The Effect Of Specific Oligonucleotides On The Proliferation Of Human Bone-marrow Mesenchymal Stem Cells

The seed cells is one of the most critical elements in research andapplication of tissue engineering, is the first material base of tissue-engineeredtissues and regeneration. According to the source, seed cells can be dividedinto autologous cells, allogeneic cells and heterogeneous cells. According tothe differentiation degree can be divided into embryonic stem cells, adult stemcells and progenitor cells and differentiation of adult cells. Although thesources of allogeneic and heterogeneous cells become more widely, but someproblems such as differences in immune rejection and cell function, directionaldifferentiation of embryonic stem cells are difficult to obtain high purity of seedcells, but also ethical problems. so autologous cell is predominantly used fortissue engineering construction and tissue defecttion.The seed cells of tissue engineering should meet the followingrequirements: capability of amplification is strong in vitro, have a good andspecific biological properties; y. The biological functions of cells of high purity,has certain cells dominate; avoided immune rejection; Biological safety. Thebone marrow mesenchymal stem cells on the meet above requirement, and itssources is abundant and renewable in the body; Based on safe andconvenient for small area wound; the technology of cell culture is simple andeasy. But the application of tissue engineering need large amount of seed cells.at present,the common culture in vitro is hard to amplify the required in alimited time, so finding an efficient method of amplification is necessary.Recent studies have shown that certain ODNs significantly promote theproliferation of human periodontal ligament cells, rat bone marrowmesenchymal stem cells, osteoblasts infected with Porphyromonas gingivalisand MG63cells. In the current study, the impact of three ODNs (MT01, FC003,Sa05f) on hBMSC proliferation was examined via CCK-8assay. The effects ofthese ODNs on the hBMSC cell cycle and the underlying mechanisms for these effects were also investigated. Elucidating this may provide a theoreticalbasis for the use of ODNs in tissue engineering and clinical practice.The content of this research is divided into five parts:Part1Isolation, culture and identification of hBMSCshBMSCs were collected from healthy adults via density gradientcentrifugation. Cell morphology was observed at different phases.Third-passage hBMSCs were seeded at1.0×104/cm2in6-well plate, andosteogenic, adipogenic or chondrogenic induction medium was added.Medium was changed every72h and after2-3weeks, the cells were subjectedto Alizarin red, Oil Red O and toluidine blue staining. Stem cell surfacemolecules were identified using flow cytometry.Part2Establishment of hBMSC optimal plating densityThird-passage hBMSCs were seeded at concentrations of3.0×103/cm2,6.0×103/cm2,1.2×104/cm2, or2.4×104/cm2in a96-well plate,200μl of completemedium was added to each well and the medium was changed on day4. TheCCK-8assay was done via reading optical density, for7consecutive days,using microplate reader (test wavelength450nm, reference wavelength630nm)and the cell growth curves were analyzed to determine the optimal platingdensity.Part3Comparison of the effects of three ODNs at four concentrations onhBMSC proliferationThird-passage hBMSCs were seeded at the optimal plating density in96-well plate and200μl of complete medium was added to each well. A total of12experimental groups were established, with four replicate wells in eachgroup, based on ODN type (MT01, FC003, Sa05f) and final concentration(0.5mg/L,1.0mg/L,2.0mg/L,4.0mg/L). An equal amount of PBS was added tothe control group. The CCK-8assay was performed as above, using theoptical density microplate reader, for three consecutive days in order todetermine which ODN had the most significant effect on the proliferation ofhBMSCs.Part4The effect of ODN MT01on the hBMSC cell cycleThird-passage hBMSCs were seeded at the optimal plating density in a6cm petri dish. After the cells adhered to the wall,2.0mg/L MT01was added tothe dish; the control cells were treated with an equal volume of PBS. On days1, 2and3, cell cycle analysis was performed. The handling of samples anddetection were conducted following the corresponding Muse Cell Analyzerkit instructions.Part5Molecular mechanism(s) underlying the effect of MT01on hBMSCproliferationhBMSCs were treated as in1.3.4. Sample handling and detection wereconducted following the corresponding LightCycler@480fluorescentquantitative real-time PCR analyzer kit instructions. The expressions of cyclinA, cyclin D1, CDK2and CDK4were measured on day2after the addition ofMT01.Through the above five experiments, the results were as follows:Part1The hBMSCs that had been isolated, purified and cultured using densitygradient centrifugation grew well. The third-passage hBMSCs were of spindleshape, and after a large amount of proliferation, the cells were arranged likevortices;After21days of osteogenic induction, the Alizarin red staining of fixedcells revealed a large amount of range-mineralized nodules in the petri dish.After15days of adipogenic induction, Red O Oil staining of fixed cells revealedthe formation of a large amount of dark red lipid droplets. After21days ofchondrogenic induction, Toluidine blue staining of fixed cells revealed light bluemetachromatic granules in the cytoplasm and the extracellular matrix was alsostained light blue. The positive expression rate of CD90, CD73, and CD105was99.6%,99.9%and96.5%, respectively. The overall positive expressionrate of CD34/45/116was0.51%.Part2The growth curve for hBMSCs was optimal at a plating density of6×103/cm2.Part3Compared to the control group, optical density was significantly higher onday1after treatment with0.5mg/L FC003(Figure J, P<0.01). Conversely,optical density was signficantly lower on day1after treatment with1.0and4.0mg/L Sa05f (Figure K, P<0.01and P<0.05, respectively). Optical densitywas significantly higher on all3days after Part4The percentage of cells in phase G0/G1was significantly lower and thepercentage of cells in phases S and G2/M was significantly higher (P<0.01) ondays1,2and3after treatment with2.0mg/L MT01, compared with the controlgroup.Part5The expressions of cyclin A, cyclin D1, CDK2and CDK4weresignificantly elevated in the group treated with MT01compared with the controlgroup (P<0.01)(Figure M).Through the above results we confirmed:BMSCs of relatively high purity can be obtained using density gradientcentrifugation for isolation, therefore, in the present study density gradientcentrifugation was used to isolate and purify hBMSCs. Identification ofhBMSCs in the present study was done per the minimum standards proposedby Dominici and published in Cytotherapy in2006. These are thought torepresent semi-official views of the Mesenchymal and Tissue Stem CellCommittee of the International Society for Cellular Therapy. These standardsare as follows:(1) Under standard culture conditions, the MSCs can be affixedto a plastic wall and grow;(2) The MSCs can express CD105, CD73and CD90,but do not express CD45, CD34, CD14, CD11b, CD79alpha, CD19orHLA-DR surface molecules; and (3) The MSCs can differentiate intoosteoblasts, fat cells and chondrocytes. The hBMSCs used in the current studymet the above standards. Their stem cell characteristics were validatedmorphologically and induced multi-differentiation. The optimal cell growthcurve was found to occur at a plating density of6.0×103/cm2. MT01at aconcentration of2.0mg/L significantly promotes hBMSC proliferation asevidenced by the decrease in the percentage of cells in phase G0/G1and theincrease in the percentage of cells in phases S and G2/M. The underlyingmolecular mechanisms may include, but is not limited to, elevated expressionsof cyclin A, cyclin D1, CDK2and CDK4.In summary, MT01appears to promote the HBMSC proliferation byregulating factors related to the cell cycle. These results shed new light on, andprovide a preliminary theoretical basis for, in vitro amplification of seed cells fortissue engineering. However, many more studies still need to be performedbefore practical application of MT01.