The Study Of Differentiation Potential Into Enteric Neuron From Bone Marrow Stromal Cells

Objective : To observe in vitro culturing condition and biological characteristics of bone marrow stromal cells(BMSC) of Sprague-Dawley (SD) rats and identify the cells. Methods : We separated bone marrow of the SD rats and cultured the BMSCs. The morphology of the cells was studied with a phase contrast microscope and the living characteristics after subculture of the cells were observed. We identified the cells by flow cytometric method.Results : Growing clones of BMSCs formed after 3-5d primary culture in L-DMEM containing 20% fetal bovine serum with whole marrow cell culture method, and cells became 90% confluence after10-14d primary culture. The living characteristics of the cultured cells were quite stable till the 6th generation. The morphorlogy of BMSCs were mainly spindle shape. Diploid time of BMSCs was about 4d, and 90% confluence time was about 10d.The 6th generation of BMSCs showed high purity— BMSC marker CD90 positive(99.7%) and hematopoietic cell marker CD45 negative on flowcytometry.Conclusion : Cultivation in L-DMEM containing 20% fetal bovine serum with wholemarrow cell culture method was a easy and quick way to isolate and culture BMSC, andthe BMSCs harvested in this way had high purity after subculture.Part II Enteric neuron differentiation from rat bone marrowstromal cells in vitroObjective To explore the differentiation potential of bone marrow stromal cells to enteric neuron in vitro and to seek proper induction methods.Methods BMSC were harvested from male rats and cultured in DMEM supplemented with 20% fetal bovine serum, and characterized by flow cytometry. At passage 6, BMSC were pre-induced by bFGF (10ng/ml) for 24h, then induced in six groups: 1)GDNF group glial cell line-derived neurotrophic factor (GDNF, 10ng/ml) in fetal gut condition medium (FGCM) for 10d; 2) VA group Vitamin A acid(VA), zinc in FGCM for 10d; 3) GDNF, VA, Zn, FGCM induction group; 4) GDNF only group; 5) FGCM only group; 6) Control group. The expressions of neuronal markers NSE (neural specific enolase), NF(neurofilament), and glial cell marker GFAP (glial fibrillary acidic protein) , enteric neuronal marker PGP9.5, nNOS were detected by fluorescent immunohistochemistry method.Results 1)The cultured BMSCs were CD90 positive and CD45 negative on flow cytometry. 2)After 10 days of induction, a certain number of cells adopted neuron-like morphological changes and showed the expressions of NSE , NF , PGP9.5 and nNOS without the expression of GFAP by fluorescent immunohistochemistry method in all the first five groups. 3)In GDNF group, the positive rate of NF , PGP9.5 and nNOS was higher than that in VA group, GDNF only group and FGCM only group significantly(p<0.05). 4) The differentiation effects of GDNF had quantity-effect and time-effect relation. 5) After induction agents withdrawed, enteric neuron-like cellswere diminished.Conclusion BMSC can be induced to differentate into enteric neuron like cell in vitroby different methods, and GDNF with FGCM can induce higher rate of enteric neuronlike cells compared with VA etc. After induction agents withdrawed, enteric neuron-likephenotype couldn't sustain for long time.Part III The expression of neurotransmittors after enteric neurondifferentiation from rat bone marrow stromal cells in vitroObjective To explore the expression and screation of enteric neurotransmitters after enteric neuronal differentiation from bone marrow stromal cells.Methods BMSC were harvested from male rats and cultured in DMEM supplemented with 20% fetal bovine serum, and characterized by flow cytometry. At passage 6, BMSC were pre-induced by bFGF (10ng/ml) for 24h, then induced in two groups: 1)GDNF group glial cell-line derived neurotrophic factor (GDNF, 10ng/ml) in fetal gut condition medium (FGCM) for 10d; 2) VA group Vitamin A acid(VA), zinc in FGCM for 10d.The expressions of neurotransmittors NOS(nitric oxide synthase), VIP(vasoactive intestinal polypeptide), ACh (acetylcholine)and SP(substance P) were detected by fluorescent immunohistochemistry method. And the concentration of nitric oxide(NO) in the culture medium was also detected by nitrate reductase method. Results 1) After 10 days of induction, a certain number of cells adopted neuron-like morphological changes and showed the positive expression of nNOS and VIP, as while as low level expressin of SP and ACh by fluorescent immunohistochemistry method in both groups; the positive rate of nNOS and VIP was higher in GDNF group than that in VA group. 2) NO was detected in both groups; the concentration of NO was higher after 10d induction than that after 6d induction; and the concentration of NO was higher inGDNF group than that in VA group.Conclusion BMSC could be induced to differentiate into enteric neuron in vitro bydifferent methods, and expressed neurotransmittors.Part IV The change of expression of neurotrophic factors— glial cell line-derived neurotrophic factor and nerve growth factor inrat bone marrow stromal cells before and after inductionObjective To investigate the change of expression of neurotrophic factors- glial cellline-derived neurotrophic factor(GDNF) and nerve growth factor(NGF) in rat bonemarrow stromal cells(BMSC) before and after induction.Methods BMSC were harvested from male rats and cultured in DMEM supplementedwith 20% fetal bovine serum, and characterized by flow cytometry. At passage 6,BMSC were pre-induced by bFGF (10ng/ml) for 24h, then induced withGDNF(10ng/ml) or Vitamin A acid, Zn in fetal gut condition medium (FGCM) for 10days. The expressions of enteric neuronal markers were detected by fluorescentimmunohistochemistry method. The expressions of mRNA of GDNF and NGF weredetected by RT-PCR method.Results (1) After 10 days of induction, a certain number of cells adopted neuron-likemorphological changes and showed the expressions of enteric neuronal markers byfluorescent immunohistochemistry method. (2) BMSC express low level NGF andGDNF mRNA, while after induction of GDNF in FGCM the expression of NGF andGDNF mRNA were highly increased.Conclusion GDNF can not only induce enteric neuronal differentiation of BMSC, butalso increase the expression of NGF and GDNF significantly.Part V The study of the mechanism of enteric neuronaldifferentiation of bone marrow stromal cellsObjective To explore the mechanism of enteric neuronal differentiation ofBMSC(bone marrow stromal cell).Methods Fetal gut cells were primarily cultivated in vitro, and the expression ofneurotrophic factors GDNF(glial cell line-derived neurotrophic factor) andNGF(nerve growth factor) was detected by RT-PCR, then fetal gut condition mediumwas made(FGCM). The effect of antibody of GDNF receptor α1(GDNFRα1) on theenteric neuronal differentiation of BMSC and the change of the exptression of RETmRNA were observed.Results Fetal gut cells in primary culture expressed neurotrophic factors GDNF andNGF. After induction of BMSCs with GDNF and FGCM, BMSCs differentiated intoenteric neuron-like cell, and the expression of RET mRNA upregulated. GDNFRα1antibody inhibited the effects of GDNF and FGCM on BMSCs differention, anddownregulated the expression of RET mRNA.Conclusion GDNF maybe induce BMSCs differentiate into enteric neuron-like cellthrough upregulation of RET and activation of GDNF-RET pathway, and FGCM mayprovide enteric neuronal differentiat micro-environment including enteric neurotrophicfactors.