| Background: When cerebral ischemia occurred, it is important for confining or deflating infarct volume and improving prognosis to remove obstacles from obstructive blood vessel and to recuperate hemoperfusion as early as possible. But reperfusion often aggravates brain tissue damage, that is reperfusion injury, which can induce neurocyte necrosis or apoptosis and nerve functional disturbance.Although supra-pristine thrombolysis, lowering brain tissue metabolism, various kinds of nerve protectants, etc may cut down the fatality of patients to some degree, the effect of improving the symptom of patients nerve function is not ideal, and problems such as transient time window of therapy and severe adverse reaction are present. The reason is that decreasing neurocyte is not avoided fundamentally.It is followed with interest by researchers to treat cerebral ischemia with stem cell transplantation. The transplanted nerve stem cells can migrate and proliferate in the ischemia brain tissue area. They also can differentiate to partly substitute and recover impaired neurons, thus to improve the symptom of loss of nerve function. But problems such as ethics issue, difficult source and immunological rejection, etc limit the clinical application of stem cell transplantation. MSCs are the most widely researched stem cells. But the low transformation of MSCs into nerve cells and the risk of becoming tumour after extraorgan amplification made the effect and safety of MSCs transplantation for cerebral ischemia be suspected.Therefore original therapy means for cerebral ischemia-reperfusion injury are needed badly to preserve and recover the function of impaired nerve cells. For the reason that the function of brain tissue relies on the quantity and quality of neurocyte to great extent, if we adopt a measure which include diminishing apoptosis of early ischemia nerve cells, preserving the quantity of neurocyte maximatily, meanwhile increasing the regeneration of postischemia neurocyte and promoting the proliferation and differentiation of nerve cells, the tissue repair and functional recovery after ischemia-reperfusion injury will profit from it inevitably. Cytokine is followed with interest by researchers because of its convenient availability, non-insult and potential effect of diminishing apoptosis and increasing the regeneration of nerve cells.Granulocyte-colony stimulating factor (G-CSF), which is granulocyte-colony hemopoietic growth factor, is used for various kinds of Neutropenia and autologous peripheral blood stem cell transplantation for immune diseases. Recent research showed that when cerebral ischemia happened G-CSF can deflate infarct volume and facilitate nerval function recovery. The possible mechanisms are : 1, promoting nerval regeneration; 2, promoting secretion of neurotrophic factor; 3, inhibiting cell apoptosis; 4, inhibiting inflammatory reaction; 5, promoting angiogenesis.Erythropoietin (EPO), which is a kind of glucoprotein hormone which can stimulate bone marrow hemopoiesis, is mainly produced by adult kidney and fetus liver. EPO is used for various kinds of anaemia, especially renal anaemia. Along with the developing of research, EPO is discovered to have neuroprotective effect in brain injury induced by ischemia through non-haemopoiesis correlated effect. The possible mechanisms are : 1, inhibiting cell apoptosis; 2, inhibiting inflammatory reaction; 3, diminishing the toxic reaction of excitatory amino acids; 4, resisting oxidization; 5, promoting neurogenesis and angiogenesis.Pathogenesy of cerebral ischemia is now considered complicated. It may be a result induced by the combined effects of multiple mechanisms. The best therapeutic efficacy can not be achieved through certain single drug treatment. It has become a new direction of research of cerebral ischemia nerve protection to combine different neuro-protectants, which have effect in different link after cerebral ischemia, to undertake multi-target and multi-link combined therapy.Therefore we attempted to combine two kinds of cytokines, which are different in structure and function, to treat cerebral ischemia-reperfusion injury. In this study, we used the model of oxygen glucose deprivation/reoxygenation of neurons of rats and the model of regional cerebral ischemia-reperfusion of rats, studied the protection effect of combining G-CSF and EPO to treat cerebral ischemia-reperfusion injury in two aspects including cell apoptosis and neural regeneration, and investigated the possible mechanism of action. This experiment content can be divided into four parts, which are summarized as follow:Part One Protection of Granulocyte-Colony