[1]蔡丽萍,孟然,张宏.眼局部应用骨髓间充质干细胞（BMSC）治疗小鼠干眼的实验研究[J].眼科新进展,2016,36(11):1024-1028.[doi:10.13389/j.cnki.rao.2016.0273]
　CAI Li-Ping,MENG Ran,ZHANG Hong.Evaluation of topical bone marrow mesenchymal stem cell therapy in experimental dry eye[J].Recent Advances in Ophthalmology,2016,36(11):1024-1028.[doi:10.13389/j.cnki.rao.2016.0273]
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眼局部应用骨髓间充质干细胞（BMSC）治疗小鼠干眼的实验研究
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《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
36卷
期数:
2016年11期
页码:
1024-1028
栏目:
实验研究
出版日期:
2016-11-05

文章信息/Info

Title:
Evaluation of topical bone marrow mesenchymal stem cell therapy in experimental dry eye
作者:
蔡丽萍; 孟然; 张宏
830011　新疆维吾尔自治区乌鲁木齐市，新疆医科大学第一附属医院眼科
Author(s):
CAI Li-Ping; MENG Ran; ZHANG Hong
Department of Ophthalmology ,the First Affiliated Hospital of Xinjiang Medical University, Urumqi 830011,Xinjiang Uygur Autonomous Region,China
关键词:
干眼; 骨髓间充质干细胞; 免疫; 炎性因子
Keywords:
dry eye ;  mesenchymal stem cell; immunology ;  cytokine
分类号:
Ｒ７７７
DOI:
10.13389/j.cnki.rao.2016.0273
文献标志码:
A
摘要:
目的　探讨局部应用骨髓间充质干细胞（ｂｏｎｅｍａｒｒｏｗｍｅｓｅｎｃｈｙｍａｌｓｔｅｍｃｅｌｌｓ，ＢＭＳＣ）对干眼小鼠的治疗作用及其机制，并比较ＢＭＳＣ滴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。利用随机数字表法将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滴眼组、ＢＭＳＣ眼眶注射组，每组７只，除正常对照组外的２８只小鼠使用２．５ｇ·Ｌ－１苯扎氯铵溶液滴双眼，连续滴２１ｄ，建立中重度干眼动物模型。造模结束后开始治疗，模型组给予磷酸盐缓冲液滴双眼；阳性对照组给予普拉洛芬眼液滴双眼；ＢＭＳＣ滴眼组给予含１×１０５ＢＭＳＣ的磷酸盐缓冲悬垂液滴双眼；ＢＭＳＣ眼眶注射组给予含１×１０５ＢＭＳＣ的磷酸盐缓冲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（ｔｅａｒｆｉｌｍｂｒｅａｋ-ｕｐｔｉｍｅ，ＢＵＴ）、角膜荧光素染色（ｃｏｒｎｅａｌｆｌｕｏｒｅｓｃｅｉｎｓｔａｉｎｉｎｇ，ＦＬＳ）评分结果。第９天过量麻醉法处死３５只小鼠，每组随机选取５眼制备ＨＥ染色、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眼用于制备冰冻切片，观察有无ＰＫ６７标记的异体ＢＭＳＣ在眼表组织迁移，５眼用于ＥＬＩＳＡ测定小鼠眼表组织中ＩＬ-１０、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Ｐ＜０．０５）；而阳性对照组、ＢＭＳＣ滴眼组、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Ｐ＜０．０５），且ＢＭＳＣ眼眶注射组低于ＢＭＳＣ滴眼组，差异有统计学意义（Ｐ＜０．０５）；仅ＢＭ-ＳＣ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（Ｐ＜０．０５）；模型组ＳＩｔ（４．００±０．３９）ｍｍ明显小于正常对照组（６．３６±０．４８）ｍｍ，差异有统计学意义（Ｐ＜０．０５）；而ＢＭＳＣ眼眶注射组ＳＩｔ（５．８６±０．５４）ｍｍ明显高于模型组、阳性对照组（３．９２±０．３８）ｍｍ、ＢＭＳＣ滴眼组（３．９０±０．３１）ｍｍ，差异均有统计学意义（均为Ｐ＜０．０５）；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（Ｐ＜０．０５），而阳性对照组（４．２０±０．２９）ｓ、ＢＭＳＣ滴眼组（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Ｐ＜０．０５）。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滴眼组、眼眶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，两组间差异不明显。ＰＡＳ染色杯状细胞数量ＢＭＳＣ眼眶注射组１３．８０±２．４８与阳性对照组１３．１７±２．０９相当，均多于模型组５．２０±１．０７，差异均有统计学意义（均为Ｐ＜０．０５）。冰冻切片：在ＢＭＳＣ滴眼组及ＢＭＳＣ眼眶注射组睑板、结膜下及角膜均未见带ＰＫＨ６７标记的ＢＭＳＣ。ＢＭＳＣ眼眶注射组ＩＬ-１０蛋白含量（５０９．８０±１９０．２１）μｇ·ｇ－１明显高于ＢＭＳＣ滴眼组（４３．６４±４３．６４）μｇ·ｇ－１，差异有统计学意义（Ｐ＜０．０５）；ＩＬ-１β蛋白含量各组间存在差异，但差异无统计学意义（Ｐ＞０．０５）。结论　眼局部应用ＢＭＳＣ能使眼表炎症减轻、ＢＵＴ延长、角膜上皮修复，从而有效缓解干眼的症状。ＢＭ-ＳＣ眼眶注射更能显著促进泪液分泌、增加杯状细胞数量，疗效优于ＢＭＳＣ滴眼。
Abstract:
Objective To investigate the therapeutic effects and explore the potential mechanism of topical application of bone marrow mesenchymal stem cells (BMSC) of dry eye rats, and compare effects of the two different methods of local transplanting BMSC. Methods BMSC were isolated from bone marrow of BALB/c rats and cultured by plastic adherence method. Thirty-five BALB/c rats were randomized into the normal control group , model group , positive control group , BMSC-dropped group and BMSC-injected group. Double eyes of the twenty-eight rats were topically treated with 2. 