冻存山羊角膜缘干细胞构造角膜上皮移植及检测研究

发布时间：2018-04-20 10:15

本文选题：冻存角膜缘干细胞 + 无饲养层　；参考：《西北农林科技大学》2008年博士论文

【摘要】： 角膜上皮完整性的维持依赖于表浅细胞不断脱落和基底层细胞不断增殖来完成,上皮细胞持续不断的水平向心运动和垂直向上运动源于角膜缘基底层的干细胞增殖和分化。因此,角膜缘干细胞对正常角膜生理功能的稳定起重要作用。许多研究报道体外培养角膜缘干细胞构建上皮植片,可以满足临床移植的需要。但是这些报道中,多采用3T3细胞做饲养层促进上皮细胞的增殖,由于3T3细胞是来自胎鼠的成纤维细胞,其在临床应用有将异种动物疾病传播给人类的危险。另外,目前培养角膜缘干细胞用于重建角膜上皮的研究与临床应用,都是在培养结束后就直接应用于临床,需要患者的身体状况与培养结束后细胞的最佳生长状态相吻合,否则,就会导致整个操作和手术失败。如果能够将培养好的角膜缘干细胞在体外保存一定时间,于不同时间根据患者的身体状况,解冻细胞再继续培养,将极大地方便其临床应用与研究。近年来成体干细胞可塑性的报道不断涌现,几乎所有哺乳动物的成体干细胞都具有横向分化潜能。但是角膜缘干细胞体外诱导分化少见报道。本研究从山羊角膜缘干细胞分离、培养入手,系统比较了原代角膜缘干细胞的分离方法,优化了角膜缘干细胞有血清培养体系,建立了角膜缘干细胞无血清培养体系。对分离富集的角膜缘干细胞体外连续传代后,液氮中长期冻存。解冻后,对其干细胞相关特性进行了研究。本研究以人羊膜为载体负载冻存角膜缘干细胞,在无饲养层无血清的培养体系中构建组织工程化角膜上皮,手术移植给角膜缘干细胞缺损模型羊眼表,结合药物使用抑制免疫排斥反应,最后对其移植治疗效果进行综合评价。实验研究主要内容包括: 1山羊角膜缘干细胞的分离培养与鉴定研究 (1)比较酶消化法与组织块培养法分离山羊角膜缘干细胞上的效率,认为酶消化法在分离所得细胞中干细胞的比例,分离效率,分离细胞增殖活性,以及分离细胞纯度上均优于组织块培养法。 (2)筛选不同浓度的IV胶原,以及不同粘附时间,对角膜缘干细胞分离的影响。结果表明:IV胶原最佳粘附浓度为20μg/mL,最佳粘附时间为20min。 (3)筛选出EGF和Insulin对角膜缘干细胞增殖影响的最佳作用浓度分别为20ng/mL和10μg/mL,优化了角膜缘干细胞有血清培养体系,建立了角膜缘干细胞无血清无饲养层培养体系,此无血清培养体系已申请国家专利,专利申请号:200710018160.9。 (4)角膜缘干细胞最高传至28代,液氮中共冻存角膜缘干细胞6×107,解冻细胞仍然保持了角膜缘干细胞的相关生物学特性。研究中所建立的冻存体系可以满足不同的实验条件下,对角膜缘干细胞的运输、保存和增殖的要求。 2山羊角膜缘干细胞体外定向诱导分化研究 (1)解冻扩增角膜缘干细胞经1m mol/Lβ-Me预诱导24h后,再用5m mol/Lβ-Me正式诱导18h,改用无血清培养基培养7d,角膜缘干细胞分化为神经样细胞,表达NSE神经细胞标志。 (2)解冻扩增角膜缘干细胞经10 u mol 5-氮胞苷诱导48h,用含心肌条件培养基的培养液体外连续培养25d,角膜缘干细胞逐渐聚集生长,最终分化为心肌样细胞,表达α-actin心肌细胞标志蛋白。 (3)解冻扩增角膜缘干细胞经50μg/mL抗坏血酸、10m mol/Lβ-磷酸甘油和0.1u mol/L地塞米松联合诱导7d后,部分细胞死亡,部分细胞呈三角形或多角形聚集生长,连续诱导21d后,诱导细胞形成细胞结节,茜素红染色阳性,呈成骨样细胞。 (4)解冻扩增角膜缘干细胞经2m mol/L谷氨酰胺、0.01%大豆胰酶抑制剂、10m mol/L烟碱、5ng/ml肝细胞生长因子(HGF)联合诱导,5d后,细胞胞体逐渐变大,继续诱导,细胞开始聚集成团,连续诱导21d后,诱导形成的细胞团Insulin抗体染色呈阳性,呈胰岛样细胞。 3山羊组织工程角膜上皮植片的构建移植与检测研究 (1)以冻存角膜缘干细胞为种子细胞,用无血清无饲养层培养体系,以去上皮羊膜为载体,体外培养12-14天,成功构建具有与正常角膜相似结构的组织工程角膜上皮。 (2)冻存角膜缘干细胞组织工程化角膜上皮移植可重建角膜缘干细胞完全缺失失明病理模型羊眼表。跟踪观察3个月,实验组100%(15/15)形成角膜型上皮(荧光素不着色);跟踪观察6个月,实验组20%(3/15)角膜上皮基本透明,80%(12/15)部分角膜开始透明;有6只实验组山羊,跟踪观察12个月,33.3%(2/6)成功修复受损角膜,角膜上皮完全透明,66.7%(4/6)部分修复,视力得到一定恢复。而角膜缘干细胞缺失未进行角膜上皮移植的对照组山羊,全部角膜结膜化失明。眼罩蒙蔽正常眼,对另一侧试验眼进行功能性检测,对试验羊进行驱赶,角膜完全修复组山羊能辨别方向正确躲避;病理模型组山羊完全无方向感,不能正确躲避甚至出现撞墙现象。 (3)提出了异体移植的角膜缘干细胞修复角膜缘干细胞完全缺失病理模型的机理可能是:外源移植的异体干细胞抑制了自身周围结膜以及血管向中央角膜上皮的生长,协同机体其他来源的前体细胞共同参与完成了角膜上皮的修复过程,实现角膜上皮重建。 (4)系统评价了组织工程化人工角膜上皮移植效果,首次通过检测SRY基因的办法,动态监测了供体细胞在受体动物机体中是否长期存在。并且采用酶标仪测定了角膜上皮透光度,此方法操作简单,结果真实可靠,可以在实验动物中很好地评价角膜上皮透光度。
[Abstract]:The maintenance of corneal epithelium integrity depends on the continuous loss of superficial cells and the continuous proliferation of basal layer cells. The continuous horizontal movement and vertical upward movement of epithelial cells are derived from the proliferation and differentiation of the stem cells in the limbal basal layer of the cornea. Therefore, the corneal limbal stem cells play an important role in the stability of normal corneal physiological function. Many studies have reported that corneal limbal stem cells are cultured in vitro to construct epithelial graft, which can meet the needs of clinical transplantation. But in these reports, 3T3 cells are used as feeder layers to promote the proliferation of epithelial cells. Because 3T3 cells are fibroblasts from fetal mice, they are in danger of spreading alien diseases to humans in clinical practice. In addition, the research and clinical application of corneal limbal stem cells for reconstruction of corneal epithelium are applied directly to the clinic after the end of culture. The condition of the patient's physical condition is in accordance with the optimal growth state of the cells after the end of culture. Otherwise, the whole operation and the operation will fail. If