本文選題：激光共焦顯微鏡 + 瞼板腺��； 參考：《復(fù)旦大學(xué)》2010年碩士論文

【摘要】： 近年來,干眼的患病率逐漸上升,已成為眼科常見疾病之一,相關(guān)研究是目前眼科研究的熱點之一。其中,瞼板腺(Meibomian glands, MGs)功能障礙所致蒸發(fā)過強型干眼占有較大比例。瞼板腺分泌多種脂質(zhì)成分,構(gòu)成淚膜的脂質(zhì)層,影響淚液蒸發(fā)及淚膜的穩(wěn)定性,因此,瞼板腺的數(shù)量及形態(tài)的變化對維持穩(wěn)定的眼表至關(guān)重要。既往研究對瞼板腺的觀察主要為病理學(xué)研究及動物實驗,目前尚無涉及正常人群的瞼板腺和瞼結(jié)膜變化的研究報道；正常人活體狀態(tài)下瞼板腺的形態(tài)及密度是否受到性別差異、年齡變化、激素水平及生活環(huán)境等內(nèi)外因素的影響而發(fā)生改變,也尚不清楚。近年來問世的激光活體共焦顯微鏡為動態(tài)觀察眼表組織在活體狀態(tài)下的情況提供了有力工具。本研究通過激光活體共焦顯微鏡(Laser confocal microscopy, LSCM)觀察四個年齡段組正常人群瞼結(jié)膜及瞼板腺的組織結(jié)構(gòu),探討瞼結(jié)膜上皮細胞(Palpebral conjunctiva epithelial cells)、杯狀細胞(Goblet cells, GCs)、樹突狀細胞(Dendritic cells, DCs)及瞼板腺腺泡細胞的密度和形態(tài),并探討其與年齡和性別的相關(guān)性。 第一部分活體共焦顯微鏡下正常人瞼結(jié)膜及瞼板腺的年齡變化 目的應(yīng)用活體LSCM觀察正常人瞼結(jié)膜及瞼板腺組織細胞結(jié)構(gòu)的年齡變化。 方法選擇正常人86例86只眼,按年齡進行分組,A組為0～20歲,B組為21～40歲、C組為41～60歲、D組為61歲以上,每組約20例。翻轉(zhuǎn)上瞼,距瞼緣約5mm使用LSCM觀察瞼結(jié)膜及瞼板腺組織細胞結(jié)構(gòu)并記錄圖像,計算結(jié)膜上皮細胞、GCs、DCs、MGs腺泡細胞密度及MGs腺泡的直徑并進行方差分析,若有統(tǒng)計學(xué)意義再行最小顯著性差異(Least significant difference, LSD)檢驗。評價瞼結(jié)膜及MGs組織細胞結(jié)構(gòu)的年齡變化。 結(jié)果(1)在共焦顯微鏡下,可見瞼結(jié)膜上皮淺表層細胞的胞體較大、胞質(zhì)低反光、周邊高反光,排列松散,細胞邊界模糊、高反光,形態(tài)似圓形、卵圓形。上皮的形態(tài)與其正常生理代謝過程關(guān)系密切,形態(tài)越扁平,面積越大,則越接近上皮表層。瞼結(jié)膜基底層在共焦顯微鏡下表現(xiàn)為周邊邊界高亮而清晰的近圓形、卵圓形細胞,細胞質(zhì)呈低反光。由于基底層較貼近瞼板腺且邊界較清晰,因此我們統(tǒng)計瞼結(jié)膜上皮細胞基底層細胞的密度,在A、B、C、D各組的平均密度分別為5357±304,4917±413,4925±521,4943±484(個／mm2)；使用方差分析,組間具有統(tǒng)計學(xué)差異(p=0.002,F=5.217),但未隨年齡增長而降低。(2)GCs是淚膜黏液層的主要來源,對于維持淚膜穩(wěn)定性起重要作用。GCs胞體較大、胞內(nèi)充滿透亮顆粒、成團或散在分布的卵圓形或圓形細胞,體積為周圍的上皮細胞的約3倍,四組中GCs密度分別為1422±590,1409±711,791±457,779±546(個／mm2),用方差分析,差異具有統(tǒng)計學(xué)意義(p=0.000,F=14.168)；(3)DC是目前所指的機體內(nèi)功能最強的抗原提呈細胞,在共焦顯微鏡下形態(tài)呈胞體小,呈高亮反光,散在分布,可為缺乏或長形或鐵絲網(wǎng)狀的樹突狀形態(tài)。各組平均的DCs細胞密度為29±15,30±12,41±23,36±15(個／mm2),采用方差分析,未見明顯的年齡相關(guān)變化(p=0.06,F=2.573)。(4)MGs腺泡細胞因光學(xué)切面不同,呈現(xiàn)不同形態(tài),腺泡外圈為輪胎樣上皮細胞,伴有高亮反光顆粒,胞腔內(nèi)呈灰色偶伴點狀高反光分泌物,團狀聚集分布,排列不規(guī)則。A～D組細胞密度分別為92±26,86±27,62±20,56±16(個／mm2),運用方差分析,差異有統(tǒng)計學(xué)意義(p=0.000,13.133)。