發(fā)布時間：2018-07-29 15:50

【摘要】：研究背景缺血性腦血管病(ischemic cerebrovascular disease,ICVD)是目前在臨床上常見的一種神經(jīng)系統(tǒng)疾病。ICVD發(fā)生時,缺血再灌注引起的繼發(fā)性損傷的關(guān)鍵環(huán)節(jié)是急性炎癥反應(yīng)。此時細胞因子被大量的釋放、粘附分子的表達明顯升高,白細胞及其它免疫細胞被激活,從而釋放各種炎性介質(zhì)如細胞因子包括TNF-α、IL-1β、IL-6、IL-8、趨化因子及粘附分子等,TNF-α被認為是炎癥網(wǎng)絡(luò)中的關(guān)鍵因子,是全身炎癥反應(yīng)的始動介質(zhì),同時,缺血再灌注時脂質(zhì)過氧化反應(yīng)明顯加速,自由基的平衡被打破,其產(chǎn)生過量而清除減少,這些因素都嚴重損傷了神經(jīng)細胞。因此,減輕炎癥反應(yīng)、減少自由基就對保護缺血再灌注后的神經(jīng)功能具有重要意義。單核細胞移動抑制因子(monocyte locomotion inhibitory factor,MLIF)是一種抗炎性反應(yīng)的多肽,它能抑制人單核細胞、多形核白細胞的移動,抑制炎癥反應(yīng)的發(fā)生、逃避宿主免疫,同時它還對細胞因子TNF-α、IL-1β等的分泌以及相應(yīng)受體的表達有明顯的抑制作用,從而影響炎癥反應(yīng)的整個過程。研究表明MLIF的抗炎活性基團是Cys-Asn-Ser。本實驗試圖通過對MLIF的活性基團Cys-Asn-Ser的N端進行修飾,了解新的修飾后的MLIF類似物對大鼠腦缺血再灌注后神經(jīng)功能改變及TNF-α、IL-1β表達的影響,來研究這種新的MLIF類似物可能的藥理作用機制,從而為臨床水平的研究提供理論依據(jù),以便將來更好地造福缺血性腦病的患者。目的本實驗共分為兩個部分,第一部分對抗炎肽MLIF的活性肽段Cys-Asn-Ser的N端進行了全新設(shè)計和化學(xué)結(jié)構(gòu)改造。為了提高Cys-Asn-Ser的抗氧化活性,我們在Cys-Asn-Ser的N端連接一個極性不帶電荷的半胱氨酸。采用固相合成法合成一條全新設(shè)計的MLIF類似物Cys-Cys-Asn-Ser以及MLIF活性基團小分子肽段Cys-Asn-Ser。第二部分建立大鼠局灶性腦缺血再灌注損傷模型,以MLIF活性基團小分子肽段Cys-Asn-Ser作為對照,考察這個新的MLIF類似物對大鼠缺血性腦病是否具有治療作用,并初步探討這種作用可能的發(fā)生機制。方法1.本研究第一部分的化學(xué)合成實驗中,采用固相合成法,合成一個新的半胱氨酸改造的MLIF類似物Cys-Cys-Asn-Ser和MLIF的活性基團小分子肽段Cys-Asn-Ser,然后對合成的粗肽進行脫鹽處理選用的是sephadexg10凝膠柱,并結(jié)合制備型hplc純化粗肽,最后采用ms對純化產(chǎn)物進行鑒定。2.本研究嘗試用線栓來阻塞大腦中動脈,從而構(gòu)建SD大鼠腦缺血再灌注損傷的模型,待造模成功、缺血再灌注24h的時候,采用Longa法對大鼠神經(jīng)功能缺損進行評分;大鼠腦切片進行TTC染色,觀察腦缺血再灌注后腦梗死體積的變化,探討這個新的半胱氨酸改造的MLIF類似物Cys-Cys-Asn-Ser對缺血性腦病的治療作用。3.本研究第二部分動物實驗中,在大鼠腦缺血再灌注24h后,取大鼠腦組織勻漿測定TNF-α和il-1β的含量,觀察這個新改造的MLIF類似物Cys-Cys-Asn-Ser的抗炎活性。結(jié)果1.本研究第一部分的化學(xué)合成實驗中,采用固相合成法獲取的新的由半胱氨酸改造的MLIF類似物Cys-Cys-Asn-Ser和MLIF的活性基團小分子肽段Cys-Asn-Ser,前者的產(chǎn)率是40.5%,后者的產(chǎn)率是61.2%,hplc分離純化后,證明二者的純度均達到95%以上,且經(jīng)質(zhì)譜對產(chǎn)物的鑒定,結(jié)構(gòu)完全相符。2.本研究第二部分動物實驗的結(jié)果表明,模型組大鼠均出現(xiàn)了嚴重的神經(jīng)功能受損的癥狀,神經(jīng)功能評分的結(jié)果為(2.20±0.45);大鼠缺血側(cè)的腦組織有非常明顯的梗死灶,測得梗死體積為(152.85±28.83)mm3;而與模型組大鼠不同的是,各藥物治療組均在不同程度上改善了MCAO大鼠的病理改變,包括減小腦梗死的體積和降低神經(jīng)功能評分。與活性基團小分子肽段Cys-Asn-Ser相比,新的MLIF類似物Cys-Cys-Asn-Ser的活性有所提高。3.本實驗從TNF-α和il-1β的檢測數(shù)據(jù)來看,用一個半胱氨酸對MLIF活性基團小分子肽段Cys-Asn-Ser修飾后,新的MLIF類似物Cys-Cys-Asn-Ser的抗炎活性有所增強。由此可見,新的MLIF類似物Cys-Cys-Asn-Ser有抑制炎癥反應(yīng)的作用,同時它自身又有著抗氧化的作用,這兩方面的原因使得它對于缺血性腦損傷有保護作用。結(jié)論一種新的MLIF類似物Cys-Cys-Asn-Ser對缺血性再灌注腦損傷有一定的神經(jīng)保護作用,同時其修飾后的抗炎活性比MLIF活性基團小分子肽段Cys-Asn-Ser有所增強。背景和研究目的呼吸系統(tǒng)疾病是嚴重危害人民健康的常見病、多發(fā)病,已經(jīng)構(gòu)成影響公共健康的重大問題。其不僅發(fā)病率高,許多疾病起病隱襲,肺功能損害嚴重,致殘率高,給社會和國民經(jīng)濟造成了巨大損失。因此,呼吸系統(tǒng)疾病的早診斷、早治療、早預(yù)防就顯得尤為重要。在呼吸系統(tǒng)疾病的檢查方法中,呼吸功能的測定已經(jīng)越來越受到人們的重視,但肺功能檢查常規(guī)項目的信號源均是受試者自己的呼吸,檢查結(jié)果可靠與否與受試者自己的理解能力和配合能力密切相關(guān),對于一些特殊人群如年老、體弱、幼兒等患者,就不能進行這樣的檢查,而脈沖振蕩法(impulse oscillometry,IOS)測定技術(shù)的突出優(yōu)點是受試者僅需平靜呼吸,操作簡便、適用對象廣泛,目前在臨床上越來越受到青睞。影響肺功能檢查的正常值的因素非常多,主要與受試者的種族、年齡、性別、體重、身高等因素密切相關(guān),其次還與受試者的生存環(huán)境、工種、體力活動及吸煙與否等因素也有關(guān)。因此,臨床上選取合適的預(yù)計值,才是提高診斷準確率的關(guān)鍵。中國目前仍然沒有統(tǒng)一的脈沖振蕩法(IOS)測定的各參數(shù)的參考預(yù)計值及正常值公式,現(xiàn)在臨床沿用的還是歐美國家(主要是歐洲)提供的正常值范圍,這些預(yù)計值不一定符合中國人的實際,而且國內(nèi)各地區(qū)在應(yīng)用IOS法做肺功能測定時的標(biāo)準亦不一致,這樣就不利于對疾病做出準確診斷。因此,建立我國自己的IOS肺功能預(yù)計值公式顯得尤為重要。蘭州地處中國西北部地區(qū),由于特殊的地理位置和工業(yè)城市的特點,制定出我們蘭州地區(qū)IOS正常值及預(yù)計值公式勢在必行。