本文選題：視神經(jīng) + 視網(wǎng)膜神經(jīng)節(jié)細(xì)胞�。� 參考：《第三軍醫(yī)大學(xué)》2015年博士論文

【摘要】：研究背景和意義視神經(jīng)是由視網(wǎng)膜神經(jīng)節(jié)細(xì)胞(retinal ganglioncells,RGCs)的軸突匯聚而成,RGCs的狀態(tài)一定程度上反應(yīng)了視神經(jīng)的功能。作為中樞神經(jīng)的一部分,視神經(jīng)傷后恢復(fù)亦成為研究的熱點(diǎn)和難點(diǎn)。眾多的調(diào)控因素使視神經(jīng)損傷后再生修復(fù)變得異常困難、復(fù)雜。損傷區(qū)抑制性蛋白的存在、膠質(zhì)瘢痕形成以及神經(jīng)營養(yǎng)因子缺乏等因素是中樞神經(jīng)再生障礙的主要原因,其中抑制性因素處于重要地位。Sema3A作為重要的軸突抑制物,在神經(jīng)損傷后誘導(dǎo)生長(zhǎng)錐塌陷,阻止神經(jīng)修復(fù),扮演著重要的調(diào)控角色,受到廣泛的關(guān)注。神經(jīng)纖毛蛋白(Neuropilin1,NRP1)和叢狀蛋白(Plexin A1-A4,PlexA1-A4)被證實(shí)為Sema3A的共受體,與Sema3A結(jié)合后發(fā)揮對(duì)神經(jīng)生長(zhǎng)的抑制作用,當(dāng)配體Sema3A不存在時(shí),Plex以一種自我抑制的狀態(tài)存在,共受體NRP1與Plex結(jié)合使得這種抑制狀態(tài)更加穩(wěn)定,在Sema3A與NRP1結(jié)合后,Plex的構(gòu)象發(fā)生了改變,從自我抑制狀態(tài)釋放,進(jìn)一步與Sema3A、NRP1結(jié)合,啟動(dòng)下游的信號(hào)傳導(dǎo),最終引起神經(jīng)生長(zhǎng)錐塌陷,抑制軸突延伸。p38絲裂原活化蛋白激酶(mitogen activated protein kinases p38MAPK)和caspase-3信號(hào)通路參與了多種細(xì)胞凋亡進(jìn)程。文獻(xiàn)報(bào)道,Sema3A能激活p38MAPK,使其磷酸化后將促進(jìn)神經(jīng)祖細(xì)胞凋亡。而且,Sema3A還能減少巨噬細(xì)胞對(duì)髓磷脂的吞噬作用,抑制中樞神經(jīng)髓鞘再生,妨礙神經(jīng)營養(yǎng)因子運(yùn)輸,阻止損傷神經(jīng)恢復(fù)。microRNA(mi RNA)是一種獨(dú)特的內(nèi)源性小分子,它通過與靶mRNA的3’UTR的互補(bǔ)配對(duì)抑制靶基因活性或使其降解,導(dǎo)致相應(yīng)蛋白表達(dá)水平降低,從而調(diào)節(jié)細(xì)胞的增殖、分化與凋亡,在生物體生長(zhǎng)發(fā)育的不同階段發(fā)揮不同功能,實(shí)現(xiàn)其生物調(diào)節(jié)作用。而且,許多mi RNAs在哺乳動(dòng)物大腦、視網(wǎng)膜中特異性存在并且在其各個(gè)發(fā)育階段動(dòng)態(tài)表達(dá),表明了這些miRNAs與哺乳動(dòng)物神經(jīng)發(fā)育密切相關(guān)。這些研究結(jié)果為找尋抑制Sema3A表達(dá)的mi RNAs、促進(jìn)損傷視神經(jīng)修復(fù)提供了新的思路。目的:通過對(duì)視神經(jīng)損傷后視網(wǎng)膜中Sema3A及其下游信號(hào)通路的研究,初步探討Sema3A抑制視神經(jīng)再生的作用機(jī)制,明確Sema3A是mi R-30b的靶基因,證實(shí)mi R-30b可通過調(diào)控sema3a表達(dá)并抑制其信號(hào)通路、發(fā)揮保護(hù)視網(wǎng)膜神經(jīng)節(jié)細(xì)胞、促進(jìn)軸突生長(zhǎng)的作用,為臨床治療視神經(jīng)損傷提供新的靶點(diǎn)和思路。方法:1、建立sd大鼠視神經(jīng)鉗夾損傷模型,于正常及傷后第1、3、7、14、21d行qrt-pcr和westernblotting檢測(cè),觀察視網(wǎng)膜中mir-30b、sema3a、nrp1、plexa1、p-p38mapk、和activecaspase-3表達(dá)變化,免疫組織化學(xué)觀察正常及傷后第7d視網(wǎng)膜上sema3a的表達(dá)情況。2、雙熒光素酶報(bào)告基因系統(tǒng)驗(yàn)證mir-30b與sema3a3’utr結(jié)合并抑制其表達(dá);將重組腺相關(guān)病毒(raav)包裝的mir-30bmimic、mir-30binhibitor、mirnanc和pbs分別轉(zhuǎn)染到體外培養(yǎng)的rgcs;培養(yǎng)第7d,westernblotting檢測(cè)sema3a、nrp1、plexa1、p-p38mapk和activecaspase-3表達(dá)情況;制作sd大鼠視神經(jīng)損傷模型,將raav-mir-30bmimic、raav-mir-30binhibitor、raav-mirnanc和pbs分別注射入損傷眼玻璃體腔內(nèi),分別于傷后3、7d用qrt-pcr和westernblotting檢測(cè)視網(wǎng)膜中mir-30b、sema3a的表達(dá)量。3、構(gòu)建以sema3a為靶標(biāo)的sirna,lipofectamine2000將其轉(zhuǎn)染到體外培養(yǎng)的rgcs,westernblotting檢測(cè)sema3a表達(dá)量,選取最強(qiáng)抑制效果的sirna,再將其轉(zhuǎn)染到體外培養(yǎng)的rgcs,培養(yǎng)第5d再分別轉(zhuǎn)染raav-mir-30bmimic和raav-mir-30binhibitor,培養(yǎng)第9dwesternblotting再檢測(cè)nrp1和p-p38mapk的表達(dá)情況,初步明確mir-30b調(diào)控sema3a的信號(hào)通路。