發(fā)布時間：2018-07-03 04:07

  本文選題：干眼 + 間充質(zhì)干細胞��； 參考：《新疆醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:探討眼局部應(yīng)用BMSC對干眼小鼠的治療作用及其機制,并比較BMSC點眼與眼眶注射兩種不同應(yīng)用途徑的療效差異。方法:收集BALB/c小鼠雙側(cè)股骨、脛骨骨髓,采用貼壁培養(yǎng)法純化BALB/c小鼠BMSC。采用隨機數(shù)字表法將35只健康6-8周齡BALB/c小鼠分為正常對照組、模型組、陽性對照組、BMSC點眼組、BMSC眼眶注射組,每組7只,除正常對照組外的28只小鼠使用0.25%苯扎氯銨溶液滴雙眼,連續(xù)滴21天,建立中重度干眼的動物模型。造模結(jié)束后開始治療,模型組給予磷酸鹽緩沖液(phosphate buffer solution,PBS)滴雙眼;陽性對照組給予普拉洛芬眼液滴雙眼;BMSC點眼組給予含1x105BMSC PBS懸垂液滴雙眼;BMSC眼眶注射組給予含1x105 BMSC PBS懸垂液分別于治療當(dāng)天、第4天注射。治療的第8天檢測并比較各組小鼠眼表炎癥指數(shù)、淚液分泌試驗(SchirmerⅠtest,SⅠt)、淚膜破裂時間(tear film break-up time,BUT)、角膜熒光染色corneal fluorescein staining,FLS)評分結(jié)果。第9天麻藥過量法處死35只小鼠,隨機選取每組5眼用以制備蘇木精-伊紅(hematoxylin-eosin,HE)染色、過碘酸-希夫(periodic acid-schiff,PAS)染色切片,行眼表組織病理學(xué)觀察,并進行杯狀細胞計數(shù);4眼用以制備冰凍切片,觀察有無PK67標(biāo)記的異體BMSC在眼表組織遷移,5眼用酶聯(lián)免疫吸附法(enzyme-linked immunosorbent assay,ELISA)測定小鼠眼表組織中白細胞介素-10(interleukin-10,IL-10)、白細胞介素-1β(interleukin-1β,IL-1β)蛋白含量。結(jié)果:模型組眼表炎癥指數(shù)0.31±0.05、FLS評分8.80±1.21分別高于正常對照組0.01±0.01、0.14±0.14,差異均有統(tǒng)計學(xué)意義(均為p0.05);而陽性對照組、BMSC點眼組、BMSC眼眶注射組炎癥指數(shù)分別為0.15±0.03、0.18±0.03、0.06±0.02均明顯低于模型組,差異均有統(tǒng)計學(xué)意義(均為p0.05),且BMSC眼眶注射組低于BMSC點眼組,差異有統(tǒng)計學(xué)意義(p0.05);僅BMSC眼眶注射組FLS評分3.93±0.74明顯低于模型組,差異有統(tǒng)計學(xué)意義(p0.05);模型組SIt(4.00±0.39)mm明顯少于正常對照組(6.36±0.48)mm,差異有統(tǒng)計學(xué)意義(p0.05);而BMSC眼眶注射組SIt(5.86±0.54)mm明顯多于模型組、陽性對照組(3.92±0.38)mm、BMSC點眼組(3.90±0.31)mm,差異均有統(tǒng)計學(xué)意義(均為p0.05);模型組BUT(3.00±0.21)s較正常對照組(6.00±0.21)s明顯縮短,差異有統(tǒng)計學(xué)意義(p0.05),而陽性對照組(4.20±0.29)s、BMSC點眼組(4.40±0.27)s、BMSC眼眶注射組(4.79±0.02)s均較模型組明顯延長,差異均有統(tǒng)計學(xué)意義(均為p0.05);眼表組織HE染色:模型組角膜上皮細胞腫脹、大量炎性細胞浸潤,基質(zhì)層膠原纖維排列紊亂、腫脹,BMSC點眼及注射組角膜上皮表面平整,基質(zhì)層膠原纖維排列緊密形態(tài)接近正常小鼠,兩組間無明顯差異。PAS染色BMSC注射組(13.80±2.48)個與陽性對照組(13.17±2.09)個杯狀細胞數(shù)量相當(dāng),均多于模型組(5.20±1.07)個,差異有統(tǒng)計學(xué)意義(均為p0.05)。冰凍切片:在BMSC點眼組及BMSC眼眶注射組瞼板、結(jié)膜下及角膜均未見帶PKH67標(biāo)記的BMSC。ELISA BMSC注射組IL-10蛋白含量(509.80±190.21)明顯高于BMSC點眼組(43.64±43.64),差異有統(tǒng)計學(xué)意義(p0.05);IL-1β蛋白含量各組間存在差異,但差異無統(tǒng)計學(xué)意義(均為p0.05)。結(jié)論:眼局部應(yīng)用BMSC能使眼表炎癥減輕、淚膜破裂時間延長、角膜上皮修復(fù),從而有效緩解干眼的癥狀。BMSC眼眶注射組更能顯著促進淚液量分泌、增加杯狀細胞數(shù)量,療效優(yōu)于BMSC點眼組。
[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.
【學(xué)位授予單位】：新疆醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R777.34

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