我們的結(jié)果將為蘭州地區(qū)乃至西北五省的臨床醫(yī)生尤其是呼吸科醫(yī)生提供一個參考,也將為國內(nèi)其他地區(qū)的臨床醫(yī)生提供一個線索,為爭取早日建立我國自己的、統(tǒng)一的預(yù)計值公式及正常值標(biāo)準奠定基礎(chǔ)。材料和方法研究對象共計920例,其中女470例,男450例。選擇對象是(1)2008年2月至2011年12月在蘭州大學(xué)第二醫(yī)院進行體檢的18歲及以上的健康成人;(2)外科非心肺和非氣道疾病擇期手術(shù)的術(shù)前檢查者。研究方法歐洲呼吸學(xué)會(ERS)推薦的IOS測量標(biāo)準是本研究所采用的檢查方法。檢查前至少24 h內(nèi)未用過膽堿能受體拮抗劑、抗過敏類藥物、茶堿類藥物、β2受體激動劑、腎上腺素能受體抑制劑及激素類藥物等。測定的指標(biāo)包括:IOS參數(shù)、結(jié)構(gòu)參數(shù)圖、頻譜分析圖、阻抗容積圖(Z-V圖)等。其中實驗測定的IOS的主要參數(shù)為:呼吸總阻抗(Zrs)、中心氣道阻力(R_20)、總氣道阻力(R_5)、中心阻力(Rc)、周邊阻力(Rp)、周邊粘性阻力(R_5-R_20)、共振頻率(Fres)及周邊彈性阻力(X_5)。統(tǒng)計學(xué)分析使用SPSS 17.0軟件進行數(shù)據(jù)分析。結(jié)果1.對蘭州地區(qū)920例18歲及以上健康成人嚴格按照歐洲呼吸學(xué)會(RES)推薦的IOS測量標(biāo)準進行IOS測定,IOS參數(shù)Zrs、Fres、R_5、R_20、X_5、Rc、Rp均不呈正態(tài)分布。2.各參數(shù)正常參考值范圍與目前臨床上一直在采用的國外提供的正常參考值范圍,二者的配對t檢驗(P0.001)有顯著性差異。3.在蘭州地區(qū),分別建立了當(dāng)?shù)爻扇私】的行院徒】蹬訧OS主要參數(shù)(Zrs、Fres、R_5、R_20、X_5、)的正常參考值預(yù)計公式。4.共振頻率(Fres)均值及95%可信區(qū)間均大于目前國內(nèi)外采用的10Hz正常值,甚至大于15Hz,男女相比無顯著性差異。5.根據(jù)建立的蘭州地區(qū)健康成人的IOS各參數(shù)的正常參考值預(yù)計公式,表明粘性阻力(R)主要與體重、身高相關(guān),與體重呈正相關(guān),而身高的影響最大,呈負相關(guān)。6.在本研究所涉及的年齡范圍內(nèi)(18歲~80歲),IOS各參數(shù)值(除外X_5)隨著年齡的增長無明顯變化,而X_5與年齡成負相關(guān)。結(jié)論蘭州地區(qū)健康成人脈沖振蕩(IOS)肺功能各參數(shù)的正常參考值范圍及預(yù)計值公式與國內(nèi)、外均有差異,建議中國構(gòu)建適合國人自己的IOS各參數(shù)的正常參考值范圍及預(yù)計值公式。
[Abstract]:Background ischemic cerebrovascular disease (ischemic cerebrovascular disease, ICVD) is a common clinically common nervous system disease.ICVD. The key link of secondary injury caused by ischemia-reperfusion is acute inflammatory reaction. At this time, the cytokines are released, the expression of adhesion molecules is significantly increased, white blood cells are increased. And other immune cells are activated to release various inflammatory mediators such as cytokines including TNF- alpha, IL-1 beta, IL-6, IL-8, chemokines and adhesion molecules. TNF- is considered to be the key factor in the inflammatory network and is the starting medium of systemic inflammatory reaction. At the same time, the lipid peroxidation obviously accelerates and the free radical is flat at the time of ischemia-reperfusion. Monocyte locomotion inhibitory factor (MLIF) is a polypeptide of anti-inflammatory response. It inhibits the movement of mononuclear cells and polymorphonuclear leukocytes, inhibits the occurrence of inflammatory reactions and escaping host immunity, and it also inhibits the secretion of cytokines TNF- a, IL-1 beta, and the expression of corresponding receptors, thus affecting the whole process of the inflammatory reaction. The study shows that the anti inflammatory active group of MLIF is Cys-Asn-Ser. By modifying the N end of the active group Cys-Asn-Ser of MLIF, this experiment is to understand the effect of the new modified MLIF analogue on the nerve function changes and the expression of TNF- alpha and IL-1 beta after cerebral ischemia reperfusion in rats, to study the possible pharmacological mechanism of this new MLIF analogue, so as to provide a theoretical basis for the study of clinical level. The purpose of this experiment is to be divided into two parts. The first part of this experiment is divided into two parts. The first part is a new design and chemical structure modification against the N terminal of the active peptide