| ProfaceSpinal Cord Injury ( SCI) is always emphasis study direction interested by medical field. Before the necrosis was thought a main reason, ultramodern studies showed, the cell apoptosis may participate in pathophysiologic process. Apoptosis was thought one of important paths of cell death in second Spinal Cord Injury, the effect of it become more and more important. The mechanism of apoptosis is unknown, it may priming suicide process through many kinds signal transmission. After SCI, the inside and external environment which the neuron to exist happened changes, many factors, such as the concentration of ecto - excitatory amino acids increased, multiplicity production of free radical, calcium overload in cell and abnormal express of gene et al, interlaced each other, it had many access to regulate the cell apoptosis. Along with the development molecular biology, it may be one of main tasks to study the mechanism of apoptosis and apoptosis related gene applying the molecular biology technique and inhibited the occurrence of apoptosis applying many means.For the past few years, the study of apoptosis regulation has gained tremendous progress, found the genes participating regulation were divided two kinds according to its function;promotion apoptosis genes and inhibition apoptosis genes. The inhibition apoptosis genes were Bel -2,Bcl - X1 and Mcl - 1;Promotion apoptosis genes were Bcl -2 related gene Bax and Bcl-Xs,P53,C -myc, especially bcl - 2 and bax gene had more close relation with the regulation of apoptosis.Pathophysiologic processes would happened after SCI, included primary injury and secondary injury, primary injury was determined by environment of external injury. The studies showed that the apoptosis acted as lead effect in spinal secondary injury, but the mechanism was unknown. Cell factors and inflammatory factors might participate onset.Myeloperoxidase ( MPO) exists in neutrophil ( PMN) as an indicator which reflects the degree of PMN infiltration sensitively. For this reason we have determined the change of TNF - aand MPO before and after SCI to evaluate mechanism of action of them in spinal secondary injury in rats.Objective presence of cell apoptosis after SCI furthermore deeply meaningful and roundly revealed pathophysiologic process of SCI, I. e, not only it had cell necrosis of acute stage, but also had subacute cell necrosis, it made people have a new recongnition about pathophysiologic process of SCI. Utilized apoptosis rule, effectually regulated apoptosis, which was new route, among of it, medicine intervention and gene therapy were a more promising therapeutic tool.VEGF is a very specific promotion vascular growth factor, it could promote dissociation , proliferation and calcium aggregation in cytoplasm of vascular en-dothelial cell in vivo, and induced vascularization. VEGF or its gene treatment on coronary artery disease (CAD) or limbs arterial occlusion disease had gained a better effect;Some one used the exogenous VEGF protein to treat cerebral infarction of rats, found that the death of nerve cell decreased, volume of cerebral infarction deflated. IN addition, recently the studies showed that the VEGF could relieve ischemic - reperfusion injury. It offered a new think and theory guidance, we believed that it had a wide application perspective in the treatment of spinal secondary injury.Materials and Methods1.1 Experimental animals and grouping129 healthy Wistar rats, female and male were not distinguished, weight (280 ±30) g, the Experimental Animals Breeding Department of China Medical University, supplement animal according to experiment condition. According tothe experimental objective group. Primary part had 28 rats, were randomly divided into 7 groups, 6 groups were experimental groups, 1 group was control group, every group removal vertebral plate to damage spinal cord, andThe control group only removal vertebral plate not to damage spinal cord;Second part had 48 rats, were randomly divided into 2 groups, the control group only removal vertebral plate not to damage spinal cord, the experimental groups removal vertebral plate to damage spinal cord;The third part had 53 rats, were randomly divided into three groups, the control group had five rats only removal vertebral plate not to damage spinal cord, the experimental groups removal vertebral plate to damage spinal cord;normal sodium ( NS) group had 24 rats, after modeled NS was infused into subarachnoid space. The VEGF treatment group had 24rats, after modeled VEGF was infused into subarachnoid space ( VEGF 5ul/100g) o Every group drew the material after SCI 2h>4h>8 hNldN3dN7d,every time point took 4 rats.1. 2 Preparation of SCI rat model,used Allen's method.1. 3 Preparation of materials and sliceAfter drew the materials ,routine paraffin imbedding, serial sections at 5um to detect1.4 Reagent and equipmentMain reagent :TNF - a NBaxxBcl - 2antibody, immunohistochemistry kit of sheep anti - Rbt and DAB kit,MPO kit,apoptosis detection kit.Main equipment: 24 hole cultivation plate, incubaton, LEICAQ550 IW image analysis system, hypothermy supercentrifuge, homogenation meter, spectro-photometer and balance weigh et al.1.5 Study contentObserved the change of gene express of Bax^Bcl -2 in SCI, investigated the effect and regulation mechanism of apoptosis in SCI;Observed the change of TNF - a and MPO, investigated the effect of TNF - a and neutrophil on apoptosis;investigated the effect of treatment method of VEGF on apoptosis.1. 