本文選題：凍存角膜緣干細(xì)胞 + 無飼養(yǎng)層　； 參考：《西北農(nóng)林科技大學(xué)》2008年博士論文

【摘要】： 角膜上皮完整性的維持依賴于表淺細(xì)胞不斷脫落和基底層細(xì)胞不斷增殖來完成,上皮細(xì)胞持續(xù)不斷的水平向心運動和垂直向上運動源于角膜緣基底層的干細(xì)胞增殖和分化。因此,角膜緣干細(xì)胞對正常角膜生理功能的穩(wěn)定起重要作用。許多研究報道體外培養(yǎng)角膜緣干細(xì)胞構(gòu)建上皮植片,可以滿足臨床移植的需要。但是這些報道中,多采用3T3細(xì)胞做飼養(yǎng)層促進(jìn)上皮細(xì)胞的增殖,由于3T3細(xì)胞是來自胎鼠的成纖維細(xì)胞,其在臨床應(yīng)用有將異種動物疾病傳播給人類的危險。另外,目前培養(yǎng)角膜緣干細(xì)胞用于重建角膜上皮的研究與臨床應(yīng)用,都是在培養(yǎng)結(jié)束后就直接應(yīng)用于臨床,需要患者的身體狀況與培養(yǎng)結(jié)束后細(xì)胞的最佳生長狀態(tài)相吻合,否則,就會導(dǎo)致整個操作和手術(shù)失敗。如果能夠?qū)⑴囵B(yǎng)好的角膜緣干細(xì)胞在體外保存一定時間,于不同時間根據(jù)患者的身體狀況,解凍細(xì)胞再繼續(xù)培養(yǎng),將極大地方便其臨床應(yīng)用與研究。近年來成體干細(xì)胞可塑性的報道不斷涌現(xiàn),幾乎所有哺乳動物的成體干細(xì)胞都具有橫向分化潛能。但是角膜緣干細(xì)胞體外誘導(dǎo)分化少見報道。 本研究從山羊角膜緣干細(xì)胞分離、培養(yǎng)入手,系統(tǒng)比較了原代角膜緣干細(xì)胞的分離方法,優(yōu)化了角膜緣干細(xì)胞有血清培養(yǎng)體系,建立了角膜緣干細(xì)胞無血清培養(yǎng)體系。對分離富集的角膜緣干細(xì)胞體外連續(xù)傳代后,液氮中長期凍存。解凍后,對其干細(xì)胞相關(guān)特性進(jìn)行了研究。本研究以人羊膜為載體負(fù)載凍存角膜緣干細(xì)胞,在無飼養(yǎng)層無血清的培養(yǎng)體系中構(gòu)建組織工程化角膜上皮,手術(shù)移植給角膜緣干細(xì)胞缺損模型羊眼表,結(jié)合藥物使用抑制免疫排斥反應(yīng),最后對其移植治療效果進(jìn)行綜合評價。實驗研究主要內(nèi)容包括: 1山羊角膜緣干細(xì)胞的分離培養(yǎng)與鑒定研究 (1)比較酶消化法與組織塊培養(yǎng)法分離山羊角膜緣干細(xì)胞上的效率,認(rèn)為酶消化法在分離所得細(xì)胞中干細(xì)胞的比例,分離效率,分離細(xì)胞增殖活性,以及分離細(xì)胞純度上均優(yōu)于組織塊培養(yǎng)法。 (2)篩選不同濃度的IV膠原,以及不同粘附時間,對角膜緣干細(xì)胞分離的影響。結(jié)果表明:IV膠原最佳粘附濃度為20μg/mL,最佳粘附時間為20min。 (3)篩選出EGF和Insulin對角膜緣干細(xì)胞增殖影響的最佳作用濃度分別為20ng/mL和10μg/mL,優(yōu)化了角膜緣干細(xì)胞有血清培養(yǎng)體系,建立了角膜緣干細(xì)胞無血清無飼養(yǎng)層培養(yǎng)體系,此無血清培養(yǎng)體系已申請國家專利,專利申請?zhí)?200710018160.9。 (4)角膜緣干細(xì)胞最高傳至28代,液氮中共凍存角膜緣干細(xì)胞6×107,解凍細(xì)胞仍然保持了角膜緣干細(xì)胞的相關(guān)生物學(xué)特性。研究中所建立的凍存體系可以滿足不同的實驗條件下,對角膜緣干細(xì)胞的運輸、保存和增殖的要求。 