Stimulating Factor Combined with Erythropoietin for the Model of Oxygen Glucose Deprivation of Neurons of RatsObjective: To establish the model of oxygen glucose deprivation/reoxygenation (OGD/R) of cortical neuron of neonate rats in vitro culture, and to observe the protection of therapy of G-CSF or EPO only or G-CSF combined with EPO.Method: Cortical neuron of neonate rats were cultured in vitro, oxygen glucose deprivation (4h)/reoxygenation (24h) were established; G-CSF and EPO were used in accordance with different concentration to study the protection for cortical neuron; optimized therapeutic strength was determined for the protection for cortical neuron which was gained by using G-CSF only or G-CSF combined with EPO; CCK-8 assay was used to determine cell viability and LDH outleakage was detected to measure the degree of cell injury; apoptosis rate was detected by means of Annexin V/PI method and the expression of apoptosis-associated protein such as Bcl-2, Bax and Caspase-3 was detected by means of Western-blot method.Results:1. The model of oxygen glucose deprivation/reoxygenation (OGD/R) of cortical neuron of neonate rats was established successfully.2. Treatment with G-CSF of 0.1μg/ml,1μg/ml and 10μg/ml can enhance cell viability,reduce LDH outleakage; the protection of G-CSF(1μg/ml subgroup) was strongest and G-CSF(10μg/ml subgroup) was a little lower.3. Treatment with EPO of 1U/ml,10U/ml and 100U/ml can enhance cell viability,reduce LDH outleakage; the protection of EPO(10U/ml subgroup) was similar to EPO(100U/ml subgroup).4. Treatment with G-CSF combined with EPO, compared with treatment with G-CSF or EPO only, obviously enhanced cell viability,reduced LDH outleakage and apoptosis rate (P<0.05). In comparison with G-CSF, EPO intervention obviously enhanced cell viability,reduced LDH outleakage and apoptosis rate (P<0.05).5. Treatment with G-CSF combined with EPO, compared with treatment with G-CSF or EPO only, obviously augmented the expression of Bcl-2, depressed the expression of Caspase-3. In comparison with G-CSF, EPO intervention obviously augmented the expression of Bcl-2, depressed the expression of Caspase-3 (P<0.05). Treatment with G-CSF or EPO only or G-CSF combined with EPO depressed the expression of Bax, but there was no significant difference in the three subgroups.Conclusions: G-CSF can lessen the injury of cortical neuron after OGD/R, the optimal treatment concentration is 1μg/ml. EPO can lessen the injury of cortical neuron after OGD/R, the optimal treatment concentration is 10U/ml. G-CSF combined with EPO can obviously inhibit cortical neuron apoptosis after OGD/R, augment the expression of Bcl-2, depress the expression of Bax and Caspase-3. EPO intervention can inhibit cortical neuron apoptosis obviously.Part Two Protection of Granulocyte-Colony Stimulating Factor Combined with Erythropoietin for the cerebral ischemia-reperfusion in RatsObjective: To establish the model of focal cerebral ischemia-reperfusion in rats, at the whole level to observe the protection of therapy of G-CSF or EPO only or G-CSF combined with EPO against the cerebral ischemia-reperfusion in rats, and to evaluate the validity and safety of therapeutic alliance.Method: The model of focal cerebral ischemia-reperfusion in rats was established by middle cerebral artery occlusion. Therapy of G-CSF or EPO only or G-CSF combined with EPO was administered for 5 days. After reperfusion for corresponding time, the neurological function score was evaluated, the infarct volume was measured with TTC stain, the brain tissue pathological changes were observed with HE stain, the brain edema was detected with dry-wet weight method, the peripheral hemogram was determined, and when the experiment completed the fatality of each group was calculated.Results:1. The model of focal cerebral ischemia-reperfusion in rats was established successfully, and the success rate of mold is 73.9%.2. The neurological function score of the three treatment groups after reperfusion for 3d, 7d,and 14d obviously exceeded that of the I/R group, and the difference was significant(P<0.05). Compared to G-CSF or EPO group, the neurological function score was significantly higher in G-CSF+EPO group (P<0.05). There was no significant difference, after each corresponding time, in the neurological function score of G-CSF group compared with that of EPO group.3. The cerebral infarction focus was mainly situated in cerebral cortex and basal ganglia area. After reperfusion for 3d and 7d, the infarct volume of the three treatment groups was significantly reduced compared to the I/R group. The infarct volume of G-CSF+EPO group was obviously reduced compared