本文選題：視網(wǎng)膜神經(jīng)節(jié)細(xì)胞 + 巨噬細(xì)胞條件培養(yǎng)液��； 參考：《第四軍醫(yī)大學(xué)》2010年博士論文

【摘要】： 研究背景: 視網(wǎng)膜神經(jīng)節(jié)細(xì)胞(RGCs)將視覺信號傳入大腦,但是神經(jīng)損傷或疾病(如青光眼)總是會導(dǎo)致神經(jīng)元的死亡和視功能的喪失。成年哺乳動物視神經(jīng)損傷后RGCs的軸突很難再生。要修復(fù)受損的視神經(jīng)必須保護(hù)神經(jīng)元的存活和促進(jìn)軸突的再生。色素上皮衍生因子(PEDF)是一種具有抗血管生成、神經(jīng)營養(yǎng)和神經(jīng)保護(hù)作用的蛋白,能夠保護(hù)神經(jīng)元免受多種病理性的損害。另外它還具有抗炎能力。當(dāng)視神經(jīng)損傷時晶狀體同時損傷,許多RGCs可以存活,而且其軸突再生進(jìn)入遠(yuǎn)端視神經(jīng)。晶狀體損傷可以誘導(dǎo)巨噬細(xì)胞活化,巨噬細(xì)胞能夠分泌多種刺激RGCs軸突再生的細(xì)胞因子。巨噬細(xì)胞在這個過程中似乎起關(guān)鍵作用,因?yàn)閱为?dú)玻璃體注射酵母多糖而沒有晶狀體損傷,也可以誘導(dǎo)RGCs軸突再生進(jìn)入遠(yuǎn)端視神經(jīng),而酵母多糖可以刺激組織的單個核細(xì)胞。 目的: 本研究探討聯(lián)合兩種方法,即PEDF聯(lián)合巨噬細(xì)胞條件培養(yǎng)液(MCM)或晶狀體損傷是否能協(xié)同保護(hù)RGCs的存活和促進(jìn)RGCs軸突的再生。 方法: 1.細(xì)胞培養(yǎng):培養(yǎng)出生24 h的SD仔鼠的RGC細(xì)胞以及SD大鼠腹腔巨噬細(xì)胞,觀察它們的形態(tài)、進(jìn)行免疫組化鑒定,用MTT法繪制它們的生存曲線,揭示它們的培養(yǎng)規(guī)律。 2.細(xì)胞實(shí)驗(yàn):(1)巨噬細(xì)胞與酵母多糖共培養(yǎng),觀察培養(yǎng)液中血清對巨噬細(xì)胞吞噬酵母多糖顆粒的影響。(2)制備細(xì)胞爬片及MCM,觀察聯(lián)合應(yīng)用PEDF和MCM對培養(yǎng)的RGC細(xì)胞的存活數(shù)和軸突再生長度的影響。 3.動物實(shí)驗(yàn):(1)將熒光金注射到SD成年大鼠及出生2 w的SD幼鼠腦內(nèi),逆行性標(biāo)記RGC細(xì)胞,觀察RGCs的分布規(guī)律和細(xì)胞數(shù)目。(2)制備視神經(jīng)損傷模型和晶狀體損傷模型,觀察聯(lián)合應(yīng)用玻璃體注射PEDF和晶狀體損傷對視網(wǎng)膜RGC細(xì)胞的存活數(shù)、視神經(jīng)軸突再生以及GAP-43 mRNA表達(dá)的影響。 結(jié)果: 1.細(xì)胞培養(yǎng):(1)成功培養(yǎng)了SD大鼠RGC細(xì)胞,GAP-43呈陽性,培養(yǎng)第4 d時,有71.4%細(xì)胞存活,而到第6 d時,大量細(xì)胞開始死亡,46.3%細(xì)胞存活,第8 d時,只有7.1%細(xì)胞存活。(2)成功培養(yǎng)了SD大鼠腹腔巨噬細(xì)胞,CD68呈陽性,可以存活2 w左右,在培養(yǎng)第8 d時,約有一半細(xì)胞死亡。 2.細(xì)胞實(shí)驗(yàn):(1)巨噬細(xì)胞同酵母多糖共培養(yǎng)2 d,血清組細(xì)胞數(shù)多于對照組(P0.01)。(2)觀察對RGCs存活數(shù)目的影響: MCM組在細(xì)胞培養(yǎng)第3 d時細(xì)胞數(shù)目為31.4±3.7(P0.05),PEDF聯(lián)合MCM組為34.7±4.2(P0.01),而對照組為27.6±2.2,與對照組均有顯著統(tǒng)計學(xué)差異。PEDF聯(lián)合MCM組的細(xì)胞數(shù)大于MCM組,兩者有顯著統(tǒng)計學(xué)差異(P0.05)。對RGC軸突再生的影響:細(xì)胞培養(yǎng)第3 d時,MCM組細(xì)胞軸突長度為57.6±3.7μm,PEDF聯(lián)合MCM組的細(xì)胞軸突長度為88.6±9.5μm,與對照組(41.8±5.4μm)均有顯著統(tǒng)計學(xué)差異(P0.01)。PEDF聯(lián)合MCM組的軸突長于MCM組(P0.01)。 3.動物實(shí)驗(yàn):(1)成功制備熒光金逆標(biāo)模型,RGCs密度為2219±113 RGC / mm2。(2)玻璃體注射PEDF聯(lián)合晶狀體損傷可以大幅提高視神經(jīng)損傷后RGCs存活數(shù)目。