2山羊角膜緣干細(xì)胞體外定向誘導(dǎo)分化研究 (1)解凍擴(kuò)增角膜緣干細(xì)胞經(jīng)1m mol/Lβ-Me預(yù)誘導(dǎo)24h后,再用5m mol/Lβ-Me正式誘導(dǎo)18h,改用無血清培養(yǎng)基培養(yǎng)7d,角膜緣干細(xì)胞分化為神經(jīng)樣細(xì)胞,表達(dá)NSE神經(jīng)細(xì)胞標(biāo)志。 (2)解凍擴(kuò)增角膜緣干細(xì)胞經(jīng)10 u mol 5-氮胞苷誘導(dǎo)48h,用含心肌條件培養(yǎng)基的培養(yǎng)液體外連續(xù)培養(yǎng)25d,角膜緣干細(xì)胞逐漸聚集生長,最終分化為心肌樣細(xì)胞,表達(dá)α-actin心肌細(xì)胞標(biāo)志蛋白。 (3)解凍擴(kuò)增角膜緣干細(xì)胞經(jīng)50μg/mL抗壞血酸、10m mol/Lβ-磷酸甘油和0.1u mol/L地塞米松聯(lián)合誘導(dǎo)7d后,部分細(xì)胞死亡,部分細(xì)胞呈三角形或多角形聚集生長,連續(xù)誘導(dǎo)21d后,誘導(dǎo)細(xì)胞形成細(xì)胞結(jié)節(jié),茜素紅染色陽性,呈成骨樣細(xì)胞。 (4)解凍擴(kuò)增角膜緣干細(xì)胞經(jīng)2m mol/L谷氨酰胺、0.01%大豆胰酶抑制劑、10m mol/L煙堿、5ng/ml肝細(xì)胞生長因子(HGF)聯(lián)合誘導(dǎo),5d后,細(xì)胞胞體逐漸變大,繼續(xù)誘導(dǎo),細(xì)胞開始聚集成團(tuán),連續(xù)誘導(dǎo)21d后,誘導(dǎo)形成的細(xì)胞團(tuán)Insulin抗體染色呈陽性,呈胰島樣細(xì)胞。 3山羊組織工程角膜上皮植片的構(gòu)建移植與檢測研究 (1)以凍存角膜緣干細(xì)胞為種子細(xì)胞,用無血清無飼養(yǎng)層培養(yǎng)體系,以去上皮羊膜為載體,體外培養(yǎng)12-14天,成功構(gòu)建具有與正常角膜相似結(jié)構(gòu)的組織工程角膜上皮。 (2)凍存角膜緣干細(xì)胞組織工程化角膜上皮移植可重建角膜緣干細(xì)胞完全缺失失明病理模型羊眼表。跟蹤觀察3個月,實驗組100%(15/15)形成角膜型上皮(熒光素不著色);跟蹤觀察6個月,實驗組20%(3/15)角膜上皮基本透明,80%(12/15)部分角膜開始透明;有6只實驗組山羊,跟蹤觀察12個月,33.3%(2/6)成功修復(fù)受損角膜,角膜上皮完全透明,66.7%(4/6)部分修復(fù),視力得到一定恢復(fù)。而角膜緣干細(xì)胞缺失未進(jìn)行角膜上皮移植的對照組山羊,全部角膜結(jié)膜化失明。眼罩蒙蔽正常眼,對另一側(cè)試驗眼進(jìn)行功能性檢測,對試驗羊進(jìn)行驅(qū)趕,角膜完全修復(fù)組山羊能辨別方向正確躲避;病理模型組山羊完全無方向感,不能正確躲避甚至出現(xiàn)撞墻現(xiàn)象。 (3)提出了異體移植的角膜緣干細(xì)胞修復(fù)角膜緣干細(xì)胞完全缺失病理模型的機(jī)理可能是:外源移植的異體干細(xì)胞抑制了自身周圍結(jié)膜以及血管向中央角膜上皮的生長,協(xié)同機(jī)體其他來源的前體細(xì)胞共同參與完成了角膜上皮的修復(fù)過程,實現(xiàn)角膜上皮重建。 (4)系統(tǒng)評價了組織工程化人工角膜上皮移植效果,首次通過檢測SRY基因的辦法,動態(tài)監(jiān)測了供體細(xì)胞在受體動物機(jī)體中是否長期存在。并且采用酶標(biāo)儀測定了角膜上皮透光度,此方法操作簡單,結(jié)果真實可靠,可以在實驗動物中很好地評價角膜上皮透光度。
[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.

【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R329

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