MG腺泡單位的直徑,A～D組分別為99.06±16.64,98.22±18.77,85.91±15.55,88.26±16.71(μm),使用方差分析,差異有統(tǒng)計學(xué)意義(p=0.022,F=3.385),但未隨著年齡而明顯縮小 結(jié)論LSCM可清晰觀察正常人群瞼結(jié)膜和瞼板腺的微觀組織結(jié)構(gòu)。瞼結(jié)膜上皮細胞結(jié)構(gòu)及DC密度無明顯年齡改變；GCs及MG腺泡細胞密度及DC分支形態(tài)隨年齡增長有退化趨勢,提示瞼板腺功能降低。 第二部分活體共焦顯微鏡下正常人瞼結(jié)膜及瞼板腺的性別差異 目的應(yīng)用活體LSCM觀察正常人瞼板腺瞼結(jié)膜及瞼板腺組織細胞結(jié)構(gòu)的性別差異。 方法選擇正常人86例86只眼,按性別、40歲上下進行分組,A組為男性、年齡0～40歲,B組為女性、年齡0～40歲,C組為男性、年齡41歲以上、D組為女性、年齡41歲以上,每組約20例。翻轉(zhuǎn)上瞼,距瞼緣約5mm使用LSCM觀察瞼結(jié)膜及瞼板腺組織細胞結(jié)構(gòu)并記錄圖像,計算結(jié)膜上皮細胞、杯狀細胞、樹突狀細胞、瞼板腺腺泡細胞密度及瞼板腺腺泡的直徑并進行t檢驗,評價瞼結(jié)膜及瞼板腺組織細胞結(jié)構(gòu)的性別差異。 結(jié)果(1)瞼結(jié)膜上皮細胞,在LSCM下,A、B、C、D各組的基底層細胞平均密度分別為5162±324,5042±608,4902±374,5047±345(個／mm2)；使用t檢驗,40歲以下(p=0.469,t=0.827)及41歲以上(p=0.607,t=-1.295)組間不具有統(tǒng)計學(xué)差異。(2)GCs,于四組中密度分別為1406±496,1426±367,840±375,744±307(個／mm2),使用t檢驗,不論40歲以下(p=0.288,t=-0.135)及41歲以上(p=0.406,t=0.801)皆未發(fā)現(xiàn)具有統(tǒng)計學(xué)意義。(3)DCs各組平均的DC密度為28±12,30±13,46±20,34±18(個／mm2),使用t檢驗,40歲以下(p=0.551,t=-0.310)及41歲以上(p=0.149,t=2.010)未見明顯的統(tǒng)計學(xué)差異。(4)MG腺泡細胞,A～D組細胞密度分別為86±26,95±24,59±17,58±19(個／mm2),使用t檢驗,40歲以下(p=0.566,t=-1.130)及41歲以上(p=0.550,t=0.285)未見明顯的統(tǒng)計學(xué)差異。MGs腺泡單位的直徑,A～D組分別為96.30±14.98,99.54±19.45,92.72±16.79,84.88±17.03(μm),使用t檢驗,40歲以下(p=0.270,t=-0.622)及41歲以上(p=0.950,t=1.473)皆未見明顯的統(tǒng)計學(xué)差異。結(jié)論正常人群瞼結(jié)膜和瞼板腺組織細胞結(jié)構(gòu)未見性別差異。LSCM可清晰觀察正常人群瞼結(jié)膜和瞼板腺的微觀組織結(jié)構(gòu),為瞼板腺相關(guān)疾病和蒸發(fā)過強型干眼的診斷提供組織學(xué)依據(jù)。
[Abstract]:In recent years, the prevalence of dry eyes has gradually increased and has become one of the common diseases in the ophthalmology. The related research is one of the hotspots in the current ophthalmology research. Among them, the dry eyes of Meibomian glands (MGs) have a large proportion of evaporated dry eyes. The secretion of multiple lipid components in the eyelid gland, the lipid layer of the tear film, affects the evaporation of tears. With the stability of the tear film, the changes in the number and morphology of the palpebral glands are essential to maintain a stable eye surface. The previous