| Introduction:Natural killer cells are very important immune cells in the body,play an important role in the immune system.NK cells can kill targets cells directly by synthesizing and secreting a variety of cytokines,such as perforin and granzyme,etc.NK cells can regulate the immune function by the interaction with dendritic cells(DC)and immune cells,NK cells can also mediate antibody-dependent cytotoxicity(ADCC)by binding CD16 to the antibody.Thus,NK cells have a variety of powerful effects,there are many researches on NK cells,including the following aspects,the activity of anti-tumor and antiviral infection,the immunotherapy of NK cells,etc.Aging is an unavoidable natural phenomenon.It refers to the progressive decline of the body's adaptability to the social and physiological environment,which gradually leads to death.With the progress of society,the number of aging people has gradually increased.Therefore,more and more researches on anti-aging are being conducted.However,how to construct an aging animal model successfully become the key to our research.A number of experiments have shown that the subacute aging rat model induced by D-galactose can suit our demand.Therefore,it is widely used in scientific experiments.Referred to the studies of relevant experiments,genetically K562 cells are used to induce umbilical cord blood NK cells in vitro culture and amplification to obtain a large number and high purity NK cells.D-galactose is used to constructed subacute aging rat liver injury model by continuous subcutaneous injection into the rat's neck.NK cells obtained from the experiment are injected into the rat models,study the effects and possible mechanisms of NK cells on subacute aging rat liver injury.Objective:1.To obtain high quality NK cells from fresh umbilical cord blood in vitro and find the best time point of NK cells culture.2.To detect the general activity and cytotoxicity to tumor cells of NK cells.3.To investigate the effect of umbilical cord blood NK cells on the treatment of hepatic injury induced by D-gal in sub-acute aging rat model and its possible mechanism.Methods:1.The culture of umbilical cord blood NK(UB-NK)cells:MNCs were isolated from fresh cord blood by healthy,full-term maternal women,MNCs were placed in culture medium to amplified NK cells from the cord blood.Flow cytometry analysis was performed at different time points to detect the expression of CD3 and CD56,the apoptosis,the ability of anti-tumor of NK cells.2.The sub-acute aging rat model:20 healthy mature male Wistar rats were divided into 2 groups randomly:Normal group served as N,Model group served as M.A sub-acute aging rat model was constructed by subcutaneous injection of D-gal into the neck,phosphate buffer(PBS)was served as the control group.The general states were observed and recorded during the modeling process.The liver and spleen index were calculated,the activity of superoxide dismutase(SOD)and the concentration of malondialdehyde(MDA)in blood and liver were examined by test kits.Hematoxylin-eosin staining(HE)was used to observe the morphology of liver,Masson staining was used to determine the liver fibrosis.The expression of Bax,Bcl-2,etc.in liver tissue were measured by qRT-PCR,VEGFA and VEGFR2 protein levels in liver were detected by Western-blot.3.The effects and mechanisms of umbilical cord blood NK cells on liver injury of sub-acute aging rat model induced by D-gal,20 healthy mature male Wistar rats were divided into 2 groups randomly,control group served as C,test group served as T.Constructed the model as above,108/Kg NK cells obtained in the study were infused into test group by tail vein,and rats in control group were infused by PBS,twice a week for a total of four times.One week after all the infusion,rats were sacrificed by intraperitoneal injection of pentobarbital sodium,liver and spleen were weighed and the indexes were calculated.The activity of SOD and the concentration of MDA in blood and liver were detected by test kits.Morphological changes of liver were determined by HE staining,fibrosis of liver was determined by Masson staining.The levels of Bax,Bcl-2,etc.in liver were detected by qRT-PCR.VEGFA and VEGFR2 in liver were measured by Western-blot.Results:1.A large number(108)and high purity(>92%)of NK cells were obtained under the vitro co-culture of genetically engineered K562 cells,IL-2 and MNCs.2.Sub-acute aging rat model and liver injury were constructed successfully by subcutaneous injection of D-gal into the neck of Wistar rats:2.1 Compared with normal group,rats in model group had obvious shedding of hair during the process of model construction,they were weak,sluggish and insensitive to stimulation,their weight was decreased,one rat sacrificed in model group while lively in normal group.After the sacrifice,complete liver and thymus were removed and wet weights were weighed,the indexed were calculated,it was found that the liver index increased(P=0.007)and thymus index decreased(P=0.0043).2.2 Compared with normal group,the activity of SOD in model group was significantly decreased(blood P = 0.02,liver P<0.01)while the concentration of MDA was significantly increased(blood P=0.008,liver P=0.004).2.3 HE staining:Compared with normal group,the congestion of liver tissues,edema of liver cells and eosinophilic cells were obviously increased in model group,part of the hepatic lobule central vein was blocked,the arrangement of surrounding hepatic cord was disorganized,the inflammatory cells were infiltrated.The proliferation of liver cells was significantly reduced(as determined by the number of binuclear cells)(P=0.001),and lipofuscin was observed in cells.2.4 Masson staining:Compared with normal group,there were more blue dyeing areas in hepatic tissue of the model group,the color was deeper.2.5 qRT-PCR:Compared with normal group,the expression of Bax(P=0.0047)and Caspase-3(P=0.0029)in model group was more while Bcl-2(P=0.0018)and FGF21(P=0.0039)were less compared with normal group.2.6 Western-blot:Compared with normal group,the expression of VEGFA(P=0.045)and VEGFR2(P=0.0041)was decreased in model group.3.The effects and mechanisms of umbilical cord blood NK cells on improving liver damage in sub-acute aging rat models induced by D-gal:3.1 Compared with control group,rats in test group showed an increase in body weight(P=0.0053),their liver index was decreased(P=0.0053)while spleen index increased(P=0.0015);3.2 SOD activity in blood and liver tissue of test group was higher than that in control group(blood P = 0.0049,liver P = 0.0013),while MDA concentration was lower(blood P<0.001,liver P=0.004);3.3 HE staining:Compared with control group,the hepatic tissue congestion and cell edema were lighter,eosinophilic cells were less,central hepatic lobule atresia was decreased,hepatic cord arranged better,hepatic sinusoidal dilation and inflammatory cell were less while the proliferation of liver cells increased in test group(P=0.0294);3.4 Masson staining:Compared with control group,the liver cells had less blue-stained area and the color was lighter in the test group.3.5 qRT-PCR:The expression of Bax(P=0.0008)and Caspase-3(P<0.001)in test group was less while Bcl-2(P=0.003)and FGF21(P=0.0008)were more compared with control group.3.6 Western-blot:Compared with control group,the expression of VEGFA(P=0.0006)and VEGFR2(P=0.0161)was increased in test group.Conclusions:1.The best value and purity of NK cells could be achieved at day 15 under the co-culture of umbilical cord blood MNCs,genetically engineered K562 cells and IL-2.2.UC-NK cells have powerful anti-tumor activity and can eliminate abnormal cells such as senescence cells.3.UC-NK cells can improve the general state of sub-acute aging rats induced by D-gal,increase the activity of inhibiting senescence enzymes,reduce the level of aging-promoting substances.They can improve the liver injury at gene and protein level,which could be achieved by cytokine factors,self-killing activity and interaction with other immune cells of the body. |
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