| Objectives:This study as a whole, the molecular and cellular level observation after cerebral ischemia SDF-1/CXCR4signaling pathways for the effect of endogenous neural stem cell migration and the mechanism of action of radix rehmanniae YinZi. By observing the rehmannia YinZi on focal cerebral ischemia in rats after migration of neural stem cells and the effect of SDF-1/CXCR4, clear SDF-1/CXCR4signaling pathway to promote the mechanism of action of endogenous neural stem cell migration. Clarify SDF-1/CXCR4signaling pathways for the effect of nerve regeneration and rehmannia YinZi mechanisms that promote nerve regeneration, revealing the ischemic cerebral apoplexy nerve regeneration new regulatory mechanisms and targets.Methods:Experiment1: radix rehmanniae YinZi on focal cerebral ischemia in rats after SDF-1the effect of protein content. By using middle cerebral artery line bolt copy cerebral ischemia model. Building successful rats were randomly divided into model group, radix rehmanniae YinZi high dose group, low dose group, radix rehmanniae YinZi in radix rehmanniae YinZi dose, nim ground group, each group of15only. Setting up15only pseudo surgery group, surgery only separation on the right side of the carotid artery, embolism, step by step after suture, postoperative intraperitoneal injection of penicillin to prevent infection3days. Blank group of normal diet, illumination. Dosage:radix rehmanniae YinZi high-dose group irrigation suits rehmannia YinZi soup120g, kg, rehmannia YinZi filling and high dose group take rehmannia YinZi soup36g, kg, rehmannia YinZi filling and high dose group take12g, kg, radix rehmanniae YinZi soup for three weeks. Nim horizon group rats to fill every day take nim horizon, dissolved the nim horizon in water for injection, the dose of12.5mg/kg, treatment for3weeks. Model group take ordinary tap water, free food, for three weeks. Measurement of ethology score, area of cerebral infarction, immunohistochemical staining, detection in SDF-1protein detection.Experiment2:radix rehmanniae YinZi drug-containing serum affect hippocampal slices of SDF-1. Middle cerebral artery line tied method is used to copy the cerebral ischemia model. Made after the success of the mold filling and uniform rehmannia YinZi each2ml of liquids, dose conversion method according to the surface area is equivalent to three times the dose of normal adults,70kg weight, the conversion of the dosage of the rat is about per kilogram of body weight to fill the14.5g content. Treatment dosing beheaded executed after7days of animals, collect the rat blood, serum, centrifugal separation inactivated in the56℃water bath temperature for30minutes, use0.22mu m filter for filtering, repackaging and placed in the refrigerator-20℃. Set aside. With ether anesthesia in rats, as for a sterile environment, break out the brain tissue, put into the sugar concentration is7.5g/L HBSS solution, temperature kept at4℃, a small local branch hippocampal tissue. On the surface covered by3%AGAR on biopsy, brain tissue using coronal incision and cut into slices of thickness of about400microns. Then slices into6hole training on the microporous membrane filter plate, soak in the nutrient solution culture plate. Each culture plate contains6holes, each hole placed6slices, nutrient solution containing5%CO2, temperature of37℃. Will culture plate into the standard balance humidity incubator for culture, every other day replacement culture, training time is2weeks. Cultivation group:2weeks later, divided into the control group (group HOTC) and low oxygen lack of sugar group (OGD group). The culture medium composition:25%HBSS,50%MEM,23%horse serum,0.75%glucose,0.5%glutamine,2%double resistance. Experiments in two groups of brain slices, respectively is HOTC group, OGD group, two groups of slices per100mg of slices, respectively, add1ml of well slurry and fully stir the mix slurry, the temperature of4℃centrifuge with12000r/min, centrifugal10minutes, take that and packaging of supernatant fluid deposit-80℃in the fridge for later use. According to the method of Lowry et al, USES the calf serum albumin as a standard reference protein, further determination of protein content.Experiment3: radix rehmanniae YinZi drug-containing serum on fetal rat hippocampal neural stem cell migration. Experiment using fetal rats born15 days break out after the brain tissue, using monoclonal cultivation of neural stem cells, methods and limit dilution method with DMEM/F12cell culture medium. Join B27additive20ml. L-1, add10g.L bFGF20g.L-1-1, EGF. Growth of nerve cells, such as nerve cells in the group, with the method of the mechanical blowing repeatedly percussion, after blowing cell subculture,1batches every2-3days. Then neural stem cells develop. Cell culture experiment is divided into three groups, including the treatment group, model group and blank group. Treatment group cell culture to give rehmannia YinZi drug-containing serum; Cell culture model group give equal0.01mol/LPBS conditioned medium; Blank group of cells to normal cells. Each nutrient solution for2hours after injury. Add in sugar cultures, nutrient solution concentration was15%, the five experiments respectively. Groups of cells with low sugar2hours after injury in the temperature of37℃and5%CO2incubator for culture, in time for24hours. Collect all training hole of cloned cells, add methanol fixed15minutes, and then to go on after centrifugal clear liquid, add0.5mL0.01mol/LPBS beat evenly, move a small cells placed in counting board, with10times under a microscope objective observation and counting, statistical cell number greater than50the