An Experimental Study On The Therapeutic Effect Of Transplanted Neural Stem Cells In Aganglionic Megacolon Mouse Model

PartⅠ: Culture and identification of neural stem cells derived from new born mouseObjective: To isolate and culture the purified monoclonal neural stem cells from the cerebral cortex of new born mice. To offer an ideal cell donor for the treatment of Hirschsprung disease with neural stem cells graft.Method: The cerebral cortex of new born mice was isolated and dissociated to single-cell suspension by mechanical trituration. The dissociated single cells were cultured in serum-free medium with B27, bFGF and EGF after trypan-blue dye exclusion test. The growth curve of neural stem cells was draw with MTT assay. After the formation of neurospheres, single-cell clone culture was performed by limiting dilution and the proliferated single-cell clones were harvested for subculture. To evaluate the potential differentiation ability of neural stem cells, the cells were cultured in medium containing 10%fetal bovine serum (FBS). NGF was supplemented into the culture medium to induce the differentiation of the neural stem cells into cholinergic neurone. Immunocytochemistry was used to detect the specific marker of neural stem cells—Nestin of the primary, passage culture and monoclonal neurospheres. The specific antigens NF-200, GFAP and MBP were detected to identify the neurons, astrocyte and oligodendrocyte. The ChAT was detected to identify the positive rate of cholinergic neurone.Results: The dissociated cells formed floating neuraspheres in suspension cultures. The primary and passaged neurospheres expressed Nestin antigen positively. By limiting dilution, we cultured the cell lines from single-cell clone and the monoclonal neurospheres expressed Nestin too. All the neurospheres had capabilities of self-renew, proliferation and the potentiality of differentiation into NF-200, GFAP and MBP positive cells. NGF can induce 15.48% of neural stem cells into cholinergic neurone. The difference between FBS and NGF group had statistical significance.Conclusion: Monoclonal neural stem cells which have the ability of proliferation and multi-directional differentiation can be isolated and cultured from the cerebral cortex of new born mice by limiting dilution. The positive rate of cholinergic neurone can significantly increased by adding NGF into the culture medium. PartⅡ: Study on cotransfection of GDNF and EDNRB into neural stem cells mediated by JetPEIObjective: To investigate the optimizing method for neural stem cells (NSCs) transfection and the expression of extrinsic genes in neural stem cells.Method: neural stem cells derived from new born mouse was cultured in Vitro.Extrinsic genes GDNF and EDNRB were cotransfected into primary cultured neural stem cells by using JetPEI. The expression of green fluorescent protein (GFP) was measured with fluorescence microscope and flow cytometer. The relative expression of GDNF and EDNRB mRNA was detected by RT-PCR.Results: Bright green fluorescence of the transfected cells could be observed underfluorescence microscope after 24h of transfection. Flow cytometer analysis showed that the efficiency of cotransfection was 17.56%,26.38%, 27.53 % in 24h, 48h, 72h respectively. Semi-quantitative RT-PCR confirmed that GDNF and EDNRB mRNA expressed successfully in the neural stem cells.Conclusions: The tartet genes were successfully cotransfected into neural stem cellsby using JetPEI. This provides a feasible technological platform for the polygene therapy of neural degenerative diseases. PartⅢ: Establishment and identification of aganglionosis mouse modelObjective: To establish an aganglionosis mouse model suitable for neural stem cells transplantation study and explore its histopathologic characteristics.Method: 90 male Kunming mice were randomly divided into normal control group, Normal saline (NS) group and benzalkonium chloride (BAC) group. In BAC group, 0.5% benzalkonium chloride was applied onto the serous layer of colon descendens for 15 minutes and normal saline was used instead of BAC in NS group. No treatment was applied to the normal control group. The change of colon in each group was observed by gross anatomy. The observation of histologic characteristics and neuron count was performed by HE staining. Acetylcholinesterase histochemical stain and Immunohistos-taining for NF-200 was used to evaluate the denervation of myenteric nerve plexus. The mRNA expression of NF-200,GFAP,ChAT and nNOS was analyze by semiquantitative RT-PCR.Results: Gross anatomy observation showed that there was no abnormality in normal control group, little conglutination and no stenosis of colon in NS group. In BAC group, the treated segment was presented as stenosis and obstruction. The Proximal part was presented as compensatory enlargement with the retention of excrement. HE staining showed no visible change in normal control group and NS group. No pathological change was observed in the mucous membrane and submucous layer of BAC group. The number of myenteric neurons decreased obviously compared with the other groups and the differences had statistical significance. Immunohistochemical staining showed that there were no NF-200 positive cells in the myenteron. Semiquantitative RT-PCR showed the down regulation of mRNA expression of NF-200,GFAP,ChAT and nNOS in BAC group compared with the other groups and the differences had statistical significance. Conclusion: The aganglionosis mouse model was established by selective chemical ablation of the myenteric nerve plexus of colon descendens. The chemically-induced colonic aganglionosis in this model provides the basis for future studies of neural stem cell transplantation therapy for HD. PartⅣ: Study on the survival and differentiation of neural stem cells in the colonic myenteron of aganglionic miceObjective: To study the survival and differentiation of neural stem cells in the colonic myenteron of aganglionic mice. To elucidate the possibility and the biological significance of intracolonic grafting of neural stem cells (NSCs) as a therapeutic strategy for aganglionosis.Methods: The descending colon serous layer of Kunming mice, 8 weeks old, was treated by 0.5% benzalkonium chloride (BAC) to selectively ablate the myenteric nerve plexus. Neural stem cells derived from the cerebral cortex of neonatal mice was cultured and labeled by Hoechst33342. The labeled NSCs were transplanted into the denervated colon by using the microinjector. The biocharacteristics of mice and the survival and differentiation of grafted cells was observed by gross anatomy, HE staining, immunohistofluorescence and RT-PCR.Results: The primary cultured NSCs were characterized as Nestin positive and can differentiate into neurons and glial cells. HE and immunohistochemistry staining showed that the myenteric plexuses of colon disappeared after treated by 0.5% BAC. The grafted cells were visualized in colon sections under fluorescence microscope. Immunohistofluorescence assay showed that there were Nestin positive cells 7 days after transplantation and NSE, GFAP positive cells 21 days after transplantation. Neurons count showed that there were 137.50 neurons per mm in NSCs group and only 54.00 neurons per mm in NS group. The differences had statistical significance. Semiquantitative RT-PCR showed the up-regulation of mRNA expression of ChAT and nNOS in NSCs group compared with the NS group and the differences had statistical significance.Conclusions: Neural stem cells can survive and differentiate into neurons and glial cells after transplanted into the colonic myenteron of aganglionic mice and partially regulate the neuromuscular modulation of colon. This provides the experimental basis for further studies of neural stem cell transplantation therapy for HD.