人角膜缘干细胞保存和结膜杯状细胞诱导分化的初步研究

发布时间：2018-05-14 03:32

本文选题：结膜 + 上皮　；参考：《济南大学》2011年硕士论文

【摘要】：目的体外分离培养人结膜杯状细胞,观察其形态结构并对其性质进行鉴定;观察正常人角膜缘组织与结膜上皮组织在羊膜上培养的区别;探讨γ-分泌酶抑制剂对体外培养的人结膜杯状细胞增殖及特异性基因表达的影响;观察羊膜为载体的重组结膜上皮与正常结膜上皮的区别。方法一、人结膜上皮细胞的体外培养及鉴定 1.人结膜上皮细胞的体外培养取眼库提供的正常人眼结膜组织,离体后6小时内采用组织块法培养原代人结膜上皮细胞。 2.体外培养的人结膜杯状细胞的鉴定采用阿利新蓝/过碘酸-希夫试剂染色(AB-PAS染色)培养的结膜上皮细胞;采用细胞免疫化学染色检测悬浮细胞中Muc5Ac的表达;采用细胞免疫荧光染色检测CK7的表达,鉴定分化的杯状细胞。二、正常人角膜缘组织与结膜上皮组织在羊膜上培养的区别取眼库提供的保存于角膜中期保存液的正常人角膜缘组织,用消化培养法培养于羊膜载体上;取眼库提供的正常人眼结膜组织,用组织块法培养于羊膜载体上;采用PAS染色观察比较二者的差别。三、γ-分泌酶抑制剂诱导人结膜杯状细胞分化及其相关检测 1.结膜杯状细胞的诱导分化原代培养的结膜上皮细胞汇合度达到30～40%时,分别加入含100nM、200nM、400nMγ-分泌酶抑制剂的培养液,处理72h,观察细胞形态变化。 2.诱导分化的结膜杯状细胞的性质检测原代培养的人结膜上皮细胞处理72h后,采用AB-PAS双染法染色,以比较相同时间、不同浓度γ-分泌酶抑制剂处理后杯状细胞密度的差异;分别提取相同时间、不同浓度γ-分泌酶抑制剂处理后人结膜上皮细胞的总RNA,反转成cDNA后行实时荧光定量PCR(RT-Q-PCR)检测,观察CK7的mRNA表达量在相同时间、不同浓度处理组结膜上皮细胞中的变化;提取阴性对照及200nMγ-分泌酶抑制剂处理72h后的人结膜上皮细胞的总蛋白,行Western-Blot检测CK7蛋白在人结膜上皮细胞中的表达情况。 3.统计学分析:对上述结果采用方差分析(Analysisi of Variance,ANOVA)检验进行统计学分析,比较不同浓度γ-分泌酶抑制剂处理组与阴性对照组的差异性,以P0.05视为差异具有统计学意义。四、羊膜为载体构建重组结膜上皮与正常结膜上皮的比较取眼库提供的正常人眼结膜组织,用组织块法培养于羊膜载体上,行PAS染色;对正常结膜上皮采用石蜡切片PAS染色,观察并比较二者的差别。结果一、培养的人结膜上皮细胞及鉴定 1.组织块培养法培养正常人结膜上皮细胞,次日补液后,12小时内可见细胞从组织块周围迁出,细胞贴壁生长,15天左右可长满培养皿,倒置显微镜下观察,可见较多悬浮状态的杯状细胞。 2.体外培养的人结膜杯状细胞的鉴定 (1)AB-PAS染色结果显示:胞浆呈蓝色及红色颗粒者为杯状细胞,可以初步鉴定体外培养的人结膜上皮细胞中含有杯状细胞。 (2)细胞免疫化学结果显示:原代培养的人结膜上皮细胞中的悬浮细胞,以小鼠单克隆Muc5Ac抗体进行染色,呈阳性反应者为杯状细胞。证明体外培养的人结膜上皮细胞中的悬浮细胞中含有杯状细胞。 (3)细胞免疫荧光结果显示:原代培养的人结膜上皮细胞,以小鼠单克隆CK7抗体进行染色,呈阳性反应者为杯状细胞。可以证明体外培养的人结膜上皮细胞中含有杯状细胞。二、正常人角膜缘组织与结膜上皮组织在羊膜上培养的区别 PAS染色结果显示:培养于羊膜上的角膜缘上皮细胞及结膜上皮细胞中均含有染色阳性的杯状细胞;角膜缘上皮细胞中的杯状细胞较结膜上皮细胞中密度低,且分化程度低。三、体外培养的人结膜杯状细胞经γ-分泌酶抑制剂诱导分化及相关检测 1.倒置显微镜下观察:加入γ-分泌酶抑制剂组杯状细胞密度较阴性对照组增高,其中200nM组增高最明显。 2. AB-PAS染色结果显示:加入γ-分泌酶抑制剂处理72h后的人结膜上皮细胞中的杯状细胞密度较阴性对照组高;其中200nM组中杯状细胞密度最高。 3. RT-Q-PCR结果显示:100nMγ-分泌酶抑制剂处理组与阴性对照组间CK7mRNA的表达量差异具有显著性。 