number of clones. Will cultivate good upper neural stem cells inoculated in transwell little room, in the small room of the upper surface as chemotactic membrane, the cell culture in transwell upper chamber,8hours incubation in the incubator, with methanol (membrane on the back of the neural stem cells, under the microscope count after dyeing, and calculate the migration index:under the condition of calculation per10high power field of vision cells was used to calculate the cell migration index.Results:(1) in the rat ethology score: Longa score is to evaluate the nervous system damage in rats, the experiment7days,14days,21days to nerve function score, respectively. Blank group rats normal nerve function, rehmannia YinZi high dose group and middle dose group in the7days,14days has significant difference compared with model group (P<0.05); In21days, compared with model group has a very significant difference (P<0.01). Rehmannia YinZi low dose group and nim horizon group was no significant difference.(2) the determination of cerebral infarction area, blank group rats infarction area is0, rehmannia YinZi high dose group and middle dose group compared with model group has a very significant difference (P<0.01), rehmannia YinZi low-dose group with significant difference compared with model group (P<0.05), nim horizon group compared with model group, no significant difference.(3) cerebral ischemia rats BrdU positive cells number changes:the BrdU positive cells of rats, compared to the number of CA1and CA3area, DG area, rehmannia YinZi compared with model group, high dose group has a very significant difference (P<0.01). YinZi in r. dose group, radix rehmanniae YinZi low dose group compared with model group there were significant differences (P<0.05); Nim horizon group compared with model group, no significant difference. (4) in cerebral ischemia rats SDF-1protein:the influence of each group, the content of SDF-1protein in rats with rehmannia YinZi high dose group and middle dose group compared with model group has a very significant difference (P<0.01), rehmannia YinZi low-dose group with significant difference compared with model group (P<0.05), nim horizon group compared with model group, no significant difference.(5) the change of rat hippocampal slice of SDF:each piece of SDF-1rats, the content of protein, CXCR4rehmannia YinZi drug-containing serum training rats, can significantly improve the ischemia and hypoxia of brain slice of SDF-1and CXCR4protein content, significant differences compared with control group (P <0.05).(6) Western blot test groups of SDF-1protein, OGD group after rehmannia YinZi drug-containing serum culture obviously improved SDF-1protein content, better than the control group (group HOTC).(7) nerve cell migration change:normal cultivation of neural stem cells, the cells to stick wall after gather around a large number of cells, the distance between the cells is less than0.5mm, low sugar, low oxygen damage cause cells to narrow spacing. Rehmannia YinZi drug-containing serum neural stem cell migration occurs after intervention, the cell spacing increases, the migration distance of1.8-1.8mm.(8) the influence of hippocampal neural stem cell cloning count:radix rehmanniae YinZi drug-containing serum on the third day,7days neural stem cell clone number compared with the model group with significant difference (P <0.05), compared with model group in the first days there was no significant difference (P>0.05).(9)Neural stem cells in the hippocampus cell migration: the influence of the experimental observation of migration of neural stem cells by Transwell method, the treatment group by Transwell Chambers staining after migration, migration cell number number significantly higher than the model group, the results showed that radix rehmanniae YinZi drug-containing serum can obviously promote the migration of neural stem cells.Conclusions:(1) rehmannia YinZi can effectively improve neurologic deficits after cerebral ischemia rats. High-dose group and middle dose group curative effect is better than that of low dose group and nim horizon.(2) rehmannia YinZi high dose and middle dose group can improve the SDF-1protein, curative effect is better than that of nim horizon group. Showed that radix rehmanniae YinZi can obviously promote the endogenous nerve regeneration. SDF-1content improve enough to help promote the migration of neural stem cells after cerebral ischemia and ischemia area, further differentiation into neurons.(3) rehmannia YinZi can promote neural functional recovery after cerebral ischemia rats, reduce brain edema, reduce the cerebral infarction area. The increase of protein content, promote the SDF-1through the SDF-1/CXCR4 signaling pathways to further promote the hippocampus neural stem cell proliferation, and migration to ischemic injury area to differentiate into neurons, repair ischemic injury.(4) rehmannia YinZi drug-containing serum can effectively improve the ischemia anoxic zone slices SDF-1content and further improve the cerebral ischemic anoxia ability, through the SDF-1/CXCR4signaling pathway to promote brain nerve regeneration, to further promote the low oxygen sugar area brain nerve regeneration ability.(5) rehmannia YinZi drug-containing serum can promote rat hippocampal neural stem cells cloned count, promote the migration of neural stem cells number and migration rate, promote the migration of neural stem cells to ischemic area, differentiate into neurons, which may be one of the mechanism of radix rehmanniae YinZi promote nerve regeneration. |
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