6 Study method1. 6. 1 Observation of histomorphology: after HE stained , observed the change of cell appearance feature at light microscope.1.6.2 The detection of TNF - a used double antibody sandwich method.1.6.3 Express of Bax and Bel -2 gene used immunohistochemistry method.1.6.4 Myeloperoxidase (MPO) was detected with MPO kit.1. 6.5 Apoptosis was detected with TUNEL in site end labeling. 1.7 Statistics analysis: All data were expressed with mean ± standard differentiation (X ± S) , and made t test.Results2. 1 Histology stainingH - E staining show myeloid tissue oedema, along with the advancement of time appear progression hemorrhage necrosis, cavitation and expansion.2. 2 The result of TUNEL in site end labelingThere are no apoptosis cell in myeloid tissue In normal control groups. In NS groups, appear positive cells at post injury 2 h,mainly appear in gray substance area, the numbers of positive cells increase at post injury 4 h, the numbers of positive cells get peak at post injury 8 h, afterward gradually decrease, the positive cells still exist until post injury a week. Contrast to NS groups, at the same time stage the positive cells numbers are obviously decrease in VEGF groups,the difference is significant.2. 3 Immunohistochemistry result of Bel -2 and BaxThe kytoplasm of positive cells of Bel - 2 are yellow, appear in neuron and colloid cells. There have a small amounts express at posy injury 2h, the numbers increase at post injury 4 - 8h,it get peak at post injury 24 h, afterward gradually decrease, there still have a small amounts express at seven day. The kytoplasm of positive cells of Bax are also yellow, there have a small amounts express at post injury 2h, get peak at 8h, maintain to24h,soon gradually decrease, there still have a small amounts express at seven day. Contrast to the control groups, the difference is significant ( P <0.01).2.4 The change of levels of MPOThe levels of MPO began obviousl increase at post injury 4 8h, get peak at one day ( P <0. 01) ,began decrease at post injury 3h,but still higher than the control group ( P < 0. 05).DiscussionEver since a long time ago, SCI is always a tough problem to puzzle the medicine realm, especially the acute spinal cord injury. In the pristine experimental study, the attention of people concentrated necrosis pattern, not found apoptosis. li et al firstly preferred that the phenomenon cell apoptosis existed after SCI, the method which detected the apoptosis cell with TUNEL method had became a common molecular biology detection method, offered a precise method for the study of SCI. In our study, cell apoptosis were all detected in the grey matter and white matter after SCI, it show that after SCI, neuron and gliocyte all take place apoptosis, and confirm that the cell apoptosis certainty happen after SCI. The concept of apoptosis was introduced by Kerr et al in 1972, it was that in the determination physiology or pathology condition, the cell initiative ended life process following personal procedure. Apoptosis also called programmed cell death, namely it pointed that the nucleated cells through priming itself interior mechanism at necessarily condition , main reason was a activation of endogenous DNA incision enzyme, happened the death process, which was a initiative cell death process regulated by gene, had a essential difference compared with necrosis. Now we know many genes such as bcl - 2, bax, p53, c - myc, fas et al have relation with apoptosis. Our findings show, on the time relatively of apoptosis , the peak time of apoptosis is post injury 8h, the peak of express of bax gene is also post injury 8h, it indicate the Bax gene may promote cell apoptosis, and at this time the express of bcl - 2 gene began increase, the express get peak atpost injury 24h, afterwards slowly decrease. On the relative time the cell apoptosis began gradually decrease. Ii et al found macrofiber bundle axon express bcl - 2 after SCI, and apoptosis oligodendroglia did not appear the up - regulation of bcl -2. In the conduction process of apoptosis signal, usually considered bcl - 2 effect on upstream of caspase - 3, produced effect through inhibited the activation of caspase - 3. Bcl - 2, which located in mitochondrial outer membrane %endocytoplasmic reticulum membrane and nuclear membrane,has effects on inhibit mitochondrial membrane permeability x prevent the releasing of cyto-chrome C, thus prevent the activation of caspase - 3. For the past few years the studies showed, bcl - 2 not only effect on the upstream of caspase - 3, but also was a direct substrate of caspase — 3, after specificity enzymolysis by caspase — 3 form bcl - 2 fragment, which the function had happened fundamental change, from inhibit apoptosis to promote apoptosis. Bax is bcl -2associated protein,has 45% homology with bcl -2, bax can direct bind to mitochondrial membrane, form mitochondrial across membrane channel, promote releasing of cytochrome C. The adjustment of bax/bcl -2 heterodimerzation is very important link of cell apoptosis, the ratio of bax/bcl - 2 determine the live or die of cells, the ratio of bax/bcl - 2 increased after SCI. The study found, bcl - 2 protein had only a small quantity express after SCI,but bax protein had a great quantity express. Indicated the promotion apoptosis factors gained advantage, and prevention factors obviously insufficiency