VERIFICATION BY REAL TIME PCR AND WESTERN BLOT FOR IMMUNE RELATED GENES DETECTED FROM CDNA MICROARRAY DURING LIVER REGENERATION

Cell mediated immune response,
including increasing of CD4, CD8
lymphocytes, NK cells, γIFN and
macrophage were noted during liver
regeneration after partial hepatectomy.
Whether immune response is the termination
factor for regeneration is not clear. The study
about the relationship between immune
related gene expressions and liver
regeneration could be very important for
gene therapy to increase liver regeneration
and survival rate.
The main purpose of this project was to
find out the variation of immune related
genes during liver regeneration after partial
hepatectomy. Male Wistar rats around 200g
were used as subject. Partial hepatectomy
around 70% and 40% were be performed and
they will be sacrificed at 2, 4, 6, 12, 24, 48,
72 hours and 5, 7days after hepatectomy. We
measured: (1) weight of remnant liver; (2)
mitotic index; and (3) genomic survey of the
expression for many proto-oncogenes by
cDNA microarray on nylon membrane,
labeling of liver mRNA hybridization and
image analysis of the cluster of immune
related genes; (4) the mRNA expression of
immune related genes by real time
polymerase chain reaction (RT-PCR), and
compare the compatibility between the genes
and the mRNA expression ; (5) the protein
product of immune related genes by Western
Blot analysis, and compare the compatibility
between the genes expressions and the
protein products; (6) Compared the
difference of all positive immune related
gene expression in the variation degree,
changing pattern, specific timing and genes
subgrouping cluster between 70% and 40%
partial hepatectomy.
The results were: (1) the remnant liver
weight recovered slower, but can reach 90%
in 72h after partial hepatectomy; (2) the
mitosis of hepatocytes also increased
markedly at 48h although not so high as 70%
group rats, and also decreased at 72h after
partial hepatectomy; (3) there were 8
immune related genes identified with marked
changes in 3 different patterns after partial
hepatectomy; (4) histocompatibility 2, O
region alpha locus gene and early B-cell
factor gene showed a marked elevated peak
at early PH period, suggesting as initiative
genes for regeneration; (5) histocompatibility
2, Q region alpha locus gene,
immunoglobulin J chain precursor gene,
histocompatibility 2, complement component
factor gene, and histocompatibility 2, L
region locus gene showed a down-regulated
pattern at mid-term PH period, suggest as
differentiative genes for regeneration; (6)
interferon gamma receptor 2 gene and mast
cell growth factor gene showed a late
increasing peak suggesting as terminated
genes for regeneration; (7) the changing
curves of IL-6 and IL-10 genes cloning by
microarray and their related m-RNA detected
by RT-PCR were similar after 70% vs 40%
partial hepatectomy, suggesting IL-6 and
IL-10 genes play more important roles in
regeneration mechanism; (8) protein products
detected by Western Blotting showed no similar changes when compared with
microarray and RT-PCR, suggesting protein
products were affected by more factors. It is
suggested that some immune related genes
did play important roles in the mechanism of
initiation, differentiation and termination
during liver regeneration after partial
hepatectomy.