The Effection Of Peripherally Adopted NGF Produced By Microcapsulated Cells On Neonatal Rats With HIBD

Neonatal hypoxic and ischemic encephalopathy (HIE) is a centre nervous system damaging disease caused by various perinatal factors and always leads to serious nervous system sequelae.Concerns about how to cure HIE and reduce its sequelae have been the major focus area of perinatal medicine research at home and abroad.The mechanism of HIE occurring is very complicated involving energy metabolism, intracellular calcium ion overloading,nervous toxicity of excitable amino acid,NO and the damage of oxygen free radical during ischemic re-perfusion. Nervous cells apoptosis causes by multifactor,so it's impotent to delay and reduce its occurring by adopting interfering measurements which have both clinical and social significance on healing HIE.As one of the most impotent bioactive factors in the nervous system,Nerve Growing Factor(NGF) has functions of maintaining existence of sympathetic nerve cells and sense nerve cells,speeding differentiation of nerve cells,deciding growing direction of axon,stimulating rehabilitation and regeneration of damaged centre nerves and surrounding nerves,maintaining survival and inducing growing of apophysis.Under physiological circumstances,the content of NGF in brain is little, and many studies at home and abroad have confirmed that the increased expression of NGF is resulted from any of brain damages.Increased expression of stressed NGF may have relationship with its nerves protective function;however,the elevated expression of endogenous NGF is too short-lasting to produce any effective protection.The rehabilitative and regenerative function of exogenous NGF is well known now,and a domestic study has shown that exogenous NGF administered by ventricular injection can reduce apoptosis in the brain of neonatal rats with hypoxic ischemic brain damage,however, clinical adoption of NGF has the following problems at present:as protein,NGF can't be taken via alimentary canal;NGF must be administered repeatedly due to its short half life;high damage and small space on local administering at centre system. There are several approaches for using exogenous NGF in the treatment of central nervous system:diseases, including NGF infusion into brain,transplantation of NGF-producing cells into damaged neurocytes,and gene therapy.These methods,however,have not been widely used clinically due to various disadvantages. Research on transplantation of micro capsule using immune separation technology to decrease the antigen reaction,especially transplantation of excretive cells can overcome those drawbacks.Micro capsule can protect its inner tissue cells by preventing harmful antibody macro molecule existing in host from infiltrating into it.At the same time,it allows nutrient needed getting into it while active materials getting out of it.In this experiment,we used alginate-poly-l-lysine - alginate(APA) as micro-capsule to enclose the NIH 3T3 cells transfected by rat NGF gene and cultivated it outside. After a period time,the 3T3 cells congregated into small cell lumps with the cell number increasing.A month later,small lumps got together to become a large lump.The cell nucleus emited yellow-green fluorescence and the cytoplasm was green via which indicate that the 3T3 cells still remain well condition of growing and multiplication after a month of cultivation.Then we enclosed the 3T3 cells with pheochronocytoma cells(PC12) having excretive potential and cultivated them together.Before cultivation,the PC12 cells were circle or ellipse with apophysis on its surface.Due to the NGF,the apophysis grew a little longer only at the 2^nd day after cultivation,and it reached to icm at 4^th day and 2.0—2.8cm at 7^th day and cells differentiated into polygon or taper.Comparatively,PC12 cells cultivated without NGF did not have obvious differentiation which indicates that the differentiation is accomplished under the effection of NGF excreted by the NGF-3T3 cells and not by natural.ELISA test indicated that fresh made microcapsulized NGF-3T3 cells excrete little NGF and this may due to the departure of their original environment when the micro capsules were made. However,this affection was transient and the cells can perform normal metabolism once the cultivating environment was settled down.Cellular activity increased and the excretion of NGF accelerated at the beginning of cultivation while both of them tended to be comparatively stable at medium of cultivation.This study aims to probe into the protection of exogenous NGF administering on neonatal rats with HIE and discuss the possibility of this method on treatment of human NHIE and expect to provided a new potential means for the clinical therapy of NHIE.Our experimental result indicates:1.After:72h of hypoxemia,the water content of neonatal rats' brain organism were much higher than that of normal rats'.For those treated with NGF,their brain organism water content had not remarkable difference with normal rats' and were much lower than untreated ones'.This indicate that treatment with NGF can delay the occurring of cerebral edema and reduce its degree in HIE therapy.2.TUNEL staining positive cells of frontal cortex and CAl subregion of hippocampus in the brains of NGF-treated neonatal rats were much lower than those untreated ones',suggesting that NGF can decrease cell apoptosis and alleviate brain damage and protect brain organism.To sum up,this experiment investigated the apoptosis of centre nervous cells after hypoxemic and ischemic damage by measuring hippocampus and frontal cortex with TUNEL method.We also observed the effect of abdominal transplantation of microcapsulated genetically modified cells expressing NGF gene on the apoptosis of frontal cortex and CAl subregion of hippocampus in neonatal rats with HIBD.Exogenous NGF administering can release clinical symptom of neonatal rats with HIE, delay the occurring of cerebral edema and reduce its degree,stimulate rehabilitation and regeneration of brain organism and reduce,the degree of cellular apoptosis.All of these indicate that peripherally administered NGF can penetrate the blood brain barrier and be absorbed by brain organism to reduce hypoxemic and ischemic brain damage in neonatal rats.This research provides a reasonable,effective and safe method on treatment of centre nervous system damage by using exogenous NGF administering and is meaningful to clinical therapy.The original findings and novelty of present study: Most domestic studies on NHIE are mainly focused on inner centre nerve of neonatal rats and little on peripherally treatment at present.For the first time, this research investigate the treatment of neonatal rats with HIE by adopting exogenous NGF administering with immune separation technology and provide theoretical evidence for clinical treatment of human NHIE.