仿生理状态组织工程角膜上皮、基质层的构建
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摘要
目的：(1)探讨C02激光打标机处理的有序排列微模板培养兔角膜基质细胞的生长特征及其细胞生物学变化。(2)探索角膜上皮、口腔黏膜上皮细胞在UpCellTM温敏型表面培养皿上的合适接种密度及生长特性,利用温敏特性获得无载体细胞片以及构建口腔黏膜上皮细胞片的意义。(3)探讨重组Maxadilan(MAX)对角膜基质微环境下培养的口腔黏膜上皮细胞的CK3表达的影响；不同浓度的MAX对角膜基质细胞的影响。
方法：(1)用CO2激光打标机刻制的聚苯乙烯有序排列微模板培养兔角膜基质细胞,模板沟槽划线间隔距离0.25mmm者为窄间隔组,间隔距离1mm者为宽间隔组,平板组作为对照组。将角膜基质细胞悬液1×105/ml细胞密度接种于培养板,倒置显微镜下每日观察细胞生长情况、HE染色观察细胞排列的形态学差异、免疫荧光法检测培养板上细胞的波形蛋白、实时监测显微镜下观察24h窄间隔组细胞的接触指引特性及细胞的动态生长过程。(2) UpCellTM温敏型表面培养皿及普通培养皿上接种不同密度的原代兔角膜上皮、口腔黏膜上皮细胞,置于37℃,5％CO2培养箱培养。通过倒置显微镜观察两种细胞在温敏培养皿上生长状况、形态学差异,同时寻求合适的接种密度；吖啶橙染色显示即将获取细胞片的活性；获取细胞片后石蜡切片HE染色以及扫描电镜观察细胞片的结构。(3)用RT-PCR检测PAC1受体在角膜基质细胞中的表达情况；Western-Blot检测PACl受体在角膜上皮细胞、角膜基质细胞及口腔粘膜上皮细胞中的表达检测；采用普通培养法及24h饥饿法分别添加不同浓度的MAX,培养兔角膜基质细胞,运用CCK8检测MAX对角膜基质细胞的促增殖作用；用免疫荧光定量Real-TimePCR检测以下4组细胞角蛋白CK3的表达差异：第一组,口腔黏膜上皮细胞+角膜基质细胞(通过插入式培养皿)。第二组,口腔黏膜上皮细胞+角膜基质细胞+100nM MAX。第三组,单纯口腔黏膜上皮细胞。第四组,单纯角膜上皮细胞。
结果：(1)培养第1天倒置显微镜下见角膜基质细胞贴壁生长,窄间隔组和宽间隔组细胞逐渐围绕沟槽接触指引生长,表现为有序排列,且窄间隔组较宽间隔组明显。HE染色与免疫荧光染色显示培养细胞在窄间隔组沟槽的接触指引下呈有序排列,呈现10余层细胞排列的平行板层结构。3组细胞波形蛋白免疫荧光染色均呈阳性反应。实时监测显微镜下可见窄宽间隔组细胞体部首先贴壁,在接触指引下生长,而细胞在干燥环境下的凋亡始于细胞伪足突起处。(2)不同密度接种于UpCellTM温敏型表面培养皿及普通培养皿中的原代兔角膜上皮、口腔黏膜上皮细胞,早期没有表现出形态学上的差别,且在温敏型表面培养皿及普通培养皿中生长状况一致。随时间延长5×104/ml密度细胞生长缓慢,2×105/ml密度的细胞容易出现凋亡态,1×105/ml密度接种细胞出现铺路石状细胞,有较多细胞克隆中心形成,细胞呈复层,8天后生长状态最佳。20℃-25℃低温培养后见培养皿的局部有细胞片脱离温敏材料的表面,呈卷曲状,随时间延长发现脱离的细胞片越来越大,石蜡切片HE染色细胞呈复层,扫描电镜观察细胞片细胞间交错连接明显,排列紧密。(3)RT-PCR证实角膜基质细胞上有PAC1受体的表达,Western-Blot证实角膜上皮细胞、角膜基质细胞及口腔黏膜上皮细胞上PAC1受体的表达阳性；CCK8细胞增殖实验显示MAX对角膜基质细胞有促进作用；Real-Time PCR证实MAX促进与角膜基质细胞共培养的口腔黏膜上皮细胞的CK3表达。
结论：(1)利用CO2激光打标机制备的有序排列微模板可获得有序排列角膜基质细胞,有利于在接近于生理状态条件下角膜基质层的构建。(2)UpCellTM温敏型表面培养皿获取无载体细胞片,符合构建生理状态的角膜上皮层的要求。(3)角膜上皮细胞、角膜基质细胞及口腔黏膜上皮细胞均表达]PAC1受体；PAC1受体激动剂Maxadilan对角膜基质细胞具有促增殖作用,并促进口腔黏膜上皮细胞CK3的表达,Maxadilan有利于组织工程角膜种子细胞的生长。
Objective:(1) In order to investigate the property of rabbit corneal keratocytes cultured in physiological status, keratocyte alignment cultured on microgrooved patterns of polystyrene plates and cellular biology were studied. (2) To explore the proper density and the cellular biological characteristics when corneal epithelial cells and oral mucosal epithelial cells were seeded on temperature-responsive Nunc UpCell Surface. The carrier-free cell sheets fabricated on the temperature-responsive culture surfaces. (3) To investigate the influence of recombinant maxadilan to keratin CK3 expression of oral mucosal epithelial cells when co-culturing keratocytes. The proliferation effects of different concentration maxadilan to keratocytes were also studied.
