| Growing incidences of age-related pathologies with aging has become to the majorscientific issues of population in the field of health sciences and has a severe challenge onlife science and medicine fields. The biological properties of tissues and organs will bechanged with age, leading to disorders of the body structure and function, as well as theoccurrence of related diseases.The liver is the largest solid organ of organism, represented2%of total body weightin humans, performing plenty of essential body function, such as synthesis of serumprotein, bile secretion, lipids and amino acids and detoxification of xenobiotic compounds.The major of parenchymal cells in liver are hepatocytes, which build up approximatelysixty per cent of the total cell count and ninety per cent of the liver-cell mass. However, theliver is also one of the most vulnerable organs in the aging progress. Recently, severalstudy demonstrate that older individuals are more susceptible to acquire liver disorders andmore vulnerable to the consequences of liver disease, including hepatitis B or C virusinfection, non-alcoholic fatty liver disease, liver fibrosis and so on. Moreover, the capacityof recovery liver disease in the older is weaker when compared to the young individuals.Changes in the liver and hepatocytes have been known to occur with age. Hepatocytestructure changes with increased age including the decreases of liver size and the numbersof hepatocytes. The ability of the liver to regenerate is reduced in the older organism.Senscent hepatocytes increased with aging, followed by the accumulatation of senescenceassociated-β-galactosidase (SA-β-Gal) positive cells and the expression of cell cycleinhibitor such as p16ink4a, p53, p21increases, as well as the accumulation of foci ofphosphorylation of Ser-139of histone H2A.X molecules (γ-H2A.X). Ployploidization isthe characteristics of mature hepatocytes. The extent of polyploid hepatocytes in adultmice is about80-90%and approximately30-40%human hepatocytes are polyploidy.Depending on the DNA content of each nucleus plus the number of nuclei per cell,polyploid hepatocytes can define as mononucleated or binucleated polyploidy. However,the function of polyploidy is not well understood, which is generally considered to indicateterminal differentiation and liver development. Polyploidy has been reported to increasewith aging, implying it may be relative to cellular senescence. Therefore, understanding ofboth development and the mechanism for liver aging has the special significance, whichcould be used to delay or reverse liver senescence. Although hepatocytes are always quiescent and turn over occur slowly, they sustain astrong potent proliferation. Hepatocytes are the only highly differentiated cells withproliferation, which can proliferate fast and regenerate liver when the liver is injury. Two-thirds partial hepatectomy model indicates that the parenchyma cells of the remaining livercan quickly enter the cell cycles and rapidly expands to compensate for the lost tissue andphysiological functions. And even up to12times of partial hepatectomy, the remaininghepatocytes still have the capacity of regeneration. Serial transplantation revealed thatisolated donor hepatocytes transplanted into Fah-/-mouse model can proliferate more than69rounds without lose their capacity of regeneration. It is suggested that the occurrence ofsenescence is inhibited by some special mechanism when hepatocytes re-entry into cellcycles in vivo proliferation, and this unlimited proliferation may breaks the traditionalconcept of noncancerous cells having a limited proliferation capacity. Furthermore,octaploid hepatocytes will generate tetraploid and diploid daughter cells and diploidy cangenerate tetraploidy and octaploidy in proliferated hepatocytes, called ploidy convey.Whether the ploidy conveyor to sustain the stable distribution in three ploid is important toliver function and is contributed to inhibit or reverse of hepatocyte senescence has not yetbeen studied.Recently, owing to lacking the idea mouse model, the hepatocytes cannot beproliferated at long-term in vivo. Meanwhile, the proliferated hepatocytes even theoctaploidy cannot be isolated and analyzed, which is a big problem for studying thebiology and genetics of hepatocytes and mechanisms of senescent inhibition and reversal.First, senescence markers such as the size of cell, telomere length and activity oftelomerase, senescence associated-β-galactosidase, γ-H2A.X, expression level of p16ink4a,p21, p53and capacity of proliferation are used to identify the senescence of hepatocytes inour study. Analysis of liver section in four indicated age by senescent markers indicatedthat hepatocytes from older mice are more likely to be senescent and less capable ofproliferation. For example, the percentages of SA-β-Gal-positive hepatocytes increasedwith age