4、將raav包裝的mir-30bmimic、mir-30binhibitor、mirnanc和pbs分別轉(zhuǎn)染到體外培養(yǎng)的rgcs;培養(yǎng)第5d流式細(xì)胞儀檢測(cè)其凋亡率,培養(yǎng)第7d采用gap-43著染,免疫熒光顯微鏡觀察各組rgcs軸突長(zhǎng)度情況。5、制作sd大鼠視神經(jīng)鉗夾傷模型,將raav-mir-30bmimic、raav-mir-30binhibitor、raav-mirnanc和pbs分別注射入損傷眼玻璃體腔內(nèi),利用熒光金逆行標(biāo)記觀察傷后7、14、28d時(shí)4組rgcs存活率變化;閃光視覺誘發(fā)電位檢測(cè)傷后28d各組視功能恢復(fù)情況。結(jié)果:1、sd大鼠視神經(jīng)鉗夾傷后,視網(wǎng)膜中mir-30b表達(dá)先升高后下降,傷后3d達(dá)高峰,傷后7d又逐漸下降,視網(wǎng)膜中sema3a、nrp1、plexa1、p-p38mapk和activecaspase-3也先升高后下降,傷后7d達(dá)高峰;免疫組化檢測(cè)發(fā)現(xiàn),sema3a表達(dá)在正常視網(wǎng)膜中內(nèi)核層和節(jié)細(xì)胞層,傷后7d,節(jié)細(xì)胞層和內(nèi)核層的陽性細(xì)胞明顯增多。2、雙熒光素酶報(bào)告基因系統(tǒng)檢測(cè)發(fā)現(xiàn)處理組相對(duì)熒光素酶活性明顯低于對(duì)照組和突變組,mi R-30b與Sema3A的結(jié)合位點(diǎn)為:UGUUUACA;轉(zhuǎn)染miR-30b到體外培養(yǎng)的RGCs,mi R-30b mimic組中Sema3A、NRP1、PlexA1、p-p38MAPK和active caspase-3表達(dá)量最低,而mi R-30b inhibit組最高;大鼠視神經(jīng)鉗夾傷后,玻璃體腔內(nèi)分別注射r AAV-miR-30b mimic、rAAV-mi R-30b inhibitor、rAAV-mi RNA NC和PBS,傷后3d和7d,視網(wǎng)膜中,miR-30b表達(dá)量在mimic組最高、inhibitor組最低,而Sema3A正好相反。3、在體外培養(yǎng)的RGCs中,si RNA2組內(nèi)Sema3A蛋白表達(dá)量最低,用siRNA2敲低Sema3A后,再分別轉(zhuǎn)染r AAV-mi R-30b mimic和r AAV-miR-30b inhibitor,兩組中Sema3A、NRP1和p-p38MAPK表達(dá)量無明顯差異。4、轉(zhuǎn)染mi R-30b到體外培養(yǎng)的RGCs,流式細(xì)胞儀檢測(cè)發(fā)現(xiàn)mi R-30b mimic組RGCs凋亡率最低,mi R-30b inhibit組最高,RGCs軸突長(zhǎng)度mi R-30b mimic組最長(zhǎng),mi R-30b inhibit組最短。5、大鼠視神經(jīng)鉗夾傷后,玻璃體腔內(nèi)分別注射rAAV-mi R-30b mimic、r AAV-miR-30b inhibitor、rAAV-mi RNA NC和PBS,雖然,傷后四組RGCs存活率都不同程度下降,但是在傷后7、14、28d,mimic組的RGCs存活率都最高,inhibitor組都最低;FVEP顯示,mimic組中P1波幅也是最寬,inhibitor組最窄。全文結(jié)論1、離體及在體實(shí)驗(yàn)證實(shí),Sema3A是miR-30b的靶基因;2、miR-30b可通過抑制Sema3A表達(dá)改變p-p38MAPK和active caspase-3的水平,減少RGCs凋亡,提高視神經(jīng)損傷后RGCs存活率;3、miR-30b可通過抑制Sema3A表達(dá)改變NRP1、Plex A1的水平,促進(jìn)RGCs軸突生長(zhǎng),并加快視神經(jīng)損傷后視功能恢復(fù),這可能是mi R-30b促進(jìn)損傷后視神經(jīng)修復(fù)的主要分子機(jī)制。
[Abstract]:The study background and significance optic nerve are formed by the axons of the retinal ganglion cells (retinal GanglionCells, RGCs). The state of the RGCs reacts to the function of the optic nerve to some extent. As a part of the central nervous system, the recovery of the optic nerve after injury is also a hot and difficult point. The existence of inhibitory proteins, the formation of glial scar and the lack of neurotrophic factors are the main causes of central nerve regeneration. The inhibitory factors are in the important position of.Sema3A as an important axon inhibitor, inducing conical collapse and blocking the nerve after nerve injury. Repair, playing an important regulatory role, is widely concerned. Neuropilin1 (NRP1) and