The Study Of The Therapeutic Effect Of Transplanting Genetic Engineering Neural Stem Cells NSCs-IGF-1 For Neonatal Rats With Hypoxic-ischemic Brain Damage

BackgroundNeonatal HIBD (Hypoxic-ischemic brain damage) is a common cause of acute neonatal death and long-term neurological disability in children. It does harm to neonate seriously, for the severity state of the illness and the high mortality and morbidity. With the advance in neonatal rescue techniques, premature children, apneic children and children with brain damage were saved, making the prevalence of neonatal HIE (hypoxic ischemic encephalopathy) increase Instead of decrease. HIE has become one of the major diseases of childhood disability, severely degrading quality of life and physical and mental development for children, and also bringing a great deal of psychological stress and economic burden to individuals, families and society. Despite advances have achieved in supportive care, such as medications and hyperbaric oxygen, no treatments for HIE are available at present. Because the traditional treatments can only relieve symptoms to some extent, it is difficult to promote nerve regeneration to restore fundamental nervous system function.The discovery of stem cells, especially NSCs (neural stem cells) has brought new hope to the HIE patients. A large number of studies have confirmed that NSCs, due to their capacity for self-renewal, multilineage differentiation potential, provide unique material for the cell therapy of a wide spectrum of neurological diseases. The researches of NSCs have currently become the focuses in the biomedical domain. And NSCs transplantation provides a new direction for the treatment of neurological damage. The study of the application of NSCs at present mainly in the following two aspects:First, transplanting NSCs cultured and expanded in vitro directly to the injury site of nervous system, to achieve repair. Second, NSCs can be genetically modified to efficiently transfer therapeutic genes to diseased CNS, and so cell replacement and gene therapy can both be achieved.Some studies showed that IGF-1 (Insulin-like growth factor-1) plays important roles for brain growth, development, myelin formation, and it also is a potential mitotic factor, which can induce the proliferation, differentiation of neural cells and vascular endothelial cells and regulate cell survival. At the same time, in vivo model of multiple trauma has demonstrated its important neurotrophic and neuroprotective effects. It is reported that, IGF-1 was administered immediately after brain injury can significantly reduce neuron death, and can effectively improve the adverse consequences of brain injury.Previous studies have shown that transplantation of NSCs or IGF-1 alone could play an important role for the treatment of animal models of HIBD in promoting neurological recovery. But preparation of IGF-1 gene-modified neural stem cells and transplantation such cells for the treatment of HIBD animal have not been reported yet. Therefore our studies have focused on seeking new sources of NSCs, establishing genetic engineerjing neural stem cells NSCs-IGF-1, By transplanting NSCs-IGF-1 to the HIBD rats, the feasibility of genetic engineering modification Neural stem cells NSCs-IGF-1 treatment for HIBD is explored to lay the theoretical foundation of clinical application of NSCs-IGF-1 for HIBD therapy.Objective1.To investigate the isolation, cultivation, differentiation and determination of neural stem cells from human umbilical cord blood in vitro, and to observe the proliferation, passage and differentiation of neural stem cells, to build stable culture system for experimental and clinical use.2. To construct eukaryotic expression vector containing IGF-1, and to establish genetic engineering neural stem cells NSCs-IGF-1 by transfecting pcDNA3.1-IGF-1 into NSCs and to provide the basis for the application of NSCs-IGF-1 in treating HIBD.3. To observe curative effects of NSCs-IGF-1 transplantation on new-born rat models with HIBD.Method1. Under sterile conditions, Umbilical cord blood was obtained from full-term, normal or cesarean deliveries of healthy child-bearing age maternal. Diluted by an equal volume of phosphate buffered saline (PBS), mononuclear cells in them were isolated in the way of density gradient centrifugation. Living cells counted by Trypan blue staining the cord blood, mononuclear cells were seeded into culture flask with