成人结膜干细胞定位特征的免疫组织化学研究

目的:研究成人结膜上皮细胞中,粘蛋白5AC(MUC5AC),角蛋白7,p63和增殖细胞核抗原(PCNA)的表达及分布情况,探讨成人结膜上皮干细胞的定位特征。方法:获取角膜移植供者的睑球结膜,采用HE免疫组化染色法,观察MUC5AC,角蛋白7,p63和PCNA的表达及分布情况。结果:PCNA及p63表达情况一致,主要表达在睑缘皮肤黏膜交界处及近角膜缘结膜上皮深层细胞。角蛋白7在穹窿部球结膜的固有层中的腺体细胞有阳性表达,MUC5AC在各部位结膜上皮的杯状细胞的胞质阳性表达。结论:MUC5AC细胞角蛋白7和p63及PCNA在成人结膜上皮细胞的特征性表达情况可以推测具有分化潜力的结膜干细胞存在于睑缘皮肤黏膜交界处及近角膜缘球结膜上皮深层细胞。     

Pax6在翼状胬肉上皮鳞状化生中的异常表达

目的:观察翼状胬肉上皮细胞中Pax6的表达情况.方法:应用免疫组织化学染色法检测15例翼状胬肉上皮细胞Pax6、K10、K19以及MUC5AC的表达,并与正常结膜对比.结果:在正常结膜上皮,K19全层表达,Pax6在全层细胞核内表达,K10染色阴性,MUC5AC阳性细胞在上皮表皮层呈单个或簇状散在分布;而翼状胬肉组织Pax6、K19和MUC5AC表达下降甚至阴性表达,且比较差异有统计学意义,而K10在上皮的部分表层细胞中阳性表达.结论:Pax6基因在翼状胬肉上皮细胞表达下调,提示其上皮细胞发生鳞状上皮化生.     

模拟角膜缘干细胞微环境诱导人多潜能干细胞分化为角膜上皮细胞的研究

目的：探讨模拟角膜缘干细胞(LSCs)微环境诱导人多潜能干细胞(hiPSCs)分化为角膜上皮细胞的可行性。方法：体外建立hiPSCs细胞系，利用transwell体系将hiPSCs与角膜基质细胞共培养模拟角膜缘干细胞微环境，添加小分子骨形态发生蛋白4(BMP4)和特异性转化生长因子β抑制剂(SB431542)，诱导hiPSCs向角膜上皮细胞分化。采用免疫荧光染色、流式细胞方法检测角膜上皮细胞特异标志物 CK3和CK12，角膜上皮细胞前体CK15，角膜缘干细胞标志物ABCG5的表达。结果：hiPSCs体外培养增殖活跃，免疫荧光染色显示干细胞特异性标志物OCT4、SOX2、TRA-1-60、NANOG呈阳性。采用transwell体系将hiPSCs与角膜基质细胞共培养，免疫荧光染色结果显示角膜缘干细胞标志物ABCG5及角膜上皮细胞前体标志物CK15阳性，角膜上皮细胞标志物CK3及CK12阴性； 在共培养的基础上添加小分子BMP4和SB431542，免疫荧光染色及流式细胞检测结果显示角膜上皮细胞特异性标志物CK3阳性表达，且随分化时间延长表达比例增高。结论：模拟角膜缘干细胞微环境同时添加小分子SB431542及BMP4，可成功诱导体外培养的hiPSCs向角膜上皮细胞分化。     

