诱导人羊膜上皮细胞分化用于结膜重建的实验研究
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摘要
目的:
探讨人羊膜上皮细胞作为新的种子细胞,在兔去上皮结膜基质和结膜匀浆培养液下定向诱导分化为结膜上皮,并进一步研究分化细胞的生物学特性及构建组织工程结膜用于眼表重建的可能性。
方法:
1.人羊膜上皮细胞在去上皮结膜基质和结膜匀浆培养液诱导环境下培养,免疫组化鉴定角蛋白CK4、CK8、CK13、CK18及MUC5AC,PAS染色;ELISA法检测不同时间培养液中MUC5AC浓度;RT-PCR检测分化细胞MUC5AC基因表达。
2.运用MTT、群体倍增时间、量子点标记示踪等方法,探讨分化细胞的生物学特性。
3.以脱细胞羊膜作为载体,量子点标记的分化细胞接种于载体上,借助transwell培养皿进行气-液界面培养,构建组织工程结膜;分别于培养7天和14天取出,苏木素-伊红(HE)染色、透射电镜观察细胞复层情况;共聚焦激光扫描显微镜观察量子点标记的分化细胞生长状态;培养14天后行免疫组化鉴定角蛋白CK4、CK8、CK13及MUC5AC。
4.量子点标记的组织工程结膜移植于结膜缺损模型兔眼表,对侧眼新鲜羊膜作对照,观察植片的存活情况;14天后取材,行HE染色、PAS染色、透射电镜、免疫组化及共聚焦激光扫描显微镜检查。
结果:
1.人羊膜上皮细胞经诱导培养后,免疫组化结果显示角蛋白CK4、CK8、CK13、CK18及MUC5AC表达阳性,PAS染色见紫红色阳性细胞;ELISA检测1-7天内培养液中均有微量MUC5AC且逐渐升高;RT-PCR检测分化细胞MUC5AC基因表达阳性。
2.在培养1-5天内,MTT法显示分化细胞活性在1-4天内呈上升趋势,第5天开始下降;1-5天内细胞生长曲线及群体倍增时间均显示分化细胞增殖能力强;量子点孵育分化细胞2h后,成功标记于细胞,在随后7天内,标记细胞荧光强度无明显衰减,细胞形态正常,呈现良好的生长分裂状态。
3.量子点标记的分化细胞接种于脱细胞羊膜,气-液界面培养7天后,HE染色和透射电镜显示2-3层细胞,培养14天,细胞增至4-5层,分化细胞与脱细胞羊膜连接紧密,成功构建类似正常结膜结构的组织工程结膜;共聚焦激光扫描显微镜显示14天内标记细胞荧光强度不衰减,细胞形态规则,整齐;免疫组化显示分化细胞表型未发生改变。
4.组织工程结膜和新鲜羊膜移植术后2周内,双组眼表临床观察无明显区别,植片在位、完整;2周后取材,HE染色显示植片有2-5层细胞,PAS染色见散在紫红色阳性细胞,细胞角蛋白CK4、CK8、CK13及MUC5AC表达阳性,量子点标记的细胞排列整齐、形态规则。
结论:
1.在兔结膜匀浆液和去细胞基质诱导条件下,人羊膜上皮细胞可分化为结膜上皮样细胞和杯状细胞,而且具有合成和分泌MUC5AC功能。证实羊膜上皮细胞具有干细胞的分化潜能。
2.分化细胞具有良好的生物学特性。本实验首次应用量子点来标记示踪结膜上皮,量子点是一种理想细胞示踪标记物,它适合长期实时观察活体细胞。
3.脱细胞羊膜是较好的组织工程化载体;分化细胞接种于此载体上,借助transwell培养皿,成功构建与正常结膜结构相似的组织工程结膜;在构建过程中,分化细胞生理状态良好,细胞表型未发生改变。
4.实验观察期间,组织工程结膜与模型兔眼具有良好的组织相容性,重建模型兔完整眼表,预示工程化结膜在根治许多难治性眼表疾病方面提供一新途径。
Objective:
To trans-differentiate to conjunctival epithelium from human amniotic epithelial cells as a new kind of seed cells under the condition of denuded conjunctival matrix and conjunctival homogenate,then to study the biological characteristics of differentiated cells and the tissue engineering conjunctival for ocular surface reconstruction.