5 g . L-1 benzalkonium chloride for 21 days to establish moderate or severe dry eye model. And then the model group was topically applied phosphate buffer solution (PBS) . The positive control group was treated with pranoprofen. I x 10i BMSC suspended in PBS was applied to the conjunctiwal sac of the BMSC-dropped group and was injected into the intraorbital gland around main lacrimal gland of the BMSC-injected group every 3 days. Dry eye-related clinical tests of every set were performed ,including ocular surface inflammation index , Schirmer I test (SIt) . tear frlm break-up time (BUT ) and scores on the 8th day after treatment. 5 eyeballs of the rats in each group were randomly sacrificed to observe histological changes of ocular surface and to count goblet cells with hematoxylin-eosin (HE) and periodic acid-schiff (PAS) . 4 eyes were used to prepare frozen section in order to evaluate the engraftment of the PKH67 labeled BMSC in ocular surface tissue. 5 eyes were used to detect interleukin-10 (IL-IO ) and interleukin1[3 (IL-1[3) concentrations in ocular surface by enzyme-linked immunosorbent assay (ELISA) . Results Inflammation index in the model group was 0. 31 + 0. 05 , and FLS scores was 8. 80 + 1. 21 , which was significantly increased in comparison with 0. 01 + 0. 01 and 0. 14 + 0. 14 in the normal control group (all P < 0. 05 ) . However .inflammation index of every treated group (0. 15 +0. 03 ,0. 18 +0. 03 ,0. 06 +0. 02) were considerably lower than that in the model group (all P < 0. 05 ) . The BMSC-injected group of inflammation index was mirumum(P < 0. 05 ) . Only FLS scores of the BMSC-injected group (3. 93 +0. 74) was sigruficantly lower than that in the model group (P < 0. 05 ) . SIt in the model group was (4. 00 + 0. 39 ) mm , which was significantly declined in comparison with (6. 36 + 0. 48 ) mm in the normal control group (P < 0. 05 ) . However. SIt of the BMSC-dropped group (5. 86 + 0. 54 ) mm was obviously longer than that in the model group (4. 00 + 0. 39 ) mm ,positive control group (3. 92 + 0. 38 ) mm and BMSC-droped group (3. 90 + 0. 31 ) mm(all P < 0. 05 ) . BUT in the model group was (3. 00 + 0. 21 ) seconds .which was significantly declined in comparison with (6. 00 + 0. 21 ) seconds in the normal control group (P < 0. 05 ) . However , BUT of all treated group were considerably longer than that in the model group (all P < 0. 05 ) . HE staining of the model group showed that the corneal epithelial cell layer was edema , lymphocytic infrltration , and the stromal layer was edema and thickening. But there was no significant difference in the BMSC-dropped group and BMSC-injected group compared with the normal control group. PAS assay revealed similar goblet cell number in BMSC-injected group (13. 80 + 2. 48 ) and positive control group (13. 17 +2. 09) , but goblet cells was remarkable lessened in the model group (5. 20 + 1. 07 ) (all P < 0. 05 ) . PKH67 labeled BMSC was not shown to infiltrate the meibomian glands , conjunctival epithelium and cornea in frozen section of the BMSC-dropped group and BMSC-injected group. ELISA assay resolved that significantly augmentation in the concentrations of IL-IO in the BMSC-injected group (509. 80 + 190. 21) yg . g -l in comparison with the BMSC-dropped group (43. 64 + 43. 64 )

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更新日期/Last Update: 2016-11-29