Rat embryonic neural stem cells in culture , differentiation and stem cell transplantation in the treatment of severe traumatic brain injury in rat studies
|Keywords||Severe brain injury Transplantation therapy Stem Cell Culture Rat Neural stem Immunocytochemistry Astrocytes Neurons Self-renewal Subventricular zone|
The first part of the rat embryonic neural stem cell culture and differentiation of neural stem cells was observed (Neural stem cell, NSC) is the central nervous system with self-renewal capacity and the ability to differentiate to produce the mature brain cells (including neurons, astrocytes and oligodendrocytes) pluripotent cells. Has confirmed that neural stem cells exist in the adult animal embryo or a wide area of ??the central nervous system from embryonic and adult neural stem cells of animals on the property, in essence, is basically the same, while the embryonic neural stem cells cultured and amplified more convenient. In this study, the 14-day gestation fetal rat brain subventricular zone, hippocampus and other structures to isolated neural stem cells. I. Materials and methods Animals: sprague an Dawley pregnant rats 3 (E-4). 2, the main reagents: 0 * 25% trypsin, B27 additives, DMENfF12 serum-free medium, basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF); Anti neuroepithelial stem cell protein antibody (Anti an estin IgG); anti-glial fibrillary acidic protein antibody (A knife ti a GFAPIgG), neuron specific enolase antibody (A knife ti an SE IgG); immunohistochemistry reagents. 3, the neural stem cells in primary culture and subculture: E14d SD rats were taken from embryos removed under sterile conditions subventricular zone, hippocampus and other structures, 0.25% trypsin and 0.02? TA (l: l) digested into single cell After serum-free suspension culture of neural stem cells, culture medium: DMEM/F12 (l: l), 2? 7, EJ Zong / ml, bFGF 20ng/ml, 37 ℃, 5% Co: suspension culture conditions. Every 2 or 3 days for fluid passage about a week. 4, the identification of neural stem cells and differentiation: Nestin immunocytochemistry neural stem cells. Taking neural stem cells passaged with 5 xl security / piece inoculated cell density poly-lysine-coated glass coverslips in DME containing 10% fetal bovine serum F12 medium stare into adherent culture, differentiation culture 5 days, as immunocytochemistry, neurons, glial cells were used anti-NSE antibody, anti-GF wonderful antibody labeling. Second, the results of a primary culture of neural stem cells have fewer visible first day of training a large number of adherent cells, a few cells extending protrusions, the fourth one from the culture day 5, adherent cells rupture, death, without adherent cells were suspended in culture medium, the number of cells increased, the formation of small neurospheres (Neurosphere), gradually increasing neurospheres. Passaged cells were suspended in culture flasks spherical cell morphology rules, no significant neurite outgrowth, neurospheres passaged seven days has grown to become the largest of hundreds of cell clones. Nestin confirmed by identification of neural stem cells. 2, anti-Nestin neurospheres done directly immunohistochemical staining, all cells were positive. Passaged God surg Zhejiang University doctoral thesis by the differentiation of stem cells cultured for 5 days, respectively, for anti-NSE, GFAP immunocytochemistry showed that some cells positive for NSE, GFAP positive. Under the microscope you can see a few of neural stem cells to differentiate into neurons NSE-positive cells differentiate into most of GFAP-positive astrocytes. Third, the conclusion of neural stem cells in primary culture and subculture success, further experiments, including the application of neural stem cell transplantation in the treatment of Neurosurgical Disease provide a solid foundation. Under natural conditions, the vast majority of neural stem cells to glial cell differentiation into neurons was relatively small. The second part of the neural stem cell transplantation in the treatment of severe traumatic brain injury in rats with severe brain injury research is neurosurgery critical illness, high mortality, the survivors are often left sequelae. In recent years, along with pre-hospital care, neurosurgery, intensive care and other technological advances, the mortality of severe brain injury declined. However, extensive and severe traumatic brain injury causes severe nerve cell necrosis, apoptosis, brain self-repair capacity is extremely limited, so damage caused by neurological deficits difficult to restore. The current rehabilitation therapy on the efficacy of sequelae of traumatic brain injury is very limited. Neural stem cell self-renewal capacity and multi-differentiation potential, under certain conditions, can be differentiated into various cells of the nervous system, so the repair of damaged nerve tissue has a good prospect. Through transplantation of neural stem cells to repair damaged brain tissue necrosis, is to reduce brain injury morbidity and effective way. In this study, the embryonic neural stem cells transplanted into rats with severe brain injury brain injury site, from other aspects of behavior and morphological study of neural stem cell transplantation in the treatment of severe traumatic brain injury efficacy of neural stem cells in future clinical applications provide a theoretical basis. I. Materials and methods an animal: Snrague an Dawley rats 150, male and female, weighing 180 a 2209, aged 3 months or so. 2, the neural stem cell marker: neural stem cells passaged after a day in the medium BrdU, final concentration of 30 mixed pair. 24 hours the medium was changed, neural stem cells can be used for transplant. 3, severe brain injury rat model was made: an improved device Feeney law to combat free-fall injury, the rats were anesthetized and placed on a stereotactic apparatus, heavy anvil 509 yards from 25cm height drop, hit the top left epidural bone window, impact area IcmZ, the impact force of 1250 Sand m,. 4, transplantation of neural stem cells: This step is divided into three phases: the first will first go through BrdU labeled neural stem cells were collected by centrifugation, Kwong 12 medium DMEM were added to a concentration of 1 xl nursing / ml, 1x 10v/ml, 1 x 10s/ml neural stem cell suspension stand. In the first day after traumatic brain injury, the rats were anesthetized and opened the original bone window, with the micro-injector injection of neural stem cell suspension. Three experimental groups with different concentrations of rats were transplanted neural stem cell suspension 5 Chuan, the control group was injected with saline, to determine the optimal concentration of transplantation. The second stage is divided into three time points identified in the first phase of transplantation optimal concentrations of neural stem cell transplantation, the time difference is that the damage immediately after injury one day, the third day after injury, to determine the best time of transplantation. The third stage of the previous two phases as determined by the concentration and the best of the best transplant transplant time, press three ways (the site of injury, intraventricular and intravenous) transplantation of neural stem cells in order to determine the best migration path. 5, behavioral judgment: rat nerve regeneration after injury using Bederson scoring method to detect signs of recovery of neurological function. 6, immunohistochemical staining: HE staining were performed after surgery, anti-BrdU, 15 rats were sacrificed on day all, cut brain tissue paraffin sections, anti-NSE immunohistochemical staining.