本文選題：骨髓間充質(zhì)干細(xì)胞　切入點：堿性成纖維細(xì)胞生長因子　出處：《福建醫(yī)科大學(xué)》2013年博士論文　論文類型：學(xué)位論文

【摘要】：隨著對間充質(zhì)干細(xì)胞（mesenchymal stem cells, MSCs）認(rèn)識的深入，MSCs的定義已經(jīng)超出了作為一種具有多向分化潛能成體干細(xì)胞的概念。MSCs被認(rèn)為是多源的細(xì)胞群體，廣泛參與了組織修復(fù)。MSCs可能通過細(xì)胞因子分泌和調(diào)節(jié)機(jī)體微環(huán)境促進(jìn)損傷修復(fù)過程，然而其確切的機(jī)制尚不清楚。本研究采用體外模型探討視網(wǎng)膜光損傷對MSCs細(xì)胞因子表達(dá)變化的影響，并進(jìn)一步闡述細(xì)胞因子表達(dá)變化在MSCs參與視網(wǎng)膜變性過程的作用。通過體內(nèi)移植研究MSCs在視網(wǎng)膜光損傷中的作用及移植介導(dǎo)的宿主細(xì)胞因子表達(dá)變化。 第一部分視網(wǎng)膜光損傷動物模型研究 目的：建立視網(wǎng)膜光損傷動物模型，探討光損傷對視網(wǎng)膜組織結(jié)構(gòu)的影響及光損傷誘導(dǎo)的內(nèi)源性細(xì)胞因子表達(dá)變化。 方法：6~8W SD大鼠經(jīng)散瞳后，進(jìn)行12h光照（5klux），12h避光，，每日反復(fù)。并于強(qiáng)光暴露后1d、2d、5d、7d、14d摘除眼球。采用透射電鏡觀察光照早期視網(wǎng)膜超微結(jié)構(gòu)的改變。HE染色觀察強(qiáng)光暴露對視網(wǎng)膜組織結(jié)構(gòu)的影響。末端脫氧核苷酸標(biāo)記法（TUNEL）檢測光照后視網(wǎng)膜細(xì)胞凋亡情況。采用墨汁灌注及視網(wǎng)膜鋪片法評估光照后視網(wǎng)膜微循環(huán)通透性改變。免疫熒光檢測視網(wǎng)膜細(xì)胞因子bFGF、BDNF、CNTF的表達(dá)變化。 結(jié)果：強(qiáng)光暴露24h后，視網(wǎng)膜超微結(jié)構(gòu)出現(xiàn)改變，光感受器細(xì)胞外節(jié)膜盤大量脫落，線粒體膜腫脹，核電子密度增高。視網(wǎng)膜組織學(xué)結(jié)構(gòu)隨光照時間的延長而持續(xù)破壞，視網(wǎng)膜外核層進(jìn)行性變薄。視網(wǎng)膜細(xì)胞凋亡于光照后持續(xù)存在，主要分布于外核層及視網(wǎng)膜色素上皮層。強(qiáng)光暴露視網(wǎng)膜微血管通透性增強(qiáng)，墨汁灌注出現(xiàn)點狀滲出，光照14d后出現(xiàn)片狀滲漏，位于深層。強(qiáng)光暴露引起視網(wǎng)膜bFGF、BDNF、CNTF及GFAP表達(dá)上調(diào)。其中CNTF及GFAP表達(dá)上調(diào)隨光照時間延長而增強(qiáng)。 結(jié)論：強(qiáng)光暴露可以引起視網(wǎng)膜組織結(jié)構(gòu)破壞并出現(xiàn)反應(yīng)性細(xì)胞因子表達(dá)上調(diào)，視網(wǎng)膜光損傷伴隨膠質(zhì)細(xì)胞反應(yīng)增強(qiáng)。 第二部分視網(wǎng)膜光損傷對骨髓間充質(zhì)干細(xì)胞神經(jīng)保護(hù)功能的影響 目的：探討光損傷視網(wǎng)膜勻漿上清液對MSCs細(xì)胞因子表達(dá)的影響及細(xì)胞因子表達(dá)變化在MSCs參與視網(wǎng)膜變性過程的作用。 方法：全骨髓貼壁法培養(yǎng)MSCs，并進(jìn)行流式細(xì)胞術(shù)、成骨及成脂分化鑒定。制備正常及光損傷視網(wǎng)膜勻漿上清液，分別誘導(dǎo)第3～5代MSCs2d及5d。采用RT-qPCR及Western-blotting檢測bFGF、BDNF、CNTF表達(dá)情況并與未誘導(dǎo)組MSCs比較。收集未誘導(dǎo)及誘導(dǎo)后MSCs培養(yǎng)上清液，制備條件培養(yǎng)液，培養(yǎng)視網(wǎng)膜片24h。收集視網(wǎng)膜片培養(yǎng)液，通過LDH釋放水平評估不同條件培養(yǎng)液對視網(wǎng)膜細(xì)胞膜完整性的影響。RT-qPCR檢測不同條件培養(yǎng)液對視網(wǎng)膜bcl-2及bax表達(dá)的影響。構(gòu)建含bFGF干擾序列的慢病毒載體，轉(zhuǎn)染MSCs并進(jìn)一步誘導(dǎo)轉(zhuǎn)染后的MSCs，觀察bFGF表達(dá)下調(diào)對MSCs發(fā)揮視網(wǎng)膜神經(jīng)保護(hù)作用的影響。 結(jié)果：MSCs可以表達(dá)bFGF、BDNF、CNTF。經(jīng)光損傷視網(wǎng)膜勻漿上清液誘導(dǎo)后，MSCs表達(dá)bFGF及CNTF顯著增強(qiáng)（P＜0.05）。MSCs條件培養(yǎng)液上調(diào)視網(wǎng)膜片bcl-2表達(dá)，抑制bax表達(dá)及LDH釋放。