本文選題：大鼠　切入點：骨髓單個核細(xì)胞　出處：《鄭州大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：背景與目的 腦出血（intracerebral hemorrhage,ICH）其定義是原發(fā)性的非外傷性的腦實質(zhì)內(nèi)出血，是一種常見的急性腦血管疾病，它的特點是發(fā)病率、病死率和致殘率都很高。然而目前尚缺乏有效的治療手段。腦出血后腦損傷機(jī)制主要包括血腫壓迫、腦水腫、炎癥和細(xì)胞凋亡等多個方面，，其中炎癥在腦出血的繼發(fā)性腦損傷中起關(guān)鍵作用。因此，防治腦出血后炎癥反應(yīng)引起的繼發(fā)性腦損傷，對于減少后遺神經(jīng)功能損害，提高患者生存質(zhì)量，具有非常重要的臨床意義。近年來干細(xì)胞移植已成為神經(jīng)科疾病治療的熱點。骨髓來源的單個核細(xì)胞(Bonemarrow mononuclear cells,BMMNCs)是由多種細(xì)胞組成的異質(zhì)群，包含造血干細(xì)胞、間充質(zhì)干細(xì)胞、內(nèi)皮祖細(xì)胞等多種干、祖細(xì)胞成分。BMMNCs用于腦血管疾病治療有許多優(yōu)勢，如獲取分離方便，避免了免疫排斥反應(yīng)，無需培養(yǎng)、可自體異體移植、無道德倫理學(xué)爭議；已較多應(yīng)用于心臟、下肢缺血性疾病方面上的動物實驗和Ⅰ期臨床研究且已證實其有效性和安全性。骨髓單個核細(xì)胞對腦出血是否有神經(jīng)保護(hù)作用，到底發(fā)揮怎樣的作用，目前未見相關(guān)報道。本研究將觀察評估骨髓單個核細(xì)胞移植后腦出血大鼠的神經(jīng)行為功能的恢復(fù)情況、腦水腫程度及神經(jīng)元細(xì)胞凋亡的情況，并檢測移植后在出血灶側(cè)周圍炎癥細(xì)胞即小膠質(zhì)細(xì)胞活化和中性粒細(xì)胞浸潤情況；進(jìn)一步探討骨髓單個核細(xì)胞對腦出血后繼發(fā)性腦損傷治療的效果，及其在繼發(fā)性腦損傷炎癥反應(yīng)中可能治療機(jī)制。 方法 應(yīng)用立體定位技術(shù)將Ⅳ型膠原酶注入大鼠尾狀核紋狀體內(nèi)制作實驗性腦出血模型，假手術(shù)組在同等條件下注射等量生理鹽水。體重250-300g的雄性SD大鼠建模后按隨機(jī)化原則分為假手術(shù)組、ICH組、PBS組、BMMNCs移植組；對用于提取細(xì)胞的大鼠連續(xù)腹腔注射Brdu,14天后收集大鼠骨髓細(xì)胞，用Fi-coll密度梯度離心法提取、分離、純化大鼠骨髓單個核細(xì)胞；Brdu標(biāo)記BMMNCs用以體內(nèi)示蹤。于ICH6h后經(jīng)股靜脈輸注3×107BMMNCs或等量PBS。采用改良神經(jīng)功能損傷評分（modi-fied neurological severity score,mNSS）評估腦出血后1d、3d、7d、14d神經(jīng)行為功能改善情況；干濕重法測定血腫周圍腦組織含水量；FJC(Fluoro-JadeC)染色法檢測出血灶周圍神經(jīng)元細(xì)胞凋亡情況；免疫熒光染色法檢測腦出血側(cè)小膠質(zhì)細(xì)胞（IBA-1+）活化及中性粒細(xì)胞浸潤(MPO+)情況；同時對標(biāo)記的Brdu行免疫熒光染色觀察BMMNCs腦內(nèi)的分布。 結(jié)果 1.假手術(shù)組在術(shù)后1d、3d、7d、14d各個時間點均未見明顯神經(jīng)功能缺損癥狀，各時間點mNSS評分均為0分，ICH組和PBS組神經(jīng)功能缺損癥狀在術(shù)后1d時達(dá)高峰，7d時均可見較明顯的恢復(fù)，14d時有進(jìn)一步的恢復(fù)。BMMNCs組神經(jīng)功能評分在7d、14d各個時間點較ICH組和PBS組明顯降低，差異有統(tǒng)計學(xué)意義(P0.05)；在腦出血后3d，BMMNCs移植組的大鼠腦組織含水量明顯降低，及神經(jīng)元凋亡數(shù)目也顯著降低，與ICH組及PBS組比較，差異有統(tǒng)計學(xué)意義(P0.05)； 2.在腦出血后1d，BMMNCs移植組可見Brdu陽性細(xì)胞散在分布于出血灶周圍，3d時達(dá)高峰，7d時呈下降趨勢；ICH組及PBS組在出血灶周圍無Brdu陽性細(xì)胞。腦出血后3d，BMMNCs移植組出血側(cè)活化增殖的小膠質(zhì)細(xì)胞數(shù)和浸潤的中性粒細(xì)胞數(shù)均較ICH組與PBS組明顯減少，差異有統(tǒng)計學(xué)意義(P0.01)。 結(jié)論 1．移植的骨髓單個核細(xì)胞能夠遷移、歸巢至腦出血灶周圍區(qū)域并存活，可顯著減輕出血后腦水腫程度，減少神經(jīng)元細(xì)胞凋亡，顯著促進(jìn)神經(jīng)行為功能的恢復(fù)。 2．骨髓單個核細(xì)胞移植可減輕出血側(cè)小膠質(zhì)細(xì)胞的增殖、活化和中性粒細(xì)胞的浸潤。 3．骨髓單個核細(xì)胞對實驗性大鼠腦出血的治療作用，其機(jī)制可能與其抑制腦出血后繼發(fā)性腦損傷中相關(guān)的炎癥反應(yīng)有關(guān)。
[Abstract]:Background and purpose
Cerebral hemorrhage (intracerebral hemorrhage ICH) is defined as the primary non traumatic intracerebral hemorrhage is a common acute cerebrovascular disease, which is characteristic of the incidence, fatality rate and mutilation rate are very high. However, there is still lack of effective treatment. The cerebral blood brain mechanism damage including hematoma oppression, brain edema, inflammation and apoptosis in many aspects, including inflammation plays a key role in the secondary brain injury of intracerebral hemorrhage. Therefore, the secondary brain injury caused by inflammatory reaction after cerebral hemorrhage prevention, to reduce the sequela of nervous function damage, improve the quality of life of patients, with clinical significance very important. In recent years, stem cell transplantation has become a hot topic in treatment of neurological diseases. Mononuclear cells derived from bone marrow (Bonemarrow mononuclear cells, BMMNCs) is composed of a variety of cells including hematopoietic stem cell heterogeneity group. , a variety of mesenchymal stem cells, endothelial progenitor cells stem progenitor cells, components of.BMMNCs for the treatment of cerebral vascular disease has many advantages, such as convenient for separation, avoid immunological rejection, without culture, autologous transplantation, no moral and ethical disputes; has more should be used for heart, hand on the ischemic disease of lower extremity animal experiment and clinical study of stage I and confirmed its effectiveness and safety. Bone marrow mononuclear cells have neuroprotective effect on cerebral hemorrhage, in the end how to play the role of, at present has not been reported. This study will evaluate the neurobehavioral function of bone marrow mononuclear cells transplantation cerebral hemorrhage rat brain recover. The degree of edema and neuronal apoptosis, and detected after transplantation in inflammatory cells around the hemorrhage side that microglia activation and neutrophil infiltration; further study of bone The effect of medullary mononuclear cells on secondary brain injury after intracerebral hemorrhage and the possible mechanism of treatment in secondary brain injury.
