活体共焦显微镜下正常人脸结膜及脸板腺的年龄变化及性别差异

发布时间：2018-05-13 22:18

本文选题：激光共焦显微镜 + 睑板腺　；参考：《复旦大学》2010年硕士论文

【摘要】： 近年来,干眼的患病率逐渐上升,已成为眼科常见疾病之一,相关研究是目前眼科研究的热点之一。其中,睑板腺(Meibomian glands, MGs)功能障碍所致蒸发过强型干眼占有较大比例。睑板腺分泌多种脂质成分,构成泪膜的脂质层,影响泪液蒸发及泪膜的稳定性,因此,睑板腺的数量及形态的变化对维持稳定的眼表至关重要。既往研究对睑板腺的观察主要为病理学研究及动物实验,目前尚无涉及正常人群的睑板腺和睑结膜变化的研究报道；正常人活体状态下睑板腺的形态及密度是否受到性别差异、年龄变化、激素水平及生活环境等内外因素的影响而发生改变,也尚不清楚。近年来问世的激光活体共焦显微镜为动态观察眼表组织在活体状态下的情况提供了有力工具。本研究通过激光活体共焦显微镜(Laser confocal microscopy, LSCM)观察四个年龄段组正常人群睑结膜及睑板腺的组织结构,探讨睑结膜上皮细胞(Palpebral conjunctiva epithelial cells)、杯状细胞(Goblet cells, GCs)、树突状细胞(Dendritic cells, DCs)及睑板腺腺泡细胞的密度和形态,并探讨其与年龄和性别的相关性。第一部分活体共焦显微镜下正常人睑结膜及睑板腺的年龄变化目的应用活体LSCM观察正常人睑结膜及睑板腺组织细胞结构的年龄变化。方法选择正常人86例86只眼,按年龄进行分组,A组为0～20岁,B组为21～40岁、C组为41～60岁、D组为61岁以上,每组约20例。翻转上睑,距睑缘约5mm使用LSCM观察睑结膜及睑板腺组织细胞结构并记录图像,计算结膜上皮细胞、GCs、DCs、MGs腺泡细胞密度及MGs腺泡的直径并进行方差分析,若有统计学意义再行最小显著性差异(Least significant difference, LSD)检验。评价睑结膜及MGs组织细胞结构的年龄变化。结果(1)在共焦显微镜下,可见睑结膜上皮浅表层细胞的胞体较大、胞质低反光、周边高反光,排列松散,细胞边界模糊、高反光,形态似圆形、卵圆形。上皮的形态与其正常生理代谢过程关系密切,形态越扁平,面积越大,则越接近上皮表层。睑结膜基底层在共焦显微镜下表现为周边边界高亮而清晰的近圆形、卵圆形细胞,细胞质呈低反光。由于基底层较贴近睑板腺且边界较清晰,因此我们统计睑结膜上皮细胞基底层细胞的密度,在A、B、C、D各组的平均密度分别为5357±304,4917±413,4925±521,4943±484(个／mm2)；使用方差分析,组间具有统计学差异(p=0.002,F=5.217),但未随年龄增长而降低。(2)GCs是泪膜黏液层的主要来源,对于维持泪膜稳定性起重要作用。GCs胞体较大、胞内充满透亮颗粒、成团或散在分布的卵圆形或圆形细胞,体积为周围的上皮细胞的约3倍,四组中GCs密度分别为1422±590,1409±711,791±457,779±546(个／mm2),用方差分析,差异具有统计学意义(p=0.000,F=14.168)；(3)DC是目前所指的机体内功能最强的抗原提呈细胞,在共焦显微镜下形态呈胞体小,呈高亮反光,散在分布,可为缺乏或长形或铁丝网状的树突状形态。各组平均的DCs细胞密度为29±15,30±12,41±23,36±15(个／mm2),采用方差分析,未见明显的年龄相关变化(p=0.06,F=2.573)。(4)MGs腺泡细胞因光学切面不同,呈现不同形态,腺泡外圈为轮胎样上皮细胞,伴有高亮反光颗粒,胞腔内呈灰色偶伴点状高反光分泌物,团状聚集分布,排列不规则。A～D组细胞密度分别为92±26,86±27,62±20,56±16(个／mm2),运用方差分析,差异有统计学意义(p=0.000,13.133)。MG腺泡单位的直径,A～D组分别为99.06±16.64,98.22±18.77,85.91±15.55,88.26±16.71(μm),使用方差分析,差异有统计学意义(p=0.022,F=3.385),但未随着年龄而明显缩小结论LSCM可清晰观察正常人群睑结膜和睑板腺的微观组织结构。睑结膜上皮细胞结构及DC密度无明显年龄改变；GCs及MG腺泡细胞密度及DC分支形态随年龄增长有退化趋势,提示睑板腺功能降低。第二部分活体共焦显微镜下正常人睑结膜及睑板腺的性别差异目的应用活体LSCM观察正常人睑板腺睑结膜及睑板腺组织细胞结构的性别差异。方法选择正常人86例86只眼,按性别、40岁上下进行分组,A组为男性、年龄0～40岁,B组为女性、年龄0～40岁,C组为男性、年龄41岁以上、D组为女性、年龄41岁以上,每组约20例。翻转上睑,距睑缘约5mm使用LSCM观察睑结膜及睑板腺组织细胞结构并记录图像,计算结膜上皮细胞、杯状细胞、树突状细胞、睑板腺腺泡细胞密度及睑板腺腺泡的直径并进行t检验,评价睑结膜及睑板腺组织细胞结构的性别差异。结果(1)睑结膜上皮细胞,在LSCM下,A、B、C、D各组的基底层细胞平均密度分别为5162±324,5042±608,4902±374,5047±345(个／mm2)；使用t检验,40岁以下(p=0.469,t=0.827)及41岁以上(p=0.607,t=-1.295)组间不具有统计学差异。(2)GCs,于四组中密度分别为1406±496,1426±367,840±375,744±307(个／mm2),使用t检验,不论40岁以下(p=0.288,t=-0.135)及41岁以上(p=0.406,t=0.801)皆未发现具有统计学意义。(3)DCs各组平均的DC密度为28±12,30±13,46±20,34±18(个／mm2),使用t检验,40岁以下(p=0.551,t=-0.310)及41岁以上(p=0.149,t=2.010)未见明显的统计学差异。(4)MG腺泡细胞,A～D组细胞密度分别为86±26,95±24,59±17,58±19(个／mm2),使用t检验,40岁以下(p=0.566,t=-1.130)及41岁以上(p=0.550,t=0.285)未见明显的统计学差异。MGs腺泡单位的直径,A～D组分别为96.30±14.98,99.54±19.45,92.72±16.79,84.88±17.03(μm),使用t检验,40岁以下(p=0.270,t=-0.622)及41岁以上(p=0.950,t=1.473)皆未见明显的统计学差异。结论正常人群睑结膜和睑板腺组织细胞结构未见性别差异。LSCM可清晰观察正常人群睑结膜和睑板腺的微观组织结构,为睑板腺相关疾病和蒸发过强型干眼的诊断提供组织学依据。
[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.

【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R777.1

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