Cys-Asn-Ser of the peptide MLIF. In order to improve the antioxidant activity of Cys-Asn-Ser, we connect a polar uncharged at the N end of Cys-Asn-Ser. Cysteine. A new designed MLIF analogue Cys-Cys-Asn-Ser and MLIF active group small molecular peptide Cys-Asn-Ser. second were synthesized by solid phase synthesis, and the focal cerebral ischemia reperfusion injury model was established in rats. The small molecular peptide Cys-Asn-Ser of the MLIF active group was used as the control, and the new MLIF analogue was investigated. Whether the ischemic encephalopathy of rat has a therapeutic effect, and preliminarily discuss the possible mechanism of this effect. Method 1. the first part of the study, in the first part of the chemical synthesis experiment, the solid phase synthesis method was used to synthesize a new cysteine modified MLIF analogue, Cys-Cys-Asn-Ser and MLIF, the small molecule peptide Cys-Asn-Ser of the active group, and then the synthesis was made. The crude peptide is used for desalination, which is sephadexg10 gel column, combined with prepared HPLC to purify the crude peptide, and finally uses MS to identify the purified product. This study attempts to block the middle cerebral artery with a thread to construct a model of cerebral ischemia reperfusion injury in SD rats. The model is successful. When 24h is reperfusion, Longa is used in Longa. The method was used to score the nerve function defect in rats. The rat brain slices were stained with TTC to observe the changes of cerebral infarction volume after cerebral ischemia-reperfusion. The effect of this new MLIF analogue of cysteine on the treatment of ischemic encephalopathy was discussed in the second part of the study, after the cerebral ischemia and reperfusion of 24h in the rat, the rats were treated with 24h. The content of TNF- alpha and IL-1 beta in the rat brain homogenate was measured and the anti-inflammatory activity of the newly transformed MLIF analogue, Cys-Cys-Asn-Ser was observed. Results 1. the first part of the study was a chemical synthesis of a new MLIF analogue of cysteine, Cys-Cys-Asn-Ser and MLIF, obtained by solid phase synthesis. Peptide Cys-Asn-Ser, the yield of the former was 40.5%, the yield of the latter was 61.2%. After HPLC isolation and purification, the purity of the two were all above 95%. And the structure of the product was identified by mass spectrometry. The structure of the second part of the animal experiment in the second part of the study showed that the rats in the model group had serious neurological impairment symptoms. The result of the functional score was (2.20 + 0.45); the cerebral tissue of the ischemic side of the rat had a very obvious infarct and the infarct volume was (152.85 + 28.83) mm3, but different from the model group, the pathological changes of the MCAO rats were improved to varying degrees, including the reduction of the volume of cerebral infarction and the reduction of the neurological function score. Compared with the small molecule peptide segment Cys-Asn-Ser of the active group, the activity of the new MLIF analogue Cys-Cys-Asn-Ser has been improved in.3. experiment, from the detection data of