【摘要】：視網(wǎng)膜色素變性(retinitis pigmentosa,RP)是一類可遺傳的視網(wǎng)膜退行性病變(retinal degeneration,RD),以進(jìn)行性視網(wǎng)膜感光細(xì)胞的死亡、視野缺損和視力喪失為特征。目前,全球RP患病率約為1/3000~1/7000。由于RP的遺傳學(xué)機(jī)制復(fù)雜,使得該病的具體發(fā)病機(jī)制尚未完全明確,且缺乏有效的防治措施。一般認(rèn)為感光細(xì)胞的凋亡是RP的共同終末途徑,而活性氧(reactive oxygen species,ROS)則被認(rèn)為是RP發(fā)病過程感光細(xì)胞凋亡的重要促成因素。氫分子是近年來被發(fā)現(xiàn)的一種可選擇性清除ROS的抗氧化劑。文獻(xiàn)和我們前期的研究發(fā)現(xiàn)氫分子可通過抗氧化損傷、調(diào)節(jié)凋亡、減輕炎癥等通路對(duì)眼內(nèi)炎、白內(nèi)障及視網(wǎng)膜光損傷等眼疾起到保護(hù)作用。據(jù)此,我們提出研究假說:氫分子對(duì)RP視網(wǎng)膜損傷具有保護(hù)作用。為了驗(yàn)證研究假說,我們選擇N-甲基-N-亞硝基脲(N-Methyl-N-nitrosourea,MNU)誘導(dǎo)RP大鼠模型進(jìn)行研究。該動(dòng)物模型具有造模快、可重復(fù)性高等優(yōu)點(diǎn),是一種常用于RP發(fā)病機(jī)制和防治措施研究的動(dòng)物模型。但是,以往的研究僅對(duì)該模型的病程時(shí)間特征進(jìn)行研究,而很少涉及該模型視網(wǎng)膜變性的空間特征(如:視網(wǎng)膜變性在中央部與周邊部是否存在差異?鼻側(cè)與顳側(cè)是否存在差異?上側(cè)與下側(cè)是否存在差異?外層視網(wǎng)膜變性的同時(shí),內(nèi)層視網(wǎng)膜是否也發(fā)生病變?視網(wǎng)膜不同神經(jīng)環(huán)路的病變是否一致?)。本研究旨在深入研究mnu誘導(dǎo)的rp大鼠視網(wǎng)膜病變時(shí)空特點(diǎn)的基礎(chǔ)上,驗(yàn)證氫分子對(duì)該模型視網(wǎng)膜功能和結(jié)構(gòu)的保護(hù)作用,并從抗氧化和抗凋亡角度初步探索其保護(hù)機(jī)制。本研究將對(duì)mnu誘導(dǎo)rp動(dòng)物模型的發(fā)病機(jī)制理論進(jìn)行補(bǔ)充,并為rp的防治提供新思路,同時(shí)也為氫分子制劑的臨床應(yīng)用轉(zhuǎn)化提供一定的實(shí)驗(yàn)依據(jù)。材料與方法實(shí)驗(yàn)一:mnu誘導(dǎo)rp大鼠視網(wǎng)膜時(shí)空特點(diǎn)評(píng)估將90只sd大鼠隨機(jī)等分5個(gè)組:1個(gè)對(duì)照組和4個(gè)mnu干預(yù)組。其中各mnu干預(yù)組大鼠僅接受1次腹腔注射mnu,劑量為60mg/kg,而對(duì)照組腹腔注射等量的生理鹽水。四個(gè)mnu干預(yù)組分別在干預(yù)后第一天(p1)、p3、p5、p7天進(jìn)行各項(xiàng)指標(biāo)的檢測。(1)利用視網(wǎng)膜電圖(electroretinogram,erg)從在體水平觀察mnu誘導(dǎo)rp大鼠視網(wǎng)膜功能隨病程變化的特點(diǎn);(2)利用多電極陣列(multielectrodearray,mea)從離體水平觀察該模型大鼠不同局部(中央/周邊、鼻側(cè)/顳側(cè)、上側(cè)/下側(cè))視網(wǎng)膜的功能;(3)利用光學(xué)相干斷層掃描(opticalcoherencetomography,oct)從在體水平觀察該模型大鼠視網(wǎng)膜結(jié)構(gòu)隨病程變化的特點(diǎn);(4)利用視網(wǎng)膜石蠟切片he染色從離體水平觀察該模型大鼠不同局部視網(wǎng)膜的組織結(jié)構(gòu);(5)利用視網(wǎng)膜鋪片免疫熒光染色觀察該模型大鼠不同局部視網(wǎng)膜感光細(xì)胞的存活情況;(6)利用westernblotting檢測該模型大鼠視網(wǎng)膜視桿細(xì)胞和視錐細(xì)胞的存活情況。(7)利用定量反轉(zhuǎn)錄聚合酶鏈反應(yīng)(quantitativereversetranscriptionpolymerasechainreaction,qrt-pcr)檢測促凋亡基因bax、calpain2、caspase3和抗凋亡基因bcl2的表達(dá)情況。實(shí)驗(yàn)二:氫飽和生理鹽水對(duì)mnu誘導(dǎo)rp大鼠視網(wǎng)膜保護(hù)作用的研究將96只sd大鼠隨機(jī)等分4個(gè)組:正常對(duì)照組(normalcontrol,nc)、氫飽和生理鹽水(hydrogen-richsaline,hrs)對(duì)照組、生理鹽水干預(yù)組(mnu+生理鹽水)和hrs干預(yù)組(mnu+hrs)。mnu的給藥方式及劑量同實(shí)驗(yàn)一。hrs和生理鹽水的干預(yù)方式均為腹腔注射,劑量為10ml/kg/d,于mnu造模前14天開始干預(yù)。在mnu造模后第三天(p3)和第七天(p7)天進(jìn)行各項(xiàng)指標(biāo)的檢測。(1)利用erg和mea場電位從外層視網(wǎng)膜細(xì)胞功能角度觀察hrs的保護(hù)作用;(2)利用mea記錄視網(wǎng)膜神經(jīng)節(jié)細(xì)胞(retinalganglioncells,rgcs)放電情況,從內(nèi)層視網(wǎng)膜細(xì)胞功能角度觀察hrs的保護(hù)作用;(3)利用視網(wǎng)膜石蠟切片he染色從形態(tài)學(xué)角度觀察hrs的保護(hù)作用;(4)利用視網(wǎng)膜切片tunel染色和qrt-pcr檢測凋亡相關(guān)基因bax、calpain2、caspase3、bcl2的表達(dá)情況,從抗凋亡角度研究hrs保護(hù)作用機(jī)制;(5)利用羥胺法測定視網(wǎng)膜總超氧化物歧化酶(totalsuperoxidedismutase,t-sod)活性和硫代巴比妥酸法測定視網(wǎng)膜丙二醛(malondialdehyde,mda)含量,從抗氧化角度研究hrs保護(hù)作用機(jī)制。