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Proteomic Analysis of Rat Focal Cerebral Ischemia and Orientation Transplant Human Bone Marrow Mesenchymal Stem Cells to Therapy the Focal Cerebral Ischemia in Rats

Author: MaXueLing
Tutor: JiangXinMei;LiuKangDing
School: Jilin University
Course: Neurology
Keywords: proteome bone marrow mesenchymal stem cells rat cerebral ischemia transplant cell α-tubulin Angiopoietins -1,2
CLC: R743.3
Type: PhD thesis
Year: 2008
Downloads: 262
Quote: 0
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Abstract


1、The study on the focal cerebral ischemia of rat with DIGECerebral ischemia is one of the clinical common and frequently-occurring diseases. It has characteristic of high incidence, disable rate and high mortality rate so it severely impairs human health. At present, many researches are focus on morbility risk factors, while there are seldom studies about pathogenesy of cerebral ischemia on protein level. Because the focal brain blood is occluded or insufficient, focal ischemia leads to the strong stimulus for cerebral gene expression that plays a role in cerebral ischemic damage. To identify disease-relevant proteins and illuminate the pathophysiological mechanisms is very important for stroke therapy. To provide the clue for identification the putative candidate proteins that can confer diagnostic markers and neuroprotection against stroke for the future.The protein is the physiological function performer, and the biological phenomena manifests. The study about protein structure and the function will directly clarify the change mechanism under physiological or the pathology condition. So a new subject, proteomics, has been produced, and the important task is of proteomics to discover change the protein.Because some cells or organizations had any normal or abnormal changes which caused protein to change correspondingly, these created the subject of differential proteome. it is for the purpose of resting on the cell either the organization sample the protein difference which in the different organization, the different time, the different condition or under the different ambient condition function expresses, the observation function related protein expression parallel change, achieves the appraisal, the screening protein, the plan difference protein mass spectrum, to seek for a difference dystopia goal. Two-dimensional differential in-gel electrophoresis (2D-DIGE) ingeniously has solved the traditional 2DE duplication badly and the system error. It mixed the difference samples respectively by the different fluorescent dye mark, and different fluorescent dye was excited by different specific wave length laser. Since DIGE fluorescence Cy dye may mark the two-dimensional electrophoresis gelatin and with highly sensitivity, DIGE may examine the low abundance proteins.The differential proteome provides the powerful evidence to study some hypothesis. It has been established the very important status in degencrative disease of nervous system, however, it was used in ischemia cerebrovascular disease research with not long period. We can apply DIGE to explore the pathogenesy of cerebral ischemia. At present, domestic and foreign scientists used the proteomics method to research cerebral ischemia on the blood plasma and the body fluid,but there was just started to study brain tissue of cerebral ischemia.We used a new improved DIGE technique for proteomic analysis, introduced by Amersham Biosciences, Inc recently. Paired samples were labeled with fluorescent Cy3 and Cy5 dyes. A Cy2-labeled sample pool was together electrophoresed with all Cy3-labeled and Cy5-labeled sample pairs in the same 2D gel as an internal standard to provide inter-gel comparisons and reliable statistics analysis of the data. Following Isoelectric focusing (IEF) was carried out, scanning the gels at specific wavelengths for each dye. And the differences in abundance of specific protein spots were determined by the Amersham Biosciences DeCyder Differential In-gel Analysis DIA software. 2D DIGE was applied to quantify the differences in protein expression between the left hemisphere cerebral cortex tissues following 6h MCAO and the normal left hemisphere cerebral cortex as control to define cerebral ischemia specific protein markers. And we selected protein spots expression levels which showed a statistically significant (p<0.05) and AR>1.4 on every gel. Peptide mass fingerprinting was used by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry to identify difference protein spots. The results of this research was that there were 13 protein spots consistent with our statistics requests compared 6h after MCAO with normal control group. These protein spots were all identified and one protein was identified asα-tubulin that was significant decreased in the ischemia rats compared with the controls.α-tubulin as one of the cytoskeletal proteins whose expression levels is decreased in the early stage after MCAO focal ischemia. And it is relevant-protein of cerebral ischemia at early stage. It supplies some clues to understand the molecular characterization of cerebral ischemia progression.2、The effects of orientation transplant human bone marrow mesenchymal stem cells into focal cerebral ischemia of rats to neurologic impairment.Cerebral ischemia is a major cause of frequently-occurring, disability and death in the neurology disease. Therefore, how to reduce the ischemia area damage and the retrieval ischemic penumbra nerve cell, becomes the key of treatment cerebral ischemia. However, there is no effective treatment to prevent the death of neural cell and the neurological deficits in stroke patients at present.In recent years, therapeutic angiogenesis (TAG) gradually became the new research hot spot, which refers to urge the new blood capillary in the original capillaries foundation through methods of medicine, gene or stem cell transplant to improve the blood supplied for ischemia tissues [1]. The people discovered that has nerve stem cell (NSC) in the central nervous system, it may differentiate into the neuron as well as the neuroglia cell. But because its quantity is few, the selection difficulty, the heterotransplantation existence immune repelling and ethics morals question, caused it to be restricted in the treatment application.Recently some studies suggest