本文關(guān)鍵詞： 多糖 間充質(zhì)干細(xì)胞 角膜堿燒傷 角膜上皮細(xì)胞 炎癥反應(yīng) 新生血管大鼠　出處：《天津醫(yī)科大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：研究目的 本研究通過建立離體角膜上皮細(xì)胞(corneal epithelial cells, CECs)損傷模型和大鼠角膜堿燒傷的動(dòng)物模型,利用局部應(yīng)用多糖結(jié)合間充質(zhì)干細(xì)胞(mesenchymal stem cells, MSCs)的聯(lián)合療法作用于兩種模型,觀察CECs的增殖和遷移變化和大鼠角膜堿燒傷損傷的修復(fù)過程,比較多糖結(jié)合MSCs的聯(lián)合治療與單獨(dú)使用多糖及單獨(dú)使用MSCs治療效果的差異。探討和評(píng)估聯(lián)合多糖和MSCs治療對(duì)大鼠角膜堿燒傷的療效,為角膜堿燒傷的臨床治療提供一種全新的治療策略。 1.多糖聯(lián)合MSCs能否比單獨(dú)使用多糖及單獨(dú)使用MSCs治療更能促進(jìn)原代培養(yǎng)的CECs增殖和遷移。 2.多糖聯(lián)合MSCs能否比單獨(dú)使用多糖及單獨(dú)使用MSCs治療更能促進(jìn)大鼠角膜堿燒傷后眼表的修復(fù)和重建。 研究方法 1.培養(yǎng)大鼠原代CECs后,分別用多糖溶液、MSCs、多糖聯(lián)合MSCs作用于CECs,并利用MTT法檢測(cè)24h,48h,72h各濃度對(duì)原代培養(yǎng)CECs增殖能力的影響。 2.培養(yǎng)大鼠原代CECs后,建立離體上皮細(xì)胞損傷模型(細(xì)胞劃痕實(shí)驗(yàn)),分別用多糖溶液、MSCs、多糖聯(lián)合MSCs作用于CECs,并檢測(cè)24h各組對(duì)原代培養(yǎng)的CECs遷移能力的影響。 3.建立大鼠角膜堿燒傷動(dòng)物模型,分別用多糖水凝膠、MSCs、多糖水凝膠聯(lián)合MSCs治療角膜堿燒傷,分別于治療后3d,7d,14d,28d觀察和評(píng)估角膜透明度、角膜上皮愈合度、新生血管數(shù)量指標(biāo),并運(yùn)用RT-PCR和ELISA方法檢測(cè)各種治療方法對(duì)大鼠角膜堿燒傷后角膜分泌的炎癥相關(guān)因TNF-α、TGF-beta,趨化因子MIP-1α、MCP-1,新生血管相關(guān)因子VEGF、MMP-2、TSP-1變化的影響。 研究結(jié)果 1.在多糖濃度200ug/ml時(shí),各濃度多糖對(duì)CECs的增殖能力的差異沒有統(tǒng)計(jì)學(xué)意義。在多糖濃度400ug/ml,CECs逐漸死亡。 2.細(xì)胞增殖實(shí)驗(yàn)顯示,多糖組、MSCs組、多糖MSCs聯(lián)合治療組與對(duì)照組在24h,48h時(shí)對(duì)CECs的增殖能力的差異沒有統(tǒng)計(jì)學(xué)意義,而MSCs組和多糖MSCs聯(lián)合治療組在72h時(shí)與對(duì)照組相比促進(jìn)CECs增殖能力的差異具有統(tǒng)計(jì)學(xué)意義。 3.細(xì)胞劃痕實(shí)驗(yàn)顯示,多糖、MSCs組、多糖MSCs聯(lián)合治療組與對(duì)照組在24h促進(jìn)CECs遷移能力差異均具有統(tǒng)計(jì)學(xué)意義,聯(lián)合療法與單治療組促進(jìn)CECs遷移的能力差異具有統(tǒng)計(jì)學(xué)意義。 4.多糖水凝膠應(yīng)用于正常大鼠眼表未見明顯的副反應(yīng)存在。 5.大鼠角膜堿燒傷后,結(jié)膜下注射MSCs7天后可見MSCs大量存活,14天后仍可見少量存活。 6.動(dòng)物模型實(shí)驗(yàn)發(fā)現(xiàn),多糖組、MSCs組、多糖MSCs聯(lián)合治療組與未治療組7天、14天、28天時(shí)提高角膜透明度差異均具有統(tǒng)計(jì)學(xué)意義,而聯(lián)合治療組與單治療組相比提高角膜透明度的差異具有統(tǒng)計(jì)學(xué)意義。 7.動(dòng)物模型實(shí)驗(yàn)發(fā)現(xiàn),多糖組、MSCs組、多糖MSCs聯(lián)合治療組與未處理組在3天、7天、14天時(shí)促進(jìn)上皮的愈合能力的差異均具有統(tǒng)計(jì)學(xué)意義,聯(lián)合治療組與單治療組在7天、14天時(shí)促進(jìn)角膜上皮愈合能力的差異具有統(tǒng)計(jì)學(xué)意義。 8.動(dòng)物模型實(shí)驗(yàn)發(fā)現(xiàn),多糖組、MSCs組、多糖MSCs聯(lián)合治療組與未處理組在7天、14天、28天時(shí)抑制新生血管能力的差異均具有統(tǒng)計(jì)學(xué)意義,聯(lián)合治療組與單治療組在14天、28天時(shí)抑制新生血管能力的差異具有統(tǒng)計(jì)學(xué)意義。 9.大鼠角膜堿燒傷后各組角膜組織HE組織化學(xué)染色顯示：MSCs組傷后第3天,第7天時(shí),角膜上皮缺損區(qū)可見大皰狀改變,膠原纖維排列疏松,可見上皮層及基質(zhì)內(nèi)炎癥細(xì)胞浸潤。第14天時(shí)角膜上皮愈合,基質(zhì)水腫消失,可見基質(zhì)內(nèi)少量新生血管生成。多糖組傷后第3天,第7天時(shí),角膜上皮缺損區(qū)未見大皰狀病變,可見上皮層及基質(zhì)內(nèi)炎癥細(xì)胞浸潤。第14天時(shí)角膜上皮愈合,可見基質(zhì)內(nèi)新生血管生成。