basis for further study of the functions of chicken Iglambda.

[Analysis of changes about hsbp1, hsf1, hsf2 AND hsp70's expression levels in rat's regenerating liver].

Heat shock factor binding protein 1 (HSBP1), a recently discovered protein, weakens and blocks transcription of the heat shock protein 70 (HSP70) gene when binding to HSF1, but HSBP1 can promote cell growth, cell development and cell differentiation when binding to HSF2. Partial hepatectomy (PH) in rat creates injury stimulation and induces liver regeneration. How does hsbp1 coordinate two processes sequently is extremely interesting. This paper, based on cloning the full-length cDNA of hsbpl in rat, applied in situ hybridration and Rat Genome 230 2.0 Array to analyze hsbp1, hsf1, hsf2 and hsp70 expression in liver after PH and sham-operation. The results indicated that the hsbp1 expression level was down-regulated meaningfully at 0.5-2h and up-regulated meaningfully at 8-16h after sham-operation, while hsf2 expression level did not meaningfully change at 0-144h after sham-operation. hsbp1 expression level was up-regulated meaningfully at 6h and 66-144h,and hsf1 at 8-16h, hsf2 at 2-16h, hsp70 at 0.5-24h after PH. Our data suggested that up-regulated expression of the hsp70 at 0.5-12h after sham-operation was controlled by intracellular HSF1, and then controlled by hsbp1 down-regulated at 0.5-2h and hsf1 up-regulated at 8-16h. In the early phase of liver regeneration in rats, hsbp1 and hsf2 expression levels were up-regulated, which promoted cell proliferation through HSBP1 and HSF2 up-regulating,upa activating,c-jun enhancing, intracellular matrix (ECM) degradation, activating the hepatocyte-like growth factor (HGF) etc. In the late phase of liver regeneration (66-144h), hsbp1 expression level was up-regulated, which promoted reconstruction of liver structure and recovery of liver function through HSBP1 inhibiting hsp70 expression, up-regulating genes related to growth, development, differentiation. In conclusion, down-regulating of hsbp1 contributed to interaction between HSF1 and HSE,increased hsp70 expression and enhanced anti-injured capacity of liver and rats. HSBP1 and HSF2 activated the genes related to growth, development, differentiation and then promoted liver regeneration in rats.

Expressed genes in regenerating rat liver after partial hepatectomy.

AIM: To reveal the liver regeneration (LR) and its control as well as the occurrence of liver disease and to study the gene expression profiles of 551 genes after partial hepatectomy (PH) in regenerating rat livers. METHODS: Five hundred and fifty-one expressed sequence tags screened by suppression subtractive hybridization were made into an in-house cDNA microarray, and the expressive genes and their expressive profiles in regenerating rat livers were analyzed by microarray and bioinformatics. RESULTS: Three hundred of the analyzed 551 genes were up- or downregulated more than twofolds at one or more time points during LR. Most of the genes were up- or downregulated 2-5 folds, but the highest reached 90 folds of the control. One hundred and thirty-nine of them showed upregulation, 135 displayed downregulation, and up or down expression of 26 genes revealed a dependence on regenerating livers. The genes expressed in 24-h regenerating livers were much more than those in the others. Cluster analysis and generalization analysis showed that there were at least six distinct temporal patterns of gene expression in the regenerating livers, that is, genes were expressed in the immediate early phase, early phase, intermediate phase, early-late phase, late phase, terminal phase. CONCLUSION: In LR, the number of down-regulated genes was almost similar to that of the upregulated genes; the successively altered genes were more than the rapidly transient genes. The temporal patterns of gene expression were similar 2 and 4 h, 12 and 16 h, 48 and 96 h, 72 and 144 h after PH. Microarray combined with suppressive subtractive hybridization can effectively identify the genes related to LR.

Identification of expressed genes in regenerating rat liver in 0-4-8-12 h short interval successive partial hepatectomy.

AIM: To identify the genes differentially expressed in the regenerating rat liver of 0-4-8-12 h short interval successive partial hepatectomy (SISPH) and to analyze their expression profiles. METHODS: Five hundred and fifty-one elements screened from subtractive cDNA libraries were made into a cDNA microarray (cDNA chip). Extensive gene expression analysis following 0-4-8-12 h SISPH was conducted by microarray. RESULTS: One hundred and eighty-three elements were selected, which were either up- or down-regulated more than 2-fold at one or more time points after SISPH. Cluster analysis and generalization analysis showed that there were five distinct temporal patterns of gene expression. Eighty-six genes were unreported, associated with liver regeneration (LR). CONCLUSION: Microarray analysis shows that the down regulated genes are much more than the up-regulated ones in SISPH; the numbers of genes expressed consistently are fewer than that expressed immediately; the genes expressed in high abundance are much fewer than that increased 2-5-fold. The comparison of SISPH with partial hepatectomy (PH) shows that the expression trends of most genes in SISPH and in PH are similar, but the expression of 43 genes is specifically altered in SISPH.

Gene expression differences of regenerating rat liver in a short interval successive partial hepatectomy.

AIM: To identify the genes expressed differentially in the regenerating rat liver in a short interval successive partial hepatectomy (SISPH), and to analyze their expression profiles. METHODS: Five hundred and fifty-one elements selected from subtractive cDNA libraries were conformed to a cDNA microarray (cDNA chip). An extensive gene expression analysis following 0-36-72-96-144 h SISPH was performed by microarray. RESULTS: Two hundred and sixteen elements were identified either up- or down-regulated more than 2-fold at one or more time points of SISPH. By cluster analysis and generalization analysis, 8 kinds of ramose gene expression clusters were generated in the SISPH. Of the 216 elements, 111 were up-regulated and 105 down-regulated. Except 99 unreported genes, 117 reported genes were categorized into 22 groups based on their biological functions. Comparison of the gene expression in SISPH with that after partial hepatectomy (PH) disclosed that 56 genes were specially altered in SISPH, and 160 genes were simultaneously up-regulated or down-regulated in SISPH and after PH, but in various amount and at different time points. CONCLUSION: Genes expressed consistently are far less than that intermittently; the genes strikingly increased are much less than that increased only 2-5 fold; the expression trends of most genes in SISPH and in PH are similar, but the expression of 56 genes is specifically altered in SISPH. Microarray combined with suppressive subtractive hybridization can in a large scale effectively identify the genes related to liver regeneration.