【摘要】：目的評價羧甲基殼聚糖(Carboxymethyl chitosan,CMCTS)共混膜片的理學(xué)及生物學(xué)性能,并研究其在角膜堿燒傷修復(fù)中的作用。方法將CMCTS與明膠(Gelatin,Gel)和透明質(zhì)酸(Hyaluronic acid,HA)按照20∶1∶2的比例制備CMCTS共混膜片,使用全波長酶標儀于400~800 nm波長下檢測該膜片的透光性;將膜片植入大鼠皮下及肌肉組織,組織切片HE染色評價其組織相容性和炎癥反應(yīng);通過大鼠肝臟和腎臟的體重質(zhì)量比評估膜片在體內(nèi)的降解毒性。將兔原代角膜上皮細胞接種于CMCTS共混膜片上構(gòu)建角膜上皮細胞移植體,CFSE染色和掃描電鏡觀察細胞在膜片上的活性和生長狀態(tài);將該細胞移植體用于新西蘭大白兔角膜堿燒傷眼表的治療,通過外眼相觀察、裂隙燈觀察和組織免疫熒光染色與空白對照組、模型組及沒有細胞移植的膜片組相比較,評價其修復(fù)效果。結(jié)果本研究制備的CMCTS共混膜片在400~800 nm波長下透光率均高于80%;大鼠肌肉及皮下植入膜片2 w后,組織切片HE染色可見周圍組織炎癥反應(yīng)基本消失;膜片在皮下及肌肉組織中能夠穩(wěn)定降解,在皮下組織中降解時間超過8 w,而在肌肉組織中4 w完成降解;膜片在降解過程中對肝、腎均無明顯毒性;CFSE染色和掃描電鏡觀察兔原代角膜上皮細胞能夠在膜片上貼附生長并連接成片;將其移植到角膜堿燒傷的兔眼表,發(fā)現(xiàn)其在術(shù)后30 d內(nèi)基本完成創(chuàng)面修復(fù),而單純膜片組的修復(fù)能力較弱,模型組僅靠角膜自身修復(fù)作用不能進行角膜堿燒傷的完全修復(fù)。結(jié)論本實驗中制備的CMCTS共混膜片具有良好的物理及生物學(xué)性能,對兔角膜堿燒傷具有明顯的治療效果,為今后角膜組織工程方法治療角膜堿燒傷提供了可靠的實驗依據(jù)。
[Abstract]:Objective to evaluate the physical and biological properties of Carboxymethyl chitosan (CMCTS) blend film and to study its role in corneal alkali burn repair. Methods CMCTS, gelatinine and hyaluronic acid (HA) were used to prepare the CMCTS blend film at 20:1:2. The transmittance of the film was detected by using a full-wavelength enzyme label at 400nm, and the film was implanted into the subcutaneous and muscle tissues of rats. The histocompatibility and inflammatory response were evaluated by HE staining and the biodegradation toxicity of the membrane was evaluated by the body mass ratio of liver and kidney in rats. Rabbit primary corneal epithelial cells were inoculated on CMCTS blend membrane to construct corneal epithelial cell graft. The activity and growth state of corneal epithelial cells on the membrane were observed by scanning electron microscope (SEM). The cell grafts were used in the treatment of alkali burn of cornea in New Zealand white rabbits. The results were compared with those of control group, model group and membrane group without cell transplantation by external eye phase observation, slit lamp observation and tissue immunofluorescence staining. The effect of restoration was evaluated. Results the transmittance of the CMCTS blend films prepared in this study was higher than that of 80% at the wavelength of 400 ~ 800nm, and the inflammatory reaction of the surrounding tissues was basically disappeared by HE staining in the tissue sections of the rats after 2 weeks of muscle and subcutaneous implantation. The membrane could degrade steadily in subcutaneous and muscle tissues, and the degradation time in subcutaneous tissue was more than 8 ws, but in muscle tissue for 4 weeks. CFSE staining and scanning electron microscopy were used to observe the primary corneal epithelial cells attached to the membrane and connected to the rabbit corneal surface, which were transplanted to the surface of the cornea after alkali burn, and the wound repair was basically completed within 30 days after operation. However, the repair ability of the simple membrane group was weak, and the model group could not completely repair the corneal alkali burn only by the corneal self-repair. Conclusion the CMCTS blend film prepared in this experiment has good physical and biological properties and has obvious therapeutic effect on alkali burn of cornea in rabbits. It provides a reliable experimental basis for the treatment of alkali burn of cornea by corneal tissue engineering in the future.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R779.1

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