而聯(lián)合治療組第3天時(shí),角膜上皮缺損區(qū)未見大皰狀病變,可見極少量炎癥細(xì)胞浸潤。第7天時(shí)角膜上皮愈合。第14天時(shí)可見基質(zhì)內(nèi)極少量新生血管生成。 10.利用RT-PCR方法對(duì)大鼠角膜堿燒傷治療后各組相關(guān)細(xì)胞因子的基因表達(dá)檢測(cè)顯示：多糖組、MSCs組、多糖MSCs聯(lián)合治療組與未處理組相比在各時(shí)間段下調(diào)TNF-α、MIP-1α、MCP-1, VEGF、MMP-2基因表達(dá)的差異均具有統(tǒng)計(jì)學(xué)意義,同時(shí)促進(jìn)TSP-1、TGF-beta的表達(dá)上調(diào)的差異均具有統(tǒng)計(jì)學(xué)意義。多糖MSCs聯(lián)合治療與單治療組相比下調(diào)TNF-a, MIP-1α、MCP-1及VEGF、MMP-2的基因表達(dá)的差異具有統(tǒng)計(jì)學(xué)意義,上調(diào)TGF-beta.TSP-1的基因表達(dá)差異具有統(tǒng)計(jì)學(xué)意義。 11.利用ELISA方法對(duì)大鼠角膜堿燒傷治療后各組相關(guān)細(xì)胞因子蛋白表達(dá)檢測(cè)顯示：多糖組、MSCs組、多糖MSCs聯(lián)合治療組與未處理組相比減少VEGF蛋白表達(dá)、促進(jìn)TGF-beta蛋白表達(dá)的差異具有統(tǒng)計(jì)學(xué)意義。而多糖MSCs聯(lián)合治療組與單治療組相比減少VEGF蛋白表達(dá),增加TGF-beta蛋白表達(dá)的差異具有統(tǒng)計(jì)學(xué)意義。結(jié)論 1.在多糖濃度200ug/ml時(shí),在體及離體實(shí)驗(yàn)均表明,不同濃度多糖與CECs均具有良好的生物相容性。 2.200ug/ml多糖溶液對(duì)原代培養(yǎng)的CECs的形態(tài)和增殖沒有影響,而MSCs在與CECs作用72h時(shí)能促進(jìn)CECs的增殖。 3.多糖與MSCs均能促進(jìn)CECs的24h遷移,而聯(lián)合處理組具有更佳的促進(jìn)效果。 4.原代培養(yǎng)的MSCs能在大鼠堿燒傷后的結(jié)膜下存活14天左右。 5.多糖和MSCs均能提高大鼠角膜堿燒傷后角膜透明度,促進(jìn)角膜上皮的愈合,并抑制角膜新生血管的形成。而多糖MSCs聯(lián)合治療組提高角膜堿燒傷后角膜透明程度、角膜上皮愈合能力和抑制角膜新生血管的能力要顯著優(yōu)于MSCs或多糖單治療組。 6.多糖和MSCs均能通過下調(diào)炎癥因子TNF-a,趨化因子MIP-1α、MCP-1,新生血管因子VEGF、MMP-2的基因表達(dá)和VEGF蛋白表達(dá),上調(diào)抗炎因子TGF-beta基因和蛋白表達(dá)、抗新生血管因子TSP-1的基因表達(dá)而達(dá)到抑制炎癥反應(yīng)和新生血管的作用。多糖MSCs聯(lián)合治療組抑制炎癥反應(yīng)和新生血管的作用要顯著優(yōu)于MSCs或多糖單治療組。
[Abstract]:research objective
In this study, through the establishment of in vitro corneal epithelial cells (corneal epithelial cells, CECs) animal model and damage model of rat corneal alkali burn, the use of topical application of polysaccharide combined with mesenchymal stem cells (mesenchymal stem cells, MSCs) of the combined therapy for the two model, to observe the repair process of CECs proliferation and migration and the change of rat corneal alkali burn injury, combined treatment with MSCs and polysaccharide alone difference of polysaccharide and the therapeutic effect of MSCs used alone. To evaluate the curative effect of combined treatment of polysaccharide and MSCs on corneal alkali burn in rats, to provide a new therapeutic strategy for the treatment of corneal alkali burns.
Whether 1. polysaccharide combined with MSCs can promote the proliferation and migration of CECs in primary culture more than using polysaccharides alone and using MSCs alone.
Whether 2. polysaccharide combined with MSCs can promote the repair and reconstruction of ocular surface after alkali burn of cornea in rats compared with the use of polysaccharides alone and the use of MSCs alone.