Methods:(1) The alignment patterns were designed as alternating grooves and ridges, including 0.25mm lineation space (narrow groove group), lmm lineation space (wide groove group), and no alignment pattern worked as control group. The suspension of rabbit P2 keratocytes was inoculated on the culture plates at a density of 1×105 cells/ml. Morphology of cultured cells was observed under the phase contrast microscope daily. The cellular growth and contact guidance property were investigated with HE staining and immunofluorescence staining of vimentin. Cells in narrow groove group was incubated at 37℃in a closed dry 5% CO2 environment and the processes of cellular adherence, contact guidance proliferation, and apoptosis of 24 hour time-lapse were observed under real-time microscope. (2) Different densities of primary corneal epithelial cells and oral mucosal epithelial cells were seeded on Nunc UpCell surface and tissue culture plate (TCP) surface of 6 well plates and incubated. Phase contrast microscope examination was performed daily to observe morphological differences in different cellular densities. Acridine orange (AO) staining was used to evaluated the cell viability. HE staining was carried to observe the paraffin longitudinal section of the cell sheets. SEM examination was carried out to observe the cell junction and the structure. (3) RT-PCR assay was used to detect the expression of PAC1 receptor in keratocytes. Western-Blot assay was used to detect the expression of PAC1 receptor in corneal epithelial cells, keratocytes and oral mucosa epithelial cells. The proliferation effects of keratocytes in 1nM～500nM maxadilan using DMEM medium with 0.3% FBS or 10% FBS were assessed by CCK-8 method. Real-Time PCR assay was established for detection of CK3 gene expression of four groups as bellow:rabbit mucosal epithelial cells co-culture with keratocytes through transwell culture (group one), group one +100nM recombinant maxadilan (group two), rabbit mucosal epithelial cells (group three) and corneal epithelial cells (group four) as control group.
Results:(1) Keratocytes in groove groups grew along the grooves, but cells in control group showed no alignment pattern. Histological evaluation of HE staining and immunofluorescence staining showed that keratocytes in groove groups had contact guidance feature and elongated on the surface with microgrooved patterns and aligned along that patterns, the cells of three groups presented the positive response for Vimentin. Comparison to the wide group, the cells in narrow group showed more obvious growing characteristics of alignment. The images of cells in narrow group under the real-time microscope confirmed that cells proliferated along the arranged grooves to abide by the contact guidance. The cellular adherence was firstly observed in cell body, but its apoptosis occurred firstly in cell foot processes. (2) There were not obvious discrimination in different densities of primary corneal epithelial cells and oral mucosal epithelial cells seeded on Nunc UpCell surface and TCP surface during the early stage. With the time prolongs, the cells of 5×104/ml density presented retardation, the cells of 2×105/ml density displayed apoptosis state, while the cells of 1×105/ml density were easy to form cobble-like morphology and colony formation. There were multilayer cells and cells reached confluence on day 8. Cell sheets were harvested by reducing the culture temperature to 20℃-25℃. HE staining showed that the cell sheets were carrier-free. SEM showed tight junction of the cells. (3)RT-PCR assay demonstrated the expression of PAC1 receptor in keratocytes. Western-Blot detected the expression of PAC1 receptor in corneal epithelial cells, keratocytes and oral mucosa epithelial cells at protein level. The CCK8 assay revealed that maxadilan promoted the proliferation of keratocytes. Real-Time PCR demonstrated that maxadilan promoted the expression of CK3 in oral mucosal epithelial cells when co-culture keratocytes.
Conclusions:(1) Keratocytes cultured on microgrooved surfaces have alignment pattern, especially in the pattern of 0.25mm lineation space. Keratocytes show contact guidance feature in alignment growth and close to the physiological status, which is suitable to the three-dimensional corneal stroma reconstruction in vitro. (2) Cell sheets obtained from Nunc UpCell surface show carrier-free without exogenous materials, which corresponds the requirements of reconstructing physiological condition of cornea cellular layers. (3) There is the expression of PAC1 receptor in corneal epithelial cells, keratocytes and oral mucosa epithelial cells. Maxadilan, an agonist of PAC1 receptor, can promote the proliferation of keratocytes and the expression of CK3 of oral mucosa epithelial cells. So Maxadilan is to the benefit of seeding cells of corneal tissue engineering.