the cultured cornea can be cultivated, the cornea can be cultivated. The stem cells are preserved for a certain time in vitro, and at different time according to the body condition of the patients, the thawing cells will continue to be cultured again. The clinical application and research of the stem cells will be great. In recent years, the reports of the plasticity of adult stem cells have emerged continuously. Almost all the adult stem cells in mammals have the potential of lateral differentiation, but the limbus stem of the cornea is dry. Cell differentiation in vitro is rarely reported.
In this study, from the separation and culture of the limbal stem cells of the goats, the isolation method of the primary corneal limbus stem cells was systematically compared, the serum-free culture system of the limbal stem cells was optimized, and the serum-free culture system of the limbal stem cells was established. After the separation and enrichment of the limbal stem cells in vitro, the liquid nitrogen was frozen for a long time. After thawing, The stem cell related characteristics of its stem cells were studied. In this study, human amniotic membrane was used as a carrier to freeze corneal limbal stem cells. The tissue engineered corneal epithelium was constructed in a serum-free culture system without a feeder layer. The corneal limbal stem cell defect model was transplanted to the eye surface of the cornea, and the drug use was used to inhibit the immune rejection. Finally, the graft was transplanted to the corneal epithelium. The main contents of the experimental study include:
Isolation, culture and identification of goat corneal limbal stem cells 1
(1) the efficiency of separating the goats' limbal stem cells by enzyme digestion method and tissue mass culture method was compared. It was concluded that the ratio of stem cells, separation efficiency, cell proliferation activity and the purity of separated cells were superior to that of tissue mass culture.
(2) to screen the effects of different concentrations of IV collagen and different adhesion time on the separation of limbal stem cells. The results showed that the best adhesion concentration of IV collagen was 20 g/mL, and the best adhesion time was 20min.
(3) the best effects of EGF and Insulin on the proliferation of limbal stem cells were selected to be 20ng/mL and 10 g/mL respectively. The serum-free culture system of limbal stem cells was optimized and a serum-free and non feeder culture system of limbal stem cells was established. This serum-free culture system has applied for national patent, patent application number: 200710018160.9.
(4) the corneal limbal stem cells spread to 28 generations, and the corneal limbal stem cells of liquid nitrogen were frozen in 6 * 107. The thawing cells still kept the related biological characteristics of the limbal stem cells. The cryopreservation system established in this study could meet the requirements for the transport, preservation and proliferation of limbal stem cells under different experimental conditions.
In vitro differentiation of 2 goat limbal stem cells in vitro
(1) after 1m mol/L beta -Me preinduced 24h by thawing and expanded limbal stem cells, 18h was formally induced by 5m mol/L beta -Me, and 7d was cultured in serum-free medium. The corneal limbal stem cells were differentiated into neurolike cells, and NSE nerve cell markers were expressed.