結(jié)果1.大鼠腹腔單次注射60mg/kg的mnu后,視網(wǎng)膜病變表現(xiàn)出以下幾個(gè)時(shí)空特點(diǎn):(1)mnu干預(yù)一周內(nèi)病變逐漸加重。erg反應(yīng)及mea場電位反應(yīng)幅值均逐漸降低,到p7時(shí),幾乎都表現(xiàn)為熄滅型反應(yīng)。oct和he染色測量視網(wǎng)膜外核層(outernuclearlayer,onl)層厚度結(jié)果也顯示,onl層厚度逐漸變薄,到p7時(shí),onl層細(xì)胞基本消失。(2)感光細(xì)胞凋亡高峰發(fā)生在p3。qrt-pcr檢測結(jié)果顯示,促凋亡基因bax、calpain2和caspase3在p3時(shí)表達(dá)上調(diào)達(dá)到峰值,同時(shí)抗凋亡基因bcl2的表達(dá)也在p3時(shí)達(dá)到最大程度的下調(diào)。(3)病變遵循視桿細(xì)胞→視錐細(xì)胞的規(guī)律。反映視錐細(xì)胞功能的明視erg反應(yīng)幅值下降(p3)要晚于反映視桿細(xì)胞功能的暗視erg反應(yīng)。視網(wǎng)膜鋪片感光細(xì)胞熒光染色及視桿細(xì)胞/視錐細(xì)胞特異蛋白westernblotting檢測結(jié)果也顯示,病變?cè)缙谝晽U細(xì)胞丟失程度要重于視錐細(xì)胞。(4)病變遵循中央部→周邊部的規(guī)律。mea場電位結(jié)果顯示,中央部幅值下降最早,隨后發(fā)展到近周部,最后累及周邊部。he染色onl層厚度變薄也表現(xiàn)出這一順序。(5)病變遵循鼻下→鼻上、顳下→顳上的規(guī)律。mea場電位分象限分析結(jié)果顯示,視網(wǎng)膜鼻下象限幅值最早發(fā)生下降,隨后是鼻上和顳下,最后發(fā)展到顳上象限。視網(wǎng)膜鋪片感光細(xì)胞熒光染色及qrt-pcr檢測視網(wǎng)膜各個(gè)象限凋亡水平結(jié)果也表現(xiàn)出這一特點(diǎn)。(6)病變遵循on通路→off通路的規(guī)律。mea記錄rgcs光誘發(fā)放電結(jié)果顯示,on型反應(yīng)的放電頻率明顯下降發(fā)生在p3,要早于off型反應(yīng)(p5),病變?cè)缙诠庹T發(fā)rgcs總反應(yīng)下降與on通路病變密切相關(guān)。2.hrs對(duì)mnu誘導(dǎo)rp大鼠視網(wǎng)膜具有保護(hù)作用。在功能學(xué)和形態(tài)學(xué)方面均可見其保護(hù)作用:(1)功能學(xué)保護(hù)作用:hrs干預(yù)組較生理鹽水干預(yù)組而言,其明視erg、暗視erg及mea場電位幅值均明顯改善,表明hrs對(duì)外層視網(wǎng)膜細(xì)胞功能有保護(hù)作用。而mea記錄rgcs動(dòng)作電位結(jié)果則顯示,hrs可有效降低mnu引起的rgcs自發(fā)放電頻率,提高其對(duì)有效視覺信息的反應(yīng),表明hrs對(duì)內(nèi)層視網(wǎng)膜細(xì)胞功能及視覺通路的完整性也具有保護(hù)作用。(2)形態(tài)學(xué)保護(hù)作用:he染色測量視網(wǎng)膜onl層厚度,發(fā)現(xiàn)hrs可有效延緩onl層細(xì)胞的丟失。3.hrs的這種保護(hù)作用無視網(wǎng)膜區(qū)域性差異。mea場電位結(jié)果及he染色結(jié)果均顯示,hrs對(duì)中央部、近周部和周邊部的視網(wǎng)膜功能和結(jié)構(gòu)均有保護(hù)作用,無區(qū)域性差異。4.hrs對(duì)mnu誘導(dǎo)rp大鼠視網(wǎng)膜保護(hù)作用與抗氧化相關(guān)。hrs干預(yù)組較生理鹽水干預(yù)組而言,視網(wǎng)膜t-sod活性升高,mda含量下降。5.hrs對(duì)mnu誘導(dǎo)rp大鼠視網(wǎng)膜保護(hù)作用與抗凋亡相關(guān)。tunel染色和qrt-pcr結(jié)果顯示,hrs可以有效降低視網(wǎng)膜組織的凋亡水平,減少視網(wǎng)膜細(xì)胞因凋亡而導(dǎo)致的丟失。6.hrs對(duì)正常大鼠視網(wǎng)膜結(jié)構(gòu)及功能無不良影響。hrs對(duì)照組與正常對(duì)照組在各項(xiàng)功能學(xué)和形態(tài)學(xué)檢查中,均無統(tǒng)計(jì)學(xué)差異。結(jié)論1.單次腹腔注射60mg/kg的mnu誘發(fā)的rp大鼠視網(wǎng)膜病變存在特定的時(shí)間和空間特點(diǎn)。因此,在利用該模型進(jìn)行rp相關(guān)研究時(shí),應(yīng)選擇合適的時(shí)間點(diǎn)進(jìn)行研究,并選擇合適的、統(tǒng)一的病變區(qū)域進(jìn)行檢測,以達(dá)到更加精準(zhǔn)的研究目的。2.hrs對(duì)mnu誘發(fā)rp大鼠的視網(wǎng)膜具有保護(hù)作用,可以有效延緩視網(wǎng)膜病變的發(fā)展。其保護(hù)作用與抗氧化和抗凋亡機(jī)制相關(guān)。3.HRS對(duì)正常視網(wǎng)膜功能及結(jié)構(gòu)無不良影響,對(duì)生物體的安全性較高,具備向臨床應(yīng)用轉(zhuǎn)化的潛質(zhì)。