DispaseⅡ消化法原代培养兔结膜上皮细胞及鉴定

目的研究DispaseⅡ消化法原代培养兔结膜上皮细胞,观察其体外生长特性,并采用免疫组织化学染色鉴定培养的上皮细胞。方法 DispaseⅡ消化法原代培养兔结膜上皮细胞,观察不同时期细胞的生长情况、细胞形态,做广谱角蛋白、MUC5AC免疫荧光染色及PAS染色鉴定;并比较不同部位结膜上皮细胞增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)表达阳性情况。结果 DispaseⅡ消化法能够获得大量的较纯净的结膜上皮细胞,细胞生长增殖快,能传代4～5代;绝大多数细胞广谱角蛋白免疫荧光染色阳性,少数细胞MUC5AC表达阳性及PAS染色阳性。睑结膜、穹隆部结膜和球结膜细胞PCNA阳性率分别为:(32.14±1.69)%、(27.67±1.20)%、(22.76±1.73)%,兔睑结膜上皮细胞PCNA阳性率较穹隆部及球结膜上皮细胞的PCNA阳性率高,各部位PCNA阳性率间比较差异有统计学意义(P<0.05)。结论 DispaseⅡ消化法是进行体外结膜上皮细胞培养的理想方法,兔眼睑结膜处可能存在结膜干细胞。     

大鼠骨髓间充质干细胞体外可诱导分化为角膜上皮细胞

目的:探讨骨髓间充质干细胞 (mesenchymal stem cell,MSC) 分化为角膜上皮细胞的可塑性及其重建角膜上皮的可能性.方法:用密度梯度离心法结合贴壁培养法分离纯化大鼠骨髓MSC,经体外与角膜基质细胞共培养诱导分化,免疫荧光法检测角膜上皮细胞特异标志物K12的表达.结果:体外培养的大鼠骨髓MSC表现出很强的增殖潜能,原代培养的骨髓MSC CD29免疫荧光染色阳性,CD34和CD45为阴性,符合骨髓MSC的特征.MSC与角膜基质细胞共培养1wk后大部分细胞分化为间质细胞,少部分细胞形态上相对偏小,免疫荧光检测这部分细胞表达角膜上皮细胞特异性标志角蛋白K12.结论:体外培养的MSC在角膜基质细胞的诱导下可横向分化为角膜上皮细胞.     

体外共培养诱导人羊膜间充质干细胞分化为眼表上皮细胞的研究

背景 人羊膜间充质干细胞（hAMSCs）在体外能诱导分化为多种体细胞,但目前有关hAMSCs分化为眼表细胞的研究鲜有报道. 目的 探讨体外共培养诱导hAMSCs向眼表细胞分化的可能性及其分化机制.方法 本研究经广州医科大学第二附属医院伦理委员会批准,在健康产妇的知情同意下从人胎盘中分离hAMSCs并进行培养,采用流式细胞术计数分析CD44、CD45、CD73、CD90在培养细胞中的表达以鉴定细胞,此外通过成骨诱导分化实验和成脂诱导分化实验对培养细胞进行体外分化鉴定.在患者知情同意下获取眼科手术中弃用的人眼球结膜组织,用组织块培养法分离和培养人球结膜成纤维细胞（hBCFs）,将hAMSCs与hBCFs在Transwell培养体系中进行共培养,分为hAMSCs培养组和hAMSCs与hBCFs共培养组,用含质量分数5％胎牛血清（FBS）的DMEM高糖/F12培养基培养7d,采用免疫荧光技术检测hAMSCs中对上皮细胞角蛋白19（CK19）的表达,评价hAMSCs向眼表细胞分化的程度,并检测hAMSCs中α-平滑肌肌动蛋白（α-SMA）的表达,评价其分化过程与间质上皮化转换（MET）过程的关系.结果 传代至3～7代的hAMSCs均为细长形,但随着传代增加,细胞体积增大,细胞中CD44、CD73、CD90表达呈强阳性,但不表达CD45.经成骨或成脂诱导分化后3～4周,细胞中茜素红S（ARS）染色或油红O染色阳性.hAMSCs与hBCFs共培养后1周,hAMSCs形态由细长形变为类上皮细胞型,共培养组的部分细胞中CK19表达阳性,所有细胞α-SMA表达呈微弱阳性,而hAMSCs细胞培养组可见呈绿色荧光的α-SMA阳性细胞,但不表达CK19. 结论 通过体外共培养可诱导hAMSCs向人眼表细胞分化,其分化机制可能与MET过程相关.     