Plexin A1-A4 (PlexA1-A4) are proved to be common receptors of Sema3A, and they are combined with Sema3A to play an inhibitory effect on nerve growth. When the ligand Sema3A does not exist, Plex is present in a state of self inhibition and co receptor. The combination of NRP1 and Plex makes this inhibition more stable. After the combination of Sema3A and NRP1, the conformation of Plex is changed, released from the state of self inhibition, further combining with Sema3A, NRP1, and starting downstream signal conduction, eventually causing the collapse of the nerve growth cone, and inhibiting the axon to extend.P38 mitogen activated protein kinase (mitogen activated PR). Otein kinases p38MAPK) and caspase-3 signaling pathway participate in a variety of apoptotic processes. It is reported that Sema3A can activate p38MAPK and promote the apoptosis of neural progenitor cells after phosphorylation. Moreover, Sema3A can reduce macrophage phagocytosis of myelin, inhibit the regeneration of myelin sheath, obstruct the transport of neurotrophic factors and prevent the transport of neurotrophic factors. The injured nerve recovery.MicroRNA (MI RNA) is a unique endogenous small molecule. It inhibits the activity or degradation of the target gene through the complementary pairing with the 3 'UTR of the target mRNA, resulting in the reduction of the expression level of the corresponding protein, thus regulating cell proliferation, differentiation and apoptosis, and exerting different functions in different stages of growth and development of raw materials. Moreover, many mi RNAs are specific in the mammalian brain and in the retina and are expressed dynamically at their various developmental stages, indicating that these miRNAs are closely related to the development of mammalian nerve. These results provide a new idea for the search for the MI RNAs that inhibits the expression of Sema3A and to the repair of the repair of the optic nerve. Objective: through the study of the Sema3A and its downstream signal pathway in the retina after optic nerve injury, the mechanism of Sema3A inhibition of optic nerve regeneration was preliminarily investigated, and Sema3A was a target gene for MI R-30b. It was confirmed that MI R-30b could protect retinal ganglion cells and promote axonal growth by regulating Sema3A expression and inhibiting its signal pathway. In order to provide new targets and ideas for clinical treatment of optic nerve injury. Methods: 1, a SD rat model of optic nerve clamp injury was established, and qRT-PCR and westernblotting were detected in 1,3,7,14,21d after normal and post injury. The changes of