| Decline of immune function or hibition of immune function on cells and fluids due to by western medicine,which can decrease the people standard of living and quality of life.Moreover Decline of immune function or hibition of immune function have close relations with Apoptosis.Cell proliferation can repair organational struction and fuction,which make people rehabilitation,enhance immunity and improve capacity of resistance to diseases.Thymus is the center immue organ of the body,yet a place of differention and maturation of T lymphocyte;its well or bad of function impact immue system of body directly.So detection of apoptosis and proliferation on thymus lymphocytes is a important method to deteminate immue function of body objectively,especilly in aspects of immue regulation on evaluation in some drug screening.The system first established thymic atrophy and apoptosis of lymphocytes model of mouse induced by Dex,By observing effect of Astragulus injection(AI)with recognized immune phamarcological effect on this model,the research will probe into the immune phamarcological effect and mechanism of AI therapying deseases of immue fuction decline induced by immunosuppressant.Chapter one Study on Intervention Effect and Mechanism of AI to thymus atrophy in Mice induced by Dex1 ObjectiveEstablishes model of the mice thymus atrophy induced by Dex,whith are treated 2 days and 5 days later,and By Observation of all groups of mice thymus index,pathology change of thymus,the thymus,ratio of papar meldulla,to discusse the Intervention Effect and Mechanism of AI to thymus atrophy in Mice induced by Dex.2 Methods72 KM SPF mice,male and female(half),18～20g,six randomized groups. Control group,Model group(50mg,kg-1),IL-2 group(5IU,kg-1),AI group of High-dose(2g.kg-1),AI group of High-dose(2g.kg-1),AI group of mid-dose(lg. kg-1),AI group of low-dose(lg·kg-1),12mice of per group,all group mice inject Dex except Control,qd,2days,Control group(of same capacity 0.9%saline injection). From first day treatment groups are injected AI and IL-2,five days later finish the experiment.every group select randomly six mice,firstly weighing,and then dislocation executed mice,removing thymus and calculate thymus index,then we fixed thymus in 10 percent of formaldehye solution,paraffin section,HE and immunohistochemistry staining,oberserting HE pathology change of thymus, Systems of Image-pro PLUS 5.1 anslyses ratio of cortex and medulla.The empirical datum uses carries on single factor variance analysis statistics processing.3 Results(1)Effect on thymus index of mouse by AIAfter two-days-treatment,mice thymus index of each group are group as following:control group5.01±13;model group1.17±0.26;IL-2 group1.68±0.52, AI group of High-dose1.68±0.44;AI group of mid-dose1.49±0.29;AI group of low-dose1.74±0.69.compared with model group,thymus index of AI group of High and low-dose and IL-2 group significantly increase(P＜0.05)After five-days-treatment,mice thymus index of each group are group as following:control group5.66±0.38,model group1.32±0.26;IL-2 group 1.70±0.57, AI group of High-dose1.42±0.31 AI group of mid-dose1.40±0.33,AI group of low-dose2.04±0.43,thymus index of AI group of low-dose significantly increase(P＜0.05)3.2 Effect on thymus pathology and ratio of papar meldulla of mouse by AIAfter two-days-treatment,thining of cortex of all AI and IL-2 group begin to better,to varying degrees reducing obviously of cells of medulla improve,compared with control group,ratio of papar meldulla of Model group Decrease obviously(P＜0.01),comparing to model group,ratio of papar meldulla in tow AI group increased obviously(P＜0.05)。After five-days-treatment,AI-different-dose group than Model group,boundaries Paper medulla are clear in AI low-dose group effect of treatment is most significant, boundaries of conterx-medulla are very clear,ration of them increase obviously,ratio of conterx-medulla of mice thymus decrease significantly.