A Small Molecule Of Tonic Prescription Influence With The Destiny Of The Mesenchymal Stem Cells

ObjectiveBone marrow stromal cells as a multiplex differentiation potential.this adult stem cells, relative to embryonic stem cells, because of its origin, easy, convenient proliferation ability is strong, can in vitro large-scale expand without loss of multiplex differentiation potential, be weak immunogenicity.strong immune tolerance, low immune rejection, inside the induction of specific conditions can be differentiated into bone and cartilage, fat, muscle tendon and ligament, nerve, etc. Various organizations in recent years, and has become a source of seed cells, tissue engineering of hot cells. Through various methods to enhance the proliferation of MSCs vigor and directional differentiation capacity to meet the clinical needs is the problem to be solved.Small molecules extracted from traditional Chinese medicine control the fate of stem cells, scientists abroad have been extraordinarily favored. China also has the relative reports, small molecules can control stem cell proliferation, differentiation, destiny includes several biological processes, apoptosis in tumor, degenerative diseases, and so on. have broad application prospects.The experimental group devoted for a long time to the research about Plastrum Testudinis(PT) with the efficacy of nourishing kidney.which suggests that cholesterol myristate. the organic small molecules from PT. can control MSC proliferation. By constructing a rat Idl promoter confirmed Extracts from Plastrum Testudinis promote(PTE) and active ingredients effected bone marrow stromal cells (MSCs) proliferation and differentiation and whose molecular mechanisms are primarily discussed. This experiment is based on the previous studies and further ascertain decisive core control area clips in the rat Idl promoter region.which play decisive role in proliferation.and predictions may interact with the control area of transcription factor for further research on relationship between it and small molecules extracted from traditional Chinese medicine of nourishing kidney promoted Idl gene expression.Strong muscle healthy strength drink is clinical treatment of myasthenia gravis. experimental study of classical prescription for the active ingredient in the screening and found a new compound K9 (three-ring monoterpenes). and the test results by MTT K9 (three-ring monoterpenes) that as low toxicity compounds, may launched MSCs differentiation. This experiment to determine MSCs to cartilage induced K9 differentiation. and discusses the mechanism.MethodsFirst-we constructed four different lengths of Id1 promoter taked earlier period construction's rate Id1 as 1742bp(-1765/+88) for the template.drived luciferase report gene vector of PGL3-basic, and sequenced before using correct,MSC and other cell lines would be transfected with the calcium phosphate co-precipitation method and PEI for luciferase activity measurement. The quantity of luciferase synthesis reflects Idl promoter activity the state. Analysis shows decisive role of core control zone (-1765-1164)in the promoter region of rats Id1 transcription, transcription factor DNA binding elements in its region were predicted by using TRANSFAC database. We selected the nuclear transcription factors E2F relating to the proliferation for further research.using gene mutation. Id 1 promoters lack of E2F transcription factor transfected MSCs to observe Idl promoter fragments activity.Second. Using the RT-PCR method, different concentrations of K9 (three-ring monoterpenes affects MSCs in different time points, and we extraced mRNA of MSC to detect protein related to cartilage differentiation phenotype and the SHH signaling pathways change.ResultsMeasured luciferase and the pRL enzyme activity after the plasmid luciferase transfection. demonstrated finally. PGL3-Id1 the different fragment group's luciferase expression quantity" is higher than the PGL3-basic group obviously, and transcription activeness presented the phenomenon which between the PGL3-Idl material particle span group and the PGL3-Id1-1 fragment decreased obviously, simultaneously PGL3-E2F-17. PGL3-E2F-26 of E2F flaw active relative PGL3-Idl had obvious reduction. The PGL3-basic group luciferase expression quantity is lower than the Id1 promotor different fragment and PGL3-E2F-17. PGL3-E2F-26. And PGL3-E2F-17. PGL3-E2F-26 active relative Id1 has obvious reduction. This can be seen E2F transcription factor for Id1 promoter length effects may be important, the key factor regulating promoters. The research was clear about transcription factor E2F to regulating the Id promoter active initially for further study and understanding in the mechanism of proliferation relative to Id1 promoters provides a new viewpoint, simultaneously this result also lay the foundation for the next step research on the medicine specificity regulated the Id expression,the RT-PCR result showed that the groups after K9 processes MSC. is opposite in control group's cartilage phenotype related protein SOX9, COIX. the Epiphycan three, six day of expressions strengthens:in SHH pathways Ptch compared with the control, three. six day of expressions strengthen, target gene Glil six day of expression enhancement.ConclusionRats Idl promoter (-1765～1164) is the core control region, lack of E2F transcription factor to influence Idl promoter activity, for Id1 promoter for breakthrough PTE promotes MSC proliferation mechanism provides the basis for further. Idl regulation of gene expression screening drugs provides a new method.K9 inducing MSC to cartilage differentiation and the SHH signal passage possibly participates in which.