TNF- alpha and IL-1 beta, the anti-inflammatory activity of the new MLIF analogue Cys-Cys-Asn-Ser is enhanced by the modification of the small molecule peptide Cys-Asn-Ser of the MLIF active group with one cysteine. It can be seen that the new MLIF analogue, Cys-Cys-Asn-Ser, has the effect of inhibiting the inflammatory reaction and has its own antioxidant effect. These two reasons make it protective for ischemic brain damage. Conclusion a new MLIF analogue, Cys-Cys-Asn-Ser, has a certain neuroprotective effect on ischemic reperfusion brain injury. Its modified anti-inflammatory activity is stronger than the small molecular peptide segment Cys-Asn-Ser of the MLIF active group. Background and research objective respiratory disease is a common disease which seriously endangers the health of the people. It has become a major problem in the public health. It has been a major problem affecting public health. It not only has high incidence, many diseases are hidden, lung function damage is serious, and the rate of disability. As a result, the early diagnosis, early treatment and early prevention of respiratory system diseases are particularly important. In the examination methods of respiratory system diseases, the measurement of respiratory function has been paid more and more attention, but the signal sources of the routine lung function examination are their own breathing. Whether the results are reliable or not is closely related to the ability to understand and cooperate with the subjects themselves. For some special people, such as old age, body, and young children, it is impossible to carry out such examination, and the protruding advantage of the impulse oscillometry (IOS) measurement technique is that the subjects only need calm breathing, and the operation is simple and applicable. Many factors affecting the normal value of the lung function test are closely related to the race, age, sex, weight and height of the subjects, and the following factors are also related to the living environment, work, physical activity and smoking or not. The estimated value is the key to improving the accuracy of the diagnosis. There is still no reference expected value and normal value formula of the parameters measured by the unified pulse oscillation method (IOS). Now it is still in use in the European and American countries (mainly Europe), which are not necessarily in accordance with the Chinese reality, but also in China. The criteria for the determination of lung function by the IOS method in each area are also inconsistent, which is not conducive to the accurate diagnosis of the disease. Therefore, it is very important to establish the formula of the predicted value of the lung function of our own IOS. Lanzhou is located in the northwest of China, and the special geographical position and the characteristics of the industrial city have made us Lanzhou. The normal value and the predicted value formula of the regional IOS are imperative. Our results will provide a reference for clinicians in the five provinces of Lanzhou and the northwest, especially the Department of respiration doctors, and also provide a clue for the clinicians in other parts of