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Research On The Cell Proliferation And Differentiation Of Mesenchymal Stem Cells Transfecting By PEN Loading Oligodeoxynucleotid

Posted on:2020-06-04Degree:MasterType:Thesis
Country:ChinaCandidate:W J LiFull Text:PDF
GTID:2404330572486044Subject:Stomatology
Abstract/Summary:PDF Full Text Request
Oligonucleotides DNA nucleotides(oligodeoxynucleotides,ODN)are some nucleotide sequence motif which contains less than 50 single bases and takes demethylation as the core.They usually exist in some lower organisms’ genome DNA in the nature and it can be easily abutted with their complementary strand.Previous studies have found that MT01 is designed on the basis of human mitochondrial DNA contains the specific sequence ODN of 27 bases which have clear osteogenesis effects.It enters the cell by endocytosis and via toll-like receptor 9(Toll like receptor 9,TLR9)and toll-like receptor 7(Toll like receptor 7,TLR7),MT01 can increase osteogenesis genes’ higher expression in the human or rat osteoblasts such as Runt related transcription factor 2(Runt-related transcription factor 2,Runx-2),type I Collagen(Collagen-Ⅰ,COL-Ⅰ),and bone protection element(osteoprotegerin,OPG)and then promote human or rat osteoblasts,differentiation.Because MT01 is easy to be degraded by nuclease and the electrostatic reulsion effect exists between the surface of the cell membrane and the surface of MT01 and thus reducing the cell uptake rate,some researches have used modified MT01-S.Although maybe it could block the intracellular nuclease degradation of MT01,but may cause a series of adverse reactions to the body,so this experiment aims to researching new carrier PEN for loading free MT01 and making it work better.Obj ective:This experiment aims to build efficient PEN/MT01 compound,by detecting the cell proliferation of BMSCs and the expression of intracellular osteogenesis related genes such as ALP,Runx2 and COL-Ⅰ,to explore if the PEN could become effective carrier of MT01 and influence the proliferation and differentiation of rat BMSCs conjointly.Methods:In this study,firstly between PEN and MT01 different mass ratios including 0.5:1,1:1,2:1,4:1,6:1,8:1 and 10:1 are used for electrostatic loading in order to form the PEN/MT01 compound respectively and then transfect them into rat bone mesenchymal stem cells.Then,through comparing with the Group simple PEN carrier and Group blank control,selecting the optimal mass ratio of PEN/MT01 compound by detecting BMSCs’,cell proliferation.Next the group of free MT01,Group MT01-S and Group blank control are set respectively for comparing with the screened optimal mass ratio of PEN/MT01 compound.Through the detection of intracellular ALP,expression level of Runx2 and COL-Ⅰ to evaluate the influence of PEN/MT01 compound on the osteogenic differentiation of rat BMSCs.In experiment 1,the cell proliferation rate of BMSCs is determined by MTT colorimetric method,in order to screen out the optimal loading mass ratio of PEN/MT01 compound.In experiment 2,the alkaline phosphatase(ALP)activity in BMSCs caused by the screened PEN/MT01 compound is detected by alkaline phosphatase kit.In experiment 3,Realtime-PCR is used to detect the changes in gene expression levels of osteogenic marker genes meaning Runx2 and COL-Ⅰ in the BMSCs caused by screened PEN/MT01 compound.In experiment 4,BMSCs’ osteogenic differentiation in vitro caused by screened PEN/MT01 compound is detected by alkaline phosphatase staining.Results:The result of BMSC’s cell proliferation shows that the mass ratio of 2:1 owns the least effect on the survival rate of BMSCs among all kinds of different mass ratio of PEN/MT01 compounds on the first day after transfection and with the time extends to the second and third after transfection,the group of 2:1 PEN/MT01 compound owns the highest cell proliferation rate(p<0.01),suggesting it has the best promoting effect,so the optimal loading mass ratio of PEN/MT01 compound is 2:1.The result of alkaline phosphatase activity in BMSCs shows that the group of free MT01,Group MT01-S,group of 2:1 PEN/MT01 compound,group of 6:1 PEN/MT01 compound and Group blank control.At the three time points tested namely the first day,the third day and the fifth day,the expression of alkaline phosphatase activity of BMSCs in the Group 2:1 PEN/MT01 compound is the highest(p<0.01)and is always better than that of Group MT01-S and other groups.Realtime-PCR detection results of BMSC osteogenic marker gene shows that among the group of free MT01,Group MT01-S,the group of 2:1 PEN/MT01 compound and Group blank control in the experiment,compared with the Group blank control,the expression of osteogenic marker gene Runx2 and COL-I have all increased.The expression of Runx2 in the Group MT01-S is significantly higher than that in the other three groups on day 3,As time goes on and until the day 5,the expression of Runx2 is significantly increased in the group of 2:1 PEN/MT01 compound(p<0.01),still higher than that in the other groups,and the significant gene expression advantage persists until the 7th day after transfection.On the three detecting time points including day 3,day 5 and day 7,the expression level of COL-I in the group of 2:1 PEN/MT01 compound is the highest(p<0.01)and is better than other groups.Alkaline phosphatase staining finds that the group of 2:1 PEN/MT01 compound shows the deepest staining,following by Group MT01-S,group of free MT01 and Group blank control respectively.Conclusion:PEN which is used as a carrier can effectively improve the endocytosis efficiency of the free MT01.Different mass ratios of PEN/MT01 compound can all have effects on the cell proliferation of bone marrow mesenchymal stem cells,when the MT01 and PEN quality loading proportion reached 6:1,poisonous to the cells begins to appear and with the PEN quality percentage increasing inside the compound,its cytotoxicity to the BMSCs also gradually increased.When the mass loading ratio of PEN and MT01 is 2:1,the rat BMSCs have the highest cell survival rate,and it even have the best promotion effect on the proliferation of BMSCs with the extension of time.Therefore,the 2:1 PEN/MT01 compound is selected to study whether it could affect the osteogenic differentiation of BMSCs.Then we find out that the 2:1 PEN/MT01 compound in this study can enhance the expression level of BMSCs’osteogenesis related protein such as alkaline phosphatase and bone reconstruction related gene Runx2 and COL-I,and the 2:1 PEN/MT01 compound ’s effect is better than that of Group MT01-S,group of free MT01 and Group blank control.
Keywords/Search Tags:bone marrow mesenchymal stem cells, oligonucleotide MT41, PEI derivatives PEN, cell proliferation, osteogenic differentiation
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