Preliminary Study Of GDNF And NT-3 Modified Bmscs Transplantation For Treatment Of Hirschsprun's Discase In Vivo

Objective: To establish a simple and reliable method for isolation, culture of rat bone mesenchymal stem cells(BMSC) in vitro.Methods: MSCs were isolated and culture by the attachment culture method. The morphologic changes of the cells were observed under an inverted lightmicroscope and the growth curve of the cell was determined by MTT assay. The expression of the BMSC marker CD90,CD44 and hematopoietic marker CD45 were detected by immunohistochemisty assay。Results: The cultured BMSCs were shuttle or polygonal shape, The size of rBMSCs was homogeneous and showing whirlpool-shape. The cell showed positive expression of CD90 and CD44, but no expression of CD45 by immunohistochemisty assay.Conclusions: BMSC can be successfully isolated,cultured and purified by the attachment culture method in vitro,and to provide the experimental basis for the clinical application of tissue engineeringPart II Identification and establishment of the rat model with experimental congenital megacolonObjective: To establish a rat model of experimental congenital megacolon on purpose to provide the experimental basis for stem cell transplantation.Methods: One hundred ten SD rats, weighted about200±15g, were randomly divided into two groups: Test group ( n =55) and control group ( n = 55) .In treatment group, 5 mL/L-1 benzalkonium chloride (BAC) was applied into the colon for 45 minutes by transanal method and 9mL/L-1 saline was applied in the control group. The models were examined through gross observation , colon manometry , AchE histochemistry , HE staining,neural specific enolase(NSE) and protein gene product 9.5(PGP9.5) immunofluorescence and the expression of AchE ,GDNF ,NT-3 by RT - PCR at the end of the 1st, 2nd , 4th and 8th weeks post-operation respectively.Results: After one week of BAC treatment, the rats in test group began to lose appetite and abdominal distention. Manometry showed the abolition of reflex contraction in the treatment group. Autopsy revealed a narrow segment and marked dilation of the proximal segment. Histologic examination showed lack of of ganglion cells at the site of BAC treatment. The mRNA expression ofAchE, GDNF and NT-3 decreased dramatically after BAC treatment. No visible change in bowel was observed in the control group.Conclusion:Per anum application of 5 mL/L-1 BAC thus successfully produced a aganglionic segment of bowel in normal rat.. The method has satisfactory stability and repeatability.This model provides the basis for future studies to investigate the pathophysiology of Hirschsprung's disease(HD).Part III Construction and identification of eukaryotic expression vector for pEGFP-GDNF-NT-3Objective: To construct the eukaryotic co-expression vector containing both GDNF and NT-3.Methods: The GDNF and NT-3 cDNA was acquired from neonatal rat cortex of cerebrum by RT-PCR. The products were cloned into an eukaryotic expression plasmid pEGFP-N1 , The constructed plasmid pEGFP-EGFP-GDNF-NT-3 was confirmed by double enzyme digestion and DNA sequencing .Results: The DNA fragment amplified by PCR from the total RNA was identical to GDNF and NT-3 from the gene library. The inserted target fragment was consistent with the target gene GDNF and NT-3 by sequence analysis,The identical DNA fragment was also amplified from the recombinants by double enzyme digestion, and the eukaryotic expression vector pEGFP-EGFP-GDNF-NT-3 was successfully constructed.Conclusions: The pEGFP-EGFP-GDNF-NT-3 eukaryotic co-expression vector has been successfully constructed.Part IV Expression of pEGFP-EGFP-GDNF-NT-3 eukaryotic expression vector in rat bone marrow mesenchymal stem cells and the induction of NeuronObjective: To investigate the expression of glial cell line-derived neurotrophic factor (GDNF) and neurotrophin-3(NT-3) in rat bone marrow mesenchymal stem cells (MSCs) and the feasibility of differentiate into neuron-like cells.Methods: BMSCs isolated by the whole bone marrow culture, they were characterized by flow cytometry of CD90 and CD45. BMSCs were transfected by GDNF and NT-3 gene, the expression of green fluorescent protein (GFP) and morphological change was measured with fluorescence microscope. The expression of neuronalmarkers NSE ( neural specific enolase) , NF ( neurofilament) and glial cellmarker GFAP ( glial fibrillary acidic protein) were detected by Immunofluorescence assay. The change of GDNF and NT-3 protein expression was quantified with western blot.The control group was BMSCs without GDNF and NT-3 gene.Results: BMSCs were cultured and purified in vitro. The cultured BMSCs were positive for CD90 (92. 