study of the palpebral glands is mainly pathological and animal experiments. At present, there is no study of the changes in the palpebral glands and conjunctiva in normal people; the morphology of the palpebral glands in normal human living body and the form of the palpebral glands in normal human living condition It is not clear whether the density is affected by the internal and external factors such as sex difference, age, hormone level and living environment, and it is not clear. In recent years, the laser living confocal microscopy has provided a powerful tool for dynamic observation of ocular surface tissues in living conditions. This study was conducted by laser living confocal microscopy (Laser CO). NFOCAL microscopy, LSCM) to observe the structure of the conjunctiva and the palpebral gland of the normal population of four age groups, to explore the density and morphology of the eyelid conjunctival epithelial cells (Palpebral conjunctiva epithelial cells), goblet cells (Goblet cells, GCs), dendritic cells (Dendritic cells), and the palpebral gland acinus cells, and to explore their age and age. Sex correlation.
The first part is the age change of normal human conjunctiva and meibomian gland under confocal microscopy.
Objective To observe the age changes of normal human conjunctiva and meibomian gland cell structure by living LSCM.
Methods 86 normal people were divided into 86 eyes, group A was 0~20, group B was 0~20, group B was 21~40 years old, group C was 41~60 years old, group D was over 61 years old, about 20 cases in each group. The eyelid conjunctiva and bleblepalpebral gland cell structure were observed with LSCM, and the cell structure of conjunctiva, GCs, DCs, and MGs acinar cell density were calculated. The diameter of MGs acinus and analysis of variance. If there were statistical significance, the minimum significant difference (Least significant difference, LSD) test was performed to evaluate the age changes of the cell structure of the conjunctiva and MGs tissue.