光損傷視網(wǎng)膜勻漿上清液誘導(dǎo)后的MSCs條件培養(yǎng)液較其它條件培養(yǎng)液顯著提高其上調(diào)視網(wǎng)膜片bcl-2表達(dá)，抑制bax表達(dá)及LDH釋放的能力（均P＜0.05）。bFGF表達(dá)下調(diào)后的MSCs條件培養(yǎng)液仍可上調(diào)視網(wǎng)膜片bcl-2表達(dá)，抑制LDH釋放。但各條件培養(yǎng)液組間比較無顯著性差異（P＞0.05）。 結(jié)論：損傷刺激可以促進(jìn)MSCs細(xì)胞因子表達(dá)，增強(qiáng)MSCs的視網(wǎng)膜神經(jīng)保護(hù)作用，損傷誘導(dǎo)的bFGF表達(dá)上調(diào)在MSCs應(yīng)對視網(wǎng)膜變性中具有重要作用。 第三部分經(jīng)靜脈移植骨髓間充質(zhì)干細(xì)胞對光損傷視網(wǎng)膜的影響 目的：探討MSCs靜脈移植對光損傷視網(wǎng)膜細(xì)胞凋亡的影響及移植后細(xì)胞遷移和移植介導(dǎo)的宿主細(xì)胞因子表達(dá)變化。 方法：建立視網(wǎng)膜光損傷動物模型，MSCs注射組于強(qiáng)光暴露48h后，經(jīng)靜脈移植MSCs，劑量5×106個/ml，DMEM注射組接受等體積的DMEM注射。采用TUNEL比較光照14d后單純光照組、DMEM注射組及MSCs注射組視網(wǎng)膜細(xì)胞凋亡情況，HE染色比較各組視網(wǎng)膜外核層厚度變化情況，Western-blotting檢測光照1d、2d、5d、7d、14d各組視網(wǎng)膜CNTF及GFAP的表達(dá)情況。免疫熒光雙重標(biāo)記檢測CNTF與GFAP的定位情況。采用GFP標(biāo)記的MSCs靜脈移植觀察移植后細(xì)胞在視網(wǎng)膜的橫向及縱向分布情況。 結(jié)果：經(jīng)靜脈移植的MSCs可以移行并遷移出視網(wǎng)膜血管，移植細(xì)胞主要分布于視網(wǎng)膜外核層、脈絡(luò)膜等。MSCs移植顯著抑制視網(wǎng)膜外核層厚度變薄（P＜0.05）及強(qiáng)光暴露引起的視網(wǎng)膜細(xì)胞凋亡（P＜0.05）。Western-blotting顯示MSCs注射組大鼠視網(wǎng)膜表達(dá)CNTF及GFAP顯著增強(qiáng)（P＜0.05）。免疫熒光雙重標(biāo)記顯示二者具有共同定位。 結(jié)論：靜脈移植MSCs具有對光損傷視網(wǎng)膜的神經(jīng)保護(hù)作用，其機(jī)制可能與移植介導(dǎo)的膠質(zhì)細(xì)胞反應(yīng)增強(qiáng)有關(guān)。
[Abstract]:As to the mesenchymal stem cells (mesenchymal stem cells, MSCs) the deeper understanding of the definition of MSCs has exceeded a kind of pluripotent adult stem cells are considered the concept of.MSCs multi cell population, widely involved in tissue repair.MSCs may promote injury repair process by cytokine secretion and regulate the body the micro environment, but its exact mechanism remains unclear. This study used an in vitro model of light induced retinal damage influence on the expression of MSCs cytokines, and further elaborates the changes in MSCs expression of cytokines involved in retinal degeneration process. The expression changes of host cell factor by in vivo transplantation of MSCs in retinal light injury and transplantation mediated.
The first part of the animal model of retinal light injury
Objective: to establish an animal model of retinal light injury and to explore the effect of light damage on the structure of retina and the changes of endogenous cytokine expression induced by light damage.