4. Western-blot结果显示:结果显示CK7蛋白的表达量在200nM组较阴性对照组显著升高。四、羊膜为载体构建重组结膜上皮与正常结膜上皮的比较 PAS染色结果显示:羊膜为载体构建的重组结膜上皮细胞中杯状细胞的密度较正常结膜上皮中杯状细胞的密度低;后者PAS染色显示杯状细胞嵌入结膜上皮层中,前者PAS染色显示杯状细胞位于羊膜表层,易脱落。结论 1.人结膜杯状细胞可以在体外进行培养扩增,并具有分泌粘蛋白的能力。 2.角膜缘组织培养过程中会有少量的杯状细胞,但与正常的结膜相比,密度偏低,且分化程度较低。 3.γ-分泌酶抑制剂可以诱导结膜上皮细胞分化为杯状细胞,并能促进杯状细胞粘蛋白和特异性标志基因的表达。 4.羊膜为载体构建的重组结膜上皮膜片中,存在着杯状细胞密度低、位置与正常结膜上皮不同的问题,因此需要寻找新的载体或诱导分化方法构建重组结膜上皮。目的将新鲜角膜环置于角膜中期保存液保存不同时间,通过检测细胞总量、细胞活性、干细胞相关标志物、细胞增殖及分化的能力,分析角膜中期保存液对角膜缘干细胞的影响。方法 1.角膜环的预处理取回眼库提供的新鲜角膜环共6个,显微镜下撕除角膜内皮及虹膜组织,每个平均分为3份,分别置于角膜中期保存液中1d、4d、7d。采用消化培养法将角膜缘上皮消化为单细胞悬液。 2.不同保存时间的角膜缘上皮通过细胞计数仪检测细胞总量,计算平均细胞总量;及活细胞比例。将细胞接种于3T3饲养层上,培养观察及姬姆萨染色,计算细胞克隆形成能力。采用流式细胞技术对其中的干细胞进行检测;采用细胞免疫荧光技术,检测相关干细胞标志物的表达。将细胞接种于羊膜上,通过组织病理学HE染色检测细胞的复层形成能力。结果 1.细胞总量检测随着角膜缘干细胞在中期保存液中保存时间的延长,细胞总量减少。1d、4d、7d保存组的平均细胞总量分别为:100%、42.45%、0。 2.细胞活性检测随着角膜缘干细胞在中期保存液中保存时间的延长,细胞活性降低。1d、4d、7d保存组的平均细胞活性分别为:82%、64%、0。 3.克隆形成能力随着角膜缘干细胞在中期保存液中保存时间的延长,细胞克隆形成能力降低、CFE降低。 4.干细胞标志检测随着角膜缘干细胞在中期保存液中保存时间的延长,角膜缘干细胞标志物P63、ABCG2、CK3表达降低。1d保存组P63、ABCG2、CK3阳性表达,4d保存组P63、ABCG2、CK3表达量明显降低。 5.组织病理学检测随着角膜缘干细胞在中期保存液中保存时间的延长,细胞复层形成能力降低。1d保存组可见羊膜表面覆盖有2～3层的复层上皮细胞;4d保存组羊膜载体表面光滑,无细胞贴附及生长。结论 1.应用角膜中期保存液保存角膜缘组织短期内可以维持其中角膜缘干细胞的活性及功能,但是随着保存时间的延长,角膜缘干细胞的活性和功能均有不同程度的下降。 2.角膜缘干细胞的功能与其标志物表达之间无直接关系:病理检测P63的表达,不代表干细胞具有克隆形成或在羊膜上形成单层的能力。
[Abstract]:objective
The human conjunctival goblet cells were isolated and cultured in vitro, and the morphological structure was observed and their properties were observed. The difference between the corneal limbal tissue and the conjunctival epithelial tissue in the amniotic membrane was observed. The effects of gamma secretase inhibitor on the proliferation and specific gene expression of human conjunctival goblet cells in vitro were investigated. The difference between the recombinant conjunctival epithelium and the normal conjunctival epithelium.
