【摘要】：目的探討氯化鈉法及冰凍切片法制備脫細(xì)胞豬角膜基質(zhì)膜片的可行性,并進一步復(fù)合兔角膜基質(zhì)細(xì)胞構(gòu)建組織工程角膜基質(zhì),探討脫細(xì)胞角膜基質(zhì)膜片在兔角膜基質(zhì)缺損中的修復(fù)效果,為角膜基質(zhì)的組織工程修復(fù)治療提供實驗依據(jù)。方法將去除上皮層和內(nèi)皮層的全層豬角膜基質(zhì)瓣采用氯化鈉法脫細(xì)胞,組織學(xué)檢測脫細(xì)胞效率,冰凍切片法將脫凈細(xì)胞的厚基質(zhì)瓣切成20μm厚度的脫細(xì)胞角膜基質(zhì)膜片,紫外線照射滅菌保存。采用組織塊法分離培養(yǎng)兔角膜基質(zhì)細(xì)胞,傳至第三代作為構(gòu)建組織工程角膜基質(zhì)的種子細(xì)胞。將培養(yǎng)的兔角膜基質(zhì)細(xì)胞以三明治法接種于脫細(xì)胞角膜基質(zhì)膜片支架,層層疊加6層膜片形成支架-細(xì)胞復(fù)合物,體外培養(yǎng)2周后組織學(xué)檢測組織形成狀態(tài)。構(gòu)建兔角膜基質(zhì)缺損模型,將膜片和基質(zhì)細(xì)胞按照三明治法移植至角膜基質(zhì)缺損部位,共疊加5層膜片,另設(shè)單純?nèi)睋p不修復(fù)組、厚基質(zhì)移植組(100μm)、單純膜片無細(xì)胞組(5層)為對照組,術(shù)后第1月,3月和6月進行大體拍照觀察、眼前節(jié)光學(xué)相干斷層掃描、超聲角膜測厚、角膜透光度檢測、生物力學(xué)檢測、組織學(xué)檢查、透射電鏡超微結(jié)構(gòu)觀察等,評價治療效果。結(jié)果氯化鈉法能有效脫除豬角膜基質(zhì)中的角膜基質(zhì)細(xì)胞,同時能良好地維持基質(zhì)膠原纖維的形態(tài)和排列,在脫細(xì)胞基質(zhì)的基礎(chǔ)上制作脫細(xì)胞基質(zhì)膜片簡單易行。組織塊法培養(yǎng)的兔角膜基質(zhì)細(xì)胞體外生長良好,細(xì)胞接種至脫細(xì)胞豬角膜基質(zhì)膜片支架材料上增殖良好,掃描電鏡檢測顯示細(xì)胞在膜片上貼附生長良好。體外構(gòu)建組織分層明顯,細(xì)胞存活但基質(zhì)分泌少。兔角膜基質(zhì)移植修復(fù)實驗中,單純?nèi)睋p不修復(fù)組角膜菲薄,局部白色瘢痕及新生血管長入;厚基質(zhì)移植組透明度尚可,瞳孔區(qū)可見云翳,隨時間的延長云翳略減淡;單純膜片無細(xì)胞組無明顯瘢痕,但可見少量膜片皺褶,角膜欠透明,呈斑翳形態(tài);脫細(xì)胞基質(zhì)膜片接種角膜細(xì)胞組3個月時可見縫線周圍新生血管生成,6個月時逐漸減退,角膜透明。眼前節(jié)光學(xué)相干斷層掃描顯示單純?nèi)睋p組基質(zhì)信號不均,有高信號區(qū)的疤痕組織;厚膜片組可見移植膜片與原基質(zhì)間有明顯的界限,融合欠佳;單純膜片組角膜基質(zhì)層信號欠均勻,可見裂隙狀低信號區(qū),表明膜片間粘合欠佳;膜片加細(xì)胞組基質(zhì)信號較均勻,移植膜片間融合良好。厚度檢測發(fā)現(xiàn)單純?nèi)睋p組最薄,其次為單純脫細(xì)胞基質(zhì)膜片移植組,脫細(xì)胞基質(zhì)移植組和膜片加細(xì)胞移植組的厚度均與正常組接近。脫細(xì)胞基質(zhì)膜片加角膜細(xì)胞移植組在各波長可見光下的透光率與正常角膜最為接近。與正常對照組相比,單純?nèi)睋p組的生物力學(xué)檢測值均最低,而脫細(xì)胞膜片加細(xì)胞移植組術(shù)后6個月的最大荷載,抗拉強度和彈性模量均與正常組十分接近。組織學(xué)檢查發(fā)現(xiàn)單純?nèi)睋p組的角膜厚度顯著變薄,雖然厚基質(zhì)移植組,及單純基質(zhì)膜片移植組的厚度有所增加,但未見細(xì)胞分布,而膜片加細(xì)胞移植組見細(xì)胞均勻分布。透射電鏡檢測中,膜片加細(xì)胞移植組的膠原纖維直徑與正常組無統(tǒng)計學(xué)意義的差異。結(jié)論氯化鈉法加冰凍切片法制作脫細(xì)胞豬角膜基質(zhì)膜片簡單易行。脫細(xì)胞豬角膜基質(zhì)膜片用組織塊法培養(yǎng)的兔角膜基質(zhì)細(xì)胞在脫細(xì)胞豬角膜基質(zhì)膜片支架上生長良好,采用膜片與接種細(xì)胞疊加的方法能使角膜基質(zhì)細(xì)胞較均勻地分布在基質(zhì)層中,利于新生角膜基質(zhì)的生成和修復(fù)缺損組織。為構(gòu)建組織工程角膜基質(zhì)提供新思路。
[Abstract]:Objective To study the feasibility of preparing acellular porcine corneal stroma membrane by sodium chloride method and frozen section method, and further compound rabbit corneal stroma cells to construct tissue engineering corneal stroma, to investigate the effect of acellular corneal stroma membrane on rabbit corneal stroma defect. provides experimental basis for tissue engineering restoration treatment of corneal stroma. Methods The whole-layer porcine corneal stroma flap was removed by sodium chloride method and the decellular efficiency was examined histologically by using sodium chloride method. The acellular corneal stroma membrane with thickness of 20. m u.m was cut by freeze-section method and sterilized and preserved by ultraviolet irradiation. The rabbit corneal stromal cells were isolated and cultured by tissue mass spectrometry, and transferred to the third generation as seed cells for constructing tissue engineering corneal stroma. The cultured rabbit corneal stroma cells were inoculated on the acellular corneal stroma membrane scaffold by sandwich method, and 6 layers of bone were superimposed on each layer to form the scaffold-cell complex, and the tissue formation was detected after 2 weeks culture in vitro. The rabbit corneal stroma defect model was constructed, the membrane and stromal cells were transplanted to the corneal stroma defect site according to the sandwich method, 5 layers of membrane were superimposed, the other was no repair group, the thick matrix transplantation group (100. mu.m), the simple membrane cell-free group (5 layers) was the control group. In the first month, March and June after operation, general photo observation, anterior segment optical coherence tomography, ultrasonic corneal thickness measurement, corneal topography examination, biomechanical testing, histological examination, transmission electron microscope ultrastructural observation, etc. were performed to evaluate the therapeutic effect. Results Sodium chloride can effectively remove corneal