research method
1. after training the primary CECs of rats, they were treated with polysaccharide solution, MSCs, polysaccharide and MSCs combined with MSCs respectively. The effects of 24h, 48h and 72h concentrations on the proliferation ability of primary cultured CECs were detected by MTT.
2. after training the primary CECs of rat, we established an in vitro epithelial cell injury model (cell scratch test), and used polysaccharide solution, MSCs, polysaccharide combined with MSCs to act on CECs, and detected the effect of 24h on the migration ability of primary cultured CECs.
3. establishment of rat animal model of corneal alkali burn, respectively by polysaccharide gel, MSCs polysaccharide gel combined with MSCs in the treatment of corneal alkali burn, respectively after treatment, 3D, 7d, 14d, 28d to observe and evaluate the corneal transparency, corneal epithelial healing degree, neovascularization index, and detection of various treatment methods with RT-PCR and the ELISA method on the secretion of inflammatory rat cornea after corneal alkali burn by TNF- alpha, TGF-beta, chemokine MIP-1 alpha, MCP-1, MMP-2, VEGF, angiogenesis related factors, the variation of TSP-1.
Research results
1. when the concentration of polysaccharide was 200ug/ml, there was no significant difference in the proliferation of CECs by the concentration of polysaccharides. At the concentration of polysaccharide, 400ug/ml, CECs gradually died.
2. cell proliferation experiment, polysaccharide group, MSCs group, polysaccharide MSCs treatment group and control group in 24h, the difference on the proliferation of CECs 48h were not statistically significant, while the MSCs group and the polysaccharide MSCs treatment group at 72h compared with control group was statistically significant difference in promoting the proliferation ability of CECs.
3. cell scratch test showed that polysaccharides, MSCs group, polysaccharide MSCs combined treatment group and control group had statistically significant difference in 24h promoting CECs migration ability. The difference between the combination therapy group and the single treatment group in promoting CECs migration was statistically significant.
No obvious side reaction was found in the ocular surface of normal rats by the 4. polysaccharide hydrogel.
After corneal alkali burn in 5. rats, MSCs survived a lot after subconjunctival injection of MSCs7 days, and a small amount of survival was still found after 14 days.