(P＜0.01),ratio of conterx-medulla of thymus increase obviously in AI high and low-dose group and IL-2 group than Model group(P＜0.05)4.Conclusion(1)Dex may induce the mouse thymus to wither,causes lymphocyte to apoptosis.(2)AI can enhance mice thymus index of mice induced by Dex,reverse ratio of papar meldulla,improve pathology shape of thymus,in conclusion AI has the function of protecting the mice thymus atrophy induced by Dex.Chapter two Study on Intervention Effect and Mechanism of AI to thymus Lymphocytes in Mice induced by Dex1 ObjectiveBy Observation of percentage of apoptosis of thymus lymphocytes of mouse,Expressing of apoptosis-related genes Caspase-3 and Bcl-2 and percentage of postive cells area of Caspase-3 and Bcl-2,24-hours survival and rate of thymus lymphocyte by AI in mice,to research Intervention Effect and Mechanism of AI to thymus lymphocytes in Mice induced by Dex.2 Methods(1)Animal model,grouping and having medicine like that of methods of chapter one.paraffin wax slice,TUNEL method oberserting lymphocycytes apoptosis,also immunohistochemistry staining oberserting expressing of Caspase-3 and Bcl-2, Systems of Image-pro PLUS 5.1 anslyses ratio of cortex and medulla,lymphocytes apoptosis,expressiong of Caspase-3 and Bcl-2,randomly selected five for every slice to analyse image.(2)Effects on 24-hours survival rate of thymus lymphocyte by AI in miceSeparating thymus lymphocyte from mouse of 5weeks,planting 5×105 cells /hole on the 96 holes plate.setting S0+Dex group,S1+Dex group,S2+Dex group, S3+Dex group,IL-2+Dex group,Model group,S0(200mg.ml-1);S1 group (20mg.ml-1);S2 group(2mg.ml-1);S3 group(0.2mg.ml-1),Model group(Dex final contration 10uM),six groups,three hole/group,adding 100μl.drugs 37℃, putting the plate into 5%CO2 incubator 24 hours.MTT methods test OD of 490nm site.(3)Effects on 24-hours apoptosis rate of thymus lymphocyte by AI in mice Planting 5×105/hole cells on the 96 holes plate,setting S0+ex group,S3+Dex group,S4+Dex group,S5+Dex group,IL-2+Dex group,Model group;S3 group (0.2mg.ml-1);S4 group(0.01mg.ml-1);S5:group(0.05mg.ml-1);Model group(Dex final contration 10uM),six groups,three hole/group.Adding 100μl drugs 37℃,putting the plate into 5%CO2 incubator 24hours,FCM detects apoptosis rate of lymphocytes3 Results(1)Effect on apoptosis of thymic cells of mouse by AIAfter two-day treatment,control group has more intensive ells and very few apoptosis,it is can finded more apoptosis in Model group.AI group of high-dose,AI middle-dose group and low-dose groups all have a certain amount of apoptosis.The positive percentage of apoptosis:Control group is 0.20%±0.21%,the model group 3.86%±1.42%,AI high-dose group 2.81%±0.81%,AI middle-dose group 3.01%±0.97%,AI low-dose group 2.33%±1.09%.Positive percentage of AI low-dose group has an increase significantly compared with model group(P＜0.05)After five-day treatment,control group has more intensive cells and little apoptosis cells.Model can be seen more apoptosis.AI high,medium and low-dose groups see a certain amount of apoptosis.The positive percentage of apoptosis are as following:Control group,0.20%±0.08%,the model group 6.4%±3.78%,astragalus high-dose group 1.80%±1.29%,AI middle-dose group 2.21%±0.75%,astragalus low-dose group 0.5%±0.27%.AI group of high-and low-dose have significantly compared with model group(P＜0.05).(2)The effect on apoptosis-related genes Caspase-3 and Bcl-2 by AI1)After two-days treatment,apoptosis-related protein expression on thymus in mice①Caspase-3:Compared with model group,Bcl-2 area percentage of positive expression AI low-dose group has significant differences.