| Part ?Experimental Study of Human Umbilical Cord Mesenchymal Stem Cells Combined with Human Neural Stem Cells Transplantation in the Treatment of Spinal Cord Injury in RatsBackgroundSpinal cord injury(SCI)is a severe complication of spinal fracture and dislocation,which often leads to severe disorders of sensory,motor and other somatic functions below the injured segment.Therefore,SCI not only has great impacts on the body and mind of the patients,but also imposes a heavy burden on their families and society.At present,there is no satisfactory method to treat or cure the disease.Mesenchymal stem cells(MSCs)belong to pluripotent stem cells with self-replication and multiple differentiation potential,which can secrete a variety of neurotrophic factors and cytokines and have immunomodulatory,anti-apoptosis and anti-inflammatory effects.In addition,MSCs can also promote the survival of neurons and promote nerve regeneration.Neural stem cells(NSCs)are a kind of stem cells with self-replication and a variety of neuronal differentiation potential,which can promote the repair and reconstruction of damaged neural pathways after SCI.However,due to the inflammatory environment of the injured spinal cord,the survival rate of the transplanted NSCs is low.Previous studies have shown that MSCs can regulate the microenvironment of neural stem cells and improve their survival rate.In addition,many studies have shown that MSCs can reduce tumorigenesis associated with stem cell transplantation.In this study,we used the combined transplantation of human umbilical cord mesenchymal stem cells(hUC-MSCs)and human neural stem cells(hNSCs)to treat SCI in rats in order to find a better way to treat SCI and explore its intrinsic mechanisms.In addition,the transplantation of hUC-MSCs and hNSCs could further verify the safety and effectiveness of hUC-MSCs and hNSCs transplantation,and provide the necessary verification and exploration for the clinical application of the cell transplantation in the future.Objective(1)To investigate the effects of combined transplantation of hUC-MSCs and hNSCs in the treatment of spinal cord injury in rats.(2)To study the effects of hUC-MSCs and hNSCs transplantation alone in the treatment of spinal cord injury in rats and compare their effects.(3)Immunohistochemistry,immunofluorescence,ELISA assay and LFB-CV staining were used to explore the mechanisms of hUC-MSCs and hNSCs transplantation in the treatment of spinal cord injury in rats.Methods(1)Isolation,culture and identification of hUC-MSCsFresh umbilical cord was obtained during cesarean section in healthy parturients with 38 to 40 weeks of gestation.HUC-MSCs were cultured by tissue block adherent method.The Wharton's jelly separated from the umbilical cord was cut into small pieces and transferred into the cell culture flask and cultured in the DMEM complete medium with the corresponding additives.The cells were cultured at 37 C in the incubator containing 5%CO2.After 7 to 10 days,the tissue blocks were removed,the adherent cells were re-laid,and the solution was changed every 3 days.After 3-5 passages,the cells were collected and prepared for transplantation.At the same time,the cell surface specific markers were identified by flow cytometry.After osteogenic and adipogenic induction,the cells were identified by Alizarin Red S staining and Oil Red O staining for differentiation ability detection.(2)Isolation,culture and identification of hNSCsForebrain tissues were obtained from 8-to-10-week-old fetuses via routine abortions.The cells were extracted by mechanical separation,added to the serum-free medium of NSCs and cultured at 37? in the incubator with 5%CO2.The neurospheres were formed after changing the culture medium twice a week for 7 to 10 days.After 3-5 passages,NSCs were obtained for transplantation.The cell specific markers were identified by immunofluorescence.After induction,the cell differenciation ability was identified by immuno:fluorescence with anti-?-tubulin?and anti-GFAP antibody.(3)Experimental grouping,establishment of SCI model and cell transplantationIn this study,the NYU impactor was used to establish the model of moderate SCI in adult female Wistar rats.The rats with BBB score<2 on the second day after SCI were selected.In this study,108 rats with SCI were selected.They were randomly divided into the following five groups:1)the hUC-MSCs group;2)the hNSCs group;3)the hUC-MSCs+hNSCs group;4)the PBS group(control group);5)Sham group.We performed intramedullary stem cell transplantation in T10 segment one week after SCI in rats.