视黄酸联合视网膜细胞共培养对胚胎干细胞体外分化的诱导作用

目的 探讨胚胎干细胞(ESC)在视黄酸联合视网膜细胞共培养诱导条件下的分化特征。方法 将ESC自液氮中复苏、培养，传1代后进行拟胚体培养。将部分3．5d拟胚体离心重悬后加入含有视黄酸的24孔板中进行诱导，另一部分加入已经培养有视网膜混合细胞的培养瓶中，培养液中同时也加入视黄酸。视网膜混合细胞中仅加入视黄酸未加拟胚体作为对照。倒置相差显微镜下观察细胞在整个诱导过程中形态学的改变并使用免疫细胞化学检测诱导细胞中巢蛋白、胶质纤维酸性蛋白、广谱细胞角蛋白、微管相关蛋白-2、视紫质蛋白表达情况。结果 (1)形态学改变仅由视黄酸诱导，诱导细胞呈多种形态；在共培养条件下，绝大多数拟胚体分化出来的细胞形态非常单一，呈透明圆形。部分原贴壁的视网膜细胞出现了明显的网状结构。(2)免疫细胞化学显示两种诱导条件均可见大部分诱导细胞MAP-2阳性，并可见Nestin阳性细胞。共培养诱导尚可见GFAP阳性、Cytokeratin阳性和Rhodopsin阳性的细胞。结论 视黄酸可以诱导大部分细胞成为神经细胞，在视黄酸和与视网膜细胞共培养诱导条件下，可以获得更为纯化的形态一致的神经样细胞，部分诱导细胞表达视网膜细胞的特性。     

苯扎氯铵对人结膜上皮细胞黏蛋白MUC1表达的影响

目的 探讨防腐剂苯扎氯铵(BAC)对体外培养的人结膜上皮细胞黏蛋白MUC1表达的影响及毒性作用.方法 采用培养的3～5代人结膜上皮细胞,将不同浓度(0.0100%、0.0050%、0.0010%、0.0005%、0.0001%)的BAC单次作用于结膜上皮细胞15 min,分别于处理细胞后0、6、12、24、48、72 h提取RNA及蛋白,采用RT-PCR及Western杂交方法检测MUC1表达水平.结果 0.0100%和0.0050%BAC作用后12～72 h可观察到结膜上皮细胞MUC1基因表达下调,0.0010%和0.0005%BAC作用后,24～48 h出现MUC1基因表达的下调,72 h恢复.0.0100%BAC作用后6～72 h检测到MUC1蛋白表达下降,而0.0050%BAC作用后12～72 h MUC1蛋白表达下降,0.0010%BAC作用后72 h MUC1蛋白表达减少.结论 BAC具有下调人结膜上皮细胞MUC1表达的作用,其下调作用具有时间依赖性和剂量依赖性.     

孕晚期胎儿结膜上皮干细胞分布的研究

目的 利用免疫组织化学方法 ,对孕晚期胎儿结膜上皮干细胞进行研究,推测其分布规律.方法 死亡6h内的孕晚期胎儿8例(16眼),制备标本,苏木精-伊红染色确定各部位结膜位置,SP法免疫组织化学染色,观察各部位角蛋白K7、K19及黏蛋白5AC(MUC5AC)、增生细胞核抗原(PCNA)、P63蛋白的表达情况.结果 在睑缘皮肤黏膜交界处及角膜缘处,PCNA及P63表达相对明显,其余指标表达不明显;在睑结膜、穹隆部结膜、球结膜呈大致相反的分布规律.结论 在孕晚期胎儿睑缘皮肤黏膜交界处及角膜缘处存在较高增生活性的细胞,结膜上皮干细胞可能存在于这两个部位.     