與單獨(dú)應(yīng)用玻璃體注射PEDF或晶狀體損傷相比,兩者聯(lián)合可以增加RGCs總存活數(shù)(1411±84 RGC/mm2),與前兩者均有顯著統(tǒng)計學(xué)差異(P 0.01)。單純的晶狀體損傷能夠刺激遠(yuǎn)端的RGCs軸突再生,PEDF聯(lián)合晶狀體損傷能夠強(qiáng)烈刺激遠(yuǎn)端視神經(jīng)軸突再生,與晶狀體損傷組有顯著統(tǒng)計學(xué)差異(P0.01)。PEDF聯(lián)合晶狀體損傷在同一時間點(diǎn)可以大幅提高視神經(jīng)損傷后GAP-43 mRNA的表達(dá),與兩者單獨(dú)進(jìn)行干預(yù)均有顯著統(tǒng)計學(xué)差異(P 0.01)。 結(jié)論: 1.血清能夠刺激巨噬細(xì)胞的吞噬功能。 2.單獨(dú)應(yīng)用PEDF不能保護(hù)培養(yǎng)的RGC細(xì)胞存活以及促進(jìn)軸突再生, MCM能保護(hù)RGC細(xì)胞存活以及促進(jìn)軸突再生,與PEDF可以協(xié)同保護(hù)RGC細(xì)胞存活以及促進(jìn)軸突再生。 3.玻璃體內(nèi)注射PEDF和晶狀體損傷都可以增加RGCs存活數(shù)目,兩者可以協(xié)同使這一作用增加。晶狀體損傷可以刺激軸突再生,聯(lián)合PEDF可以增強(qiáng)刺激再生作用。晶狀體損傷和PEDF都能提高GAP-43 mRNA的表達(dá),兩者聯(lián)合使這一作用加強(qiáng)。 創(chuàng)新點(diǎn): 1.首次觀察幼鼠熒光金逆行性標(biāo)記RGCs。 2.首次探討PEDF對培養(yǎng)RGCs軸突再生和視神經(jīng)損傷修復(fù)的作用。 3.首次探討聯(lián)合PEDF和MCM或晶狀體損傷對視神經(jīng)夾傷后RGCs的存活和促進(jìn)RGCs軸突再生的影響。
[Abstract]:Research background:
Retinal ganglion cells (RGCs) transmit visual signals into the brain, but nerve damage or disease (such as glaucoma) always leads to neuronal death and loss of visual function. The axon of RGCs is difficult to regenerate in adult mammalian optic nerve injury. To repair the damaged optic nerve must protect the survival of the neuron and promote the regeneration of the axon. Pigment epithelial derived factor (PEDF) is a protein with antiangiogenic, neurotrophic and neuroprotective effects that can protect neurons from a variety of pathological damage. In addition, it has the ability to resist inflammation. When the optic nerve is damaged, many RGCs can survive, and its axon regenerates into the distal optic nerve. It can induce macrophage activation and macrophages secrete a variety of cytokines that stimulate the regeneration of RGCs axons. Macrophages seem to play a key role in this process, because the single vitreous injection of Yeast Polysaccharides without lens damage can also induce the regeneration of RGCs axons into the distal optic nerve, and yeast polysaccharide can be induced. To stimulate the mononuclear cells of the tissue.