方法：(1)用CO2激光打标机刻制的聚苯乙烯有序排列微模板培养兔角膜基质细胞,模板沟槽划线间隔距离0.25mmm者为窄间隔组,间隔距离1mm者为宽间隔组,平板组作为对照组。将角膜基质细胞悬液1×105/ml细胞密度接种于培养板,倒置显微镜下每日观察细胞生长情况、HE染色观察细胞排列的形态学差异、免疫荧光法检测培养板上细胞的波形蛋白、实时监测显微镜下观察24h窄间隔组细胞的接触指引特性及细胞的动态生长过程。(2) UpCellTM温敏型表面培养皿及普通培养皿上接种不同密度的原代兔角膜上皮、口腔黏膜上皮细胞,置于37℃,5％CO2培养箱培养。通过倒置显微镜观察两种细胞在温敏培养皿上生长状况、形态学差异,同时寻求合适的接种密度；吖啶橙染色显示即将获取细胞片的活性；获取细胞片后石蜡切片HE染色以及扫描电镜观察细胞片的结构。(3)用RT-PCR检测PAC1受体在角膜基质细胞中的表达情况；Western-Blot检测PACl受体在角膜上皮细胞、角膜基质细胞及口腔粘膜上皮细胞中的表达检测；采用普通培养法及24h饥饿法分别添加不同浓度的MAX,培养兔角膜基质细胞,运用CCK8检测MAX对角膜基质细胞的促增殖作用；用免疫荧光定量Real-TimePCR检测以下4组细胞角蛋白CK3的表达差异：第一组,口腔黏膜上皮细胞+角膜基质细胞(通过插入式培养皿)。第二组,口腔黏膜上皮细胞+角膜基质细胞+100nM MAX。第三组,单纯口腔黏膜上皮细胞。第四组,单纯角膜上皮细胞。
结果：(1)培养第1天倒置显微镜下见角膜基质细胞贴壁生长,窄间隔组和宽间隔组细胞逐渐围绕沟槽接触指引生长,表现为有序排列,且窄间隔组较宽间隔组明显。HE染色与免疫荧光染色显示培养细胞在窄间隔组沟槽的接触指引下呈有序排列,呈现10余层细胞排列的平行板层结构。3组细胞波形蛋白免疫荧光染色均呈阳性反应。实时监测显微镜下可见窄宽间隔组细胞体部首先贴壁,在接触指引下生长,而细胞在干燥环境下的凋亡始于细胞伪足突起处。(2)不同密度接种于UpCellTM温敏型表面培养皿及普通培养皿中的原代兔角膜上皮、口腔黏膜上皮细胞,早期没有表现出形态学上的差别,且在温敏型表面培养皿及普通培养皿中生长状况一致。随时间延长5×104/ml密度细胞生长缓慢,2×105/ml密度的细胞容易出现凋亡态,1×105/ml密度接种细胞出现铺路石状细胞,有较多细胞克隆中心形成,细胞呈复层,8天后生长状态最佳。20℃-25℃低温培养后见培养皿的局部有细胞片脱离温敏材料的表面,呈卷曲状,随时间延长发现脱离的细胞片越来越大,石蜡切片HE染色细胞呈复层,扫描电镜观察细胞片细胞间交错连接明显,排列紧密。(3)RT-PCR证实角膜基质细胞上有PAC1受体的表达,Western-Blot证实角膜上皮细胞、角膜基质细胞及口腔黏膜上皮细胞上PAC1受体的表达阳性；CCK8细胞增殖实验显示MAX对角膜基质细胞有促进作用；Real-Time PCR证实MAX促进与角膜基质细胞共培养的口腔黏膜上皮细胞的CK3表达。
结论：(1)利用CO2激光打标机制备的有序排列微模板可获得有序排列角膜基质细胞,有利于在接近于生理状态条件下角膜基质层的构建。(2)UpCellTM温敏型表面培养皿获取无载体细胞片,符合构建生理状态的角膜上皮层的要求。(3)角膜上皮细胞、角膜基质细胞及口腔黏膜上皮细胞均表达]PAC1受体；PAC1受体激动剂Maxadilan对角膜基质细胞具有促增殖作用,并促进口腔黏膜上皮细胞CK3的表达,Maxadilan有利于组织工程角膜种子细胞的生长。
Objective:(1) In order to investigate the property of rabbit corneal keratocytes cultured in physiological status, keratocyte alignment cultured on microgrooved patterns of polystyrene plates and cellular biology were studied. (2) To explore the proper density and the cellular biological characteristics when corneal epithelial cells and oral mucosal epithelial cells were seeded on temperature-responsive Nunc UpCell Surface. The carrier-free cell sheets fabricated on the temperature-responsive culture surfaces. (3) To investigate the influence of recombinant maxadilan to keratin CK3 expression of oral mucosal epithelial cells when co-culturing keratocytes. The proliferation effects of different concentration maxadilan to keratocytes were also studied.
