| Introduction:With the coming of aged society,senile diseases(such as Alzheimer's disease,atherosclerosis,etc.) get more and more attention.The current view expressed that aging itself is an important inducement of the senile diseases.The delaying the body "aging" from happening is an important method of prevention senile diseases,so anti-aging research will not only benefit to improving the health of the elderly,but also benefit the reduce of incidence and the degree of malignancy in senile diseases and the reduction of social cost of of treating the senile diseases.Since 1961,Hayflick found human embryonic lung diploid cells grown in culture occurs replicative senescence following a limited number of population doublings.And then,preferred the suppose of cell replicability aging,successfully established an aging cell model.So that the anti-aging research in body simplified to the anti-aging cell research.Over the past three decades,biogerontology research at the cellular and molecular level has focused on the elucidation of the mechanism(s) responsible for this limited replicative potential.It is now well established that replicative senescence is an intrinsic process which is stringently controlled by the activity of various gene products.Key genes involved in this process in human cells are p53,pRB,p16INK4A and p21CIP-1.Early research proved that there are many induce factors of aging,including:free radicals oxidative damage,mitochondrial DNA damage,radiation damage,excessive intake of calories,telomere shortening,and so on.Which can be divided into two parts,telomere shortening classified as a separate sort and the others can be classified as non- telomere-dependent induce factors.Telomere is the DNA repair structure of cell chromosome.Telomere erosion at each cell division acts as a counting mechanism to measure the age of a cell in PD and that replicative senescence is induced when one or more telomeres shorten to a critical length(that is,cells lose their proliferation,but still survival for a long-term). So how is the aging induced by telomere shortening? Still now the point is that this process may bemediated by a p53-dependent pathway.The non-telomere-independent aging induce factors include:free radicals, mitochondrial DNA damage,radiation damage,all related to the intracellular accumulation of cell structural damage,and has a common senescence induced pathway,p 16-pRb pathway.Cell Senescence is a complex process,a large number of other gene activated in addition to the p53-p21 and pRb-p16 gene activation pathway,such as p19INK4D,p18INK4C,p15INK4B,MAPK,MEK,Ras,Raf,etc,much more facts are unknown.Furthermore,different cell types may utilize different senescence pathways. But now the main theory is that aging in the human cell senescence induced by various factors,including:sexual and non-telomeric of the telomere(oxidative stress,mitochondrial DNA damage,etc.) are mainly adopt the following two ways respectively: p53-p21- pRb and p16-pRb senescence pathways.Aging theoretical innovation for the anti-aging drug screening and efficacy study provided a strong basis.The current drug- studies are always choose the aspects of inhibiting the induced factors of senescence(telomere shortening,mitochondrial DNA damage and oxidative stress,et al) or medicating the expression of gene involved in senescence pathway as index to verify its anti-aging effects.At the innovative research of anti-aging medicine,Chinese traditional and herbal drugs in a unique position(the development of new drugs base two points:the effective and non-toxic).Large number of ancient Medical Books recorded the anti-aging effect of much Chinese herbal medicine,how to put these Chinese herbal medicines who was confirmed by clinical experience that there anti-aging effects of to the interna- tional market,it has become a new topic for the development of modern medicine.At present,the domestic anti-aging medicines research has increasingly mature,Astrag- alus,ginseng,Rhodiola and many other valuable traditional Chinese medicine has through the process of refined monomer,cell and animal experimental studies,and have gratifying results.The issue began in 2004,select pine pollen for the object of anti-aging research experiment.Pine pollen was named yellow pine in medicine,first described in the Eastern Han Dynasty·"Shen Nong's Herbal",bright yellow or pale yellow powder,the scent bland and non-toxic,long-time use could light-weight,reinforcing qi and prolonging natural life.In Botany,pine pollen is the evergreen pine pollen - the pine's sperm cells.It was reported abroad,scientists found the pine polyphenol extract from the pine bark is the bioactive substances in the current of most anti-aging effects. Well,pine pollen contains all the nutrients of pine reproduction,and how of its anti-aging effectsNow in domestic,the related study of Pine pollen is not much.the project received support from the National Natural Science Fund and started in 2004.This project choose the natural aging mice and natural senescence human embryonic lung diploid cell as the research model,which is with great difficulty in modeling and with high persuasion.From the levels of organs,tissues,cells,molecules,genes and other levels to study systematicly and comprehensively the effects and mechanism of pine pollen anti-aging through the aspects of morphology,free radicals,telomerase and mitochondria.Still now,it confirmed that pine pollen could promote cell proliferation, inhibit occurrence of free radicals,increase activity of telomerase and reduce the rate of mutation of mitochondrial mtDNA4977.From the aspects of suppression of telomere and non-telomeric(free radicals,mitochondrial DNA mutations) senescence induced factors and promote cell