7%) and negative for CD45 on flow cytometry. After the induction of differentiation, BMSCs became round or cone-shaped with distinctive outgrowth of protrusions. These cells revealed neuron-like morphological changes,and most of these cells were intertwined into a network structure . Immunofluorescence assay showed positive expression of NSE and NF without the expressions of GFAP in experimental group, while the cells showed no expressions of NSE,NF and GFAP in control group. The expression of GDNF and NT-3 protein was highly increased.Conclusions: The study indicates that recombinant plasmid pEGFP-EGFP-GDNF-NT-3 can express in BMSCs. The BMSCs transfected with GDNF and NT-3 were able to differentiate into neuronal-like cells and express nerve markers. This study provides an experimental basis for gene therapy to treat nervous system-related disorders.Part V Differentiation of GDNF and NT-3 Dual Gene-modified Rat Bone Marrow Mesenchymal Stem Cells into Enteric Neuron-like CellsObjective: The purpose of our study was to investigate the feasibility of using in vitro induced enteric neuron-like cells, differentiated from rat BMSCs, for the treatment of Hirschsprung's disease (HD).Methods: BMSCs isolated by the whole bone marrow culture, They were characterized by flow cytometry of CD90 and CD45. At passage 5, BMSCs were transfected with eukaryotic expression plasmids coexpressing GDNF and NT-3, differentiation induced in fetal gut culture medium (FGCM), The control group was BMSCs without GDNF and NT-3 gene. The expression of green fluorescent protein (GFP) and morphological change was measured with fluorescence microscope;The expression of the neuronal marker NSE(neural specific enolase,NSE), glial cell marker GFAP (glial fibrillary acidic protein,GFAP) and the enteric neuronal markers PGP9.5(Protein gene production 9.5,PGP9.5),VIP(vasoactive intestinal peptide,VIP) and nNOS(nitric oxide synthase,nNOS) were detected by Immunofluorescence assay.To detect the expression of mRNA of GDNF and NT-3 by RT-PCR .Results: BMSCs were cultured and purified in vitro. The cultured BMSCs were positive for CD90 (92. 7%) and negative for CD45 on flow cytometry. The transfected cells displayed neuron-like changes after the induction of differentiation, Immunofluorescence assay showed positive expression of the neuronal marker NSE and the enteric neuronal markers PGP9.5, VIP and nNOS, but no expression of GFAP in experimental group, while the cells showed no expressions of NSE,PGP9.5,VIP,nNOS and GFAP in control group. RT-PCR revealed successful expression of GDNF and NT-3 in transfected BMSCs.Conclusions: Therefore, the present stud y indicates that genetically modified BMSCs coexpressing GDNF and NT-3 were able to differentiate into enteric neuronal cells and express enteric nerve markers when induced with FGCM. This study provides an experimental basis for gene therapy to treat enteric nervous system-related disorders, such as Hirschsprung's disease.PartⅥPreliminary study of GDNF and NT-3 modified BMSCs transplantation for treatment of Hirschsprun's discase in vivo Objective: To study the survival and gene expression of transplanted GDNF and NT-3 modified BMSCs in rat colonic myenteron with experimental aganglionosis. To investigate the feasibility of the treatment of experimental aganglionosis by GDNF and NT-3 modified BMSCs transplantation in vivo.Methods: BMSCs isolated by the Adhesive-screening method ,and were transfected with eukaryotic expression plasmids coexpressing GDNF and NT-3. GDNF and NT-3 modified BMSCs were transplantated into the denervated colon by microinjection, . Macroscopic,HE staining,AchE histochemistry,protein gene product 9.5(PGP9.5) and VIP immunofluorescence and the expression of RET ,GDNF ,NT-3 by RT - PCR at the end of the 1st, 2nd , 4th and 8th weeks post- transplantation respectively.Results: 1.BMSCs were cultured and purified by Adhesive-screening method. 2. BMSCs able to differentiate into enteric neuronal-like cells by genetically modified in vitro 3. After one week of 0.5%BAC treatment, Histologic examination showed lack of ganglion cells at the site of BAC treatment. Immunohistofluorescence assay showed that there were positive cells of PGP9.5,VIP at 1w, 2w and 4w after BMSCs transplantation. No positive cells were observed after PBS transplantation. The mRNA expression of RET, GDNF and NT-3 increased dramatically in test group as compared to the control group .Conclusions: GDNF and NT-3 modified BMSCs can survive and expression elated genes in the denervated colon of experimental aganglionosis rat, partially recovering the neuromuscular modulation of colonic This study provides an experimental basis for cell transplantation therapy to treat Hirschsprung's disease.