本文選題：結(jié)膜 + 上皮�。� 參考：《濟(jì)南大學(xué)》2011年碩士論文

【摘要】：目的 體外分離培養(yǎng)人結(jié)膜杯狀細(xì)胞,觀察其形態(tài)結(jié)構(gòu)并對(duì)其性質(zhì)進(jìn)行鑒定;觀察正常人角膜緣組織與結(jié)膜上皮組織在羊膜上培養(yǎng)的區(qū)別;探討γ-分泌酶抑制劑對(duì)體外培養(yǎng)的人結(jié)膜杯狀細(xì)胞增殖及特異性基因表達(dá)的影響;觀察羊膜為載體的重組結(jié)膜上皮與正常結(jié)膜上皮的區(qū)別。 方法 一、人結(jié)膜上皮細(xì)胞的體外培養(yǎng)及鑒定 1.人結(jié)膜上皮細(xì)胞的體外培養(yǎng) 取眼庫(kù)提供的正常人眼結(jié)膜組織,離體后6小時(shí)內(nèi)采用組織塊法培養(yǎng)原代人結(jié)膜上皮細(xì)胞。 2.體外培養(yǎng)的人結(jié)膜杯狀細(xì)胞的鑒定 采用阿利新藍(lán)/過(guò)碘酸-希夫試劑染色(AB-PAS染色)培養(yǎng)的結(jié)膜上皮細(xì)胞;采用細(xì)胞免疫化學(xué)染色檢測(cè)懸浮細(xì)胞中Muc5Ac的表達(dá);采用細(xì)胞免疫熒光染色檢測(cè)CK7的表達(dá),鑒定分化的杯狀細(xì)胞。 二、正常人角膜緣組織與結(jié)膜上皮組織在羊膜上培養(yǎng)的區(qū)別 取眼庫(kù)提供的保存于角膜中期保存液的正常人角膜緣組織,用消化培養(yǎng)法培養(yǎng)于羊膜載體上;取眼庫(kù)提供的正常人眼結(jié)膜組織,用組織塊法培養(yǎng)于羊膜載體上;采用PAS染色觀察比較二者的差別。 三、γ-分泌酶抑制劑誘導(dǎo)人結(jié)膜杯狀細(xì)胞分化及其相關(guān)檢測(cè) 1.結(jié)膜杯狀細(xì)胞的誘導(dǎo)分化 原代培養(yǎng)的結(jié)膜上皮細(xì)胞匯合度達(dá)到30～40%時(shí),分別加入含100nM、200nM、400nMγ-分泌酶抑制劑的培養(yǎng)液,處理72h,觀察細(xì)胞形態(tài)變化。 2.誘導(dǎo)分化的結(jié)膜杯狀細(xì)胞的性質(zhì)檢測(cè) 原代培養(yǎng)的人結(jié)膜上皮細(xì)胞處理72h后,采用AB-PAS雙染法染色,以比較相同時(shí)間、不同濃度γ-分泌酶抑制劑處理后杯狀細(xì)胞密度的差異;分別提取相同時(shí)間、不同濃度γ-分泌酶抑制劑處理后人結(jié)膜上皮細(xì)胞的總RNA,反轉(zhuǎn)成cDNA后行實(shí)時(shí)熒光定量PCR(RT-Q-PCR)檢測(cè),觀察CK7的mRNA表達(dá)量在相同時(shí)間、不同濃度處理組結(jié)膜上皮細(xì)胞中的變化;提取陰性對(duì)照及200nMγ-分泌酶抑制劑處理72h后的人結(jié)膜上皮細(xì)胞的總蛋白,行Western-Blot檢測(cè)CK7蛋白在人結(jié)膜上皮細(xì)胞中的表達(dá)情況。 3.統(tǒng)計(jì)學(xué)分析:對(duì)上述結(jié)果采用方差分析(Analysisi of Variance,ANOVA)檢驗(yàn)進(jìn)行統(tǒng)計(jì)學(xué)分析,比較不同濃度γ-分泌酶抑制劑處理組與陰性對(duì)照組的差異性,以P0.05視為差異具有統(tǒng)計(jì)學(xué)意義。 四、羊膜為載體構(gòu)建重組結(jié)膜上皮與正常結(jié)膜上皮的比較 取眼庫(kù)提供的正常人眼結(jié)膜組織,用組織塊法培養(yǎng)于羊膜載體上,行PAS染色;對(duì)正常結(jié)膜上皮采用石蠟切片PAS染色,觀察并比較二者的差別。 