from1.37±0.64%at2months to10.55±0.78%at12months and to40±4.6%at18months, suggesting that about50%hepatocytes in18-month-old mice weresenescence. In order to conform whether polyploidy is related to senescence, diploid,tetraploid, and octoploid hepatocytes from2-and18-month-old mice were isolated byfluorescence-activated cell sorting (FACS) and analyzed the expression of p16ink4a, p21,p53and γ-H2A.X. The results demonstrated that the increasing of polyploidy especially octoploidy in18months correlated with the increasing hepatocyte senescence. However,FACS cannot distinguish the mononucleated or multinucleated hepatocytes, microscopicanalysis after F-actin and Hoechst33342staining was used to measure the DNA contentand to directly assess the ploidy of hepatocytes. Using fluorescence imaging technology,we found that both mononucleated and binucleated polyploidy were increased with age.Moreover, SA-β-Gal, γ-H2A.X, F-actin and Hoechst33342were co-stained in the sameliver section and indicated that the increase of polyploidy hepatocytes in older micecorrelated with senescence regardless of whether the mono-or binucleated hepatocytes.Until now, the limit of proliferation capacity for hapatocytes is still a long-termunsolved question. The proliferation characteristic of hepatocytes during liver regenerationhas not been studied yet for without an in vivo model system capable of keepinghepatocytes in continuous proliferation. The fumarylacetoacetatehydrolase knocked outmouse model (Fah-/-in short), a model of liver injury and liver repopulation, can be used tostudy of isolation, sorting and analysis of normal or senescent hepatocytes. We analyzedsenescence of hepatcytes undergoing continuous proliferation during serial transplantationin Fah-/-mice.2-month-old donor hepatocytes elapsed20and26months after9and12rounds of transplantation but did not have any characteristics of senescence, suggestingthat senescence may be inhibited in continuously proliferated hepatocytes. Excitedly,donor hepatocytes with50%or80%senescent cells could engraft into liver parenchymaand complete liver repopulation, while the percentage of senescent cells in repopulatedhepatocytes decreased after transplantation of senescent hepatocytes, all of that indicatedthe senescent hepatocytes could be rejuvenated after continuous cell proliferation.In order to understand the mechanism in senescent inhibition and reversal ofhepatocytes, repopulated hepatocytes from young and old donor cells were analyzed in cellbiology and molecular levels. We found that telomere shortening did not occur inhepatocytes up to18months, but telomerase activity did only exist in2-month-oldhepatocytes. However, telomerase activity was reactivated in the repopulated hepatocytesfrom2and18months donor cells undergoing serial transplantation, which implied thatrestoration of telomerase activity during continuous hepatocyte proliferation was importantfor maintenance of the regenerative capacity of hepatocytes and avoidance of senescence.More important, as we known, two main tumor suppressor pathways, p53-p21and p16ink4a-pRB, become activated when cell enter into senescence. But the expression level ofp16ink4a, p21, and p53reduced during repopulation, while promoters of cell proliferation such as CDK2, CDK4and pRB gradually increased. The deduced expression of p16ink4a,p21, and p53with reactivating of cyclin-dependent kinase may be a vital molecularmechanism for inhibition and reversal of hepatocytes senescence. In addition, it is the firstreport that senescent donor hepatocytes with high proportion of polyploidy redistribute thethree polyploidy after regeneration as to young liver by ploidy conveys, and remainedstable over time when there was continuous repopulation of hepatocytes, suggesting thatploidy convey may involve in senescence rejuvenation.Similar to mouse hepatocytes, human senescent hepatocytes could rejuvenate toregain proliferation capacity after reentry into the cell cycle, which may provide a cellresource for treatment of clinical cell transplantation, and may help to treat liver disease.According to the above results, we conclude: inhibition of senescence and senescentrejuvenation occurs during continuous cell proliferation in vivo after transplantation. Thepossible mechanism is about reactivation of telomere activity, upregulated expression ofgene about proliferated pathway and ploidy convey. Therefore, understanding themechanism of liver aging has special significance for the treatment of liver diseases thatare influenced by age. This study suggests that old hepatocytes could still be considered forcell therapy treatment of liver diseases. It also supplies a basis for strategies to the delay orreverse hepatocytes senescence for clinical applications, which have important biologicaland medical significances. |
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