眼局部应用骨髓间充质干细胞（BMSC）治疗干眼的实验研究

发布时间：2018-07-03 04:07

本文选题：干眼 + 间充质干细胞　；参考：《新疆医科大学》2017年硕士论文

【摘要】：目的:探讨眼局部应用BMSC对干眼小鼠的治疗作用及其机制,并比较BMSC点眼与眼眶注射两种不同应用途径的疗效差异。方法:收集BALB/c小鼠双侧股骨、胫骨骨髓,采用贴壁培养法纯化BALB/c小鼠BMSC。采用随机数字表法将35只健康6-8周龄BALB/c小鼠分为正常对照组、模型组、阳性对照组、BMSC点眼组、BMSC眼眶注射组,每组7只,除正常对照组外的28只小鼠使用0.25%苯扎氯铵溶液滴双眼,连续滴21天,建立中重度干眼的动物模型。造模结束后开始治疗,模型组给予磷酸盐缓冲液(phosphate buffer solution,PBS)滴双眼;阳性对照组给予普拉洛芬眼液滴双眼;BMSC点眼组给予含1x105BMSC PBS悬垂液滴双眼;BMSC眼眶注射组给予含1x105 BMSC PBS悬垂液分别于治疗当天、第4天注射。治疗的第8天检测并比较各组小鼠眼表炎症指数、泪液分泌试验(SchirmerⅠtest,SⅠt)、泪膜破裂时间(tear film break-up time,BUT)、角膜荧光染色corneal fluorescein staining,FLS)评分结果。第9天麻药过量法处死35只小鼠,随机选取每组5眼用以制备苏木精-伊红(hematoxylin-eosin,HE)染色、过碘酸-希夫(periodic acid-schiff,PAS)染色切片,行眼表组织病理学观察,并进行杯状细胞计数;4眼用以制备冰冻切片,观察有无PK67标记的异体BMSC在眼表组织迁移,5眼用酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)测定小鼠眼表组织中白细胞介素-10(interleukin-10,IL-10)、白细胞介素-1β(interleukin-1β,IL-1β)蛋白含量。结果:模型组眼表炎症指数0.31±0.05、FLS评分8.80±1.21分别高于正常对照组0.01±0.01、0.14±0.14,差异均有统计学意义(均为p0.05);而阳性对照组、BMSC点眼组、BMSC眼眶注射组炎症指数分别为0.15±0.03、0.18±0.03、0.06±0.02均明显低于模型组,差异均有统计学意义(均为p0.05),且BMSC眼眶注射组低于BMSC点眼组,差异有统计学意义(p0.05);仅BMSC眼眶注射组FLS评分3.93±0.74明显低于模型组,差异有统计学意义(p0.05);模型组SIt(4.00±0.39)mm明显少于正常对照组(6.36±0.48)mm,差异有统计学意义(p0.05);而BMSC眼眶注射组SIt(5.86±0.54)mm明显多于模型组、阳性对照组(3.92±0.38)mm、BMSC点眼组(3.90±0.31)mm,差异均有统计学意义(均为p0.05);模型组BUT(3.00±0.21)s较正常对照组(6.00±0.21)s明显缩短,差异有统计学意义(p0.05),而阳性对照组(4.20±0.29)s、BMSC点眼组(4.40±0.27)s、BMSC眼眶注射组(4.79±0.02)s均较模型组明显延长,差异均有统计学意义(均为p0.05);眼表组织HE染色:模型组角膜上皮细胞肿胀、大量炎性细胞浸润,基质层胶原纤维排列紊乱、肿胀,BMSC点眼及注射组角膜上皮表面平整,基质层胶原纤维排列紧密形态接近正常小鼠,两组间无明显差异。PAS染色BMSC注射组(13.80±2.48)个与阳性对照组(13.17±2.09)个杯状细胞数量相当,均多于模型组(5.20±1.07)个,差异有统计学意义(均为p0.05)。冰冻切片:在BMSC点眼组及BMSC眼眶注射组睑板、结膜下及角膜均未见带PKH67标记的BMSC。ELISA BMSC注射组IL-10蛋白含量(509.80±190.21)明显高于BMSC点眼组(43.64±43.64),差异有统计学意义(p0.05);IL-1β蛋白含量各组间存在差异,但差异无统计学意义(均为p0.05)。结论:眼局部应用BMSC能使眼表炎症减轻、泪膜破裂时间延长、角膜上皮修复,从而有效缓解干眼的症状。BMSC眼眶注射组更能显著促进泪液量分泌、增加杯状细胞数量,疗效优于BMSC点眼组。
[Abstract]:Objective: To explore the therapeutic effect and mechanism of BMSC on dry eye mice, and to compare the difference between two different ways of BMSC eye and orbital injection. Methods: to collect bilateral femur and tibial bone marrow in BALB/c mice, and to purify BALB/c mice by adherent culture method, BMSC. by random digital table method, 35 healthy 6-8 weeks old BALB/c The mice were divided into the normal control group, the model group, the positive control group, the BMSC eye group and the BMSC orbital injection group, with 7 mice in each group. The 28 mice, except the normal control group, used 0.25% Benzalkonium Chloride Solution drops for 21 days to establish the medium and severe dry eye animal models. The model group was treated with phosphate buffer solution (phosphate BU). Ffer solution, PBS) drops both eyes; positive control group was given eyes with Pla Lo Finn eye drops; BMSC eye group was given 1x105BMSC PBS droppout drops in the eyes; BMSC orbital injection group was given 1X105 BMSC PBS suspension solution on the day of treatment, fourth days of injection. The eighth days of treatment and comparison of the eye surface inflammation index, tear secretion test ( Schirmer I test, S i t), tear film rupture time (tear film break-up time, BUT), corneal fluorescent staining corneal fluorescein staining, 35 mice were killed at ninth days, and 5 eyes in each group were randomly selected for preparation of hematoxylin eosin staining, periodate Schiff dyeing. Color section, histopathological observation of eye surface, and cup cell count; 4 eyes were