thus made the nerve cells develop toward to apoptosis. Our study also this view. Takahashi et al through transfection bcl - 2 gene to the injury spinal cord, bcl - 2 gene retrogradatly transported to the Clarke nuclear neuron, prevented cell death and decreased cell atrophy. Lou et al trans-fectioned the contusive spinal cord with adenovirus mediated bcl - 2 gene, SCI zone decreased. Therefore, we presume, overdose express of bax gene promote apoptosis, bcl - 2 gene has a potential effect to promote SCI neuron survival and inhibit apoptosis. Abnormal express of apoptosis related gene bax and bcl -2 in SCI, may participate in the second SCI, apoptosis act as important effect in the course of SCI. The mechanism of apoptosis is not completely understand, it may priming suicide process through many signal transmission channel.The study confirmed, inflammatory reaction induced by SCI might act as aimportant effect in the secondary injury. But the specific effect was unknown. Neutrophil is a important inflammatory cell, and TNF - a is a key promotion inflammatory cell factor, both can act each other, mutually promote,enlarge inflammatory effect, form vicious cycle, aggratate tissue injury. MPO is a enzyme which mainly exist in azurophil granule of neutrophil, tall or low of its activity represent the number and activity of neutrophil. Vitro experiment confirmed the neutrophil Nmacrophage and gitter cell all produce TNF - a,the level of TNF -a increased after SCI. TNF - a promoted the activation of neutrophil and the cell aggregateion in the endotheliocyte, leading to produce more cell factors. More and more evidences indicated the aggregation of neutrophil aggratated SCI. Our finding show, the activity of MPO obviously increase after SCI, get peak at 24, the level of MPO is still higher at three day, it indicate, injury region have a great quantity activation and aggregation of neutrophil after SCI. Activated neutrophil, enlarge inflammatory reaction through release mediators of inflammatory, aggratate injury section spinal cord. Our study find, the level of TNF - a quickly increase in the acute injury early stage, get peak at 24h, this kind high level expression last longer time, at 72h is still higher than the control group. The persistence expression of TNF - a may be the result of hypoxia ischemia x in-cursive neutrophil persistence secretion. In addition, TNF - a which generated in early stage can activate platelet activeating factor ( PAF) , the latter can further stimulate the expression of TNF - a. On the time course, the peak of TNF -aandneutrophil is post injury 24h, afterwards gradually decrease, and the peak of apoptosis is post injury 24h, it indicate , from the expression change of TNF -aand neutrophil to time course of apoptosis, we presume this course just is a course to trigger apoptosiss cascade reaction.The mechanism of TNF - a to induce apoptosis possible had: (l)intra — cellular calcium overload. ?oxygen free radical injury. (3) inflammatory cell infiltration. (4)expression reinforcement of apoptosis gene. We study find that the Bel -2 expression down regulationxBax expression up regulation lead to apoptosis. The mechanism may have relation with Bax protein promote injured region neuron to happen inflammatory reaction. Recently transgenic animal study showed;Bel - 2 protein could inhibit the synthesis and releasing of TNF - a, lighten theextent of apoptosis. Further, Bel -2 protein implement this signal conduction path by the activation of nuclear factor. In addition, TNF - a induce apoptosis had another signal conduction path that it bind with TNFR1 or fatty acid syn-thetase to activate apoptosis. TNF -a not only can induce apoptosis alone, but also can aggratate the extent of apoptosis induced by hypoxia reoxygen.Objective existence of apoptosis and distribution characteristic on time and space after SCI, deeply and roundly reveal pathophysiologic process of SCI, offer a theory guidance for the treatment after SCI.We find, contrast to NS group, the number of positive apoptosis cell are obviously decrease in VEGF group, the difference is significant( P <0. 01) ,HE staining show, contrast to NS group, injury extent of spinal cord in VEGF group is obviously relieve. VEGF can inhibit the happen apoptosis after SCI. the mechanism may be the VEGF make regeneration of vascular endothelial cell increase x quicken , formation of neogenesis micrangium, improve ischemia hypoxia condition, promote the exchange of material, quickly recover the normal blood -nerve barricade, thus decrease or end apoptosis. The result show the exogenous VEGF have protection to the ischemia myeloid tissue.Conclusion1. Apoptosis related gene bax and bcl - 2 may participate the second SCI, apoptosis act as important effect in the course of SCI. After the SCI the promotion apoptosis factors gain advantage, and prevention factors obviously insufficiency thus make the nerve cells develop toward to apoptosis.2. Inflammatory reaction induced by SCI act as a important effect in the secondary injury. Neutrophil is a important inflammatory cell, and TNF - a is a key promotion inflammatory cell factor, both can interact each other, mutually promote, enlarge inflammatory effect, form vicious cycle, aggratate tissue injury-3. VEGF can inhibit the happen apoptosis after SCI. VEGF have protection to the ischemia myeloid tissue.
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