The Study Of Culture In Adipose Derived Stem Cells And The Expression Of The ACTIN In The Induction Procedure For Neuron

Background and Objective: The discovery of neural stem cells(NSCs) has brought us into an epoch of neural restoration and there is agreat progress of the transplantation therapy for the central nervoussystem disease. However, NSCs is greatly limited because large stemcells from abortive fetus whose source is limited and can give rise toethical controversy. Therefore, it is very important to find new sources forstem cells.Mesenchymal stem cells (MSCs) are derived from single layer ofextraembryonic mesoderm. They are characterized by being muti-potential and capable of self renewal. With suitable circumstances, MSCshas the capability of being induced to various kinds of tissues, includingmesoderm and even ectoderm tissues. As a new kind of seed cell, theapplication perspective is broad and the ethical problem has greatlydecreased. MSCs were initially identified in postnatal bone marrow.However, for clinical use, the isolation of such stem cells from individualis invasive and other sources such as umbilical cord blood cells, amnioticderived cells are much finite. Thus, it is meaningful to find new cell replacement without ethic issue, easily accessible and high-yield for theexperimental research and clinical application of the MSCs.Fat tissue could be easily derived and the resource is abundant. Thecontent of the stem cells in fat tissue is much higher than in bone marrow.Therefore, as an important resource of stem cells, adipose derivedmesenchymal stem cells (ADMSCs) have a important value for researchand application. In this research, ADMSCs which is derived from foldinguen fat tissue of the S-D rat is cultivated and induced to neutral cells.Through the investigation, approaching the method of the cultivation andinducing to neural cells in vitro and the function of the actin in thisprocess. Future, making a foundation for the next step of researchingMSCs.Methods: 1.ADMSCs were derived from the fold inguen fat tissueof the S-D rat. Isolated cells were maintained in L-clucose DMEMmedium supplemented with 10% fetal bovine serum (FBS) and 2mmol/LL-glutamine and in 37℃incubator.2. ADMSCs which were cultivated for five generations beimplanted in cell cultivation plate. Giving preparing- antileptic (L-clucoseDMEM/10%FBS/10ug/ml bFGF) when the cells got 60—70% moreor less. Being cultivated for 24h then observed the cell morphous.3. Part of the cells were identified by immunofluorescence with nestin antibody. Others were given antileptic (L-clucose DMEM/DMSO/6mmol/L BME), and DMSO with different dose. Being cultivated for 3h,5h, 7h,then terminated the growth separately. The cells were identified byimmunocytochemistry with actin antibody and by immunofluorescencewith nestin, neuron specific enolase (NSE) antibodies respectively.Results: 1. The adipose derived cells begun to attach in 24h. Whenobserved with phase contrast microscope at 72h, there is paratomy andthe cells displayed in regular spindle shape, the nucleus in centre, enationand 1-2 nucleoles could be seen. The cells growed in one layer anddisplayed with orientation after one week. The adipose derived cellsreached confluence about 80%-90% after one week. After passage, thecells achieved complete confluence at 3-4 d. The red blood cells werecleared during passages. After reaching to 5 th generation, cells grewactively and still displayed in spindle shape. After reaching to 8th-10thgeneration, cells grew very slowly. Most cells became fiat shape andaged.2. Giving preparing- antileptic (L-clucose DMEM/10%FBS/10ug/ml bFGF) to 5th generation cells, nestin were positive. Others weregiven antileptic (L-clucose DMEM/DMSO/6mmol/L BME), nestin,NSE and ACTIN were positive. With the different cultivated period,nestin positive cells decreased and NSE positive cells increased. The cells were identified by immunocytochemistry with ACTINantibody, and cells with small shapes and small enations after 3 hours.Positive substance were in nucleus and cytoplasm. With thedifferentiation period prolonged, ACTIN were positive in enations also. Itcan be identified that the actin proteinum make a important role to growthand development of the neuron.Conclusions: 1. ADMSCs were successfully isolated from the foldinguen fat tissue of the S-D rat by being treated with collagenaseⅠ, andwere maintained in L-clucose DMEM medium supplemented with 10%fetal bovine serum (FBS) and 2mmol/L L-glutamine and in 37℃in vitro.2. There were neural precursor cells when the ADMSCs wereinduced with bFGF, and cells could differentiate to neurocytes.3. The ADMSCs are capable of differentiating into neuro-like-cellsinduced by bFGF, DMSO and BME in vitro.4.The expression of actin proteinum make a important role to thegrowth and development of the neuron..