(4)Data detectionAll animals were evaluated by BBB score one day before and after SCI and then weekly until the end of the experiment.Two weeks after cell transplantation,some rats were sacrificed and spinal cord tissues were extracted.Then frozen sections were made and immunofluorescence and immunohistochemistry were performed to observe and evaluate the survival and differentiation of the transplanted stem cells and the expression of BDNF in injured spinal cord.Two weeks after cell transplantation,some rats were sacrificed and fresh spinal cord tissues were extracted and homogenized,then the expression of BDNF was detected by sandwich ELISA method.At the completion of the eight-week study,the experimental animals were sacrificed and the spinal cord tissues were extracted,then paraffin sections were made and Luxol Fast Blue-Cresyl Violet staining was performed to detect the number of myelin sheath and motor neurons in the anterior horn of the spinal cord.Results(1)Cell culture and identificationHUC-MSCs was successfully isolated and cultured from Wharton' s jelly by tissue block adherent culture.The surface markers of the third generation of cells were detected by flow cytometry,and the results showed high expressions of CD44,CD90,CD 105;the expressions of CD34 and CD45 were negtive.After cell differentiation induced by osteogenetic and adipogenic medium,both cell Alizarin red S staining and oil red O staining showed positive results,indicating that the cultured hUC-MSCs had the ability of osteogenetic and adipogenic differentiation.We successfully isolated and cultured hNSCs from fetal forebrain tissues via routine abortions.Further immunofluorescence staining showed that the cell markers(Nestin and SOX2)were positive in hNSCs.After cell differentiation induction,the immunofluorescence showed the positive expressions of ?-tubulin? and GFAP,confirming that the cultured cells had the ability to differentiate into neurons and astrocytes.(2)Results of BBB scoreFrom the second week after SCI to the end of the experiment,the BBB score in the hUC-MSCs+hNSCs group was significantly higher than that in the other three treatment groups.The BBB score in the hNSCs group was significantly higher than that in the PBS group from 4 weeks after SCI to the end of the experiment.At the fifth week after SCI,the BBB score in the hUC-MSCs group increased rapidly and displayed significant difference with the PBS group,and this trend continued until the eighth week(p<0.01).In the whole course of the experiment,there was no significant difference in BBB score between the hUC-MSCs group and the hNSCs group.(3)Survival and differentiation of the transplanted stem cellsThe survival number of stem cells in the hUC-MSCs+hNSCs group was significantly higher than that in the hNSCs group(p<0.05)and the hUC-MSCs group(p<0.01)at two weeks after cell transplantation.No HuNu positive cell was found in the PBS group(negative control group).In addition,there was no significant difference between the hUC-MSCs group and the hNSCs group.In the hUC-MSCs group,no double labeled HuNu-GFAP,HuNu-?-tubulin? or HuNu-CNP antibody positive cells were observed,indicating that the transplanted stem cells did not differentiate into neurons,astrocytes and oligodendrocytes.The double-labeled positive cells were observed in both the hNSCs group and the hUC-MSCs+hNSCs group,indicating that the stem cells transplanted in these two groups could differentiate into neurons,astrocytes and oligodendrocytes.According to the immunofluorescence images,we could also observe that the survived hUC-MSCs were more more converged and did not integrate well with the host.However,the transplanted stem cells in the hNSCs group and the hUC-MSCs+hNSCs group dispersed and integrated with the host tissues very well.(4)Immunohistochemistry and ELIS A testTwo weeks after stem cell transplantation,the expression of BDNF detected by immunohistochemistry in the three stem cell-treated groups was significantly higher than that in the PBS group with statistically significant differences(PBS group vs hUC-MSCs+hNSCs group,p<0.01;PBS group vs hUC-MSCs group and hNSCs group,p<0.05).However,there was no significant difference between the three stem cell-treated groups.The results of ELISA assay of BDNF expression in spinal cord homogenate were consistent with the results of immunohistochemistry.(5)Examination of myelin sheath and anterior horn motor neurons in the spinal cordAt the eighth week after SCI,Luxol Fast Blue-Cresyl Violet staining showed large amounts of cell debris,degenerated axons,and cavities in the transverse spinal cord sections of the injured groups.The amount of myelin sheath(per slide)evaluated by the I.O.D.in each of the three stem cell-treated groups was larger compared with that in the PBS group(hUC-MSCs+hNSCs group vs PBS group,P<0.01;hUC-MSCs group and hNSCs group vs PBS group,P<0.05),and the amount of residual myelin sheath in the hUC-MSCs+hNSCs group was the largest of the three(hUC-MSCs+hNSCs group vs hUC-MSCs group,P<0.01;hUC-MSCs+hNSCs group vs hNSCs group,P<0.05).The numbers of motor neurons in the