Method
In vitro culture and identification of human conjunctival epithelial cells
In vitro culture of 1. conjunctival epithelial cells
The conjunctiva tissue was supplied from the eye bank, and the primary conjunctival epithelial cells were cultured within 6 hours in vitro.
Identification of 2. human conjunctival goblet cells in vitro
The conjunctival epithelial cells were cultured with alirea Blue / periodate Schiff reagent staining (AB-PAS staining), and the expression of Muc5Ac in the suspension cells was detected by cytochemical staining, and the expression of CK7 was detected by cell immunofluorescence staining, and the differentiated goblet cells were identified.
Two, the difference between normal limbal tissue and conjunctival epithelial tissue cultured on amniotic membrane.
The normal human corneal limbal tissue preserved in the medium cornea preservation solution was obtained from the eye bank and cultured on amniotic membrane by digestion culture. The normal human eye conjunctiva tissue provided by the eye bank was cultured on the amniotic membrane by tissue block method, and the difference between the two was observed by PAS staining.
Three, the secretion of human conjunctival goblet cells induced by gamma secretase inhibitor and its related detection.
Induced differentiation of 1. conjunctival goblet cells
When the conjunctival epithelial cell confluence of primary cultured conjunctival epithelial cells reached 30 ~ 40%, the culture medium containing 100nM, 200nM, 400nM gamma secretase inhibitor was added to treat 72h, and the morphological changes of cells were observed.
Detection of the properties of 2. induced conjunctival goblet cells
The primary cultured human conjunctival epithelial cells treated 72h with AB-PAS double staining to compare the difference in the density of goblet cells treated with different concentrations of gamma secretase inhibitors at the same time. The total RNA of the conjunctival epithelial cells after the treatment of different concentrations of gamma secretase inhibitors was obtained, and the real time fluorescence was reversed to cDNA after the reversal of cDNA. Quantitative PCR (RT-Q-PCR) detection was used to observe the changes in the mRNA expression of CK7 at the same time, in the conjunctival epithelial cells of different concentration treatment groups, and to extract the total protein of the conjunctival epithelial cells of the human conjunctival epithelial cells after the negative control and the 200nM gamma secretase inhibitor treated 72h. The expression of CK7 egg white in the human conjunctival epithelial cells was detected by Western-Blot.
3. statistical analysis: the above results were statistically analyzed by Analysisi of Variance (ANOVA) test, and the difference between the different concentration of gamma secretase inhibitor treatment group and the negative control group was compared. The difference between the P0.05 and the negative control group was statistically significant.
Four, the construction of recombinant conjunctival epithelium and normal conjunctival epithelium with amniotic membrane as carrier.
The normal human eye conjunctiva tissue provided by the eye bank was cultured on amniotic membrane by tissue block method and stained with PAS. The normal conjunctival epithelium was stained with paraffin section PAS to observe and compare the difference between the two.
Result
1. Cultured human conjunctival epithelial cells and identification
The normal human conjunctival epithelial cells were cultured in 1. tissue mass culture method. After the second day, the cells were found to move out from the tissue block in 12 hours, the cells were adhered to the wall, and the culture dish could be filled at about 15 days, and the cup like cells with more suspended state were observed under the inverted microscope.
Identification of 2. human conjunctival goblet cells in vitro
(1) the results of AB-PAS staining showed that the cytosolic blue and red granules were goblet cells, and it could be preliminarily identified in the cultured human conjunctival epithelial cells with goblet cells.
(2) the results of cell immunocytochemistry showed that the primary cultured human conjunctival epithelial cells were stained with murine monoclonal Muc5Ac antibody, and the positive reaction was goblet cells.
(3) the results of cell immunofluorescence showed that the primary cultured human conjunctival epithelial cells were stained with murine monoclonal CK7 antibody, and the positive reaction was goblet cells. It was proved that the cultured human conjunctival epithelial cells contained goblet cells.
Two, the difference between normal limbal tissue and conjunctival epithelial tissue cultured on amniotic membrane.
The results of PAS staining showed that the corneal limbal epithelial cells and conjunctival epithelial cells cultured on the amniotic membrane were all stained positive goblet cells, and the goblet cells in the limbal epithelial cells were lower in density than in conjunctival epithelial cells, and the degree of differentiation was low.
Three, induced differentiation of human conjunctival goblet cells by gamma secretase inhibitors and related detection.
1. under inverted microscope, the density of goblet cells in the group of gamma secretase inhibitor was higher than that in negative control group, and the increase in group 200nM was the most obvious.
