冻存山羊角膜缘干细胞构造角膜上皮移植及检测研究
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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。
Stem cells (SC) of the corneal epithelium have been found in the limbal palisades of Vogt, between the conjunctiva and limbus. Complete loss of the corneal limbal epithelium leads to re-epithelialization by bulbar conjunctival cells, which is followed by neovascularisation, chronic inflammation, causing a pronounced decrease in visual acuity and severe discomfort. Transplantation of of limbal stem cells (LSCs) is necessary to restore vision and the corneal surface in these diseases.The isolation, in vitro cell culture, and transplantation of LSCs for clinical applications has been well documented, although there are still many unsolved problems. All cornea transplantations reported to date used freshly cultured cells, which were usually cultured on NIH 3T3 feeder cells, applied directly to cornea-damaged patients or experimental animals. A disadvantage of this approach is that it limits the clinical practice. To resolve this, we report the use of cryopreserved LSCs cultured in a feeder-free system to generate the applicable artificial corneal epithelium, and the allograft transplantation of these corneal membranes in LSCD models. We used a series of methods to determine the outcome of the ex vivo-expanded stem cell allograft for LSCD, to provide reference experiences and data for clinical application. Many reports of human adult stem cell plasticity have focused on hematopoietic, mesenchymal, skeletal muscle, and neural stem cells. In contrast, little is known about the plasticity of LSCs. In the present study, we investigated the plasticity of LSCs in vitro and in vivo to further demonstrate the potential therapeutic benefits of these cells. The details of the research include the following three points:
1. The isolation and identification of goat LSCs
(1) Comparing the methods of digestion and explant, we find the digestion was much better than explant in terms of ration of stem cell, efficiency, proliferation and purification.
(2) We selected the optimal the concentration of collagen IV and time of adherence, the results showed the optimal concentration is 20μg/mL, and the optimal adherence time is 20min.
(3)We selected the optimal concentration of EGF and Insulin on LSCs were 20ng/mL and 10μg/mL respectively. We also optimized the culture system including serum, and established the serum free and feeder free culture system of LSCs, which was applied for a patent and the applied number was 200710018160.9.
(4) The LSCs were subcultured 28 times in serum free system, and 6×107 LSCs were cryopreserved in liquid nitrogen. The studied the relative characteristics of thawed LSCs, the results showed that the stem cell characteristics were well maintained. The cryopreserved system set up in this research could satisfied with the needs of LSCs transportation, preservation and proliferation.
2. Differentiation properties of LSCs in vitro
(1)The thawed LSCs can be induced into neurons by theβ-mercaptoethanol(β-Me). Being treated with medium supplemented with 1 m mol/L pre-induced for 24h and then with 5 m mol/Lβ-Me formal induced for 18h, serum free medium cultured for 7d,LSCs differentiated towards neural cells which expressed the NSE marker of neural cells.
(2)The thawed LSCs would differentiated towards myocardium when exposed to 10μmol/L 5-Azacitidine for 24h,then cultured in myocardium conditional medium for 25d, the cells congregated together and became longer in shape which expressedα-actin myocardial protein.
(3)Differentiation of thawed LSCs towards the osteoblast lineage can be induced by supplementing with 0.1μmol/L dexamethasone,10m mol/Lβ-glycerophosphate and 50μg/mL ascorbic acid, the induced cells formed nodules after cultured 21 days, and the nodules were staining positive with Alizarin Red.
(4)The thawed LSCs were induced by 2m mol/L glutamine, 0.01% soybean trypsin inhibitor, 10m mol nicotin and 5ng/ml hepatocyte growth factor for 21d ,the cell were induced to pancreatic cells which staining positive with insulin antibody.
3. Construction, transplantation and assay of tissue engineered corneal epithelium
(1)Seed the thawed LSCs on denuded amniotic membrane, the cell membrane was cultured in serum free medium for 12-14 days, the constructed artificial epithelium with multiple layers of cells which had the similar structure as normal corneal epithelium.
(2)The artificial corneal epitheliums were transplanted into the limbal stem cells deficiency (LSCD) eyes. We surveiled the clinical outcomes, after 3 months, 100% (15/15) LSCD model transplanted with artificial epitheliums goat corneal type epithelium; after 6 months, 20% (3/15) LSCD models corneal epitheliums get transparency basically, and 80% (12/15) started to get transparency; We observed 6 transplanted goats for 12 months, 33.3%(2/6)reconstructed corneal epitheliums successfully which had transparent epitheliums and immune type as normal control, 66.7%(4/6)reconstructed partially. The negative control without transplantation corneal surface was entirely covered with conjunctiva and blood vessels, and the cornea lost vision completely.
(3)We supposed the mechanism of transplanted LSCs reconstructed the corneal epithelium as the following: the therapeutic effect of the transplantation may be associated with the inhibition of inflammation-related angiogenesis after transplantation of thawed LSCs, the allograft plays a key role to corporate with autologous progenitor cells to repair the damaged goat cornea.
(4)The effects of reconstruction were evaluated by a series methods such as clinical observation, histological and immunofluorescence examination, detected transparency of corneal epitheliums and the SRY gene of transplanted seed cells. We first used the Bio-Tek Instrument to evaluate the transparency of corneas, this is a feasible and objective method to evaluate the effect of reconstruction used for animal experiments.