6. animal model experiment found that polysaccharides group, MSCs group, polysaccharide MSCs combined treatment group and untreated group 7 days, 14 days, 28 days increased corneal transparency difference was statistically significant, while the combined treatment group compared with the single treatment group to improve corneal transparency difference is statistically significant.
7. experimental animal model, polysaccharide group, MSCs group, polysaccharide MSCs treatment group and untreated group in 3 days, 7 days, 14 days to promote epithelial healing ability of the differences were statistically significant, the combined therapy group and single treatment group in 7 days, 14 days to promote corneal epithelial healing difference the ability has statistical significance.
8. experimental animal model, polysaccharide group, MSCs group, polysaccharide MSCs treatment group and untreated group in 7 days, 14 days, differences in inhibition of angiogenesis capacity of 28 days were statistically significant, the combined therapy group and single treatment group in 14 days, the difference was statistically significant inhibition of angiogenesis ability 28 day.
9. rats after corneal alkali burn corneal tissue were HE histochemical staining showed that third days after injury in MSCs group, the seventh day, corneal epithelial defect area can be bleb shape change, loosely arranged collagen fibers, visible epithelial layer and stromal inflammatory cell infiltration. After fourteenth days of corneal epithelial healing, stromal edema disappeared, a little angiogenesis within third days. The visible matrix polysaccharide group after injury, seventh days, no corneal epithelial defect area like bullous lesions, visible epithelial layer and stromal inflammatory cell infiltration. Fourteenth days of corneal epithelial healing, angiogenesis is visible within the substrate. And the combined treatment group for third days, no corneal epithelial defect area like bullous lesions, showed very few infiltration of inflammatory cells. The seventh day healing of corneal epithelium. The matrix generates visible minimal neovascularization at fourteenth days.
10. by using the RT-PCR method of each cytokine gene expression assay showed that corneal alkali burn in rats after treatment: polysaccharide group, MSCs group, polysaccharide MSCs treatment group and untreated group compared to the down-regulation of TNF- alpha, in each period of MIP-1 alpha, MCP-1, VEGF, MMP-2 gene expression differences were statistically at the same time, promote TSP-1, differences in the up-regulated expression of TGF-beta were statistically significant. The polysaccharide MSCs combination therapy and single therapy group compared to the down-regulation of TNF-a, MIP-1 alpha, MCP-1 and VEGF, the difference was statistically significant. The expression of MMP-2 gene, upregulation of TGF-beta.TSP-1 gene expression difference was statistically significant.
11. by using the ELISA method of each related cytokine protein expression assay showed that corneal alkali burn in rats after treatment: polysaccharide group, MSCs group, polysaccharide MSCs treatment group and untreated group compared to reducing the expression of VEGF was statistically significant differences, promote the expression of TGF-beta protein. The polysaccharide MSCs treatment group and single treatment compared to decrease the expression of VEGF protein, the difference was statistically significant. Conclusion the increased expression of TGF-beta protein
1. when the concentration of polysaccharide was 200ug/ml, both in vivo and in vitro experiments showed that the different concentrations of polysaccharides and CECs had good biocompatibility.
2.200ug/ml polysaccharide solution has no effect on the morphology and proliferation of CECs in primary culture, but MSCs can promote the proliferation of CECs when it acts with 72h with CECs.
Both polysaccharide and MSCs could promote the 24h migration of CECs, and the combined treatment group had better effect on promoting the migration of 24h.
4. primary cultured MSCs can survive for about 14 days under the conjunctiva of alkali burned rats.
5. polysaccharide and MSCs can improve the rat corneal alkali burn corneal transparency, promote healing of corneal epithelium, and inhibit the corneal neovascularization. The polysaccharide MSCs treatment group increased after corneal alkali burn corneal transparency, corneal epithelial healing ability and the ability to inhibit corneal neovascularization was superior to that of MSCs or more sugar single treatment group.
6. polysaccharide and MSCs can through down-regulation of TNF-a inflammatory cytokines, chemotactic factor MIP-1 alpha, MCP-1, angiogenesis factor VEGF, gene expression and VEGF protein expression of MMP-2, up-regulated the expression of anti-inflammatory factor TGF-beta gene and protein, anti angiogenic factor TSP-1 gene expression and inhibit inflammation and angiogenesis. The polysaccharide MSCs treatment group the inhibition of inflammation and angiogenesis was significantly better than single MSCs or polysaccharide treatment group.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R779.1

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相關(guān)期刊論文 前1條

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