mir-30b, Sema3A, Nrp1, plexa1, P-P38MAPK, activecaspase-3 expression in the retina were observed, and the immunohistochemical observation was observed. The expression of Sema3A on the retina of 7D after normal and post injury.2, the dual luciferase reporter gene system verified that mir-30b was combined with sema3a3 'UTR to inhibit its expression, and the mir-30bmimic, mir-30binhibitor, mirnanc and PBS, packaged in recombinant adeno-related virus (rAAV), were transfected to the extracellular RGCs. The expression of Nrp1, plexa1, P-P38MAPK and activecaspase-3, the model of the optic nerve injury in SD rats was made, and raav-mir-30bmimic, raav-mir-30binhibitor, raav-mirnanc and PBS were injected into the vitreous cavity of the injured eye respectively. The expression of the retina in the retina was detected by qRT-PCR and westernblotting, respectively. For the target siRNA, lipofectamine2000 transfected it into the cultured RGCs, westernblotting detected the Sema3A expression, selected the strongest inhibitory effect siRNA, then transfected it into the cultured RGCs, cultured 5D and then transfected raav-mir-30bmimic and raav-mir-30binhibitor respectively. The expression of.4 was preliminarily defined by mir-30b, which was used to regulate the Sema3A, mir-30bmimic, mir-30binhibitor, mirnanc and PBS were transfected to RGCs in vitro, respectively. The apoptosis rate was detected by the flow cytometry. The GAP-43 staining was used in the culture 7d, and the length of the axon was observed by the immunofluorescence microscope. D rat model of optic nerve clamp injury, raav-mir-30bmimic, raav-mir-30binhibitor, raav-mirnanc and PBS were injected into the vitreous cavity of the injured eye respectively. The changes of the 4 groups of RGCs survival rates were observed by the fluorescent gold retrograde labeling, and the visual evoked potential of the 28d in each group after injury was detected by the flash visual evoked potential. Results: 1, SD rats were deity. After the clamp injury, the expression of mir-30b in the retina increased first and then decreased, and the 3D reached the peak after injury, and the 7d decreased gradually after injury. The Sema3A, Nrp1, plexa1, P-P38MAPK and activecaspase-3 in the retina increased first and then declined, and the 7d reached the peak. The immunohistochemical detection found that the Sema3A table reached the core layer and the ganglion layer in the normal retina, 7d after injury, slender after injury. The positive cells in the cell layer and the core layer were significantly increased by.2. The double luciferase reporter gene system detection showed that the relative luciferase