anterior horn of the gray matter in the three stem cell-treated groups was significantly higher than that in the PBS group(hUC-MSCs+hNSCs group and hUC-MSCs group vs PBS group,p<0.01;hNSCs group vs PBS group,p<0.05).The numbers of motor neurons in the anterior horn of the spinal cord in the three stem cell-treated groups was significantly lower than that in the sham group(p<0.01),but no statistically significant differences were found between the three groups.Conclusion(1)This study showed that the intramedullary transplantation of hNSCs,hUC-MSCs or hUC-MSCs+hNSCs in the subacute stage of SCI could promote the hindlimb functional recovery in rats with SCI,and the result of the combined transplantation group was the best.(2)Combined transplantation of hNSCs and hUC-MSCs could promote the survival of the transplanted stem cells.(3)HNSCs,hUC-MSCs or hNSCs+hUC-MSCs transplantation could promote the secretion of BDNF in the injured spinal cord.(4)HUC-MSCs could not differentiate into neurocytes after transplantation alone in vivo,while hNSCs could differentiate into neurons,astrocytes and oligodendrocytes after transplantation.(5)HNSCs,hUC-MSCs or hNSCs+hUC-MSCs transplantation could increase the numbers of myelin sheath and anterior horn motor neurons in the injured spinal cord.Combined transplantation had the best results on increasing the numbers of myelin sheath.Part ?Experimental Study of Human Neural Stem Cells Transplantation Combined with Electrical Stimulation in the Treatment of Spinal Cord Injury in RatsBackgroundSpinal cord injury(SCI)refers to the destruction of spinal cord tissues by various factors,resulting in temporary or permanent changes in somatic nerve function.Once the spinal cord is severely injured,it will be very difficult to repair.At present,no methods have been found to cure SCI completely.The treatment of SCI is mainly focused on two aspects,neuroprotection and nerve regeneration.Neuroprotection can prevent or slow down the secondary damage after nerve injury,and nerve regeneration is aimed at restoring the interrupted nerve pathway and nerve function.Stem cell transplantation is a research hotspot in the treatment of SCI.Because stem cells can differentiate into various types of cells lost after SCI and secrete many nutritional factors to regulate the local inflammatory environment of SCI,so stem cell transplantation is considered to be a promising approach to replace damaged spinal cord cells and promote neuroprotection and nerve regeneration in the future.As seed cells for spinal cord regeneration,neural stem cells(NSCs)have their unique advantages.SCI destroies communication between the brain and the body,causing the brain to lose control of the intact somatic neuromuscular system.At present,researchers have developed a number of neural prostheses to restore part of the body function,strengthen muscle strength and alleviate its atrophy through functional electrical stimulation of the central and peripheral nervous system.It has been found that the electrical stimulation of the injured spinal cord can also improve the local blood circulation,reduce the local endogenous current,maintain the stability of the cell membrane,activate the endogenous nerve regeneration and so on.Because of the complex pathophysiological process of SCI,it is difficult to have satisfactory curative effect by single treatment.The combination of multiple methods of treatment is currently a trend to repair the SCI.Our previous studies have confirmed that MSCs combined with electrical stimulation is superior to cell transplantation or electrical stimulation alone in the treatment of spinal cord injury.Regarding the treatment of spinal cord injury by human neural stem cells(hNSCs)combined with electrical stimulation(ES),there have been no reports at home and abroad.We conducted this study to find a better way to treat spinal cord injury and explore its internal mechanisms,laying the foundation for the future cure of spinal cord injury.Objective(1)To study the therapeutic effects of hNSCs transplantation combined with ES on rats with SCI.(2)To explore the effects of hNSCs transplantation and ES on the repair of SCI in rats and compare the two methods.(3)Immunohistochemistry,immunofluorescence and tissue western blot were used to investigate the pathophysiological mechanisms of hNSCs and ES on the repair of SCI.(4)To reveal the effects of ES on the biological behavior of transplanted hNSCs such as survival and differentiation into neurons in the injured spinal cord.Methods(1)Isolation and culture of hNSCsForebrain tissues were obtained from 8-to-10-week-old fetuses via routine abortions.The cells were extracted by mechanical separation,and then added to the serum-free medium of NSCs and cultured at 37 C in the cell incubator containing 5%CO2.The medium was changed twice a week.After 7 to 10 days,the neurospheres were formed,and the third to fifth generation of NSCs were obtained after passaged.