used to prepare frozen section, to observe the migration of BMSC in ocular surface tissue without PK67 marker, and the 5 eyes with enzyme-linked immunosorbent assay, ELISA to determine the interleukin -10 (interleukin-10, IL-10) in the ocular surface tissues of mice and white thin. The content of cytokine -1 beta (interleukin-1 beta, IL-1 beta) protein. Results: the ocular surface inflammation index in the model group was 0.31 + 0.05, and the FLS score was 8.80 + 1.21 higher than that in the normal control group (0.01 + 0.01,0.14 0.14). The difference was statistically significant (all P0.05), while the positive control group, BMSC eye group and BMSC orbital injection group were 0.15 + 0.03,0.18 + 0.03, respectively. 0.06 + 0.02 were significantly lower than the model group, the difference was statistically significant (P0.05), and the BMSC orbital injection group was lower than the BMSC eye group, the difference was statistically significant (P0.05), the FLS score of the BMSC orbital injection group was 3.93 + 0.74 significantly lower than the model group (P0.05), and the SIt (4 + 0.39) mm in the model group was significantly less than the normal control group (4 + 0.39). 6.36 + 0.48) mm, the difference was statistically significant (P0.05), but SIt (5.86 + 0.54) mm in the BMSC orbital injection group was significantly more than the model group, the positive control group (3.92 + 0.38) mm, BMSC point group (3.90 + 0.31) mm, the difference was statistically significant (all P0.05), BUT (3 + 0.21) s in the model group was significantly shorter than that in the normal control group (6 + 0.21), and the difference was statistically significant (P0.05), the positive control group (4.20 + 0.29) s, BMSC eye group (4.40 + 0.27) s, BMSC orbital injection group (4.79 + 0.02) s were significantly longer than the model group, the difference was statistically significant (all P0.05); ocular surface tissue HE staining: the model group corneal epithelial cells swelling, a large number of inflammatory cells infiltration, matrix layer collagen fiber arrangement disorder, swelling, BMSC points. The corneal epithelium surface was smooth and the collagen fibers in the matrix layer were close to the normal mice. There was no significant difference between the two groups (13.80 + 2.48) and the positive control group (13.17 + 2.09) of the two groups, all of which were more than the model group (5.20 + 1.07), and the difference was statistically significant (P0.05). In the BMSC eye group and the BMSC orbital injection group, the IL-10 protein content in the BMSC.ELISA BMSC injection group under the conjunctiva and the cornea was not found (509.80 + 190.21) was significantly higher than that in the BMSC group (43.64 + 43.64), the difference was statistically significant (P0.05), but there was a difference in the content of IL-1 beta protein in each group, but the difference was not statistically significant (p0., p0.). 05). Conclusion: local application of BMSC can reduce the inflammation of ocular surface, prolong the rupture time of the tear film and repair the corneal epithelium, thus effectively relieving the symptoms of dry eyes, the.BMSC orbital injection group can increase the secretion of tear volume and increase the number of goblet cells. The effect is better than that of the BMSC eye group.
【学位授予单位】：新疆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R777.34

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