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。
Stem cells (SC) of the corneal epithelium have been found in the limbal palisades of Vogt, between the conjunctiva and limbus. Complete loss of the corneal limbal epithelium leads to re-epithelialization by bulbar conjunctival cells, which is followed by neovascularisation, chronic inflammation, causing a pronounced decrease in visual acuity and severe discomfort. Transplantation of of limbal stem cells (LSCs) is necessary to restore vision and the corneal surface in these diseases.The isolation, in vitro cell culture, and transplantation of LSCs for clinical applications has been well documented, although there are still many unsolved problems. All cornea transplantations reported to date used freshly cultured cells, which were usually cultured on NIH 3T3 feeder cells, applied directly to cornea-damaged patients or experimental animals. A disadvantage of this approach is that it limits the clinical practice. To resolve this, we report the use of cryopreserved LSCs cultured in a feeder-free system to generate the applicable artificial corneal epithelium, and the allograft transplantation of these corneal membranes in LSCD models. We used a series of methods to determine the outcome of the ex vivo-expanded stem cell allograft for LSCD, to provide reference experiences and data for clinical application. Many reports of human adult stem cell plasticity have focused on hematopoietic, mesenchymal, skeletal muscle, and neural stem cells. In contrast, little is known about the plasticity of LSCs. In the present study, we investigated the plasticity of LSCs in vitro and in vivo to further demonstrate the potential therapeutic benefits of these cells. The details of the research include the following three points:
1. The isolation and identification of goat LSCs
(1) Comparing the methods of digestion and explant, we find the digestion was much better than explant in terms of ration of stem cell, efficiency, proliferation and purification.
(2) We selected the optimal the concentration of collagen IV and time of adherence, the results showed the optimal concentration is 20μg/mL, and the optimal adherence time is 20min.
(3)We selected the optimal concentration of EGF and Insulin on LSCs were 20ng/mL and 10μg/mL respectively. We also optimized the culture system including serum, and established the serum free and feeder free culture system of LSCs, which was applied for a patent and the applied number was 200710018160.9.
(4) The LSCs were subcultured 28 times in serum free system, and 6×107 LSCs were cryopreserved in liquid nitrogen. The studied the relative characteristics of thawed LSCs, the results showed that the stem cell characteristics were well maintained. The cryopreserved system set up in this research could satisfied with the needs of LSCs transportation, preservation and proliferation.
2. Differentiation properties of LSCs in vitro
(1)The thawed LSCs can be induced into neurons by theβ-mercaptoethanol(β-Me). Being treated with medium supplemented with 1 m mol/L pre-induced for 24h and then with 5 m mol/Lβ-Me formal induced for 18h, serum free medium cultured for 7d,LSCs differentiated towards neural cells which expressed the NSE marker of neural cells.
(2)The thawed LSCs would differentiated towards myocardium when exposed to 10μmol/L 5-Azacitidine for 24h,then cultured in myocardium conditional medium for 25d, the cells congregated together and became longer in shape which expressedα-actin myocardial protein.
(3)Differentiation of thawed LSCs towards the osteoblast lineage can be induced by supplementing with 0.1μmol/L dexamethasone,10m mol/Lβ-glycerophosphate and 50μg/mL ascorbic acid, the induced cells formed nodules after cultured 21 days, and the nodules were staining positive with Alizarin Red.
(4)The thawed LSCs were induced by 2m mol/L glutamine, 0.01% soybean trypsin inhibitor, 10m mol nicotin and 5ng/ml hepatocyte growth factor for 21d ,the cell were induced to pancreatic cells which staining positive with insulin antibody.
3. Construction, transplantation and assay of tissue engineered corneal epithelium
(1)Seed the thawed LSCs on denuded amniotic membrane, the cell membrane was cultured in serum free medium for 12-14 days, the constructed artificial epithelium with multiple layers of cells which had the similar structure as normal corneal epithelium.
(2)The artificial corneal epitheliums were transplanted into the limbal stem cells deficiency (LSCD) eyes. We surveiled the clinical outcomes, after 3 months, 100% (15/15) LSCD model transplanted with artificial epitheliums goat corneal type epithelium; after 6 months, 20% (3/15) LSCD models corneal epitheliums get transparency basically, and 80% (12/15) started to get transparency; We observed 6 transplanted goats for 12 months, 33.3%(2/6)reconstructed corneal epitheliums successfully which had transparent epitheliums and immune type as normal control, 66.7%(4/6)reconstructed partially. The negative control without transplantation corneal surface was entirely covered with conjunctiva and blood vessels, and the cornea lost vision completely.
(3)We supposed the mechanism of transplanted LSCs reconstructed the corneal epithelium as the following: the therapeutic effect of the transplantation may be associated with the inhibition of inflammation-related angiogenesis after transplantation of thawed LSCs, the allograft plays a key role to corporate with autologous progenitor cells to repair the damaged goat cornea.
(4)The effects of reconstruction were evaluated by a series methods such as clinical observation, histological and immunofluorescence examination, detected transparency of corneal epitheliums and the SRY gene of transplanted seed cells. We first used the Bio-Tek Instrument to evaluate the transparency of corneas, this is a feasible and objective method to evaluate the effect of reconstruction used for animal experiments.
引文
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