“去上皮”羊膜对体外培养的结膜上皮细胞的作用

目的：探讨羊膜基底膜对结膜上皮细胞形态功能的影响。方法：把“去上皮”羊膜按照基底膜或实质面向上分为两组，接种结膜上皮细胞，同样条件下培养，取培养14天的两组标本进行组织学（HE和PAS染色），透射电镜，以及免疫组化检查（包括抗角蛋白和抗波形蛋白染色）。结果：细胞72小时内贴附在羊膜上，基底膜面接种的上皮细胞顶端见微绒毛，单层排列，细胞间有丰富桥粒，基底膜完整，可见半桥粒，抗角蛋白染色阳性，抗波形蛋白染色阴性；实质面接种的上皮细胞浆内有大量脂滴，表面未见微绒毛，细胞松散堆积，与羊膜连接不紧密，未见桥粒和半桥粒，抗角蛋白染色弱阳性，抗波形蛋白染色强阴性。结论：羊膜基底膜是结膜上皮细胞体外培养的良好载体，有利于上皮细胞生长，黏附，分化，并能减缓上皮细胞老化。     

Niflumic Acid Reduces Histamine-Induced MUC5AC Expression in Human Conjunctival Epithelial Cells

Background/Aims: Histamine is an important inflammatory mediator in allergic conjunctivitis. The aim of the present study was to explore whether histamine induced MUC5AC production in human conjunctival epithelial cells (HCECs), and to describe the relationship between human calcium-activated chloride channel 1 (hCLCA1) activity and MUC5AC expression. Methods: HCECs were isolated from human conjunctiva and cultured at an air-liquid interface. MUC5AC and hCLCA1 mRNA expression was examined by real-time PCR after exposure of the cells to histamine. MUC5AC secretion was quantitated by ELISA. hCLCA1 protein was detected by Western blot analysis. Results: Histamine upregulated MUC5AC expression and MUC5AC secretion in a dose-dependent manner. Histamine also upregulated hCLCA1 mRNA expression in a dose-dependent manner. Preincubation of histamine receptor 1 antagonist reduced histamine-induced MUC5AC and hCLCA1 mRNA expression. Niflumic acid, a chloride channel inhibitor, reduced histamine-induced MUC5ACand hCLCA1 expression. Conclusion: Histamine-induced MUC5AC production could be decreased by niflumic acid, a calcium-activated chloride channel inhibitor.     

Expansion of conjunctival epithelial progenitor cells on amniotic membrane

Amniotic membrane (AM) reconstructed human conjunctival surfaces recover a goblet cell density higher than normal. Cultured rabbit conjunctival epithelial cells (RCE) on AM preferentially exhibit non-goblet epithelial differentiation. It was thus wondered if conjunctival progenitor cells that might have been preserved during ex vivo expansion on AM can still differentiate into conjunctival non-goblet epithelial and goblet cells under the influence of mesenchymal cells. Fourteen day old AM cultures of RCE were subcutaneously implanted in Balb/c athymic mice for 11 days and processed for PAS staining and immunostaining with monoclonal antibodies to conjunctival goblet cell mucin (MUC5AC, AM3), glycocalyx (AMEM2), cornea specific cytokeratins K3 (AE5) and K12 (AK2) and basal cell specific cytokeratin K14. Cell cycle kinetics were measured by BrdU labelling for 1 or 7 days. The 7 day labelled RCE were chased for 14 days in the same primary culture. After subcutaneous implantation, conjunctival non-goblet epithelial cells increased stratification and formed occasional cysts. The resultant epithelial phenotype was conjunctival with many PAS-positive, MUC5AC-positive, and AM3-positive goblet cells, AMEM2-positive suprabasal and superficial cells, and K14-positive basal cells, but was not corneal (negative to AE5 and AK2 staining). Twenty four hr BrdU labelling showed a labelling index of 42.5%. A higher labelling index or 69% was noted after continuous BrdU labelling for 7 days. A large number of label retaining basal cells with a labelling index of 84% were noted following 14 days of chase. Conjunctival epithelial progenitor cells for goblet and non-goblet cell differentiation are preserved by AM in vitro as evidenced by being able to differentiate into goblet cells in a permissive stromal environment, and being slow-cycling, and label retaining. This information is useful for future ex vivo expansion of conjunctival epithelial stem cells for conjunctival surface reconstruction.     