(2) thawing and amplified corneal limbal stem cells were induced by 10 u mol 5- azacytidine, and 25d was cultured with the culture medium containing the myocardial conditioned medium. The corneal limbal stem cells gradually gathered and grew, and finally differentiated into myocardial like cells and expressed the alpha -actin cardiac myocyte marker protein.
(3) some cells died after 50 mu g/mL ascorbic acid, 10m mol/L beta glycerol phosphate glycerol and 0.1U mol/L dexamethasone induced 7d, and some cells were killed in a triangle or polygon. After continuous induction of 21d, the cells were induced to form cell nodules, alizarin red staining was positive for osteoblast like cells.
(4) thawing and amplified corneal limbal stem cells were induced by 2m mol/L glutamine, 0.01% soybean trypsin inhibitor, 10m mol/L nicotine and 5ng/ml hepatocyte growth factor (HGF). After 5D, the cell body gradually became larger and continued to be induced, and the cells began to gather into a group. After continuous induction of 21d, the staining of Insulin antibody in the induced cell group was positive and islet like. Cells.
Construction, transplantation and detection of 3 tissue engineered corneal epithelium in goat
(1) using the frozen limbal stem cells as the seed cells, using the serum-free feeder layer culture system, the epithelial amniotic membrane as the carrier and in vitro culture for 12-14 days, the tissue engineering corneal epithelium with the similar structure of normal cornea was successfully constructed.
(2) corneal limbal stem cell tissue engineered corneal epithelial transplantation could reconstruct the eye surface of the corneal limbal stem cell completely missing blindness pathological model. After 3 months, the experimental group 100% (15/15) formed the corneal epithelium (fluorescein non coloring), followed by 6 months, the experimental group 20% (3/15) corneal epithelium was basically transparent, 80% (12/15) part of the cornea was opened. 6 experimental groups of goats were observed for 12 months, and 33.3% (2/6) repaired the damaged cornea successfully, the corneal epithelium was completely transparent, 66.7% (4/6) was repaired, and the visual acuity was restored. The corneal limbal stem cells were absent from the control group of the corneal epithelium and all cornea conjunctiva blindness. The eye mask blinded the normal eye and the other side The experimental eyes were tested with functional test, driven by the experimental sheep, and the complete repair group of the cornea was able to distinguish the direction correctly. The pathological model group goats had no sense of direction, and could not avoid the correct avoidance or even the phenomenon of wall collision.
(3) the mechanism of the allograft corneal limbal stem cells to repair the complete corneal limbal stem cell deletion may be that exogenous stem cells inhibit the growth of the surrounding conjunctiva and the growth of the blood vessels to the central corneal epithelium, and the precursor cells of other sources of the body are involved in the repair process of the corneal epithelium together. To reconstruct the corneal epithelium.
(4) the effect of tissue engineered artificial corneal epithelial transplantation was evaluated systematically. For the first time, the SRY gene was detected to dynamically monitor the long-term existence of donor cells in the recipient animal body. And the corneal epithelial photometry was measured by an enzyme labeled instrument. This method is simple and reliable, and can be very good in experimental animals. Evaluation of corneal epithelial photometry.

【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R329

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