| Objective: To observe the influence of the micture that contain platelet rich plasma(PRP)and cyclic adenosine monophosphate(cAMP)to the proliferation of rabbit tendon stem cell and the content of type I and III collagen,type-I metalloproteinase and its antagonist.Preliminarily Analyze the influential mechanism of mixture and explore the possible reason for the changed content of type I and III、collagen type-I metalloproteinase and its antagonist.Laying the foundation for further research of platelet rich plasma(PRP)and cyclic adenosine monophosphate(cAMP)blend to the diseased tendon tissue.Methods:(1)The part of vitro cell experiment.(1)Observation of cell proliferation:Taking a cell Petri dish(covered with tendon stem cells)with a diameter of 10 cm to passage,then assign 0.5ml tendon stem cells suspension into 4 Petri dishes with a diameter of 3.5cm.After the passaging,add 3ml complete medium to each little culture dishes.The Petri dishes were labeled with 4 kinds,A dish was intervened by cAMP,B dish was intervened by PRP,C dish was intervened by the mixture of PRP and cAMP,D dish was intervened by nothing.After cell attaching on the wall of Petri dish,we added 1ml cAMP into A dish,1ml platelet rich plasma into B dish,0.5ml PRP and 0.5ml c AMP into C dish.then another 200 ul Heparin sodium was added to dish B and C.After 2days we use Microscope to observe the cell proliferation.(2)CCK8 cell proliferation/apoptosis experiment:During the cell passage,assign 200 ul tendon stem cells suspension into 16 holes in 24 well-plate and mark the hole.then we assign 800 ul complete medium into the 16 hole.After 1 day,the cell has attached on the wall of Petri dish,we added 500 ul cAMP suspension into A1-A4 hole,added 500 ul PRP into B1-B4 hole,added 250 ul cAMP and 250 ul PRP into C1-C4 and added noting into D1-D4.another 100 ul Heparin sodium was added to B1-B4 and C1-C4.After 1 day,we abandon all complete medium in the 16 holes,using PBS slightly wash the B1-B4 and C1-C4 3-4 times in a bid to clean the platelet on the wall but we must avoid wiping cell on the buttom,As A1-A4 and D1-D4 just washing 1 time.Finish washing,we add 1000 ul complete medium and 100 ul CCK8 into the 16 hole with dark condition,then put the 24 well-plate into CO2 ncubator for 3 hours.After the time we use Microplate Reader to test the OD underthe 450 nm light.(2)The part of Animal experiment.36 new zealand white rabbits(6-8 months old),weight 2.25±0.19 kg,were randomly divided into 6 groups After a week of adaptive feeding.Group A was treated with cAMP,Group B was treated with PRP,Group C was treated with mixture of PRP and cAMP,Group D was treated with noting,Group E was treated with normal saline,Group F is normal control group.According to the chemical modeling method,we use Prostaglandin E2 with the concentration of 500ng/0.2ml on A,B,C,D,E group rabbits to establish Achilles tendon disease models.After the Achilles tendon disease models have been established for 1 week.we use different method to treat group A,B,C,D and E.1ml cAMP were injected into the Damaged part on Achilles tendon in group A,1 time/3 weeks,a total of 2 times.1ml PRP were injected into the Damaged part on Achilles tendon in group B,1 time/3 weeks,a total of 2 times,0.5ml PRP combined with 0.5ml cAMP were injected into the Damaged part on Achilles tendon in group C,1 time/3 weeks,a total of 2 times.normal saline were injected into the Damaged part on Achilles tendon in group E,1 time/3 weeks,a total of 2 times,and nothing is injected into group D.After the second treatment,the animals were sacrificed and the Achilles tendon tissue was taken out to make into histological sections and test Experimental index.(1)Taking advantage of western-blot method to detect the expression of MMP-1 and TIMP-1 in tendon tissue,and compare the differences between the two groups.(2)Judging the recovery state of Achilles tendon tissue,and comparing the differences between the groups by observing the Sirius staining of H-E staining of the Achilles tendon tissue sections and PCR’s result of colalgen expression.Results:(1)The part of vitro cell experiment.(1)From the observation in the microscope,the proliferation of C dish is the most dramatic,D dish proliferate the least,The proliferation of dish A and B was located between D dish and C dish.(2)The result of CCK8 experiment:Compared with A,B and D group,the proliferation of cells from C group are the most dramatic,the difference was statistically significant(P<0.05),the proliferation of A and B group have Increased to a certain extent,but lower than C group,There was no significant difference between the two groups(P>0.05).the proliferation of D group are the lowest,Compared with other group,the difference was statistically significant(P<0.05).(2)The part of Animal experiment.(1)The result of MMP-1 shows the content of MMP-1 in C group is very low,only higher than F group,D group and E group rabbits have the highest level of MMP-1,group A and B are located between D group and C group,the comparison between the A and B group has no significant difference(P>0.05),compared A group with C,D,E and F group,the difference was statistically significant(P<0.05),compared B group with C,D,E and F group,the difference was statistically significant(P<0.05),compared C group with D,E,F group,the difference was statistically significant(P<0.05),compared D group with E group,the difference was no statistically significant(P>0.05),compared D group with F group,the difference was statistically significant(P<0.05),compared E group with F group,the difference was statistically significant(P<0.05).(2)The result of TIMP-1 shows the content of TIMP-1 in C group is very low,only higher than F group,D group and E group rabbits have the highest level of TIMP-1,group A and B are located between D group and C group,the comparison between the A and B group has no significant difference(P>0.05),compared A group with C,D,E and F group,the difference was statistically significant(P<0.05),compared B group with C,D,E and F group,the difference was statistically significant(P<0.05),compared C group with D,E,F group,the difference was statistically significant(P<0.05),compared D group with E group,the difference was no statistically significant(P>0.05),compared D group with F group,the difference was statistically significant(P<0.05),compared E group with F group,the difference was statistically significant(P<0.05).(3)The result of the ultramicro nucleic acid’s concentration shows the concentration of ultramicro nucleic acid in C group is very low,only higher than F group,D group and E group rabbits have the highest level of ultramicro,group A and B are located between D group and C group,the comparison between the A and B groups has no significant difference(P>0.05),compared A group with C,D,E and F group,the difference was statistically significant(P<0.05),compared B group with C,D,E and F group,the difference was statistically significant(P<0.05),compared C group with D,E,F group,the difference was statistically significant(P<0.05),compared D group with E group,the difference was no statistically significant(P>0.05),compared D group with F group,the difference was statistically significant(P<0.05),compared E group with F group,the difference was statistically significant(P<0.05).Conclusion:(1)the tendon stem cells could be induced to adipogenic or chondrogenic tissues in an appropriate manner.(2)The properties and effects of PRP prepared by landesberg method are stable,which is consistent with the results reported in most literatures.(3)cAMP can reduce inflammation,improve local blood capillary permeability,provide the energy required for tendon stem cell proliferation,promote cell proliferation of tendon stem,decrease the expression of MMP-1 and TIMP-1 damaged tendon,increase damaged tendon tissue I,collagen type III,to restore the damaged tendon tissue(4)PRP can provide platelet derived growth factor,promote the proliferation of tendon stem cells and reduce the local inflammatory response,promote the proliferation of tendon stem cells,reduce the expression of MMP-1 and TIMP-1 in the injured tendon,increase the content of I and collagen type III in the injured tendon tissue,and restore the damaged tendon tissue(5)PRP and cAMP blend could promote the proliferation of tendon stem cells to a great extent,promote the recovery of damaged tendon tissue,and the therapeutic effect is better than using PRP or cAMP alone. |
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