the country to strive for the early establishment of our own, unified predicted value formula and normal value standard. The materials and methods were studied in 920 cases, including 470 women and 450 men. (1) healthy adults aged 18 and above in Second Hospital Affiliated to Lanzhou University from February 2008 to December 2011; (2) preoperative examiners of elective hand surgery for non cardiopulmonary and non airway diseases. Methods European Respiratory Society (ERS). The recommended IOS measurement standard is the method used in this study. Unused cholinergic receptor antagonists, antiallergic drugs, theophylline, beta 2 receptor agonists, adrenergic receptor inhibitors and hormone drugs, including IOS parameters, structural parameters, spectrum analysis, impedance volume, and so on at least 24 h before examination. The main parameters of the experimental IOS were: total respiratory impedance (Zrs), central airway resistance (R_20), total airway resistance (R_5), central resistance (Rc), peripheral resistance (Rp), peripheral viscous resistance (R_5-R_20), resonance frequency (Fres) and peripheral elastic resistance (X_5). Statistical analysis used SPSS 17 software to carry out data analysis. Results 1. pairs of data. 920 healthy adults aged 18 and over in Lanzhou were determined strictly according to the IOS measurement standard recommended by the European Society of respiratory (RES). IOS parameters Zrs, Fres, R_5, R_20, X_5, Rc, Rp are not normal distribution of the normal reference range of the.2. parameters and the normal range of reference values that are currently being used abroad, and the two P0.001 has significant difference.3. in Lanzhou region, the normal reference value of local adult healthy male and healthy female IOS parameters (Zrs, Fres, R_5, R_20, X_5), the mean value of.4. resonance frequency (Fres) and 95% confidence interval are greater than that of 10Hz normal values, even larger than 15Hz, and no men and women. The significant difference.5. based on the normal reference value prediction formula of the IOS parameters of healthy adults in Lanzhou area, indicating that the viscosity resistance (R) is mainly related to body weight, height and body weight, and the height has the greatest influence, and the negative correlation.6. is within the age range of 18 years (18 years old) and IOS values (except X_5). The growth of age has no significant change, but X_5 is negatively correlated with age. Conclusion the normal reference value range and prediction formula of lung function parameters of healthy adults in Lanzhou region are different from those at home and abroad. It is suggested that China construct the normal reference range and the predicted value formula suitable for the number of IOS of Chinese people.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R743.3

【參考文獻】

相關(guān)期刊論文 前10條

1 胡國平;鐘南山;冉丕鑫;吳開杰;;中國的大氣污染和COPD[J];中國胸心血管外科臨床雜志;2016年02期

2 崔國東;張雪梅;陳立杰;;IL-1β在急性腦缺血中作用機制的研究進展[J];臨床神經(jīng)病學(xué)雜志;2015年06期

3 祁生貴;吳天一;;慢性高原病診斷標(biāo)準及相關(guān)研究[J];高原醫(yī)學(xué)雜志;2015年04期

4 翟小虎;王斌;宋亞剛;沈甜;馬忠英;文愛東;;線栓法制備大鼠腦缺血模型的研究進展與思路[J];實驗動物科學(xué);2015年05期

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