(P＜0.05)②Bcl-2:Bcl-2 area percentage of positive expression:AI low-dose group compared with the model,there are significant differences(P＜0.05).Model group compared with the control group,there were significant differences(P＜0.05).2)After five-days treatment,apoptosis-related protein expression on thymus in mice①Caspase-3 expression:Caspase-3 percentage of positive cells in mice thymocyte AI tow-dose group and the IL-2 group compared with the model,there were significant differences(P＜0.05).②Bcl-2:Bcl-2 percentage of positive cells in mice thymocyte.AI high and low-dose group compared with the model,there are significant differences(P＜0.01).(3)Effects on 24-hours survival rate of thymus lymphocyte by AI in mice MTT results showed that 24-hour survival rate of lymphocyte are as follows:S0 Group 39.84%±4.41%,(S1+Dex)Group 46.42%±10.90%,(S2+Dex)Group47.88%±8.98%,(S3+Dex)Group 58.18%±4.90%,IL-2 group 46.17%±2.87%,model group, 47.96%±6.38%;S3(0.2mg.ml-1),have significant apoptosis inhibition,compared with the model group,here are significant differences(P＜0.05).(4)Effects on 24-hours apoptosis rate of thymus lymphocyte by AI in miceFCM results showed that 24 hours apoptosis rate of thymus lymphocyte:S3 Group 84.57%±5.33%,S4 Group 78.67%±0.95%,S5 Group 81.37%±2.10%,model group,77.97%±1.75%,control group,54.63%±3.09%.The control group mice thymocyte apoptosis rate compared with the model group(P＜0.05).It illustrates that in vitro model has been successfully established,Astragalusge groups compared with the model group,(P＞0.05).4 Conclusion(1)AI can inhibite apoptosis of phymus lymphocytes induced by Dex in mice.(2)Mechanism of inhibition to apoptosis may due to uping expression of Bcl-2 and downing expression of Caspase-3.(3)AI also enhance 24-hours survival rate of thymus lymphocyte by AI in mice in Vitro experiment.Chapter three Study on Effect and mechanism of thymus lymphocyte proliferation by AI in Mice1 ObjectiveFrom the body in vitro,through observation lymphocyte anti-nuclear antigen--the proliferation-related protein PCNA expression,IL-2 contents in serum and thymus,measuring 48 hours apoptosis rate in vitro,Serum pharmacology method of measuring a 24-hour survival rate,to further explore the impact and molecula mechanism of astragalus injection onlymphocyte proliferation.2 Methods(1)Animal model,grouping and having medicine like that of 1.2.1.Two hours after lasting injecting,dislocation executed mice,taking all blood, separating serum;taking thymus,make it two pieces,putting one piece into the 1.5ml glass homogenate tube Adding 1ml Protein lysis in tube making homogenate liquid about two minites,and other piece and then in low temperature centrifugal 10min, 12000speed/min,sucking supnatant,we fixed other piece thymus in 10 percent of formaldehye solution,paraffin section,immunohistochemistry staining,observing western-blot and immunohistochemistry staining check protein expression of PCNA, ELISA check content of homogenate liquid and serum of thymus.(2)Effects on 24-hours survival rate of thymus lymphocyte by AI in normal miceSeparating thymus lymphotocytes of normal 5-weeks mouse,planting 5×105/hole cells on the 96.holes plate.Grouping the sixes.:S0group(200mg.ml-1),Slgroup (20mg.ml),S2 group(2mg.ml-1),S3group(0.2mg ml-1),IL-2+Dex group,Model group;Model group(Dex final contration is 10uM),six groups,three hole/group.adding 100μ1.drugs 37℃,putting the plate into 5%CO2 incubator 24 hours.MTT methods test OD value of 490nm site.(3)Effects on 48-hours apoptosis rate of thymus lymphocyte by AI in normal miceSetting four groups,planting 5×105/hole cells on the 96 holes plate.Grouping the fours,S3group(0.2mg.ml-1),S4group(0.01mg.ml-1,S5(0.05mg.ml-1),control group three hole/group.adding 100μ1.drugs 37℃,putting the plate into 5%CO2 incubator 24 hours。FCM methods test apoptosis rate of thymic cells in mice in(4)Effect on serum containing astragulas and supnatant homogenate of thymus to proliferation of 24-hours lymphyocytes21KM mice of 4-6weeks(SPF),divide three groups:control:0.9%saline by intraperitoneal injection and AI low-dose group,Low+Dex,making homogenate liquid and separating serum are same as Experiment of in vivo.