l×106/mL and serum-free DMEM/F12 medium containing B27, EGF and bFGF. Cells were cultured at 37℃,5% CO2 half of medium was changed after 3 days. To the 7th day of culturing, neurospheres were collected by centrifugation at 800r/m for 10min using the method of combining trypsin digestion and mechanical method for passage, and the passage of neural stem cells were seeded in new flasks at 5×105/mL and cultured continuously at 37℃,5% CO2. P3 generation of NSCs were induced by 20% fetal bovine serum (FBS). The dynamic status of cell growth was observed by inverted microscope.2. P3 generation of NSCs were labeled with BudU, and after cultured for 3 days at 37℃5% CO2, BrdU-positive cells were detected by immunofluorescence to evaluate the potential proliferation ability. At the same time, immunocytochemistry was used to detect the specific markers of Nestin, NSE, GFAP and MBP of NSCs and the differentiated cells. In addition, determinate optimal selection of P3 generation of NSCs derived from cord blood in the concentration of G418.3. Total RNA was extracted from fetal liver, and the target gene IGF-1 was obtained by RT-PCR. The target gene in 1% agarose gel electrophoresis was tapped and recovered. IGF-1 genes was linked to the plasmid to recombinate eukaryotic expression plasmid pcDNA3.1-IGF- 1. Preparation of competent cells of DH5a E. coli using calcium chloride, conjugate was added into cells for transformation. Ampicillin resistant colonies were picked for amplification the next day, and a small amount of recombinant plasmid were prepared. The recombinant plasmid was identified by the enzyme of BamH I and Hind III, and the correct enzyme broth was identified by sequencing.4. The recombinant plasmid was amplified and a large number of plasmid was extracted using SDS lysis method. The recombinant plasmid pcDNA3.1-IGF-l was transfected into P3 generation of umbilical cord blood NSCs by lipofection transfection method. Empty plasmid group and control group were also set up. Resistant clone was selected with G418 and expanded in vitro. For further identification, the expression of IGF-1 was detected by RT-PCR. And 2% agarose gel electrophoresis was used to identify the products. The expression of IGF-1 and Nestin in the transfected cells were detected by immunocytochemistry, and the same method was used to detect the expression of NSE, GFAP and MBP in the cells which induced by 20% FBS.5. HIBD rat model were established by 80 SD rats in the way of ligation of the left common carotid artery and subjecting to hypoxia. The 75 survivors were randomly divided into 3 groups:HIBD control group, NSCs transplantation group and the NSCs-IGF-1 transplantation group,25 rats per group. And each group was further randomly divided into 1-day group,7-day group,14-day,21-day group and motor function testing group,5 rats per group.3 days before transplantation, the genetic engineering neural stem cells NSCs-IGF-1 and non-transfected NSCs were labeled with BrdU (final concentration 1μmol/L).24h after the animal model, cell transplantation was carried by tail vein injection at 1.0 mL/100g.25 normal SD rats were used for normal control group; genetic engineering neural stem cells NSCs-IGF-1 were transplanted by tail vein injection in NSCs-IGF-1 transplantation group; NSCs were transplanted in NSCs transplantation group, and normal saline were transplanted into HIBD control group.6.1 day,7 day,14 day and 21 day after transplantation,5 rats per group were killed respectively. Immunocytochemical staining for BrdU, Nestin, NSE and GFAP were applied to observe the survival and distribution of the transplanted cells in the HIBD rat brain; Y-maze test and motor behavioral tests were performed as well to research the recovery of brain function of rats in different groups.Results1. When cultured in serum-free DMEM/F12 medium for 7 days, the NSCs could give rise to floating primary neurospheres. After passage, the cells were scattered in the suspension, and after cultured at 37℃,5% CO2 medium-sized neurospheres were formed. With the passages, cell proliferation rate gradually slowed down.1 day after induced by 20% FBS, P3 generation of NSCs began to adhere, and small protrusions appeared at the edge of cells.3 days after induction, neurite outgrowth could be seen in the adherent cells.7 days after induction, neurons, astrocytes and oligodendrocytes could be observed.2. Having labeled the NSCs of P3 generation with BrdU, a