【作者】 赵清梅；

【导师】 窦忠英；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2008， 博士

【摘要】 各种角膜疾病和外科损伤都会导致角膜细胞受损,当损伤超过其生理代偿时,就会发生角膜病变,严重时会导致角膜盲。目前角膜移植是治疗此类角膜疾病比较成熟的方法,但受到了供体角膜缺乏、术后并发症、角膜供体老龄化等条件的限制。将自体或异体角膜细胞体外培养构建组织工程化人工角膜移植,既可解决临床应用供体短缺问题,又可为角膜体外药理或生理研究提供大量材料。本研究通过体外分离培养山羊角膜上皮细胞和角膜内皮细胞,并研究其体外生长特性和接种羊膜载体培养情况,为组织工程人工角膜的构建奠定理论基础。1.山羊角膜上皮细胞的体外分离培养与鉴定通过比较组织块和酶消化法培养山羊原代角膜缘上皮细胞,发现酶消化法更适合原代角膜缘上皮细胞的分离纯化培养。获取的山羊角膜缘上皮细胞形态为多角形,具有较强的增殖能力,体外培养能传20多代,细胞冻存后仍具有细胞冻存前的形态和活力。经免疫组化和RT-PCR检测,培养的角膜缘上皮细胞表达K3/K12、p63、Cx43、ABCG2和PCNA,具有角膜上皮细胞和角膜缘干细胞表型,说明分离培养的细胞为角膜缘上皮细胞和角膜缘干细胞混合物,可为组织工程化人工角膜上皮的构建提供种子细胞。2.组织工程化人工角膜上皮的构建在去上皮细胞人羊膜上接种关中奶山羊角膜缘组织块或培养的关中奶山羊角膜缘上皮细胞构建的组织工程角膜上皮,并对其进行组织形态学、超微结构观察和免疫组化检测。结果显示,角膜缘组织块和角膜上皮细胞在羊膜上培养15 d均可形成6～7层结构,细胞内含丰富的细胞器和糖原颗粒,细胞间有大量的桥粒连接,细胞与羊膜间形成半桥粒连接,构建的复合角膜上皮p63染色阳性。比较角膜缘组织块在去羊膜上皮细胞的新鲜羊膜、冻存30 d羊膜、冻存60 d羊膜和冻存180 d羊膜上生长情况发现,从角膜缘组织块脱出的细胞生长面积在冻存30 d羊膜上最大,而在新鲜羊膜上细胞生长面积最小。比较角膜缘组织块在冻存60 d去上皮羊膜和未去上皮羊膜上生长情况发现,从角膜缘组织块脱出的细胞生长面积在去上皮羊膜上显著大于未去上皮羊膜上。说明用组织块法和种传代细胞法均能构建组织工程化人工角膜上皮,细胞培养载体需用冻存30 d左右去上皮细胞的羊膜,为组织工程化人工角膜上皮的构建研究奠定理论基础。3.山羊角膜内皮细胞的体外分离培养与鉴定比较组织块法、胰酶消化法、dispase酶消化法和dispase酶+胰酶消化法培养山羊原代角膜内皮细胞。dispase酶+胰酶消化法为角膜内皮细胞原代分离纯化的最适方法,分离的细胞在体外能连续传20多代,细胞冻存后解冻培养能保持冻存前细胞形态和活力。培养的细胞经扫描电镜和透射电镜观察,具有正常角膜内皮细胞特征,免疫组化染色和RT-PCR检测角膜内皮细胞表达神经元标志NSE和β3-tublin、星形胶质细胞标志GFAP、细胞间连接蛋白ZO-1和Cx43,不表达神经干细胞标志物Nestin、角膜缘干细胞标志p63和角膜上皮细胞标志K3/K12。比较细胞培养板包被物对角膜内皮细胞贴壁和增殖能力影响发现,Ⅳ胶原、多聚赖氨酸和明胶均能明显促进细胞贴壁和增殖,Ⅳ胶原和明胶组强于多聚赖氨酸组,而Ⅳ胶原组和明胶组之间差异不显著,提示可选择价格便宜的明胶作为细胞培养板用包被物。角膜内皮细胞培养液筛选发现:与NBS相比,FBS能更好的促进角膜内皮细胞增殖和维持其形态;培养液中添加bFGF能显著促进细胞增殖,但能促进细胞形态向成纤维形转变;添加EGF对细胞增殖影响不大,但能促进细胞形态改变;添加硫酸软骨素显著抑制细胞增殖,但能较好的维持细胞形态;添加Vc对细胞增殖和形态均无明显影响。将传代培养的角膜内皮细胞种羊膜上培养,经组织切片、透射和扫描电镜观察发现细胞在羊膜上具有形成单层的能力。说明分离培养的角膜内皮细胞具有正常角膜内皮细胞特征,能体外传代培养,在羊膜载体上具有形成单层的能力,可为构建组织工程化人工角膜内皮和角膜内皮细胞的体外实验研究提供种子细胞。4.山羊角膜内皮细胞人端粒酶逆转录酶基因转染将人端粒酶逆转录酶基因(hTERT)转入培养至7代的角膜内皮细胞内,通过免疫组化和RT-PCR检测证明转染的hTERT在山羊角膜内皮细胞内表达。通过传代培养观察,转染hTERT的角膜内皮细胞体外能传20多代,与未转染hTERT的角膜内皮细胞相比,并未显著提高其增殖能力。说明将人端粒酶基因转入山羊角膜内皮细胞内,未能明显延长细胞寿命。更多还原

【Abstract】 All kinds of cornea diseases and traumas can result in corneal cells damaging. When the number of damaged-cells exceeds the threshold of corneal physiological compensation, pathologic changes will occur on the cornea and the sufferer can lose his sight eventually. Although corneal transplantation is regarded as an effective method curing the corneal diseases which induced the number of corneal cells by serious decreasing, the technology is not applied fully by some problems, such as the shortage of donors’cornea, the aging of donors and the syndrome after transplantation. Tissue engineering cornea which is regenerated with cultured cells from autograft or allograft in vitro can overcome the above-mentioned problems and is considered as the substitute of the cornea of dornors. It can be applied to the treatment of cornea diseases and the pharmacological or physiological research about cornea in vitro. To provide references for the rebuilding of tissue engineering artificial cornea, goat corneal epithelial cells and endothelial cells were isolated and cultured in vitro, the growth characteristic in culture dishes and on amniotic membrane were evaluated. We obtained the following results after carrying