-20℃save,reserve. MTT check survival rate of thymus lymphocytes.3 Results(1)Effect on thymus expression of PCNA in mice by Astragalus injectionExperiment 2 days later,astragalus high-dose group and low-dose IL-2 group and IL-2 group express more,and model group and low-doses less positive cells,the control group also expressed less.PCNA percentage of each group of Lymphocyte: Control groupl.79%±0.42%;model group 3.49%±0.11%;IL-2 Group 3.04%±1.29%;Astragalus high-dose group 3.69%±2.82%;astragalusmid-dose group 3.79%±1.53%;Astragalus low-dose group 6.90%±4.18%.The treatment group compared with the model goup without statistical significance.(P＞0.05)Experiment 5 days later,astragalus high-,mid,low-dose and IL-2 group express more,especially low-dose group has a lot of the positive cells with brown dying.(2)Western-blot results5 days later PCNA protein content in order are as follows:AI low-dose group→AI high-dose group→control group→model group→IL-2 Group→AI mid-dose group. (3)IL-2 level in Serum and thymus homogenate in mice by astragalus injection for 5 daysAfter five-day treatment;levels of IL-2 in serum shows that:,the control group 20.72±6.48(pg/ml),the model group 21.74±3.55(pg/ml),AI high-dose group 20.12±8.04(pg/ml),AI mid- dose group 20.93±7.61(pg/ml),AI low Dose group 47.27±15.01(pg /ml).Between astragalus injection of low-dose group and the model group had a significant difference(P＜0.05).After five-day treatment,IL-2 content of in Mouse thymus:The control group 135.75±11.15(pg/ml),the model group 82.93±11.56(pg/ml),astragalus high-dose group 192.24±12.13(pg/ml),astragalus mid-dose group 170.32±36.62(pg/ml), astragalus low -dose group 211.84±12.28(pg/ml).Compared with the model group, the treatment group has the most significant difference(P＜0.01),Astragalus high-and low-dose group compared with the control group,there is a very significant difference (P＜0.01).(4)24-hour survival rate of Lymphocyte by AI(MTT)AI concentration 0.2mg ml-1,compared with the control group,there are significant differences(P＜0.05),which note this concentration of thymus to promote the role of lymphocyte proliferation.Astragalus injection concentration 200mg.ml-1,compared with the control group,cell survival rate decreased significantly, there is a very significant difference P＜0.01.That this concentration is likely to be a certain toxic effect on mice thymocyte.(5)Apoptosis rate of 48-hour Lymphocyte by AI(FCM)AI of the all concentration groups and compared with control group,there was no significant difference(P＞0.05).(6)IL-2 level in lymphocytes by AI after 24-hours treatmentIL-2 level Of S2 group(2mg.ml-1)and S3 group(0.2mg.ml-1)are all higher than the control group(P＜0.05and P＜0.01).4 Conclusion(1)AI can increase expression of PCNA of phymus lymphocytes in mice.(2)AI can enhance IL-2 level of serum and phymus lymphocytes in mice.(3)AI can improve 24 hours survival rate of phymic cells in mice.5 At last,it would take a conclusion to the whole thesis:(1)AI can inhibit thymus atrophy of mice induced by Dex,which shows AI will be used to treat immunodeficient diseases and defects of immune function.(2)Mechanism of AI on inhibiting thymus atrophy of mice may be due to:①Increasing expression of Bcl-2 and decreasing expression of Caspase-3,and Inhibiting apoptosis.②Increasing expression of PCNA and improving contents of IL-2 in serum and thymus of mice,to promote proliferation of Thymic Cells in Mice.(3)AI can improve survival rate of Thymic Cells in Mice in vitro,which may be due to promote synthesis and release,moreover promote own proliferation of Thymic Cells and inhibit apoptosis in Mice... |
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