Multi-layered culture of primary human conjunctival epithelial cells producing MUC5AC

The purpose of our study was to establish a system for culturing normal human conjunctival epithelial (NHCE) cells under serum-free culture conditions without compromising their ability to differentiate into a mucous epithelium. To this end, small pieces of normal conjunctiva were biopsied from patients undergoing cataract surgery. Obtained NHCE cells were cultured in bronchial epithelial growth medium (BEGM) under serum free culture conditions and passage 3 cells were air-lifted. Cultured NHCE cells displayed typical epithelial morphology. Expression of cytokeratin 19 and conjunctival epithelial specific carbohydrate residue were detected. Air-lifted NHCE cells demonstrated an increase in stratification and differentiation into goblet cells up to 3weeks under air-liquid interface (ALI) culture condition. NHCE cells expressed MUC1, MUC4, MUC16, and MUC5AC mRNA, and MUC5AC production and secretion increased in a time dependent manner after culture under ALI conditions. Exposure of cells to proinflammatory cytokines (TNF-alpha or IFN-gamma) resulted in upregulation of MUC1, MUC4, MUC16, and MUC5AC gene expression. In conclusion, differentiated NHCE cells showed features of a multi-layered conjunctival epithelium, including goblet cells, and retained functional characteristics similar to those seen in vivo. This cell culture system can better facilitate investigation of conjunctival epithelial cell biology and goblet cell differentiation.     

The in vitro and in vivo proliferative capacity of serum-free cultivated human conjunctival epithelial cells

PURPOSE: To investigate the in vitro and in vivo proliferative capacity of human conjunctival epithelial cells cultured in serum-free media, and to compare this with current methods that utilize serum-containing media and 3T3 feeder layers. METHODS: Human conjunctival epithelial cells were cultivated in serum-free media alone, serum-free media with a 3T3 feeder layer, and serum-containing media with a 3T3 feeder layer. The areas of outgrowth, colony-forming efficiencies and number of population doublings were compared. The in vivo proliferative potential was assessed by analyzing the number of cells generated by the implantation of cultured cells into athymic mice. Cultured cells were evaluated for the expression of cytokeratins K3, K4, K12, K19, as well as the gel-forming goblet cell mucin, MUC5AC. RESULTS: Cells cultivated in serum-free media, serum-free media and feeder cells, and serum-containing media and feeder cells achieved colony-forming efficiencies of 14.5 +/- 4.1%, 10.1 +/- 3.1%, and 20.4 +/- 6.7%, respectively, and number of population doublings of 24.8 +/- 4.3, 14.8 +/- 3.6, and 30.0 +/- 5.0, respectively. Nine-day old athymic mice conjunctival cysts derived from serum-free cultures comprised 1.29 x 10(6) +/- 0.46 x 10(6) cells, while cysts derived from serum-containing cultures comprised 1.30 x 10(6) +/- 0.53 x 10(6) cells. The degree of epithelial stratification was similar in both conditions. Serum-free cultivated conjunctival cells retained their in vivo characteristics and expressed K4, K19 and MUC5AC. The presence of MUC5AC mRNA in these cells was confirmed by RT-PCR. CONCLUSIONS: Conjunctival epithelial cells propagated in serum-free media demonstrated a similar in vivo proliferative capability, as compared to serum-containing media with 3T3 feeder cells. This has important clinical implications, as the serum-free ex vivo expansion of cells for clinical transplantation overcomes the problems associated with the use of animal serum and cells.     