The results of 2. AB-PAS staining showed that the density of goblet cells in human conjunctival epithelial cells after 72h was treated with gamma secretase inhibitor was higher than that in negative control group, and the density of goblet cells in group 200nM was the highest.
3. RT-Q-PCR results showed that there was significant difference in the expression of CK7mRNA between the 100nM gamma secretase inhibitor group and the negative control group.
4. Western-blot results showed that the expression of CK7 protein in 200nM group was significantly higher than that in negative control group.
Four, the construction of recombinant conjunctival epithelium and normal conjunctival epithelium with amniotic membrane as carrier.
The results of PAS staining showed that the density of goblet cells in the recombinant conjunctival epithelial cells constructed by amniotic membrane was lower than that in the normal conjunctival epithelial goblet cells, while the latter PAS staining showed that goblet cells were embedded in the conjunctival upper cortex, and the former PAS staining showed that goblet cells were located in the amniotic membrane layer and were easy to fall off.
conclusion
1. conjunctival goblet cells can be cultured and amplified in vitro and have the ability to secrete mucin.
2. there were a few goblet cells in the limbal tissue culture, but compared with the normal conjunctiva, the density was low and the degree of differentiation was low.
3. gamma secretase inhibitors can induce conjunctival epithelial cells to differentiate into goblet cells, and promote the expression of mucin and specific marker genes in goblet cells.
4. amniotic membrane as a carrier, there is a low density of goblet cells and a different location from normal conjunctival epithelium. Therefore, a new carrier or differentiation method is needed to construct a recombinant conjunctival epithelium.
objective
The effect of the medium cornea preservation solution on the limbal stem cells was analyzed by placing the fresh cornea ring in the medium preservation solution of the cornea for different time, and by detecting the total amount of cells, cell activity, stem cell related markers, cell proliferation and differentiation.
Method
1. preconditioning of the cornea ring
A total of 6 fresh corneal rings were taken back from the eye bank. The corneal endothelium and iris tissue were tore out under microscope, each was divided into 3 parts, which were placed in the medium cornea preservation solution 1D, 4D, and 7d. respectively. The limbal epithelium was digested as single cell suspension by digestion culture.
2. the corneal limbal epithelium with different preservation time detected the total cell volume by cell count instrument, calculated the average total cell total, and the proportion of living cells. The cells were inoculated on the 3T3 feeder layer, cultured and observed and Giemsa staining, and the cell clone formation ability was calculated. The stem cells were detected by flow cytometry, and cell immunofluorescence was used. The expression of related stem cell markers was detected by light technology. The cells were seeded on the amniotic membrane and the ability of cell formation was detected by histopathological HE staining.
Result
1. cell total amount detection
With the prolongation of the preservation time of limbal stem cells in the medium-term preservation solution, the total amount of cells decreased by.1d. The average cell volume of 4D and 7d preservation groups was 100%, 42.45%, 0. respectively.
Detection of 2. cell activity
With the prolongation of the preservation time of limbal stem cells in the medium-term preservation solution, cell viability decreased by.1d, and the average cell viability of 4D and 7d preservation groups was 82%, 64%, 0. respectively.
3. clone formation ability
With the prolongation of the preservation time of limbal stem cells in the medium-term preservation solution, the colony forming ability of cells decreased and CFE decreased.
Detection of 4. stem cell markers
With the prolongation of the preservation time of limbal stem cells in the medium-term preservation solution, the expression of P63, ABCG2, and CK3 of the limbal stem cells decreased the positive expression of P63, ABCG2, CK3 in the.1d preservation group, and the P63, ABCG2, CK3 expression of 4D preservation group decreased significantly.
5. histopathological detection
With the prolongation of the preservation time of the limbal stem cells in the medium-term preservation solution, the formation ability of the cells was reduced in the.1d preservation group. The amniotic membrane covered 2~3 layers of lamina epithelial cells on the amniotic membrane, and the amniotic membrane carrier in the 4D preservation group was smooth, without cell attachment and growth.
conclusion
1. the corneal limbal stem cells can maintain the activity and function of the limbal stem cells in the short term by using the medium cornea preservation solution, but the activity and function of the limbal stem cells have decreased to varying degrees with the prolongation of the preservation time.
2. the function of corneal limbus stem cells has no direct relationship with the expression of its markers: the expression of P63, which is not represented by the cloning of stem cells or the ability to form a monolayer on the amniotic membrane, is not a direct relationship between the function of the corneal limbal stem cells.

【学位授予单位】：济南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R772.2

【参考文献】