[Abstract]:Retinitis pigmentosa (RP) is a kind of inheritable retinal degeneration (RD), characterized by progressive retinal photoreceptor cell death, visual field loss and loss of vision. It is generally believed that apoptosis of photoreceptor cells is the common terminal pathway of RP, while reactive oxygen species (ROS) is considered to be an important contributing factor to photoreceptor cell apoptosis in the pathogenesis of RP. Chemicals. Literature and previous studies have shown that hydrogen molecules can protect endophthalmitis, cataract and retinal light damage by antioxidant damage, regulating apoptosis and reducing inflammation. We propose a hypothesis that hydrogen molecules have a protective effect on RP retinal injury. The rat model of RP induced by N-methyl-N-nitrosourea (MNU) was studied. The animal model has the advantages of rapid modeling and high repeatability. It is an animal model commonly used in the study of pathogenesis and prevention of RP. The spatial characteristics of retinal degeneration in the model (e.g. are there differences between central and peripheral retinal degeneration? Are there differences between nasal and temporal retinal degeneration? Are there differences between upper and lower retinal degeneration? Are there pathological changes in the inner retina as well as outer retinal degeneration? Are the pathological changes in different retinal nerve circuits consistent? Based on the in-depth study of the temporal and spatial characteristics of retinopathy induced by MNU in RP rats, the protective effects of hydrogen on the retinal function and structure of the model were verified, and the protective mechanism was explored preliminarily from the perspective of antioxidation and anti-apoptosis. Materials and methods: ninety SD rats were randomly divided into five groups: one control group and four MNU intervention groups. The same amount of normal saline was injected into the abdominal cavity of the control group. Arrays, mea) were used to observe the function of the retina in different parts (central / peripheral, nasal / temporal, superior / inferior) of the model rats in vitro; (3) optical coherence tomography (oct) was used to observe the changes of retinal structure with the course of the disease in vivo; (4) HE staining of paraffin sections of the retina The histological structure of different local retinas of the model rats was observed at the level of in vitro; (5) The survival of photoreceptor cells in different local retina of the model rats was observed by immunofluorescence staining of retinal slices; (6) The survival of rod cells and cone cells in retina of the model rats was detected by Western blotting. (7) Quantitative analysis was used. Quantitative transcription polymerase chain reaction (qrt-pcr) was used to detect the expression of pro-apoptotic genes bax, calpain 2, caspase 3 and anti-apoptotic gene bcl 2. experiment 2: the protective