Establishment of a cultivated human conjunctival epithelium as an alternative tissue source for autologous corneal epithelial transplantation

PURPOSE: The corneal epithelium is essential for maintaining corneal transparency, and efforts have been made to develop improved techniques for corneal epithelial transplantation in patients with total limbal failure. We evaluated the suitability of transplanted cultivated human conjunctival epithelium (HCjE) as a corneal epithelium replacement in rabbits with total corneal and limbal deficiency. METHODS: HCjE cells, cultivated on human amniotic membrane (AM) to confluence and exposed to an air-liquid interface (air-lifted), were transplanted onto denuded rabbit corneas and monitored for 2 weeks. The cultivated HCjE sheet and the engrafted epithelium were analyzed by immunohistochemistry and transmission electron microscopy (TEM). RESULTS: The transplanted HCjE remained transparent, smooth, and without epithelial defects during the follow-up period. Both the cultivated HCjE cells and the engrafted epithelium manifested five to six layers of stratified squamous epithelium similar in morphology to normal corneal epithelium. The basal cells expressed the putative stem cell markers (ABCG2 and P63) and hemidesmosome and desmosome component proteins. The cytokeratins (CK4, CK13, CK3, and CK12) and MUC4 were found in the engrafted epithelium. However, MUC5AC was not expressed. The results indicate that HCjE cultivated on AM has the potential to be used as an alternative corneal epithelium. CONCLUSIONS: The transplantation of cultivated HCjE sheets is a promising technique for the treatment of eyes with limbal failure.     

Decellularized porcine conjunctiva as an alternative substrate for tissue-engineered epithelialized conjunctiva

PurposeThe long-term success of visual rehabilitation in patients with severe conjunctival scarring is reliant on the reconstruction of the conjunctiva with a suitable substitute. The purpose of this study is the development and investigation of a re-epithelialized conjunctival substitute based on porcine decellularized conjunctiva (PDC).MethodsPDC was re-epithelialized either with pre-expanded human conjunctival epithelial cells (PDC + HCEC) or with a human conjunctival explant placed directly on PDC (PDC + HCEx). Histology and immunohistochemistry were performed to evaluate epithelial thickness, proliferation (Ki67), apoptosis (Caspase 3), goblet cells (MUC5AC), and progenitor cells (CK15, ΔNp63, ABCG2). The superior construct (PDC + HCEx) was transplanted into a conjunctival defect of a rabbit (n = 6). Lissamine green staining verified the epithelialization in vivo. Orbital tissue was exenterated on day 10 and processed for histological and immunohistochemical analysis to examine the engrafted PDC + HCEx. A human-specific antibody was used to detect the transplanted cells.ResultsFrom day-14 in vitro onward, a significantly thicker epithelium and greater number of cells expressing Ki67, CK15, ΔNp63, and ABCG2 were noted for PDC + HCEx versus PDC + HCEC. MUC5AC-positive cells were found only in PDC + HCEx. The PDC + HCEx-grafted rabbit conjunctivas were lissamine-negative during the evaluation period, indicating epithelial integrity. Engrafted PDC + HCEx showed preserved progenitor cell properties and an increased number of goblet cells comparable to those of native conjunctiva.ConclusionPlacing and culturing a human conjunctival explant directly on PDC (PDC + HCEx) enables the generation of a stable, stratified, goblet cell-rich construct that could provide a promising alternative conjunctival substitute for patients with extensive conjunctival stem and goblet cell loss.     

Development of a conjunctival epithelial equivalent with improved proliferative properties using a multistep serum-free culture system