stroma cells in porcine corneal stroma, and can maintain the morphology and arrangement of matrix collagen fibers well, and make acellular matrix membrane simple and feasible on the basis of acellular matrix. The rabbit corneal stromal cells cultured in the tissue mass method were well-grown in vitro, and the cells were inoculated to the acellular porcine corneal stroma membrane scaffold for good proliferation. Scanning electron microscopy (SEM) showed that the cells were well-grown on the membrane. Tissue stratification was evident in vitro, but cell survival was less than that of stroma. In the rabbit corneal stroma transplantation and repair experiment, the pure defect did not repair the corneal firthin, the local white blood line and the newborn blood vessel length; the transparency of the thick matrix transplantation group was fair, the pupil area could be seen to be cloudy, and the cloud length was slightly decreased over time; the pure membrane cell-free group had no obvious effect. However, a small number of membrane wrinkles were found, the cornea was undertransparent and in the form of speckles, and when the acellular matrix membrane was inoculated with corneal cell group for 3 months, the formation of new blood vessels around the suture was seen, and the cornea was gradually decreased in 6 months, and the cornea was transparent. The anterior segment optical coherence tomography showed that the matrix signal of the simple defect group was uneven and the scar tissue in the high signal region was found; the thick film group showed obvious boundary between the graft membrane and the original substrate; the fusion was poor; the corneal stroma layer signal of the simple membrane group was under-uniform and the slit-shaped low signal region was visible. It is shown that the adhesion between the membranes is poor; the matrix signal of the membrane plus cell group is more uniform, and the fusion between the transplanted membranes is good. The thickness test showed that the pure defect group was the thinnest, and then the thickness of the acellular matrix membrane transplantation group, the acellular matrix transplantation group and the membrane plus cell transplantation group were close to that of the normal group. The light transmittance of the acellular matrix membrane plus corneal cell transplantation group at each wavelength of visible light was the closest to that of the normal cornea. Compared with the normal control group, the biomechanical detection value of the pure defect group was the lowest, while the maximum load, tensile strength and elastic modulus were very close to the normal group after 6 months after the cell membrane plus cell transplantation group. Histologic examination revealed a significant thinning of the corneal thickness in the pure defect group, although the thickness of the thick matrix graft group and the simple matrix membrane graft group increased, but no cell distribution was found, whereas the membrane plus cell transplantation group saw a uniform distribution of cells. There was no statistically significant difference between the diameter of collagen fibers in membrane plus cell transplantation group and normal group during transmission electron microscope (TEM). Conclusion The preparation of acellular porcine corneal stroma membrane by using sodium chloride method and frozen section method is simple and easy. The rabbit corneal stroma cells cultured by the tissue mass method of the acellular porcine corneal stroma membrane are well grown on the acellular porcine corneal stroma membrane support, and the method for stacking the membrane and the inoculated cells enables the corneal stroma cells to be uniformly distributed in the matrix layer, is beneficial to the generation and repair of the new corneal stroma and repair the defect tissue. and provides a new idea for constructing tissue engineering corneal stroma.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2013
【分類號】：R772.2

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