that marrow mesenchymal stem cells (MSCs) can migrate selectively and be localized to the boundary of the ischemic lesion in rats subjected to stroke and improve functional recovery. It is the mesoderm growth early mesenchymal stem cell, has the very strong self-renewal and reproductive activity, multi-directional differentiation potential. In addition, MSC also has the origin to be widespread, the selection is easy, to increase rapidly, the organization fusion is good, migration ability, the immunogenic is weak. MSCs provide the broad prospects for transplanted to treatment cerebral ischemia.Therefore, this part object was to detect behavior and pathology change of MCAO rats after hMSCs transplanted. We attempt to confirm that hMSCs improve nerve function of MCAO rats and to provide the experiment foundation for transplantation the hMSCs to treatment cerebral ischemia disease.The methods of this part research: The hMSCs were cultured, purified, and proliferated in vitro. Then identified hMSCs by flow cytometer before the BrdU-labeled hMSCs. The models of Wistar rats were prepared after model MCAO lasted 90 minutes and reperfusion 1 hour. Wistar rats were randomly divided into the normal group(A), the sham operation group(B), the no-handle group after cerebral ischemia/reperfusion(C), the group of transplantation free-serm DMEM after cerebral ischemia/reperfusion(D), the group of transplantation hMSCs after cerebral ischemia/reperfusion(E). And every group was divided into 1, 3, 7, 28 days different points. The BrdU-labeled hMSCs and serum-free DMEM were respectively injected intracerebral into ischemic boundary zone. Immunohistochemical method was used to detect the BrdU-labeled hMSCs expression and survival in vivo. Neurological functional behavioral tests, HE dyeing observation pathology change, were performed at different days after transplantation.The results of this part: the hMSCs were successfully purified and proliferated in vitro and the hMSCs expressed CD29, CD44 but no expressed CD34, CD45, CD31 identified by flow cytometer. The BrdU immunohistochemistry confirmed that transplanted hMSCs were survived and migrated in the ischemic rat brain. And behavioral tests of every group were improved with time prolong. However, group E was significantly better than any other groups (P<0.05). And at 7th day after hMSCs transplantation, neurological function was significantly improved than other time points in group E (P<0.01). HE dyes proved that the nerve cells necrosis and decrease with massive glial cells proliferation and the neogenesis blood capillary formation following the time prolonged in group E. We considered that after transplanting the hMSCs, nerve function of MCAO rats were promoted restoration.3、The discussion about the mechanism of hMSCs treatment cerebral focal ischemia.However, what mechanisms urges hMSCs to produce to the nerve function resumes the function? At present they are still not very clear. Many scientists thought that MSCs differentiate into the neurons and the neuroglia cells to substitute necrosis cells, MSCs secrete nutrition factors such as nerve growth factor, MSCs activate the NSCs to regeneration ioto nerve cells. But resumes the blood supply is an important for nerve cells in the ischemia area. And it depends on to a great extent the new vessels. Present, many factors about angiopoiesis absorbed researcher attentions, such as vascular endothelial growth factor(VEGF) and Angiopoietins (Ang). The Ang1 can maintain the stability of endangium, promote blood vessel maturity,Ang2 can antagonize Ang1,destroy the stability of blood vessels, and it relates to endothelial cell proliferation. Angiogenesis is associated with improved neurological recovery. But how hMSCs to promote the new vessels and improve blood supply, and the relation with Ang-1 and Ang-2, there are few research and explanation on them.Therefore we want to explore that treatment of stroke with hMSCs. The expression and distribution ofα-tubulin by immunohistochemistry and the immunofluorescence, and the expressions of angiogenin-1 and angiogenin-2 mRNA by RT-PCR, were performed at 1, 3, 7 and 28 days in different groups after transplantation. We explore the mechanisms of hMSCs implantation on neurofunctional recovery following focal cerebral ischemia in rats.The results of this part: the expression ofα-tubulin, cerebral ischemia relevant proteins at early stage, was increased along with the time expand, the structure of neuron was gradually distinct, and neurite were extended in group E by immunohistochemistry stain. The immunofluorescence intensityα-tubulin was increased gradually along with the transplant time prolonged, and it was increased obviously from 3th day to 7th day (P<0.01), then it was got to platform stage after 7th day maintaining in a high level. The rules of Ang-2 mRNA expression were that it reached the peak at the 3th day in group E (P<0.01) while it started to descend from the 7th to the 21th day and the expression of Ang-2 mRNA was similar with group C and group D at 21th day (P>0.05). The rules of Ang-1 mRNA expression were that it reached the peak at the 3th day in group E (P<0.01) and was continued from 3th day to 21th day in a high level.This research confirmed that the intracerebral administration of hMSCs into ischemic boundary zone can significantly promote the recovery of neurological function of rats with focal cerebral ischemia, promote the expression ofα-tubulin, revival the numbers of neuron and neurite. In addition, the ischemia oxygen deficit made Ang2 the synthesis to increase the numbers of blood capillary in ischemic penumbra. The Ang-2 expression declined, and simultaneously the Ang-1 expression increased, Ang2 was possibly one of new vessels starting links. The transplantation of hMSCs enforce vascular neogenesis in the early stage while stabilize new vessels to retrieve the blood supply.α-tubulin is neuron-protection protein at early stage and hMSCs possess neuroprotective effect. And we provided the experiment foundation and the theory basis to transplant hMSCs for treatment cerebral focal ischemia.

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CLC: > Medicine, health > Neurology and psychiatry > Neurology > Cerebrovascular disease > Acute cerebrovascular disease ( stroke)
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