on the careful experiments.1. Isolation, culture and identification of goat corneal epithelial cells in vitroIn this experiment, the explant culture and enzyme digestion method that used to isolated goat corneal epithelial cells separately were assessed. The result shows the enzyme digestion method was more suitable for isolation and culture of limbal epithelial cells than explant culture. Isolated Limbal epithelial cells gained by enzyme digestion method appear polygonal shapes and better proliferation and these cells can proliferate for 20 passages in vitro. Significant difference of morphology and proliferating capacity between pre-cryopreserved corneal epithelial cells and the ones after resuscitation was not found. The results of Immunostaining and RT-PCR showed that the cultured cells expressed some phenotype-characteristics of corneal epithelial cells and limbal stem cell, for instance, K3/K12, p63, Cx43, ABCG2 and PCNA. Therefore these isolated cells were made up of limbal epithelial cells and limbal stem cells and could be employed to rebuild tissue engineering corneal epithelium. 2. Reconstruction of tissue engineering corneal epitheliumLimbal explants and cultured limbal epithelial cells of Guanzhong dairy goats were seeded on the human denuded amniotic membrane for reconstructing tissue engineering corneal epithelium, whose histological morphology, ultrastructure and immunocytochemiacal characteristic were observed. Results showed that limbal stem cell could proliferate on amniotic membrane and form 6～7 layers of stratified epithelium. The cells of stratified epithelium contained abundant organelles and glycongenosomes, and there were a lot of desmosomes and hemidemosome between cells or cells and amnioticmembrane. Immunostaining of paraffinic slice for p63 was positive. Comparing the growth performance of limbal epithelial cells from limbal explants seeded respectively on uncryo-preserved, cryo-preserved 30-day, cryo-preserved 90-day, cryo-preserved 180-day human amniotic membrane which had been removed of epithelium, we concluded that the cellular outgrowing area from limbal explants seeded on cryo-preserved 30-day human amniotic membrane was biggest, the cellular outgrowing area from limbal explants seeded on fresh human amniotic membrane was smallest. The growth performance of limbal epithelial cells seeded on cryo-preserved 60-day human amniotic membrane removed of epithelium and unremoved of epithelium was compared, the results showed that the cellular outgrowing area from limbal explants seeded on denuded amniotic membrane were significantly larger than that of intact amniotic membrane and implicated that both limbal explants and cultured limbal epithelial cells seeded on denuded human amniotic membrane can be used to reconstruct tissue engineering epithelium, the 30-day denuded amniotic membrane were most suitable to growth of limbal epithelial cells. The results provided evidences for studying tissue engineering artificial cornea.3. Isolation, Culture and Identification of Goat Corneal Endothelial CellsFour methods including explant culture, trypsin digestion, dispase