(2)Experimental grouping,establishment of SCI model,cell transplantation and ESNYU impactor was used to establish SCI models at T10 level of rats.A total of 100 adult female Wistar rats with successful SCI modeling were selected and randomly divided into four groups:the hNSCs group(n=25),the ES group(25),the hNSCs+ES group(n25)and the PBS group(n=25).One week after the establishment of SCI models,stimulating electrode fixation and hNSCs transplantation were performed.From the second day after the installation of the stimulating electrode,the ES group and the hNSCs+ES group were given ES intervention twice a day until the end of the experiment.(3)Data detectionBBB scores of the hindlimb motor function of rats were measured one day before and after the SCI and then weekly until the end of the experiment.Then we sacrificed some rats to obtain spinal cord tissues.The spinal cord tissues were further made into frozen sections to detect the survival and differentiation of hNSCs through immunofluorescence.In addition,the expressions of GFAP and NF-H were assayed by immunohistochemistry and immunofluorescence,and HE staining was performed to observe the cavity formation in injued spinal cord.Moreover,the expressions of NF-H and NGF in injured spinal cord tissues were detected by western blot method.Results(1)BBB scoreAt the end of the experiment,the BBB scores of hindlimb function of rats in each group were ranked from high to low as follows:the hNSCs+ES group,the ES group,the hNSCs group and the PBS group,and the differences between the groups were statistically significant(p<0.01).(2)Detection of the survival of hNSCs by immunofluorescent assayThe survival number of hNSCs in the hNSCs+ES group was significantly higher than that in hNSCs group(p<0.05).In addition,the number of survival cells that had differentiated into neurons in the hNSCs+ES group was also significantly higher than that in the hNSCs group(p<0.05).(3)Immunofluorescence expression of NF-HThe NF-H expression in the injured spinal cord around glial scar tissues in the hNSCs group,the ES group and the hNSCs+ES group was higher than that in the PBS group,and the difference was statistically significant(hNSCs group vs PBS group,P<0.05;ES group and hNSCs+ES group vs PBS group,P<0.01).The expression of NF-H in the three treatment groups was ranked from high to low as follows:the hNSCs+ES group,the ES group and the hNSCs group,and the difference between each group was also statistically significant(hNSCs+ES group vs ES group,p<0.05;hNSCs+ES group vs hNSCs group,p<0.01;ES group vs hNSCs group,p<0.01).(4)Immunohistochemical detection of GFAP expressionThe GFAP expression in glial scar tissues of injured spinal cord in the three treatment groups was significantly lower than that in the control group(the PBS group),and the difference was statistically significant(hNSCs group and hNSCs+ES group vs PBS group,p<0.01;ES group vs PBS group,p<0.05).Among them,the expression of GFAP in the hNSCs+ES group was the lowest,with statistically significant differences compared with the other three groups(hNSCs+ES group vs hNSCs group,p<0.05;hNSCs+ES group vs ES group and PBS group,p<0.01).(5)HE staining resultsLocal cavity area in the injured spinal cord was detected by HE staining.The area in the hNSCs group and the hNSCs+ES group was smaller than that in the PBS group and the difference was statistically significant(hNSCs group vs PBS group,p<0.05;hNSCs+ES group vs PBS group,p<0.01).The area in the ES group was smaller than that in the PBS group,but the difference was not statistically significant.(6)Western blot analysis of NF-H and NGF expression in injured spinal cord tissue homogenateThe expression of NF-H in the three treatment groups(hNSCs+ES group,ES group and hNSCs group)was significantly higher than that in the control group(p<0.01).Moreover,the expression of NF-H in the hNSCs+ES group was significantly higher than that in the ES group and the hNSCs group(p<0.01),and that in the ES group was also significantly higher than that in the hNSCs group(p<0.01).The expression of NGF in the three treatment groups was significantly higher than that in the PBS group(p<0.01),but there was no significant difference among the three groups.Conclusion(1)Both hNSCs transplantation and ES can promote the recovery of hind limb function in rats with SCI,and the combination method works best.(2)ES can promote the survival of hNSCs and their differentiation into neurons.(3)HNSCs transplantation and ES can promote the repair of SCI through improving the expression of NGF and NF-H,down-regulating the expression of GFAP and inhibiting the formation of glial scar in injured spinal cord. |
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