Methods:
(1) Human amniotic epithelial cells were cultured under the inducing conditions of denuded conjunctival matrix and conjunctival homogenate,immunohistochemistry with cytokeratin4、cytokeratin8、cytokeratin13、cytokeratin18、muc5ac and PAS staining were performed respctively;the concentrations of muc5ac in different time were measured by ELISA;the gene expressions of intracellular muc5ac were tested by RT-PCR.
(2) Biological characteristics of differentiated cells was been studied by MTT, population doubling time and trace labelling with quantum dots.
(3) The differentiated cells with quantum dots were shifted onto denuded amniotic membrance used as a carrier and were cultured with air-lifting cultivation by transwell culture plate to contruct tissue engineering conjunctival.It was removed in 7~(th) day and 14~(th) day respectively,differentiated cells were indentified to whether compound layer by HE staining and transmission electron microscope;the growth state of differentiated cells with quantum dots were frequently studied by confocal laser scanning microscopy;cytokeratin4, cytokeratin8,cytokeratin13 and muc5ac were performed by immunohistochemistry in 14~(th) day.
(4) Tissue engineering conjunctival labelling with quantum dots was transplanted to rabbit model with conjunctival defect and fresh amniotic membrance as the control in the opposite eye.the graft were observed by slit lamp after surgery;tissue engineering conjunctival and fresh amniotic membrance were removed respectively in14~(th) day after surgery,HE staining,PAS staining,transmission electron microscope,immunohistochemistry and confocal laser scanning microscopy were performed.
Results:
(1) After induction of human amniotic epithelial cells,the expression in immunohistochemistry was positive for cytokeratin4,cytokeratin8,cytokeratin13, cytokeratin18 and muc5ac,the positive cells of purplish red color were detected in PAS staining,trace amount of mu5ac in culture medium was measured by ELISA and it increased gradually in 1-7days;gene expression of muc5ac in differentiated cells was positive by RT-PCR.
(2) MTT showed an increasing trend of differentiated cells activities in 1-4days and then decreased in 5~(th) day during1-5days;cell growth curve and population doubling time from 1~(th) day to 5~(th) day simultaneously indicated differentiated cells had favorable proliferation abilities;quantum dots successfully labelled differentiated cells after 2 hour culture,the decay of fluorescence in cells wasn't detected,cellular morphology was normal and showed a good state of growth during following 7 days.
(3) The differentiated cells with quantum dots inoculated onto denuded amniotic membrance and exposed to an air-liquid interface(air-lifted),2-3 layers of cells in 7~(th) day and 4-5 layers in 14~(th) day were detected by HE staining and transmission electron microscope,differentiated cells and denuded amniotic membrance connected tightly,which indicated it successfully established tissue engineering conjunctival;no decay of fluorescence in cells and regular cell morphology was observed by confocal laser scanning microscopy;immunohistochemistry indicated the cellular phenotype didn't change.
(4) Clinical manifestations in ocular surface both the control with fresh amniotic membrance and the experimential with tissue engineering conjunctival hadn't existed obvious difference during the postoperative 2 weeks,conjunctival grafts survived and were integrated;2 weeks after surgery,2-5 layers of cells were observed by HE staining, some positive cells of purplish red color were detected in PAS staining, immunohistochemistry showed positive for cytokeratin4,cytokeratin8,cytokeratin13 and muc5ac,orderly arrangements and regular morphologies of cells with quantum dots were found.
Conclusions:
(1) Human amniotic epithelial cells directionally differentiated to conjunctival epithelium-like cells and globet cells which synthesized and secreted muc5ac under induction of denuded conjunctival matrix and conjunctival homogenate,which proved amniotic epithelial cells possessed multi-differentiation potentials of stem cells.
(2) The differentiated cells had good biological characteristics,quantum dots were firstly used to label and trace conjunctival epithelium,which verified quantum dots was perfect as a trace marker and it fited long-term observations of living cells.
(3) Denuded amniotic membrance was a ideal carrier of tissue engineering; differentiated cells inoculated onto this carrier and it successfully constructed tissue engineering conjunctival which had similar organization structures of normal conjunctival; differentiated cells maintained a favorable physiological state and cellular phenotype didn't change in the process of construction.
(4) Tissue engineering conjunctival and rabbit model had a superior biocompatibility during experimental observation,transplantation rebuilt ocular surface integrity of rabbit model.it foretelled tissue engineering conjunctival should provide a alternative approach to cure many severe ocular surface disease.