Method
The application of stereo positioning technology of type IV collagenase injected into rat caudate nucleus striatum produced in experimental intracerebral hemorrhage model, sham operation group injected under the same conditions of saline. Male SD rats weighing 250-300g modeling after randomly divided into sham operation group, ICH group, PBS group, BMMNCs group of transplantation; for the extraction of cells in rats by intraperitoneal injection of Brdu, collected 14 days after rat bone marrow cells by Fi-coll density gradient centrifugation extraction, separation, purification of rat bone marrow mononuclear cells; Brdu marker for BMMNCs. After ICH6h in vivo via femoral vein infusion of 3 x 107BMMNCs or equivalent PBS. with improved neural function the injury score (modi-fied neurological severity score, mNSS) assessment of cerebral hemorrhage after 1D, 3D, 7d, 14d neurobehavioral function improvement; Determination of perihematomal brain tissue water content of dry wet weight method; FJC (Fluoro-JadeC) staining. Neuronal apoptosis was detected around the hemorrhagic foci. Microglia (IBA-1+) activation and neutrophil infiltration (MPO+) were detected by immunofluorescence staining. Meanwhile, labeled Brdu was used to detect the distribution of BMMNCs brain in immunofluorescence staining.
Result
1. in the sham surgery group after operation, 1D, 3D, 7d, 14d each time point showed no obvious symptoms of neurological deficits, each time point mNSS score was 0 points, ICH group and PBS group of neurological symptoms in the postoperative 1D reached the peak, 7d showed obvious recovery, when 14d recovery.BMMNCs group further neurological score in 7d, 14d each time point were significantly lower than that in ICH group and PBS group decreased, the difference was statistically significant (P0.05); at 3D after cerebral hemorrhage, brain tissue of rats with BMMNCs transplantation group water content decreased significantly, and the number of apoptotic neurons were significantly decreased, compared with group ICH and in PBS group, the difference was statistically significant (P0.05);
In 2. 1D after ICH, BMMNCs transplantation group showed Brdu positive cells scattered in the bleeding around, reached a peak at 3D, 7d decreased; ICH group and PBS group of Brdu positive cells in the cerebral hemorrhage bleeding around. After 3D, the number of neutrophil number of microglial cell transplantation group the activation and proliferation and infiltration of BMMNCs were lower than ICH group and PBS group were significantly reduced, the difference was statistically significant (P0.01).
conclusion
1. transplantation of bone marrow mononuclear cells can migrate and migrate to the surrounding areas of cerebral hemorrhage and survive. It can significantly reduce the degree of cerebral edema after hemorrhage, reduce neuronal apoptosis, and significantly promote the recovery of neurobehavioral function.
2. transplantation of bone marrow mononuclear cells can reduce the proliferation, activation and infiltration of neutrophils in the haemorrhagic microglia.
3. the therapeutic effect of bone marrow mononuclear cells on experimental intracerebral hemorrhage in rats may be related to inhibiting inflammatory reaction related to secondary brain injury after intracerebral hemorrhage.

【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R743.34

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