proliferation to verify the anti-aging effects of pine pollen.Well,What a role of the pine pollen in the medicate of the senescence pathway ? How the key Senescence relatived gene p16INK4A,p21CIP-1changed? Objective:Analyze the effects of pine pollen on the expression of p16INK4Aand p21CIP-1 in cell replicability aging,because aging cells with the characteristics of the three changes:cell area increased,β-galactosidase staining(SA-β-gal) in the blue stained,cells G1 arrest,select the three indicators of this study to validate the scientificity of the aging model,while detect the effects of pine pollen on the three targets in the aging cells.And then,according to gene-dependent theory,detect the expression changes of age-related oncogene p16INK4A and p21CIP-1 when the pine pollen added in and explore the correlationship between the pine pollen's effect of anti-aging and oncogene-related Signaling pathway,for further studies of the Molecular Mechanisms in the pine pollen anti-aging.The following are the main research topics:①Found the aging cell model, determine the single cell area,the positive rate of SA-β-gal staining and the cell cycle in aging cell model after the utilizing of pine pollen;explore the anti-aging effect from the apparent-aspects of senescent cell,such as morphology,biochemistry and DNA distribution changes.②The p16INK4A and p21CIP-1 are respectively the core components of p53-p21-p16 and pRb-pRb two senescence pathway,with the character of easy to detecting.Choose p16INK4A and p21CIP-1 as the object of study,set two pine pollen serum levels to determine the expression of p16INK4A and p21CIP-1 in the aging model cell and the pine pollen treated cell.Materials and methods:①Cell treatment:human embryonic lung diploid fibroblasts(2BS) cultured from 27 PDL.Eight-bottle cells of 30 PDL randomly selected for the younger control group,and cultured to 56 PDL divided 24-bottle cells randomly into three groups: aging model group,pine pollen light dose(120 mg / dl) group,pine pollen high dose (240 mg / dl) group,Were supplemented respectively by the general media,120 mg / dl pine pollen serum,and 240 mg / dl pine pollen serum cultured.②Preparation of pine pollen serum:This project uses pollen nutrition extraction technology,extract sterile liquid pollen nutrient solution as experimental materials,a liquid compound to study the overall pharmacodynamics mechanism of pine pollen.③the determination of the single cell area,SA-β-gal staining positive rate and cell cycle:a stereological analysis of the change of average single-cell area in each group;SA-β-gal staining in each group by immunohistochemical methods,and analysis of the ratio of the blue cells in each group.Flow cytometry analysis of the cell cycle,counts the changes of the ratio of G0/G1,S,G2 / M phases and the PI index in each group.④P16INK4A and p21CIP-1 Determination:RT-PCR method to detect the cell's P16INK4A,p21CIP-1 gene mRNA expression levels change.⑤Statistical analysis:Analysis datas by SPSS13.0.Homogeneity of variance, using single-factor analysis of variance methods,LSD method on compare each two groups;otherwise,using independent samples to non-parametric test,Dunnett's T3 method on compare each two groups,P≤0.05 difference with statistical significanceResults:①In aging model cells,single cells were significantly increased in size(P=0.023),SA-β-gal positive rate(P＜0.001)and the G1 phase ratio(P=0.001)was significantly higher,PI was significantly decreased(P＜0.001).②As the pine pollen serum-treated,size of a single cell significantly reduced(P＜0.02),SA-β-gal staining positive rate and the G1 phase ratio were significantly decreased in senescent cells(P＜0.001,P＜0.05).Between the high dose and middle dose pine pollen groups,the changes of the G1 phase ratio(P=0.003) and PI index are significant(P=0.003).③In aging model group,the mRNA expression of p16INK4A and p21CIP-1gene both significant increased(P＜0.001,P=0.044).In pine pollen serum-treated group, p16INK4AmRNA expression of a significant downturn(P＜0.001),between the high dose(240mg/dl) and the middle dose(120mg/dl) treatment group,there were significant differences in P16mRNA expression(P=0.006),under the treatment of pine pollen,the expression of p21CIP-1mRNA have no significant difference(P=0.061).Conclusion:①Natural senescence 2BS cell model to be build successfully, appearant the typical aging morphological characteristics,express typical aging associated changes in the indexes of single-cell surface area,SA-β-gal staining positive rate,the cell cycle and the expression p16INK4A,p21CIP-1mRNA in aging model cell.②Identify the anti-aging effects of pine pollen,the aging related cell changes of aging morphological characteristics appearance and the positive expression of apparent indexes,are significantly improved.③Set up two different pine pollen concentration of serum,between the high doses(240mg/dl) and the middle dose(120mg/dl) of pine pollen serum,the proportion of cells in G1 phase,PI index and p16INK4AmRNA expression have significant difference,suggesting that pine pollen anti-aging effects of a dose-dependent characteristic.④Discuss the moleculer mechanism of pine pollen anti-aging.In the aging model cells,the p16INK4A,p21CIP-1mRNA expression both of a significant increase,but under the pine pollen treatment,p16INK4AmRNA expression of a significant downward but p21CIP-1mRNA expression of no statistics Significance.Reminded that p16-pRb pathway may be the major role in pine pollen anti-aging process in 2BS.⑤Pine pollen have the effect of anti cell replicability aging in 2BS.the related molecular mechanism may be decreased the p16INK4A mRNA expression of genes to improve aging-related cell G1 arrest.
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