Effects Of Cdc6 On Liver Regeneration After Partial Hepatectomy In Mouses

Objective:The experiment was made up of two sections.The first section:The specific siRNA sequence was designed according to the Cdc6 sequence in GenBank.the sequence was cloned into pGCsi_U6 /Neo / GFP and then sequence analysis was performed.The pGCsi_U6/Neo/GFP/RNAi plasmid were transient transfected into mouse liver cells by tail vein injecting.RT-PCR and fluorescent microscope was used to detect the result.The second section:To observe the expression of Cdc6 in liver cells after partial hepatectomy in mouses, and effects of Cdc6 on liver cells' proliferation and cell cycle to prove that this gene plays a key role in liver regeneration.Methods:The specific siRNA sequence was designed according to the Cdc6 sequence in GenBank.the sequence was cloned into pGCsi U6 / Neo / GFP and then sequence analysis was performed.Plasmid were injected into mouses with 2ml PBS solution within 5 s via the tail vein.Then the express of Cdc6 in liver cell was observed.All mouses were divided into three groups:RNAi group,control group,blank vector group.The left and medium lobes of normal mouse's liver were ligatured and resected by the classics methods of partial hepatectomy established by Higgins & Aderson.Mouse hepatocytes were separated on five time points after partial hepatectomy in normal mouse, namely(0h,6h,12h,24h,72h).and the method to separate and culture hepatocytes was established using collagenase circumfusion in situ as well as in vitro.Cells' activity was measured by trypan blue dye exclusion.Extracts of the hepatocytes from remnant liver were assayed for Cdc6 protein through western blotting.The level of protein synthesis in regenerative hepatocytes was assayed by ³H-leucine.Cell proliferation was accessed through a flow cytometer.Results:1.Through sequence analysis,the template sequence of RNAi eukaryotic expression vector was the same as the designing sequence,that the constructed the pGCsi_U6/Neo/ GFP/RNAi plasmid was successful.2.The pGCsi_U6/Neo/GFP/RNAi plasmid were transient transfected into mouse liver cell by tail vein injecting.The transfected cell can be identified by the green fluorescence under the fluorescent microscope.3.The total amount of hepatocytes in a mouse was about 1.5×10⁷.The average activity of hepatocytes was 96%.The forms of separated cells were agreed well with those of classic hepatocytes.The freshly separated hepatocytes were single,round,similar in term of size,and high refraction.4h later they gradually became stretched.24h later most of them adherenced to the culture dish and became fiat.48h later they turned to the classic epithelioid shapes.The experiments were repeated by multiple trials and reproducible results were observed.4.In control group and blank vector group,the Cdc6 protein expression began to increase at 6h and peaked at 24h(P＜0.01) after hepatectomy,then decreased gradually,But the Cdc6 protein expression was greatly suppressed in RNAi group.5.Our experiments show ³H-leucine CPM of regeneration hepatocytes cultured in control group and blank vector group was higer than that in RNAi group after partial hepatectomy;Significant difference was observed at time points of 6h,12h and 72h after PH(P＜0.05),and very significant difference on the time point of 24h(P＜0.01).6.Cell cycle study by flow cytometry:Compared with RNAi group,in control group and blank vector group,the percentage of cells in G₀/G₁ phase gradually decreased while that in S and G₂/M phase gradually increased after partial hepatectomy,the peak wasobserved at 24h,with a obvious decrease at 72h.Conclusion:1.the constructed pGCsi_U6/Neo/GFP/RNAi plasmid was successful.The pGCsi_U6/Neo/GFP/RNAI plasmid were transient transfected into mouse liver cells by tail vein injecting,the green fluorescence was observed by fluorescent microscope.This demonstrated that the plasmid transfected into mouse liver cell was successful and had the action.2.The expression of Cdc6 protein increased slightly at 6h,with a peak at 24h after partial hepatectomy.Our data suggested that Cdc6 gene is rapidly activated in the residual hepatocytes at early stage of liver regeneration after partial hepatectomy. 3.Cdc6 gene activated at early stage of liver regeneration after partial hepatectomy can strongly promote protein synthesis and cell growth in hepatocytes.We think that the role of this gene contribute to body recovery after partial hepatectomy,on the other hand it also settle a substance basis for hepatocyte proliferation.4.Hepatocytes were clearly in proliferative state after partial hepatectomy.The percentage of cells in G₀/G₁ phase gradually decreased while that in S and G₂/M phase gradually increased in all points after partial hepatectomy;the peak was at 24h,with an clear decrease at 72h.The cell cycle was obstructed and stopped in G₀/G₁ phase after RNAi.Our results strongly suggest that this gene plays a pivotal pathophysiological role in hepatocyte proliferation in liver regenerative response.Further study on Cdc6 gene and the relationship between cell growth and cell proliferation will greatly enrich the theory of liver regeneration.