探讨人羊膜上皮细胞作为新的种子细胞,在兔去上皮结膜基质和结膜匀浆培养液下定向诱导分化为结膜上皮,并进一步研究分化细胞的生物学特性及构建组织工程结膜用于眼表重建的可能性。
方法:
1.人羊膜上皮细胞在去上皮结膜基质和结膜匀浆培养液诱导环境下培养,免疫组化鉴定角蛋白CK4、CK8、CK13、CK18及MUC5AC,PAS染色;ELISA法检测不同时间培养液中MUC5AC浓度;RT-PCR检测分化细胞MUC5AC基因表达。
2.运用MTT、群体倍增时间、量子点标记示踪等方法,探讨分化细胞的生物学特性。
3.以脱细胞羊膜作为载体,量子点标记的分化细胞接种于载体上,借助transwell培养皿进行气-液界面培养,构建组织工程结膜;分别于培养7天和14天取出,苏木素-伊红(HE)染色、透射电镜观察细胞复层情况;共聚焦激光扫描显微镜观察量子点标记的分化细胞生长状态;培养14天后行免疫组化鉴定角蛋白CK4、CK8、CK13及MUC5AC。
4.量子点标记的组织工程结膜移植于结膜缺损模型兔眼表,对侧眼新鲜羊膜作对照,观察植片的存活情况;14天后取材,行HE染色、PAS染色、透射电镜、免疫组化及共聚焦激光扫描显微镜检查。
结果:
1.人羊膜上皮细胞经诱导培养后,免疫组化结果显示角蛋白CK4、CK8、CK13、CK18及MUC5AC表达阳性,PAS染色见紫红色阳性细胞;ELISA检测1-7天内培养液中均有微量MUC5AC且逐渐升高;RT-PCR检测分化细胞MUC5AC基因表达阳性。
2.在培养1-5天内,MTT法显示分化细胞活性在1-4天内呈上升趋势,第5天开始下降;1-5天内细胞生长曲线及群体倍增时间均显示分化细胞增殖能力强;量子点孵育分化细胞2h后,成功标记于细胞,在随后7天内,标记细胞荧光强度无明显衰减,细胞形态正常,呈现良好的生长分裂状态。
3.量子点标记的分化细胞接种于脱细胞羊膜,气-液界面培养7天后,HE染色和透射电镜显示2-3层细胞,培养14天,细胞增至4-5层,分化细胞与脱细胞羊膜连接紧密,成功构建类似正常结膜结构的组织工程结膜;共聚焦激光扫描显微镜显示14天内标记细胞荧光强度不衰减,细胞形态规则,整齐;免疫组化显示分化细胞表型未发生改变。
4.组织工程结膜和新鲜羊膜移植术后2周内,双组眼表临床观察无明显区别,植片在位、完整;2周后取材,HE染色显示植片有2-5层细胞,PAS染色见散在紫红色阳性细胞,细胞角蛋白CK4、CK8、CK13及MUC5AC表达阳性,量子点标记的细胞排列整齐、形态规则。
结论:
1.在兔结膜匀浆液和去细胞基质诱导条件下,人羊膜上皮细胞可分化为结膜上皮样细胞和杯状细胞,而且具有合成和分泌MUC5AC功能。证实羊膜上皮细胞具有干细胞的分化潜能。
2.分化细胞具有良好的生物学特性。本实验首次应用量子点来标记示踪结膜上皮,量子点是一种理想细胞示踪标记物,它适合长期实时观察活体细胞。
3.脱细胞羊膜是较好的组织工程化载体;分化细胞接种于此载体上,借助transwell培养皿,成功构建与正常结膜结构相似的组织工程结膜;在构建过程中,分化细胞生理状态良好,细胞表型未发生改变。
4.实验观察期间,组织工程结膜与模型兔眼具有良好的组织相容性,重建模型兔完整眼表,预示工程化结膜在根治许多难治性眼表疾病方面提供一新途径。
Objective:
To trans-differentiate to conjunctival epithelium from human amniotic epithelial cells as a new kind of seed cells under the condition of denuded conjunctival matrix and conjunctival homogenate,then to study the biological characteristics of differentiated cells and the tissue engineering conjunctival for ocular surface reconstruction.