結(jié)果 一、培養(yǎng)的人結(jié)膜上皮細(xì)胞及鑒定 1.組織塊培養(yǎng)法培養(yǎng)正常人結(jié)膜上皮細(xì)胞,次日補(bǔ)液后,12小時(shí)內(nèi)可見(jiàn)細(xì)胞從組織塊周圍遷出,細(xì)胞貼壁生長(zhǎng),15天左右可長(zhǎng)滿培養(yǎng)皿,倒置顯微鏡下觀察,可見(jiàn)較多懸浮狀態(tài)的杯狀細(xì)胞。 2.體外培養(yǎng)的人結(jié)膜杯狀細(xì)胞的鑒定 (1)AB-PAS染色結(jié)果顯示:胞漿呈藍(lán)色及紅色顆粒者為杯狀細(xì)胞,可以初步鑒定體外培養(yǎng)的人結(jié)膜上皮細(xì)胞中含有杯狀細(xì)胞。 (2)細(xì)胞免疫化學(xué)結(jié)果顯示:原代培養(yǎng)的人結(jié)膜上皮細(xì)胞中的懸浮細(xì)胞,以小鼠單克隆Muc5Ac抗體進(jìn)行染色,呈陽(yáng)性反應(yīng)者為杯狀細(xì)胞。證明體外培養(yǎng)的人結(jié)膜上皮細(xì)胞中的懸浮細(xì)胞中含有杯狀細(xì)胞。 (3)細(xì)胞免疫熒光結(jié)果顯示:原代培養(yǎng)的人結(jié)膜上皮細(xì)胞,以小鼠單克隆CK7抗體進(jìn)行染色,呈陽(yáng)性反應(yīng)者為杯狀細(xì)胞�？梢宰C明體外培養(yǎng)的人結(jié)膜上皮細(xì)胞中含有杯狀細(xì)胞。 二、正常人角膜緣組織與結(jié)膜上皮組織在羊膜上培養(yǎng)的區(qū)別 PAS染色結(jié)果顯示:培養(yǎng)于羊膜上的角膜緣上皮細(xì)胞及結(jié)膜上皮細(xì)胞中均含有染色陽(yáng)性的杯狀細(xì)胞;角膜緣上皮細(xì)胞中的杯狀細(xì)胞較結(jié)膜上皮細(xì)胞中密度低,且分化程度低。 三、體外培養(yǎng)的人結(jié)膜杯狀細(xì)胞經(jīng)γ-分泌酶抑制劑誘導(dǎo)分化及相關(guān)檢測(cè) 1.倒置顯微鏡下觀察:加入γ-分泌酶抑制劑組杯狀細(xì)胞密度較陰性對(duì)照組增高,其中200nM組增高最明顯。 2. AB-PAS染色結(jié)果顯示:加入γ-分泌酶抑制劑處理72h后的人結(jié)膜上皮細(xì)胞中的杯狀細(xì)胞密度較陰性對(duì)照組高;其中200nM組中杯狀細(xì)胞密度最高。 3. RT-Q-PCR結(jié)果顯示:100nMγ-分泌酶抑制劑處理組與陰性對(duì)照組間CK7mRNA的表達(dá)量差異具有顯著性。 4. Western-blot結(jié)果顯示:結(jié)果顯示CK7蛋白的表達(dá)量在200nM組較陰性對(duì)照組顯著升高。 四、羊膜為載體構(gòu)建重組結(jié)膜上皮與正常結(jié)膜上皮的比較 PAS染色結(jié)果顯示:羊膜為載體構(gòu)建的重組結(jié)膜上皮細(xì)胞中杯狀細(xì)胞的密度較正常結(jié)膜上皮中杯狀細(xì)胞的密度低;后者PAS染色顯示杯狀細(xì)胞嵌入結(jié)膜上皮層中,前者PAS染色顯示杯狀細(xì)胞位于羊膜表層,易脫落。 結(jié)論 1.人結(jié)膜杯狀細(xì)胞可以在體外進(jìn)行培養(yǎng)擴(kuò)增,并具有分泌粘蛋白的能力。 2.角膜緣組織培養(yǎng)過(guò)程中會(huì)有少量的杯狀細(xì)胞,但與正常的結(jié)膜相比,密度偏低,且分化程度較低。 3.γ-分泌酶抑制劑可以誘導(dǎo)結(jié)膜上皮細(xì)胞分化為杯狀細(xì)胞,并能促進(jìn)杯狀細(xì)胞粘蛋白和特異性標(biāo)志基因的表達(dá)。 4.羊膜為載體構(gòu)建的重組結(jié)膜上皮膜片中,存在著杯狀細(xì)胞密度低、位置與正常結(jié)膜上皮不同的問(wèn)題,因此需要尋找新的載體或誘導(dǎo)分化方法構(gòu)建重組結(jié)膜上皮。 