Methods:(1) The alignment patterns were designed as alternating grooves and ridges, including 0.25mm lineation space (narrow groove group), lmm lineation space (wide groove group), and no alignment pattern worked as control group. The suspension of rabbit P2 keratocytes was inoculated on the culture plates at a density of 1×105 cells/ml. Morphology of cultured cells was observed under the phase contrast microscope daily. The cellular growth and contact guidance property were investigated with HE staining and immunofluorescence staining of vimentin. Cells in narrow groove group was incubated at 37℃in a closed dry 5% CO2 environment and the processes of cellular adherence, contact guidance proliferation, and apoptosis of 24 hour time-lapse were observed under real-time microscope. (2) Different densities of primary corneal epithelial cells and oral mucosal epithelial cells were seeded on Nunc UpCell surface and tissue culture plate (TCP) surface of 6 well plates and incubated. Phase contrast microscope examination was performed daily to observe morphological differences in different cellular densities. Acridine orange (AO) staining was used to evaluated the cell viability. HE staining was carried to observe the paraffin longitudinal section of the cell sheets. SEM examination was carried out to observe the cell junction and the structure. (3) RT-PCR assay was used to detect the expression of PAC1 receptor in keratocytes. Western-Blot assay was used to detect the expression of PAC1 receptor in corneal epithelial cells, keratocytes and oral mucosa epithelial cells. The proliferation effects of keratocytes in 1nM～500nM maxadilan using DMEM medium with 0.3% FBS or 10% FBS were assessed by CCK-8 method. Real-Time PCR assay was established for detection of CK3 gene expression of four groups as bellow:rabbit mucosal epithelial cells co-culture with keratocytes through transwell culture (group one), group one +100nM recombinant maxadilan (group two), rabbit mucosal epithelial cells (group three) and corneal epithelial cells (group four) as control group.
Results:(1) Keratocytes in groove groups grew along the grooves, but cells in control group showed no alignment pattern. Histological evaluation of HE staining and immunofluorescence staining showed that keratocytes in groove groups had contact guidance feature and elongated on the surface with microgrooved patterns and aligned along that patterns, the cells of three groups presented the positive response for Vimentin. Comparison to the wide group, the cells in narrow group showed more obvious growing characteristics of alignment. The images of cells in narrow group under the real-time microscope confirmed that cells proliferated along the arranged grooves to abide by the contact guidance. The cellular adherence was firstly observed in cell body, but its apoptosis occurred firstly in cell foot processes. (2) There were not obvious discrimination in different densities of primary corneal epithelial cells and oral mucosal epithelial cells seeded on Nunc UpCell surface and TCP surface during the early stage. With the time prolongs, the cells of 5×104/ml density presented retardation, the cells of 2×105/ml density displayed apoptosis state, while the cells of 1×105/ml density were easy to form cobble-like morphology and colony formation. There were multilayer cells and cells reached confluence on day 8. Cell sheets were harvested by reducing the culture temperature to 20℃-25℃. HE staining showed that the cell sheets were carrier-free. SEM showed tight junction of the cells. (3)RT-PCR assay demonstrated the expression of PAC1 receptor in keratocytes. Western-Blot detected the expression of PAC1 receptor in corneal epithelial cells, keratocytes and oral mucosa epithelial cells at protein level. The CCK8 assay revealed that maxadilan promoted the proliferation of keratocytes. Real-Time PCR demonstrated that maxadilan promoted the expression of CK3 in oral mucosal epithelial cells when co-culture keratocytes.
Conclusions:(1) Keratocytes cultured on microgrooved surfaces have alignment pattern, especially in the pattern of 0.25mm lineation space. Keratocytes show contact guidance feature in alignment growth and close to the physiological status, which is suitable to the three-dimensional corneal stroma reconstruction in vitro. (2) Cell sheets obtained from Nunc UpCell surface show carrier-free without exogenous materials, which corresponds the requirements of reconstructing physiological condition of cornea cellular layers. (3) There is the expression of PAC1 receptor in corneal epithelial cells, keratocytes and oral mucosa epithelial cells. Maxadilan, an agonist of PAC1 receptor, can promote the proliferation of keratocytes and the expression of CK3 of oral mucosa epithelial cells. So Maxadilan is to the benefit of seeding cells of corneal tissue engineering.
引文
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