activity of the treatment group was significantly lower than the control group and the mutation group. The binding site of MI R-30b and Sema3A was UGUUUACA, RGCs in the transfected miR-30b to the in vitro culture, Sema3A in the MI R-30b mimic group. The expression of Caspase-3 was the lowest, and the highest in the MI R-30b inhibit group. After the clamp injury of the optic nerve in the rat, the intravitreal R AAV-miR-30b mimic, rAAV-mi R-30b inhibitor, rAAV-mi RNA, and the retina were the highest and the lowest in the retina. In s, the expression of Sema3A protein in group Si RNA2 is the lowest. After siRNA2 knocking down Sema3A, R AAV-mi R-30b mimic and R AAV-miR-30b are respectively transfected. The 0b inhibit group was the highest, the RGCs axon length mi R-30b mimic group was the longest and the MI R-30b inhibit group was the shortest.5. After the optic nerve clamp injury in the rat, the vitreous cavity was injected respectively with rAAV-mi R-30b. Although, the survival rate of the four groups after injury decreased in varying degrees. The survival rate of Cs was the highest and that of group inhibitor was the lowest; FVEP showed that P1 amplitude in group mimic was also the most wide and inhibitor group was the narrowest. Conclusion 1, in vitro and in vivo experiments, Sema3A is the target gene of miR-30b; 2, miR-30b can be inhibited by Sema3A expression to change p-p38MAPK and active levels to reduce apoptosis and improve the optic nerve injury. The survival rate of Cs; 3, miR-30b can promote the growth of RGCs axon by inhibiting the expression of NRP1 and Plex A1 by inhibiting the expression of Sema3A, and accelerating the recovery of visual function after optic nerve injury, which may be the main molecular mechanism of MI R-30b to promote the repair of optic nerve after injury.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R774.6

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5 王文軍;閆元奎;唐羅生;;大鼠視神經(jīng)損傷視網(wǎng)膜內(nèi)p38絲裂原活化蛋白激酶活性的表達(dá)[A];中國眼底病論壇·全國眼底病專題學(xué)術(shù)研討會(huì)論文匯編[C];2008年

6 施天嚴(yán);俞頌平;朱艷霞;李霞;;輕型間接視神經(jīng)損傷的臨床特點(diǎn)分析[A];2011年浙江省眼科學(xué)術(shù)會(huì)議論文集[C];2011年

7 傅繼弟;;顱腦外傷合并視神經(jīng)損傷685例的臨床觀察[A];中國中西醫(yī)結(jié)合學(xué)會(huì)神經(jīng)外科專業(yè)委員會(huì)第一屆學(xué)術(shù)大會(huì)暨神經(jīng)外科專業(yè)委員會(huì)成立大會(huì)論文匯編[C];2014年

8 張宇強(qiáng);黃書嵐;易偉;陳強(qiáng);;顱腦損傷合并視神經(jīng)損傷的病因分析及其診治[A];中華醫(yī)學(xué)會(huì)神經(jīng)外科學(xué)分會(huì)第九次學(xué)術(shù)會(huì)議論文匯編[C];2010年

9 邱懷雨;魏世輝;;輕型間接視神經(jīng)損傷的認(rèn)識(shí)與對(duì)策[A];2011中華醫(yī)學(xué)會(huì)神經(jīng)外科學(xué)學(xué)術(shù)會(huì)議論文匯編[C];2011年

10 薛飛;曹云莉;李澤卿;王秋萍;吳昆e，

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