Objective:
The present study explored whether the combined two methods, namely PEDF combined macrophage conditioned medium (MCM) or lens injury, could synergistically protect the survival of RGCs and promote the regeneration of RGCs axons.
Method:
1. cell culture: RGC cells of 24 h born SD mice and peritoneal macrophages of SD rats were cultured. Their morphology was observed and identified by immunohistochemistry. Their survival curves were plotted by MTT, and their cultivation rules were revealed.
2. cell experiment: (1) the effect of macrophage and yeast polysaccharide co culture to observe the effect of serum on macrophage phagocytosis of yeast polysaccharide particles. (2) to prepare cell crawling tablets and MCM, to observe the effect of combined application of PEDF and MCM on the survival number of cultured RGC cells and the axon regeneration length.
3. animal experiments: (1) injection of fluorescent gold into the brain of SD adult rats and SD young rats born with 2 W, retrograde labeling of RGC cells to observe the distribution of RGCs and the number of cells. (2) to prepare the optic nerve injury model and lens injury model, to observe the survival of the retina RGC cells combined with vitreous injection of PEDF and lens injury, and to observe the deity of the retina of the retina RGC cells. The effect of the regeneration of the axon process and the expression of GAP-43 mRNA.
Result:
1. cell culture: (1) SD rat RGC cells were successfully cultured, GAP-43 was positive, and 71.4% cells survived at fourth D, while at sixth D, a large number of cells began to die, 46.3% cells survived, and eighth D, only 7.1% cells survived. (2) the peritoneal macrophages of SD rats were successfully cultured, CD68 was positive, and could survive 2 W, about eighth D, about 71.4% D. Half of the cells died.
2. cell experiment: (1) the macrophage and yeast polysaccharide co culture 2 D, the number of serum group more than the control group (P0.01). (2) the effect of observation on the number of RGCs survival: MCM group in cell culture third D cell number of 31.4 + 3.7 (P0.05), PEDF combined MCM group of 34.7 + 4.2 (P0.01), and the control group is 27.6 + 2.2, and the control group were significantly worse than the control group The number of cells in group MCM combined with.PEDF was larger than that of group MCM, and there was significant difference between the two groups (P0.05). The effect on the axon regeneration of RGC: when cell culture was third D, the cell axon length of the MCM group was 57.6 + 3.7 mu m, and the length of the cell axon in the PEDF combined MCM group was 88.6 + 9.5 micron m, and there were significant statistical differences between the control group and the control group (41.8 + 5.4). The axon of group M was longer than that of group MCM (P0.01).
3. animal experiments: (1) the successful preparation of the fluorescent gold reverse standard model, the RGCs density of 2219 + 113 RGC / mm2. (2) of vitreous injection of PEDF combined with lens injury can significantly increase the number of RGCs survival after optic nerve injury. Compared with the single use of vitreous injection of PEDF or lens injury, the combination of both can increase the total number of RGCs survival (1411 + 84 RGC/mm2), There were significant statistical differences between the two groups (P 0.01). Pure lens injury could stimulate the regeneration of RGCs axons in the distal end. PEDF combined with lens injury could strongly stimulate the regeneration of the distal optic axon. There was a significant statistical difference between the lens injury group and the lens injury group (P0.01).PEDF joint lens injury could be greatly improved at the same time point. The expression of GAP-43 mRNA after nerve injury was significantly different from that of intervention alone (P 0.01).
Conclusion:
1. the serum can stimulate the phagocytosis of macrophages.
2. PEDF alone can not protect the cultured RGC cells to survive and promote axon regeneration. MCM can protect the survival of RGC cells and promote axon regeneration, and PEDF can protect the survival of RGC cells and promote axon regeneration in collaboration with PEDF.
3. intravitreal injection of PEDF and lens injury can increase the number of RGCs survival. Both can increase the effect. Lens injury can stimulate axon regeneration, combined with PEDF can enhance stimulation of regeneration. Lens injury and PEDF can increase the expression of GAP-43 mRNA, both combined to strengthen the effect.
Innovation point:
1. the first observation of fluorescent gold retrograde RGCs. in young rats
2. to explore the effect of PEDF on axonal regeneration and repair of optic nerve injury in RGCs.
3. the effects of combined PEDF and MCM or lens injury on RGCs survival and RGCs axonal regeneration after optic nerve crush were first discussed.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R774.1

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