Methods:
(1) Human amniotic epithelial cells were cultured under the inducing conditions of denuded conjunctival matrix and conjunctival homogenate,immunohistochemistry with cytokeratin4、cytokeratin8、cytokeratin13、cytokeratin18、muc5ac and PAS staining were performed respctively;the concentrations of muc5ac in different time were measured by ELISA;the gene expressions of intracellular muc5ac were tested by RT-PCR.
(2) Biological characteristics of differentiated cells was been studied by MTT, population doubling time and trace labelling with quantum dots.
(3) The differentiated cells with quantum dots were shifted onto denuded amniotic membrance used as a carrier and were cultured with air-lifting cultivation by transwell culture plate to contruct tissue engineering conjunctival.It was removed in 7~(th) day and 14~(th) day respectively,differentiated cells were indentified to whether compound layer by HE staining and transmission electron microscope;the growth state of differentiated cells with quantum dots were frequently studied by confocal laser scanning microscopy;cytokeratin4, cytokeratin8,cytokeratin13 and muc5ac were performed by immunohistochemistry in 14~(th) day.
(4) Tissue engineering conjunctival labelling with quantum dots was transplanted to rabbit model with conjunctival defect and fresh amniotic membrance as the control in the opposite eye.the graft were observed by slit lamp after surgery;tissue engineering conjunctival and fresh amniotic membrance were removed respectively in14~(th) day after surgery,HE staining,PAS staining,transmission electron microscope,immunohistochemistry and confocal laser scanning microscopy were performed.
Results:
(1) After induction of human amniotic epithelial cells,the expression in immunohistochemistry was positive for cytokeratin4,cytokeratin8,cytokeratin13, cytokeratin18 and muc5ac,the positive cells of purplish red color were detected in PAS staining,trace amount of mu5ac in culture medium was measured by ELISA and it increased gradually in 1-7days;gene expression of muc5ac in differentiated cells was positive by RT-PCR.
(2) MTT showed an increasing trend of differentiated cells activities in 1-4days and then decreased in 5~(th) day during1-5days;cell growth curve and population doubling time from 1~(th) day to 5~(th) day simultaneously indicated differentiated cells had favorable proliferation abilities;quantum dots successfully labelled differentiated cells after 2 hour culture,the decay of fluorescence in cells wasn't detected,cellular morphology was normal and showed a good state of growth during following 7 days.
(3) The differentiated cells with quantum dots inoculated onto denuded amniotic membrance and exposed to an air-liquid interface(air-lifted),2-3 layers of cells in 7~(th) day and 4-5 layers in 14~(th) day were detected by HE staining and transmission electron microscope,differentiated cells and denuded amniotic membrance connected tightly,which indicated it successfully established tissue engineering conjunctival;no decay of fluorescence in cells and regular cell morphology was observed by confocal laser scanning microscopy;immunohistochemistry indicated the cellular phenotype didn't change.
(4) Clinical manifestations in ocular surface both the control with fresh amniotic membrance and the experimential with tissue engineering conjunctival hadn't existed obvious difference during the postoperative 2 weeks,conjunctival grafts survived and were integrated;2 weeks after surgery,2-5 layers of cells were observed by HE staining, some positive cells of purplish red color were detected in PAS staining, immunohistochemistry showed positive for cytokeratin4,cytokeratin8,cytokeratin13 and muc5ac,orderly arrangements and regular morphologies of cells with quantum dots were found.
Conclusions:
(1) Human amniotic epithelial cells directionally differentiated to conjunctival epithelium-like cells and globet cells which synthesized and secreted muc5ac under induction of denuded conjunctival matrix and conjunctival homogenate,which proved amniotic epithelial cells possessed multi-differentiation potentials of stem cells.
(2) The differentiated cells had good biological characteristics,quantum dots were firstly used to label and trace conjunctival epithelium,which verified quantum dots was perfect as a trace marker and it fited long-term observations of living cells.
(3) Denuded amniotic membrance was a ideal carrier of tissue engineering; differentiated cells inoculated onto this carrier and it successfully constructed tissue engineering conjunctival which had similar organization structures of normal conjunctival; differentiated cells maintained a favorable physiological state and cellular phenotype didn't change in the process of construction.
(4) Tissue engineering conjunctival and rabbit model had a superior biocompatibility during experimental observation,transplantation rebuilt ocular surface integrity of rabbit model.it foretelled tissue engineering conjunctival should provide a alternative approach to cure many severe ocular surface disease.
引文
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