Methods: 6~8W SD rats after mydriasis, 12h light (5klux), 12h light, and light for repeated daily. After exposure to 1D, 2D, 5D, 7d, 14d of enucleation by transmission electron microscopy to observe the light early retinal ultrastructural changes were observed by.HE staining light exposure effect on retinal structure the terminal deoxynucleotidyl labeling (TUNEL) assay after irradiation, the apoptosis of retinal cells. By ink perfusion and retinal flatmount was evaluated after illumination change. Detection of bFGF retinal microvascular permeability, retinal cell factor immunofluorescence BDNF, the expression of CNTF.
Results: after exposure of 24h light, the retinal ultrastructure changed, photoreceptor cell outer membrane disc abscission, mitochondria swelling, nucleus electron density increased. The retinal tissue structure with the prolonging of UV irradiation time and sustained damage, the outer nuclear layer of retina thinning. Persistent apoptosis of retinal cells after being exposed to light. Mainly distributed in the outer nuclear layer and retinal pigment epithelium. Light exposure of retinal microvascular permeability, ink perfusion punctate exudate, after 14d light leakage in the deep lamellar, light exposure. The retina caused bFGF, BDNF, CNTF and GFAP. The expression of CNTF and GFAP expression increased with the irradiation time increased.
Conclusion: strong light exposure can cause the destruction of retinal tissue structure and the expression of reactive cytokines.
The effect of retinal light damage on the neuroprotective function of bone marrow mesenchymal stem cells in the second part
Objective: To investigate the effect of light injured retinal homogenate supernatant on the expression of MSCs cytokines and the expression of cytokines in the process of MSCs's involvement in retinal degeneration.
Methods: whole bone marrow adherent culture method and MSCs, flow cytometry, osteogenic and adipogenic differentiation. Preparation of normal and light damage to the retina homogenate, respectively by third ~ 5 generations of MSCs2d and 5d. by RT-qPCR and Western-blotting to detect bFGF expression of CNTF and BDNF, and non induced group MSCs. Collect without induction and after MSCs culture supernatant, preparation of conditioned medium, cultured retinal slices of 24h. retinal slice culture fluid collection, evaluation of different effects of conditioned medium on retinal cell membrane integrity in different culture conditions to detect the influence of.RT-qPCR solution on the expression of retinal Bcl-2 and Bax through the release levels of LDH lentiviral vectors containing bFGF. The transfection of MSCs interference sequence, and further induce MSCs after transfection, to observe the effect of down-regulation of bFGF expression may play a protective role in retinal nerve on MSCs.
Results: MSCs expression of bFGF, BDNF, CNTF. after light damage to the retina homogenate after induction of MSCs expression of bFGF and CNTF increased significantly (P < 0.05).MSCs expression in liquid culture conditions by retinal Bcl-2, inhibit the expression of Bax and LDH. The release of light damage of retinal homogenate induced after MSCs conditioned medium than other conditions medium significantly enhanced the expression of Bcl-2 by retinal slices, inhibition of Bax expression and LDH release (P < 0.05) after downregulation of.BFGF MSCs conditioned medium can increase retinal bcl-2 expression, inhibiting the release of LDH. But the culture conditions showed no significant difference between the liquid group (P > 0.05).
Conclusion: injury stimulation can promote the expression of MSCs cytokines, enhance the retinal neuroprotective effect of MSCs, and increase the expression of bFGF induced by injury. MSCs plays an important role in the treatment of retinal degeneration.
The effect of bone marrow mesenchymal stem cells transplanted into the third part of the vein on the light damage of the retina
Objective: To investigate the effect of MSCs vein transplantation on retinal cell apoptosis induced by light injury and the changes of cell migration and transplantation mediated host cell factor expression after transplantation.
Methods: to establish the animal model of retinal light damage, MSCs injection group in light exposed 48h after intravenous transplantation of MSCs, dose of 5 * 106 /ml, DMEM injection group received equal volume of DMEM injection. With TUNEL after 14d light irradiation group, DMEM injection group and MSCs injection group of retinal cell apoptosis. Comparison of the thickness of the outer nuclear layer changes each retina HE staining, detection of Western-blotting light 1D, 2D, 5D, 7d, CNTF and 14d groups the expression of retinal GFAP. The localization of the double labeled immunofluorescence detection of CNTF and GFAP. Using GFP labeled MSCs vein graft after transplantation were observed in the horizontal and vertical distribution the retina.
Results: after intravenous transplantation of MSCs can migrate and migrate out of retinal vessels, transplanted cells were mainly distributed in the outer nuclear layer of retina, choroid transplantation.MSCs significantly inhibit the retinal outer nuclear layer thickness (P < 0.05) and exposure to light induced retinal cell apoptosis (P < 0.05).Western-blotting display MSCs retinal injection group rats the expression of CNTF and GFAP increased significantly (P < 0.05). Double immunofluorescent labeling showed the two have a common location.
Conclusion: venous transplantation of MSCs has a neuroprotective effect on light damaged retina, and its mechanism may be related to the enhanced reaction of glia mediated by transplantation.

【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2013
【分類號】：R774.1


本文編號：1556579