多糖水凝胶结合间充质干细胞治疗大鼠角膜碱烧伤的实验研究

发布时间：2018-02-27 03:16

  本文关键词： 多糖 间充质干细胞 角膜碱烧伤 角膜上皮细胞 炎症反应 新生血管大鼠　出处：《天津医科大学》2014年博士论文　论文类型：学位论文

【摘要】：研究目的 本研究通过建立离体角膜上皮细胞(corneal epithelial cells, CECs)损伤模型和大鼠角膜碱烧伤的动物模型,利用局部应用多糖结合间充质干细胞(mesenchymal stem cells, MSCs)的联合疗法作用于两种模型,观察CECs的增殖和迁移变化和大鼠角膜碱烧伤损伤的修复过程,比较多糖结合MSCs的联合治疗与单独使用多糖及单独使用MSCs治疗效果的差异。探讨和评估联合多糖和MSCs治疗对大鼠角膜碱烧伤的疗效,为角膜碱烧伤的临床治疗提供一种全新的治疗策略。 1.多糖联合MSCs能否比单独使用多糖及单独使用MSCs治疗更能促进原代培养的CECs增殖和迁移。 2.多糖联合MSCs能否比单独使用多糖及单独使用MSCs治疗更能促进大鼠角膜碱烧伤后眼表的修复和重建。 研究方法 1.培养大鼠原代CECs后,分别用多糖溶液、MSCs、多糖联合MSCs作用于CECs,并利用MTT法检测24h,48h,72h各浓度对原代培养CECs增殖能力的影响。 2.培养大鼠原代CECs后,建立离体上皮细胞损伤模型(细胞划痕实验),分别用多糖溶液、MSCs、多糖联合MSCs作用于CECs,并检测24h各组对原代培养的CECs迁移能力的影响。 3.建立大鼠角膜碱烧伤动物模型,分别用多糖水凝胶、MSCs、多糖水凝胶联合MSCs治疗角膜碱烧伤,分别于治疗后3d,7d,14d,28d观察和评估角膜透明度、角膜上皮愈合度、新生血管数量指标,并运用RT-PCR和ELISA方法检测各种治疗方法对大鼠角膜碱烧伤后角膜分泌的炎症相关因TNF-α、TGF-beta,趋化因子MIP-1α、MCP-1,新生血管相关因子VEGF、MMP-2、TSP-1变化的影响。 研究结果 1.在多糖浓度200ug/ml时,各浓度多糖对CECs的增殖能力的差异没有统计学意义。在多糖浓度400ug/ml,CECs逐渐死亡。 2.细胞增殖实验显示,多糖组、MSCs组、多糖MSCs联合治疗组与对照组在24h,48h时对CECs的增殖能力的差异没有统计学意义,而MSCs组和多糖MSCs联合治疗组在72h时与对照组相比促进CECs增殖能力的差异具有统计学意义。 3.细胞划痕实验显示,多糖、MSCs组、多糖MSCs联合治疗组与对照组在24h促进CECs迁移能力差异均具有统计学意义,联合疗法与单治疗组促进CECs迁移的能力差异具有统计学意义。 4.多糖水凝胶应用于正常大鼠眼表未见明显的副反应存在。 5.大鼠角膜碱烧伤后,结膜下注射MSCs7天后可见MSCs大量存活,14天后仍可见少量存活。 6.动物模型实验发现,多糖组、MSCs组、多糖MSCs联合治疗组与未治疗组7天、14天、28天时提高角膜透明度差异均具有统计学意义,而联合治疗组与单治疗组相比提高角膜透明度的差异具有统计学意义。 7.动物模型实验发现,多糖组、MSCs组、多糖MSCs联合治疗组与未处理组在3天、7天、14天时促进上皮的愈合能力的差异均具有统计学意义,联合治疗组与单治疗组在7天、14天时促进角膜上皮愈合能力的差异具有统计学意义。 8.动物模型实验发现,多糖组、MSCs组、多糖MSCs联合治疗组与未处理组在7天、14天、28天时抑制新生血管能力的差异均具有统计学意义,联合治疗组与单治疗组在14天、28天时抑制新生血管能力的差异具有统计学意义。 9.大鼠角膜碱烧伤后各组角膜组织HE组织化学染色显示：MSCs组伤后第3天,第7天时,角膜上皮缺损区可见大疱状改变,胶原纤维排列疏松,可见上皮层及基质内炎症细胞浸润。第14天时角膜上皮愈合,基质水肿消失,可见基质内少量新生血管生成。多糖组伤后第3天,第7天时,角膜上皮缺损区未见大疱状病变,可见上皮层及基质内炎症细胞浸润。第14天时角膜上皮愈合,可见基质内新生血管生成。而联合治疗组第3天时,角膜上皮缺损区未见大疱状病变,可见极少量炎症细胞浸润。第7天时角膜上皮愈合。第14天时可见基质内极少量新生血管生成。 10.利用RT-PCR方法对大鼠角膜碱烧伤治疗后各组相关细胞因子的基因表达检测显示：多糖组、MSCs组、多糖MSCs联合治疗组与未处理组相比在各时间段下调TNF-α、MIP-1α、MCP-1, VEGF、MMP-2基因表达的差异均具有统计学意义,同时促进TSP-1、TGF-beta的表达上调的差异均具有统计学意义。多糖MSCs联合治疗与单治疗组相比下调TNF-a, MIP-1α、MCP-1及VEGF、MMP-2的基因表达的差异具有统计学意义,上调TGF-beta.TSP-1的基因表达差异具有统计学意义。 11.利用ELISA方法对大鼠角膜碱烧伤治疗后各组相关细胞因子蛋白表达检测显示：多糖组、MSCs组、多糖MSCs联合治疗组与未处理组相比减少VEGF蛋白表达、促进TGF-beta蛋白表达的差异具有统计学意义。而多糖MSCs联合治疗组与单治疗组相比减少VEGF蛋白表达,增加TGF-beta蛋白表达的差异具有统计学意义。结论 1.在多糖浓度200ug/ml时,在体及离体实验均表明,不同浓度多糖与CECs均具有良好的生物相容性。 2.200ug/ml多糖溶液对原代培养的CECs的形态和增殖没有影响,而MSCs在与CECs作用72h时能促进CECs的增殖。 3.多糖与MSCs均能促进CECs的24h迁移,而联合处理组具有更佳的促进效果。 4.原代培养的MSCs能在大鼠碱烧伤后的结膜下存活14天左右。 5.多糖和MSCs均能提高大鼠角膜碱烧伤后角膜透明度,促进角膜上皮的愈合,并抑制角膜新生血管的形成。而多糖MSCs联合治疗组提高角膜碱烧伤后角膜透明程度、角膜上皮愈合能力和抑制角膜新生血管的能力要显著优于MSCs或多糖单治疗组。 6.多糖和MSCs均能通过下调炎症因子TNF-a,趋化因子MIP-1α、MCP-1,新生血管因子VEGF、MMP-2的基因表达和VEGF蛋白表达,上调抗炎因子TGF-beta基因和蛋白表达、抗新生血管因子TSP-1的基因表达而达到抑制炎症反应和新生血管的作用。多糖MSCs联合治疗组抑制炎症反应和新生血管的作用要显著优于MSCs或多糖单治疗组。
[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.

【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R779.1

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1 王海燕;严军;蒋建新;龙在云;张冲;黄苏娜;王正国;;大鼠角膜缘上皮细胞的体外培养以及碱烧伤后β-连环蛋白表达的实验研究[J];创伤外科杂志;2010年04期

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