Effects Of Specific Sequence Oligonucleotides On The Proliferation Of Human Bone Marrow Mesenchymal Stem Cells

The seed cells is one of the most critical elements in research and application of tissueengineering, is the first material base of tissue-engineered tissues and regeneration. Accordingto the source, seed cells can be divided into autologous cells, allogeneic cells andheterogeneous cells. According to the differentiation degree can be divided into embryonic stemcells, adult stem cells and progenitor cells and differentiation of adult cells. Although thesources of allogeneic and heterogeneous cells become more widely, but some problems such asdifferences in immune rejection and cell function, directional differentiation of embryonic stemcells are difficult to obtain high purity of seed cells, but also ethical problems. so autologous cellis predominantly used for tissue engineering construction and tissue defecttion.The seed cells of tissue engineering should meet the following requirements: capabilityof amplification is strong in vitro, have a good and specific biological properties; y. Thebiological functions of cells of high purity, has certain cells dominate; avoided immunerejection; Biological safety. The bone marrow mesenchymal stem cells on the meet aboverequirement, and its sources is abundant and renewable in the body; Based on safe andconvenient for small area wound; the technology of cell culture is simple and easy. But theapplication of tissue engineering need large amount of seed cells. at present,the commonculture in vitro is hard to amplify the required in a limited time, so finding an efficient methodof amplification is necessary.Recent studies have shown that certain ODNs significantly promote the proliferation ofhuman periodontal ligament cells, rat bone marrow mesenchymal stem cells, osteoblastsinfected with Porphyromonas gingivalis and MG63cells. In the current study, the impact ofthree ODNs (MT01, FC003, Sa05f) on hBMSC proliferation was examined via CCK-8assay.The effects of these ODNs on the hBMSC cell cycle and the underlying mechanisms for theseeffects were also investigated. Elucidating this may provide a theoretical basis for the use of ODNs in tissue engineering and clinical practice.The content of this research is divided into fiveparts:Part1Isolation, culture and identification of hBMSCshBMSCs were collected from healthy adults via density gradient centrifugation. Cellmorphology was observed at different phases. Third-passage hBMSCs were seeded at1.0×104/cm2in6-well plate, and osteogenic, adipogenic or chondrogenic induction medium wasadded. Medium was changed every72h and after2-3weeks, the cells were subjected toAlizarin red, Oil Red O and toluidine blue staining. Stem cell surface molecules were identifiedusing 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 complete medium was added to eachwell and the medium was changed on day4. The CCK-8assay was done via reading opticaldensity, for7consecutive days, using microplate reader (test wavelength450nm, referencewavelength630nm) and the cell growth curves were analyzed to determine the optimal platingdensity.Part3Comparison of the effects of three ODNs at four concentrations on hBMSC 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 wereestablished, with four replicate wells in each group, 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 wasadded to the control group. The CCK-8assay was performed as above, using the optical densitymicroplate reader, for three consecutive days in order to determine which ODN had the mostsignificant effect on the proliferation of hBMSCs.Through the above five experiments, the results were as follows: Part1The hBMSCs that had been isolated, purified and cultured using density gradientcentrifugation grew well. The third-passage hBMSCs were of spindle shape, and after a largeamount of proliferation, the cells were arranged like vortices;After21days of osteogenicinduction, the Alizarin red staining of fixed cells revealed a large amount of range-mineralizednodules in the petri dish. After15days of adipogenic induction, Red O Oil staining of fixedcells revealed the formation of a large amount of dark red lipid droplets. After21days ofchondrogenic induction, Toluidine blue staining of fixed cells revealed light blue metachromaticgranules in the cytoplasm and the extracellular matrix was also stained light blue. The positiveexpression rate of CD90, CD73, and CD105was99.6%,99.9%and96.5%, respectively. Theoverall positive expression rate 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 on day1aftertreatment with0.5mg/L FC003(Figure J, P<0.01). Conversely, optical density was signficantlylower on day1after treatment with1.0and4.0mg/L Sa05f (Figure K, P<0.01and P<0.05,respectively). Optical density was significantly higher on all3days afterThrough the above results we confirmed:BMSCs of relatively high purity can be obtained using density gradient centrifugation forisolation, therefore, in the present study density gradient centrifugation was used to isolate andpurify hBMSCs. Identification of hBMSCs in the present study was done per the minimumstandards proposed by Dominici and published in Cytotherapy in2006. These are thought torepresent semi-official views of the Mesenchymal and Tissue Stem Cell Committee of theInternational Society for Cellular Therapy. These standards are as follows:(1) Under standard culture conditions, the MSCs can be affixed to a plastic wall and grow;(2) The MSCs canexpress CD105, CD73and CD90, but do not express CD45, CD34, CD14, CD11b, CD79alpha,CD19or HLA-DR surface molecules; and (3) The MSCs can differentiate into osteoblasts, fatcells and chondrocytes. The hBMSCs used in the current study met the above standards. Theirstem cell characteristics were validated morphologically and induced multi-differentiation. Theoptimal cell growth curve was found to occur at a plating density of6.0×103/cm2. MT01at aconcentration of2.0mg/L significantly promotes proliferation of hBMSC.In summary, MT01appears to promote the HBMSC proliferation by regulating factorsrelated to the cell cycle. These results shed new light on, and provide a preliminary theoreticalbasis for, in vitro amplification of seed cells for tissue engineering. However, many morestudies still need to be performed before practical application of MT01.