Fetal Mesenchymal Stem Cells Derived From Amniotic Fluid Into Epidermic Cells In Vitro During Mid-pregnancy And The Role Of Hydroxyl Fasudil

Objective: The osteosarcoma is one of the most common malignant tumors among young people.lt is easy to palindromia and metastasize. The fatality rate and mutilation rate are exceeding high. It is severely detrimental to people's health. The occurrence and development of osteosarcoma is closely concerned with multiple oncogenes as well as anti-oncogenes. Apoptosis closely affect the occurrence of osteosarcoma, and The anti- apoptosis gene Bcl-2 and Bag-1 may are most important action in the occurrence, development, anticipation and prophylaxis of osteosarcoma. Most studies only detect the relationship of the expression of the single indicator Bcl-2 with the development of osteosarcoma ,but local authority has not been reported about the relationship of bag-1 in osteosarcoma and the interreaction relationship of Bcl-2 and Bag-1 in osteosarcoma . The research was to detect the expression of Bcl-2 and Bag-1 in osteosarcoma tissues using immunohistochemical method (SP), and to analyze the separate and associated expression of Bcl-2 and Bag-1 in osteosarcoma. Research the expression and the interreaction relationship to find a safety molecular indicatrix for patients to demonstrate the clinical Pathology characteristic, to provid theory evidence for Part I :The Culture in Vitro of Fetal Mesenchymal Stem Cells Derived from Amniotic Fluid and the induction into Epidermic like CellsObjective:To observe the possibility of the induction of fetal mesenchymal stem cells (MSCs) derived from amniotic fluid into epidermic cells during mid-pregnancy.Method: Chose pregnant women who accepted prenatal diagnosis and induction, obtained ten Amniotic fluid of pregnant women whose pregnant weeks were 16~22 by ultrasound -guided amniocentesis.Mechanical dissociate the cells and then passage culture in vitro. Generated, amplificated and amplificated to the 1st~3rd generation after culture and proliferation.Used a flow cytometry to detect surface antigen expression.Chose the 3rd generation MSCs from fetus which grew well.Used culture solution B(70% culture solution A+30% supernatant fluid from fibroblast medium+10 ng/LEGF) to induce the fetal MSCs differentiate into epidermic cells。Chose fetal MSCs which were continuous passage cultured by solution A as control roup.Observed the change of cells morphology structure after induction through inverted phase contrast microscope.After 20d, detect the express of CK19 by immunohistochemistry.Result: fetal MSCs into epidermic cells during mid-pregnancy generate fast in the culture in vitro. After 3d, we could see a single attached cell or a few colony through inverted phase contrast microscope,most of them were almost shuttle-shaped or needle tip-shaped. A part of them were round or polygon, the cell nucleus were in the middle,we could also see the nucleoli. After 8d, these were sporadic MSCs colony of fibroblas, the main of which were spindle cells,and epithelioid cell colony, the main were polygonal cells, every colony didn't contact each other. Mechanical chose fetal MSCs colony and inoculation culture, the cells grew homogeneous distribution, the shape of which were Fusiform, apiciform or Stellate.After 7~8d, the number and size of colony increase obviously, the cells ranked directivity, which were turbulent, reticulate or reticulate, and the fusion rate up to 90%, the were no obviously limit between each other. Fetal mesenchymal stem cells derived from amniotic fluid are: CD44 (+), CD34(-).After 20d Inducing cultured with culture solution B(70% culture solution A+30% supernatant fluid from fibroblast medium+10 ng/LEGF), the cells which were fusiform fibroblas became short gradually, and they ranked tight .Identify the structure by cytochemistry, there were the express of CK19,β1.Conclusion: 1 Fetal MSCs can separate in vitro, purify culture, grow stable, generate fast and serial passage.2 Fetal MSCs have the tendency to differentiate into epidermic cells under the culture with supernatant fluid from fibroblast mediumand EGF in vitro.Part II: The effect of hydrochloric Fasudil to differentiation of epidermic cells from fetal mesenchymal stem cells derived from amniotic fluid.Objective:To observe the effect of different concentrations of hydrochloric Fasudil to differentiation of epidermic cells from fetal mesenchymal stem cells derived from amniotic fluid. Hydrochloric Fasudil is blocking agent of Rho molecular signal path.Method: According to the method in the first experiment,we got amniotic fluid at mid-pregnancy and then cultured and passaged the cells in vitro. We chose the 3 rd generation of MSCs from fetus which grew well and divided them into 5 groups. Group 1 was continuously cultured for passsaging by culture solution A(90%L-DMEM+10%FBS+p enicillin100U/ml+phytomycin 100μg/ ml). As to group 2,3,4,5,we washouted solution A with sterile PBS and respectively used substitute as follows: culture solution B (70%culture solution A +30%clear supernatant liquid from fibroblast nutritive medium +10 ng/LEGF) ; culture solution C: (HA1077 culture solution B with a concentration of 10μmol/L); culture solution D (HA1077 culture solution B with a concentration of 20umol/L); culture solution E: (HA1077 culture solution B with a concentration of 40μmol/L).Puted them into incubator for culture and changed their culture solution every 2~3 days until the fusion rate rase up to 90%,and then we digested them with 0.25%parenzyme -lmmol EDTA to passage them by 1: 2.We cultured 3 generations.Group 1 was blank group, group 2 was single induced group. We observed the cell morphous and groweing status in every group regularly.20 days after inducted,we detected the expression of antibody of CK19 with flow cytometry in group 1~5. Group 2 and 4 were detected for RT-PCR.Result:The positive rate of CK19 in group 1 detected with flow cytometry was 0.37% on 20d,and group 2 was 3.08%.We observed the 3 rd fetus MSCs on 20d,which was induced respectively with solution C and E.The cells which are fusiform fibroblas become short gradually, and they rank tight . Identify the structure by cytochemistry, the positive expression of CK19 was 2.93%and 3.02%.There were changes in cell morphous 18d induced dy solution D,which occurred earlier than the blank group. Identified the structure by cytochemistry on 20d, the positive expression of CK19 was 3.40%. As to group 2 and group 4 detected with RT-PCR,we observed the tendency to differentiate into epidermic cells in Group 4 was trengthened. Conclusion: Different concentrations of hydrochloric Fasudil (HA1077 ) can influence the differentiation of epidermic cells from fetal mesenchymal stem cells derived from amniotic fluid.