large number of BrdU-positive cells could be observed by fluorescence microscope. The results of immunocytochemistry showed that primary and passaged NSCs were Nestin-positive, and that after induction for 7 days there were no Nestin-positive cells, but NSE, GFAP and MBP positive cells. Optimal selection of P3 generation of NSCs Derived from(?)d bloodin the.concentration of.G418 was 400ug/mL. (?)3. After RT-PCR test ot tetal liver total KNA, a fragment of 462bp obtained.After digestion by HindⅢand BamH I, ligation products of gene IGF-1 and the plasmid pcDNA3.1 turned to two fragments, which were about 5428bp and 462bp. Empty plasmid group only has one fragments of 5428bp. RT-PCR and sequence analysis confirmed the correct construction of the recombinant plasmid, and that showed the eukaryotic expression vector pcDNA3.1-IGF-1 containing IGF-1 was successfully constructed.4. After the recombinated plamid was transfected into NSCs, medium containing 400ug/mL G418 was used to select resistant clones.2 days later, the cells began to die, and the dead cells peaked after a week, and then cell death reduced gradually, and began to proliferate. Resistant clones were selected 2 weeks later. This showed that IGF-1 gene was transfected into neural stem cells successfully. After RT-PCR, a fragment of 462bp was obtained and the length was consistent with the target gene showing that genetic engineering neural stem cells NSCs-IGF-1 were established successfully. Positive Nestin and IGF-1 indicated that IGF-1 successfully expressed in transfected cells, and the undifferentiated state was not changed because of the expression of IGF-1. After induced by 20% FBS for 7 days, they could differentiate into typical neuron, astrocytes and oligodendrocytes detected by immunocytochemi-stry of NSE, GFAP and MBP staining.5. Subjected to ligation of the left common carotid artery and hypoxia, SD rats were found paralysis of right limbs. It showed that HIBD rat model were established successfully.6. After the transplantation of genetic engineering of neural stem cells NSC-IGF-1 into the HIBD rats, tracing with immunofluorescence for BrdU showed that, BrdU-positive cells can be observed both in the NSCs transplantation group and the NSCs-IGF-1 transplantation group, and with the observation time prolonged, BrdU-positive cells continued to increase. The BrdU-positive cells in vein graft could migrate to HIBD rat brain and further to the left of SVZa (anterior subventricular zone,) and survive.7 day after transplantation, BrdU-positive rate in the NSCs-IGF-1 transplantation group was much more than that in the NSCs transplantation group (P <0.05).7. After transplantation of NSC-IGF-1 into the HIBD rats, HE staining, immunohistochemical staining for Nestin, NSE, GFAP were applied. The results showed that transplanted cells migrated to the left side of SVZa, survived and differentiated. Within the observation time, the expression of Nestin positive cells of the NSCs-IGF-1 transplant group increased early after transplantation, and the numbers reached a peak at day 14, then declined gradually, and the expression of NSE positive cells increased gradually. But Nestin positive and NSE positive cells of the model control group decreased gradually after transplantation for 7 days. There was significant difference between the two groups (P<0.05). GFAP positive cells increased gradually in every group and among them the most was in the model group, which was significantly different from other three groups (P<0.05). There was no significant difference (P>0.05) between the NSCs-IGF-1 transplantation group and the control group. Examinations on the rat learning, memory abilities and motor behavioral tests showed that NSCs-IGF-1 transplantation group was significantly better than the control group (P<0.05).Conclusion1. The multipotential NSCs can be isolated from human umbilical cord blood.2. IGF-1 gene can be transfected into NSCs safely and effectively by Liposome transfection method.3. Genetic engineering neural stem cells NSCs-IGF-1 are available by transfection of IGF-1, and the self-renewal and multilineage differentiation potential will not be changed due to the expression of IGF-1.4. NSCs-IGF-1 can survive, migrate, proliferate and differentiate into neurons in HIBD rat brain and the nervous function recovery is improved prominently by transplantation of NSCs-IGF-1, which may become a potentially effective method to treat HIBD.