digestion and dispase with trypsin digestion method were employed to isolate goat corneal endothelial cells (GCECs)in this experiment. The effectiveness of the methods to isolate GCECs was assessed. It was an optimum method to isolate GCECs in vitro using dispase with trypsin digestion. The obtained cells by the method could be cultured for about 20-passage. The resuscitated cryo-preserved GCECs performed the same morphology and proliferating capacity as that of cryo-preserved GCECs. Resorting to scan and transmission electron microscope, we saw that the morphology of isolated GCECs resembled that of GCECs in vivo. The cultured GCECs in vitro were measured by immunocytochemical staining and RT-PCR, of which results showed that the cells expressed NSE andβ3-tublin of neuronal marker, GFAP of astroglial cell marker, ZO-1 and Cx43 of cell connecting protein marker,but not expressed Nestin of neuronal marker, p63 of limbal stem cell marker, K3/K12 of corneal epithelial cell marker. The influence of different substances coating plastic ware,Ⅳcollagen, gelatin and poly-L-lysine, on the adherence and proliferation of GCECs was assessed in these experiments. It was found thatⅣcollagen , gelatin and poly-L-lysine greatly improved the adherence and proliferation of GCECs.Ⅳcollagen and gelatin could promote GCECs adherence and proliferation more remarkably than poly-L-lysine, but there was no distinct difference betweenⅣcollagen and gelatin. While gelatin is less expensive thanⅣcollagen and more appropriate for GCECs culture in vitro. Basing on the experiment about optimizing culture medium for culturing GCECs, we have obtained following conclusions. Both FBS and NBS can promote cell proliferation and maintain cellular morphology, but FBS was more appropriate. Although bFGF could also promote cell proliferation remarkably, it could also change the morphology of GCECs into fibroblast-like. EGF could not promote cell proliferation, while also influenced cellular morphology. Chondroitin sulfate could inhibit cell proliferation and maintain cellular morphology. Vitamin C could not influence cell proliferation and cellular morphology. By optical microscope, scan and transmission electron microscope, we could conclude that cultured GCECs in vitro were seeded on the human denuded amniotic membrane could form a monoptychial tissue on amniotic membrane. The result also implicated the isolated GCECs had the characteristics of native GCECs and could be cultured in vitro to form a monoptychial on amniotic membrane. The cells can be used for reconstrcting tissue engineering epithelium.4. Transfection of hTERT gene in GCECsThe human telomerase reverse transcriptase(hTERT) gene was transferred into GCECs. The results of immunocytochemical staining and RT-PCR showed that hTERT gene was expressed in GCECs, which could proliferate for 20 passages after being transfected by hTERT gene. But there was no significant difference of cellular proliferation between transfected cells and untransfected cells. The study implicated that the proliferation of GCECs could not be promoted by transfecting hTERT gene into GCECs.更多还原