PURPOSE: To investigate the use of a multistep serum-free culture system in developing a conjunctival epithelial equivalent with improved in vitro and in vivo proliferative properties and to evaluate the effect of serum supplementation and culture conditions on the proliferative capacity of these cells. METHODS: Conjunctival epithelial cells were cultivated on human amniotic membrane (HAM) in a multistep serum-free culture system, under submerged and air-lifted conditions. The bromodeoxyuridine (BrdU) ELISA proliferation assay, colony-forming efficiency (CFE), and number of cell generations were compared with those in serum-containing medium. The in vivo proliferative capability of the tissue-constructs were evaluated by xenotransplantation to SCID mice. Cultured cells were evaluated for the expression of keratin-4, -19, and -3, as well as MUC5AC goblet cell mucin. RESULTS: The epithelial cells cultivated in serum-free medium (BrdU absorbance, 1.91 +/- 0.08; cell generations, 25.6 +/- 4.5) were more proliferative than those cultivated in serum-containing medium (BrdU absorbance, 1.06 +/- 0.08; cell generations, 12.1 +/- 3.0). The serum-free-derived epithelial equivalents demonstrated a significant increase in proliferation and stratification after transplantation. Cells that were air lifted for 6 and 12 days had a reduced proliferative capacity in vitro and in vivo compared with submerged cultures. Cultured cells expressed keratin-4 and -19, and MUC5AC mRNA was detected by RT-PCR. Electron microscopy demonstrated a basal lamina with numerous hemidesmosomes. CONCLUSIONS: This is a multistep serum-free culture system for developing a conjunctival epithelial equivalent with improved proliferative and structural properties, which are crucial for enhancing graft survival and regeneration of the conjunctival surface after clinical transplantation.     

MUC19 expression in human ocular surface and lacrimal gland and its alteration in Sjögren syndrome patients

This study investigated the expression of MUC19, a newly discovered gel-forming mucin gene, in normal human lacrimal functional unit components and its alteration in Sj?gren syndrome patients. Real-time PCR and immunohistochemistry were performed to determine the expression of MUC19 and MUC5AC in human cornea, conjunctiva, and lacrimal gland tissues. Conjunctival impression cytology specimens were collected from normal control subjects and Sj?gren syndrome patients for Real-time PCR, PAS staining, and immunohistochemistry assays. In addition, conjunctiva biopsy specimens from both groups were examined for the expression differences of MUC19 and MUC5AC at both mRNA and protein level. The MUC19 mRNA was found to be present in cornea, conjunctiva and lacrimal gland tissues. The immunohistochemical staining of mucins showed that MUC19 was expressed in epithelial cells from corneal, conjunctival, and lacrimal gland tissues. In contrast, MUC5AC mRNA was only present in conjunctiva and lacrimal gland tissues, but not in cornea. Immunostaining demonstrates the co-staining of MUC19 and MUC5AC in conjunctival goblet cells. Consistent with the significant decrease of mucous secretion, both MUC19 and MUC5AC were decreased in conjunctiva of Sj?gren syndrome patients compared to normal subjects. Considering the contribution of gel-forming mucins to the homeostasis of the ocular surface, the decreased expression of MUC19 and MUC5AC in Sj?gren syndrome patients suggested that these mucins may be involved in the disruption of the ocular surface homeostasis in this disease.     

神经生长因子对人结膜杯状细胞增殖与黏蛋白基因表达的影响

目的探讨神经生长因子（NGF）对体外培养人结膜杯状细胞增殖与黏蛋白MUC5AC基因表达的影响。方法利用抗NGF高亲和力受体（TrkA）的单克隆抗体进行免疫组织化学染色,初步检测杯状细胞膜表面是否存在此受体。在传代的人结膜杯状细胞中加入含20、40、60及80ng／ml NGF的RPMI-1640培养液,加入不含NGF的培养液作为阴性对照。于加药后1、3、5d分别用四甲基偶氮唑盐（MTT）法观察细胞增殖情况。加入含80ng／ml NGF的培养液后第5天,通过逆转录聚合酶链反应（RT-PCR）方法检测NGF对杯状细胞中MUCSAC基因表达的影响。结果人结膜杯状细胞膜表面存在NGF高亲和力受体（TrkA）。加入NGF后不同时间（F=12．766,P=0．000）、不同浓度组（F=892．406,P=0．000）之间的吸光度值比较,差异有统计学意义,时间和浓度这两个因素之间无交互作用（F=1．778,P=0．095）。NGF组MUC5AC的mRNA表达量明显高于对照组（t=4．963,P=0．000）。结论体外培养的人结膜杯状细胞膜表面存在NGF高亲和力受体;NGF有促进杯状细胞增殖的作用,并随浓度增加和时间的延长而促增殖作用增强;NGF能够促进杯状细胞黏蛋白MUC5AC基因表达。