effect of hydrogen-saturated saline on retina of MNU-induced RP rats Normal control group (nc), hydrogen-saturated saline (hrs) control group, normal saline intervention group (mnu + normal saline) and HRS intervention group (mnu + hrs). the mode of administration and dosage of MNU were the same as experiment 1. the intervention mode of HRS and normal saline were intraperitoneal injection, the dosage was 10ml / kg / d, and the intervention began 14 days before the modeling of mnu. The protective effect of HRS was observed by ERG and mea field potentials from the perspective of the function of outer retinal cells; (2) the discharge of retinal ganglion cells (rgcs) was recorded by mea, and the protective effect of HRS was observed from the perspective of the function of inner retinal cells; (3) the protective effect of HRS was observed by means of mea. (4) the expression of apoptosis-related genes bax, calpain-2, caspase-3 and Bcl-2 was detected by TUNEL staining and qrt-pcr, and the protective mechanism of HRS was studied from the angle of anti-apoptosis; (5) the total superoxide dismutase (total superoxide dismutase) was determined by hydroxylamine method. Superoxide dismutase (t-sod) activity and malondialdehyde (mda) content in the retina were measured by thiobarbituric acid method, and the protective mechanism of HRS was studied from the angle of antioxidation. Results 1. After intraperitoneal injection of 60 mg / kg mnu, the retinopathy showed the following spatial and temporal characteristics: (1) the pathological changes of the retina were gradually aggravated within one week after the intervention of mnu. The results of OCT and he staining also showed that the thickness of the outer nuclear layer (onl) of the retina became thinner gradually, and the onl layer cells disappeared basically at p7. (2) The apoptosis peak of photoreceptor cells occurred at p3.qrt-pcr. Bax, calpain 2 and caspase 3 were up-regulated at p3, and Bcl 2 was down-regulated at p3. (3) the pathological changes followed the rule of rod cells to cone cells. the amplitude of light-vision ERG which reflected the function of cone cells decreased later than that of dark-vision ERG which reflected the function of rod cells. The results of fluorescence staining of photoreceptor cells and Western blotting of rod/cone specific protein also showed that the loss of rod cells was more serious than that of cone cells in the early stage of the lesion. (4) The lesion followed the rule of central to peripheral part. The results of MEA field potential showed that the amplitude of central part decreased first, and then developed to near. The thickness of onl layer thinned by HE staining also showed this