to that of the G-CSF or EPO group, which has significant difference (P<0.05 repectively). There was no significant difference, after each corresponding time, in the infarct volume of G-CSF group compared with that of EPO group.4. In I/R group, tissue HE staining showed that the brain tissue edema was obvious, the neurocyte was rarefied, the inter-space between cells became wide, the cell volume became small, the nucleus was pyknotic, the chromatospherite became not clear, cell necrosis changes such as nuclear fragmentation and caryolysis etc appeared in the center of ischemia. In each treatment group, neurocyte damage was obviously lessened, cell necrosis was reduced, and tissue edema was improved.5. In I/R group, the body weight of rats was reduced obviously, after 7d the weight appeared to increase and afterward continued to increase. After reperfusion for 1d and 3d, there was no significant difference in the weight of the three treatment groups compared with that of the I/R group. After reperfusion for 7d and 14d, the weight of the three treatment groups increased significantly compared to that of the I/R group (P<0.05). There was no significant difference, after each corresponding time, in the body weight of the three treatment groups when compared with each other.6. The fatality of the I/R group was 31.91%. The fatality of the three treatment group (G-CSF group 20.0%, EPO group 17.94% and G-CSF+EPO group 17.94%) was significantly reduced compared to that of the I/R group (P<0.05), but there was no significant difference in the fatality of the three treatment groups when compared with each other.7. There was no significant difference in the water content of brain tissue of the three treatment groups after reperfusion for 1d, and 3d compared with that of the I/R group. After reperfusion for 7d, the water content of brain tissue of the three treatment groups was reduced significantly compared to that of the I/R group (P<0.05). There was no significant difference, after each corresponding time, in the water content of brain tissue of the three treatment groups when compared with each other.8. There was no significant difference, after each corresponding time, in HB, RBC and PLT of each group when compared with each other. The WBC of I/R group began to increase after 1d, peaked after 7d and then began to decline after 14d. There was no significant difference, after each corresponding time, in the WBC of the three treatment groups when compared with each other (P>0.05); but the WBC of each treatment group was obviously more than that of control group (P<0.05).Conclusions: Therapy of G-CSF combined with EPO can obviously improve the neurological function symptoms of rats, reduce the infarct volume, lessen the pathological changes of brain tissue, reduce the fatality of rats and has protection against cerebral ischemia-reperfusion injury. Temporarily combining G-CSF with EPO does not has obvious effect on the blood parameters, and the dosage of the used drug is safe.Part Three Research of the effect of Granulocyte-Colony Stimulating Factor Combined with Erythropoietin on the neurocyte apoptosis of the cerebral ischemia-reperfusion in RatsObjective: To observe the effect of G-CSF or EPO only or combined treatment on the neurocyte apoptosis of the cerebral ischemia-reperfusion in rats and its mechanism.Method: After each corresponding time of reperfusion, the quantity of neurocyte apoptosis in the ischemia brain tissue of rats was determined by tissue TUNEL staining. The expression of apoptosis-associated protein such as Bcl-2, Bax and Caspase-3 was detected by means of Immunohistochemistry method. The activity of p-Akt was detected by means of Western-blot method.Results:1. The cell apoptosis of I/R group occurred considerably after 1d, peaked after 3d and then began to decline after 7d. There was no significant difference in the cell apoptosis quantity of the three treatment groups after reperfusion for 1d compared with that of the I/R group. After reperfusion for 3d and 7d, the cell apoptosis quantity of the three treatment groups was reduced significantly compared to that of the I/R group (P<0.05), and the cell apoptosis quantity of G-CSF+EPO group was obviously reduced compared to that of the G-CSF or EPO group, which has significant difference (P<0.05 respectively). After reperfusion for 3d and 7d, the cell apoptosis quantity of the EPO group was reduced obviously compared to that of the G-CSF group (P<0.05 respectively).2. The expression of Bcl-2 and Caspase-3 of the G-CSF+EPO group was obviously augmented and obviouly depressed respectively compared to that of the G-CSF group or EPO group. The