山羊角膜缘干细胞荧光蛋白（Venus）细胞株建立及其移植研究

发布时间：2018-02-26 12:39

本文关键词： 角膜缘干细胞 Venus荧光蛋白荧光角膜上皮植片角膜缘干细胞移植　出处：《西北农林科技大学》2010年硕士论文　论文类型：学位论文

【摘要】： 角膜缘干细胞(LSCs)是角膜上皮再生与修复的来源。许多角膜疾病是由于角膜缘干细胞缺失(LSCD)引发的。目前国内外许多研究者通过角膜缘干细胞移植术(LSCT)治愈受损的角膜上皮,并且取得良好的治愈效果,但是关于角膜缘干细胞移植受损机体后的修复机理并不明确。为了揭示角膜缘干细胞移植后移行及修复变化,本研究分离培养原代山羊角膜缘干细胞(GLSCs),并标记venus荧光基因建立转基因细胞株(GLSC-V),以角膜缘干细胞荧光蛋白(Venus)细胞株为种子细胞组织工程化构建成荧光角膜上皮植片并移植于缺失角膜上皮山羊眼表,最后对重建的角膜上皮进行相关性检测,为研究角膜缘干细胞修复严重受损角膜上皮机理奠定基础。本研究主要内容如下: 1.山羊角膜缘干细胞分离、培养及鉴定本研究首先体外分离山羊角膜缘上皮组织,依次使用Dispase II酶及胰酶处理上皮组织获得角膜缘上皮单细胞,再使用20 min Collagen IV快速粘附法部分富集GLSCs。体外扩增的GLSCs的生长特性与国内外报道一致,免疫化学及RT-PCR检测结果表明分离纯化的GLSC未分化且具有高增殖能力,可用于后期转基因工作。 2.山羊角膜缘干细胞荧光蛋白(Venus)细胞株的建立本研究使用脂质体转染方法对GLSCs进行pVenus转染,并使用最佳浓度的G418 (500μg/mL)培养液筛选获得稳定传代的Venus荧光蛋白细胞株(GLSC-V)。GLSC-V具有与GLSCs相同的生理特性。荧光倒置显微镜下观察Venus在细胞质和核中均表达绿色荧光,荧光在体外培养可至少持续3个月。免疫荧光及半定量RT-PCR检测GLSC-V低分化且有高增殖能力,可作为组织工程化角膜上皮的种子细胞。 3.荧光角膜上皮植片的构建、移植及检测本研究以GLSC-V为种子细胞接种于去上皮羊膜上,体外培养15~21天构建成荧光角膜上皮植片。HE染色观察该荧光角膜上皮植片具有与正常角膜上皮相似的组织结构。透射电镜观察荧光角膜上皮细胞间形成紧密桥粒联结,细胞核大,核仁突出,细胞核附近富集张力丝。免疫荧光检测荧光角膜上皮组织仅基底部最基层细胞表达P63,上表皮及中间层细胞不表达P63,这与正常角膜缘中干细胞分布特征相似。将构建成功的荧光角膜上皮植片移植于山羊角膜上皮缺失模型上。术后实行4个月临床观察:实验组山羊眼表的鼻、颞两侧角膜边缘最先复明,然后透亮区域逐渐从鼻、颞两侧角膜周边向中央及上、下两侧周边扩张。实验组山羊眼表67%有效复明(2/3),33%部分复明(1/3),阴性对照组山羊眼表,角膜结膜化、血管化。HE染色观察,重建的角膜缘、角膜周边及角膜中央修复部分细胞与正常角膜上皮组织结构相似,但重建的角膜上皮层数分布不均匀,中央角膜上皮未修复的部分细胞形态不规则。免疫荧光检测角膜缘及中央角膜部的P63表达情况,结果表明重建的角膜和正常角膜上皮一样最表层细胞是终末分化细胞;重建角膜缘基底层和中间层干细胞分布不均匀;正常角膜中央细胞基底层和中间层细胞仅有少量干细胞存在,而重建中央角膜上皮基底层和中间层干细胞较多。对重建角膜上皮的不同区域细胞进行RT-PCR及免疫荧光检测Venus,结果表明供体细胞在角膜中央部最多,角膜缘部最少,结膜部也能检测到Venus表达。综合以上研究结果可推测,角膜上皮供体细胞移植后不仅存在于重建角膜上皮中,而且也向结膜上移行。角膜修复机制可能是供体细胞主要完成角膜上表皮的重建,并在一段时间内不被受体细胞替代;受体与供体高增殖细胞共同参与角膜上皮的修复,随着角膜修复进行,受体细胞从角膜缘开始逐渐替代供体细胞,并且角膜上皮的修复与更新是共同发生的。
[Abstract]:Corneal limbal stem cells ( LSCs ) are the source of corneal epithelium regeneration and repair . Many corneal diseases are caused by the absence of corneal limbal stem cells ( LSCD ) . Many researchers at home and abroad have successfully cured the damaged corneal epithelium by limbal stem cell transplantation ( LSCT ) . 1 . Isolation , Culture and Identification of Goat Cornea Stem Cells The results showed that GLSCs isolated and purified were undifferentiated and had high proliferative capacity and could be used in post - transgenic work . 2 . Establishment of fluorescence protein ( Venus ) cell line of goat corneal limbal stem cells GLSC - V was transfected with pVenus by liposome transfection method , and the optimal concentration of G418 ( 500 渭g / mL ) was used to screen the same physiological characteristics . GLSC - V had the same physiological characteristics as GLSCs . 3 . Construction , transplantation and detection of fluorescent corneal epithelium graft In this study , we observed the expression of P63 , epithelial and central corneal epithelium in the corneal epithelium of the experimental group . The results showed that the reconstructed cornea and the normal corneal epithelium were the same as the normal corneal epithelium . The results showed that the reconstructed cornea and the normal corneal epithelium were not uniform . The results showed that the reconstructed cornea and the normal corneal epithelium were not uniform . The basal and middle layer cells of the normal cornea were only in the presence of small amount of stem cells , but the central corneal epithelium and the middle layer stem cells were reconstructed . The expression of Venus was detected by RT - PCR and immunofluorescence in different regions of corneal epithelium . It is speculated that the corneal epithelium donor cells are not only present in the reconstruction of the corneal epithelium but also to the conjunctiva . The mechanism of corneal repair may be that the donor cells mainly finish the reconstruction of the upper epidermis of the cornea and are not replaced by somatic cells for a period of time ; the receptor and the donor high - proliferative cells are involved in the repair of the corneal epithelium , and the recipient cells gradually replace the donor cells from the corneal margin with the repair of the cornea , and the repair and renewal of the corneal epithelium occur together .

【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R779.6

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