目的 將新鮮角膜環(huán)置于角膜中期保存液保存不同時(shí)間,通過(guò)檢測(cè)細(xì)胞總量、細(xì)胞活性、干細(xì)胞相關(guān)標(biāo)志物、細(xì)胞增殖及分化的能力,分析角膜中期保存液對(duì)角膜緣干細(xì)胞的影響。 方法 1.角膜環(huán)的預(yù)處理 取回眼庫(kù)提供的新鮮角膜環(huán)共6個(gè),顯微鏡下撕除角膜內(nèi)皮及虹膜組織,每個(gè)平均分為3份,分別置于角膜中期保存液中1d、4d、7d。采用消化培養(yǎng)法將角膜緣上皮消化為單細(xì)胞懸液。 2.不同保存時(shí)間的角膜緣上皮通過(guò)細(xì)胞計(jì)數(shù)儀檢測(cè)細(xì)胞總量,計(jì)算平均細(xì)胞總量;及活細(xì)胞比例。將細(xì)胞接種于3T3飼養(yǎng)層上,培養(yǎng)觀察及姬姆薩染色,計(jì)算細(xì)胞克隆形成能力。采用流式細(xì)胞技術(shù)對(duì)其中的干細(xì)胞進(jìn)行檢測(cè);采用細(xì)胞免疫熒光技術(shù),檢測(cè)相關(guān)干細(xì)胞標(biāo)志物的表達(dá)。將細(xì)胞接種于羊膜上,通過(guò)組織病理學(xué)HE染色檢測(cè)細(xì)胞的復(fù)層形成能力。 結(jié)果 1.細(xì)胞總量檢測(cè) 隨著角膜緣干細(xì)胞在中期保存液中保存時(shí)間的延長(zhǎng),細(xì)胞總量減少。1d、4d、7d保存組的平均細(xì)胞總量分別為:100%、42.45%、0。 2.細(xì)胞活性檢測(cè) 隨著角膜緣干細(xì)胞在中期保存液中保存時(shí)間的延長(zhǎng),細(xì)胞活性降低。1d、4d、7d保存組的平均細(xì)胞活性分別為:82%、64%、0。 3.克隆形成能力 隨著角膜緣干細(xì)胞在中期保存液中保存時(shí)間的延長(zhǎng),細(xì)胞克隆形成能力降低、CFE降低。 4.干細(xì)胞標(biāo)志檢測(cè) 隨著角膜緣干細(xì)胞在中期保存液中保存時(shí)間的延長(zhǎng),角膜緣干細(xì)胞標(biāo)志物P63、ABCG2、CK3表達(dá)降低。1d保存組P63、ABCG2、CK3陽(yáng)性表達(dá),4d保存組P63、ABCG2、CK3表達(dá)量明顯降低。 5.組織病理學(xué)檢測(cè) 隨著角膜緣干細(xì)胞在中期保存液中保存時(shí)間的延長(zhǎng),細(xì)胞復(fù)層形成能力降低。1d保存組可見(jiàn)羊膜表面覆蓋有2～3層的復(fù)層上皮細(xì)胞;4d保存組羊膜載體表面光滑,無(wú)細(xì)胞貼附及生長(zhǎng)。 結(jié)論 1.應(yīng)用角膜中期保存液保存角膜緣組織短期內(nèi)可以維持其中角膜緣干細(xì)胞的活性及功能,但是隨著保存時(shí)間的延長(zhǎng),角膜緣干細(xì)胞的活性和功能均有不同程度的下降。 2.角膜緣干細(xì)胞的功能與其標(biāo)志物表達(dá)之間無(wú)直接關(guān)系:病理檢測(cè)P63的表達(dá),不代表干細(xì)胞具有克隆形成或在羊膜上形成單層的能力。
[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.

【學(xué)位授予單位】：濟(jì)南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R772.2

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本文編號(hào)：1886164