expression of Bcl-2 and Caspase-3 of the EPO group was obviously augmented and obviouly depressed respectively compared to that of the G-CSF group, which has significant difference (P<0.05). Compared with I/R group, the expression of Bax of G-CSF group, EPO group and G-CSF+EPO group was depressed, but there was no significant differance in the expression of Bax of the three treatment groups when compared with each other.3. The activity of the brain tissue p-Akt was obviously increased in I/R group. In the three treatment groups, the activity of p-Akt was increased respectively. The activity of p-Akt of the G-CSF+EPO group was obviously increased compared to that of the G-CSF group and the EPO group (P<0.05). There was no significant difference in the activity of p-Akt when compared the G-CSF group with the EPO group.Conclusions: Therapy of G-CSF combined with EPO can obviously reduce the neurocyte apoptosis of the cerebral ischemia-reperfusion, augment the expression of Bcl-2 and depress the expression of Bax and Caspase-3. Combined treatment can have synergetic effect of inhibiting apoptosis partly by means of activating PI-3K/Akt signaling pathway. EPO has significant effect in respect of inhibiting neurocyte apoptosis.Part Four Research of the effect of Granulocyte-Colony Stimulating Factor Combined with Erythropoietin on the neural regeneration of the cerebral ischemia-reperfusion in RatsObjective: To observe the effect of G-CSF or EPO only or combined treatment on the neural proliferation, differentiation and the expression of neurotrophic factors of the cerebral ischemia-reperfusion in rats.Method: The proliferative cells were marked by Brdu. After each corresponding time of reperfusion, the Brdu positive cells of cerebral ischemia in rats of each group were detected by single fluorescent staining. The Brdu/NeuN and Brdu/GFAP both positive cells were detected by double-label immunofluorescence assays. The expression of neurotrophic factors including BDNFmRNA and NGFmRNA are detected by means of RT-PCR.Results:1. In I/R group sporadic BrdU positive cells were observed after reperfusion for 1d, the quantity of the BrdU positive cells peaked after reperfusion for 7d and began to decline after reperfusion for 14d. There was no significant difference in the BrdU positive cell quantity of the three treatment groups after reperfusion for 1d and 3d when compared with each other or compared to that of the I/R group. After reperfusion for 7d and 14d, the BrdU positive cell quantity of the three treatment groups respectively exceeded that of the I/R group significantly (P<0.05) and the BrdU positive cell quantity of G-CSF+EPO group significantly exceeded that of the G-CSF group or the EPO group respectively (P<0.05). After reperfusion for 7d and 14d, the BrdU positive cell quantity of the G-CSF group was obviously increased compared to that of the EPO group (P<0.05 respectively).2. The Brdu/NeuN and Brdu/GFAP both positive cells were observed in the I/R group. The Brdu/NeuN and Brdu/GFAP both positive cell quantity of the three treatment groups respectively exceeded that of the I/R group significantly (P<0.05); and the Brdu/NeuN and Brdu/GFAP both positive cell quantity of G-CSF+EPO group significantly exceeded that of the G-CSF group or the EPO group respectively (P<0.05). The Brdu/NeuN and Brdu/GFAP both positive cell quantity of the G-CSF group was obviously increased compared to that of the EPO group (P<0.05).3. The expression of BDNFmRNA in the I/R group was augmented obviously. The expression of BDNFmRNA of the three treatment groups respectively was augmented significantly compared to that of I/R group (P<0.05 respectively). But there was no significant differance in the expression of BDNFmRNA of the three treatment groups when compared with each other.4. The expression of NGFmRNA in the I/R group was augmented obviously. The expression of NGFmRNA of the three treatment groups respectively was augmented significantly compared to that of I/R group (P<0.05 respectively). The expression of NGFmRNA of the G-CSF+EPO group was augmented significantly compared to that of the G-CSF group or the EPO group. The expression of NGFmRNA of the G-CSF group was augmented significantly compared to that of the EPO group (P<0.05)Conclusions: Therapy of G-CSF combined with EPO can obviously promote the neural proliferation and the differentiation to neurons and glial cell of the cerebral ischemia-reperfusion in Rats, augment the expression of neurotrophic factors including BDNFmRNA and NGFmRNA. G-CSF has significant effect in respect of promoting neural regeneration.
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