sequence. (5) The lesion followed the rule of inferior nasal to supratemporal and infratemporal to supratemporal. The results of MEA field potential quadrant analysis showed that the amplitude of inferior nasal quadrant of retina decreased first, then the upper nasal and infratemporal quadrants, and finally the upper temporal quadrant. The results of cell fluorescence staining and qRT-PCR also showed this characteristic. (6) the pathological changes followed the rule of on-pathway off-pathway. Hrs has protective effects on MNU-induced RP rat retina: (1) functional protection: compared with saline intervention group, the amplitudes of open-vision erg, dark-vision ERG and mea field potential of HRS intervention group were significantly improved, indicating that hrs has protective effects on outer retinal cells. The results of MEA recording of RGCs action potential showed that hrs could effectively reduce the spontaneous discharge frequency of RGCs induced by MNU and increase its response to effective visual information, indicating that hrs also had protective effects on the function of inner retinal cells and the integrity of visual pathway. (2) Morphological protective effect: HE staining was used to measure retinal onl. Hrs can effectively delay the loss of onl layer cells. 3. there is no regional difference in this protective effect of hrs. mea field potential and he staining results show that hrs can protect the function and structure of the central, peripheral and peripheral retina. 4. hrs has no regional difference in MNU-induced retinal protection in RP rats. The protective effect of hrs on retina of RP rats induced by MNU is related to anti-apoptosis. TUNEL staining and qRT-PCR results showed that hrs can effectively reduce the level of retinal apoptosis and reduce the retinal cell apoptosis induced by apoptosis. Hrs had no adverse effects on the structure and function of the retina in normal rats. there was no significant difference between the HRS control group and the normal control group in all functional and morphological examinations. conclusion 1. the retinopathy of RP rats induced by intraperitoneal injection of 60mg / kg MNU has specific temporal and spatial characteristics. therefore, the model was used. Hrs can protect the retina of RP rats induced by MNU and delay the development of retinopathy. its protective effect and anti-oxidation and anti-apoptosis mechanism Correlation. 3. HRS has no harmful effect on normal retinal function and structure. It has high safety to organisms and has the potential to transform into clinical application.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R774.1

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