Results (1) under the confocal microscope, the cell body of superficial layer of conjunctival epithelium of the eyelid is larger, the cytoplasm is low reflecting light, the surrounding light is high, the boundary is loose, the cell boundary is blurred, the shape is round and oval. The morphology of the epithelium is closely related to the normal physiological metabolism process, the flatter the shape, the larger the area, the closer to the epithelia surface. Palpebral. The conjunctival basement layer showed a bright and clear near circle on the periphery of the confocal microscope. The oval cells and cytoplasm showed low reflectivity. Because the basal lamina was closer to the palpebral gland and the boundary was clearer, we counted the density of the basal layer cells in the eyelid conjunctival epithelial cells. The average density of the cells in the A, B, C and D groups were 5357 + 413, respectively, respectively. 4925 + 5214943 + 484 (mm2); using variance analysis, there were statistical differences between groups (p=0.002, F=5.217), but did not decrease with age. (2) GCs was the main source of the mucous layer of tear film, and it played an important role in maintaining tear film stability..GCs was larger, and the cell was filled with bright particles. The cell volume was about 3 times that of the surrounding epithelial cells, and the GCs density in the four groups was 1422 + 5901409 + 711791 + 457779 + 546 (mm2). The difference was statistically significant (p=0.000, F=14.168) with variance analysis. (3) DC was the most powerful anti proactive cell in the body at present, and the morphology was small and high under confocal microscope. Light reflection, scattered in the distribution, can be a lack or long form or wire reticulate dendritic shape. The average density of DCs cells in each group is 29 + 15,30 + 12,41 + 23,36 + 15 (mm2). Using variance analysis, no obvious age related changes (p=0.06, F=2.573). (4) MGs acinar cells are different form of optical cutting surface, and the outer ring of acinus is a tire. The epithelial cells were accompanied by high light reflective particles, and the cellular lumen was gray even with high reflective exudates and cluster distribution. The cell density in the irregular.A ~ D group was 92 + 26,86 + 27,62 + 20,56 16 (mm2). The difference was statistically significant (p=0.000,13.133) with the diameter of the.MG acinus unit, and the A to D group was 99.06, respectively. + 16.64,98.22 + 18.77,85.91 + 15.55,88.26 + 16.71 (m), using variance analysis, the difference was statistically significant (p=0.022, F=3.385), but did not decrease significantly with age.
Conclusion LSCM can clearly observe the microstructure of the conjunctiva and the palpebral gland in normal population. There is no obvious age change in the structure of the conjunctival epithelial cells and the density of DC in the conjunctiva. The density of GCs and MG acinar cells and the morphology of the DC branches degenerate with age, suggesting the decrease of the function of the palpebral gland.
The second part is the sex difference between normal human conjunctiva and meibomian gland under confocal microscopy.
Objective To observe the sex differences in the cell structure of palpebral conjunctiva and meibomian gland in normal human eyes by living LSCM.
Methods 86 normal people were divided into 86 eyes, group A was male, age 0~40 years old, group B was female, age 0~40, group C was 0~40 years old, group C was male, age 41 years old, group D was female, age 41 years old, each group was over 20 cases. The upper eyelid was overturned and 5mm using LSCM to observe the eyelid conjunctiva and palpebral gland cell structure with LSCM. The images were recorded to calculate the conjunctival epithelial cells, goblet cells, dendritic cells, the cell density of the palpebral gland acinus and the diameter of the palpebral gland acinus by t test, and to evaluate the sex difference between the conjunctiva and the structure of the palpebral gland.
Results (1) the average density of the basal layer cells of the palpebral conjunctiva cells at LSCM, A, B, C, and D were 5162 + 3245042 + 6084902 + 3745047 + 345 (mm2), respectively. Using t test, there was no statistical difference between 40 years old (p=0.469, t=0.827) and 41 years old (p=0.607, t=-1.295). (2) GCs, the density of four groups was 1406 + 496142, respectively. 6 + 367840 + 375744 + 307 (mm2), using t test, no matter under 40 years of age (p=0.288, t=-0.135) and over 41 years (p=0.406, t=0.801) were not found to have statistical significance. (3) the average DC density of each group was 28 + 12,30 + 13,46 + 20,34 + 18 (mm2), using the test, under 40 years of age, and over 41 years of age (4) the cell density of MG acinar cells in A ~ D group was 86 + 26,95 + 24,59 + 17,58 + 19 (mm2), and t test showed that there was no significant statistical difference between 40 years old (p=0.566, t=-1.130) and 41 years old (p=0.550, t=0.285). The diameter of acinus unit was 96.30 + 96.30 + 16.79, respectively. 84.88 + 17.03 (mu m), t test, under 40 years of age (p=0.270, t=-0.622) and above 41 years of age (p=0.950, t=1.473) no significant statistical difference. Conclusion the normal population of the eyelid conjunctiva and the palpebral gland cell structure no sex difference.LSCM can clearly observe the normal human conjunctiva and the microscopic structure of the palpebral gland, for the palpebral gland associated disease The diagnosis of dry eye and evaporative eyes provides histological basis.

【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R777.1

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