Amniotic membrane covering promotes healing of cornea epithelium and improves visual acuity after debridement for fungal keratitis

AIM:To investigate the effect of amniotic membrane covering(AMC) on the healing of cornea epithelium and visual acuity for fungal keratitis after debridement.METHODS:Twenty fungal keratitis patients were divided into two groups randomly, the AMC group and the control group, ten patients each group. Both debridement of the infected cornea tissue and standard anti-fungus drugs treatments were given to every patients, monolayer amniotic membrane were sutured to the surface of the entire cornea and bulbar conjunctiva with 10-0 nylon suture for patients in the AMC group.The diameter of the ulcer was determined with slit lamp microscope and the depth of the infiltration was determined with anterior segment optical coherence tomography. Uncorrected visual acuity(UCVA) was tested before surgery and three month after healing of the epithelial layer. The healing time of the cornea epithelium, visual acuity(VA) was compared between the two groups using t- test.RESULTS:There was no statistical difference of the diameter of the ulcer, depth of the infiltration, height of the hypopyon and VA between the two groups beforesurgery(P 】0.05). The average healing time of the AMC group was 6.89 ±2.98 d, which was statistically shorter than that of the control group(10.23±2.78d)(P 【0.05).The average UCVA of the AMC group was 0.138 ±0.083,which was statistically better than that of the control group(0.053±0.068)(P 【0.05).CONCLUSION:AMC surgery could promote healing of cornea epithelium after debridement for fungal keratitis and lead to better VA outcome.

Comment on amniotic membrane covering promotes healing of cornea epithelium and improves visual acuity after debridement for fungal keratitis

【正】Dear Editor,We congratulate Zeng et al[1]for their study entitled"Amniotic membrane covering promotes healing of cornea epithelium and improves visual acuity after debridement for fungal keratitis".The authors endeavored to present an alternative method for ophthalmologists in the treatment of a challenging case.We would like to express our reservations and ask for the attitudes of the authors about

Regulation of interleukin 33/ST2 signaling of human corneal epithelium in allergic diseases

AIM:To identify the function of ST2 and explore the role of IL-33/ST2 signaling in regulating the pro-allergic cytokine production in human corneal epithelial cells (HCECs). METHODS:Human corneal tissues and cultured primary HCECs were treated with IL-33 in different concentrations without or with different inhibitors to evaluate the expression, location and signaling pathways of ST2 in regulating production of pro-allergic cytokine and chemokine. The expression of mRNA was determined by reverse transcription and real time PCR, and protein production was measured by enzyme-linked immunosorbent assay (ELISA), immunohistochemical and immunofluorescent staining. ST2 protein was detected in donor corneal epithelium, and ST2 signal was enhanced by exposure to IL-33. ·RESULTS:IL-33 significantly stimulated production of pro-allergic cytokines thymic stromal lymphopoietin (TSLP) and chemokine (CCL2, CCL20, CCL22) in HCECs at both mRNA and protein levels. These stimulated productions of pro-allergic mediators by IL-33 were blocked by ST2 antibody or soluble ST2 protein(P 【0.05). Interestingly, the IκB-α inhibitor BAY11-7082 or NF-κB activation inhibitor quinazoline blocked NF-κB p65 protein nuclear translocation, and also suppressed the productions of these pro-allergic cytokines and chemokine induced by IL-33. CONCLUSION:These findings demonstrate that IL-33/ ST2 signaling plays an important role in regulating IL-33 induced pro-allergic responses. IL-33 and ST2 could become novel molecular targets for the intervention ofallergic diseases in ocular surface.

Hyperosmolarity disrupts tight junction via TNF-α/MMP pathway in primary human corneal epithelial cells

AIM:To investigate the mechanism of the tight junction(TJ) disruption and the association between tumor necrosis factor(TNF)-α and matrix metalloproteinase(MMPs) under hyperosmotic condition in primary human corneal epithelial cells(HCECs).METHODS:The cultured HCECs were exposed to media which adding sodium chloride(Na Cl) for hyperosmolar stress or adding rh-TNF-α(10 ng/m L). NF-κB inhibitor(5 μmol/L) or GM-6001(potent and broad spectrum MMP inhibitor, 20 μmol/L)was added 1 h before that treatment. The integrity of TJ proteins was determined by immunofluorescent(IF) staining. The m RNA levels of TNF-α and MMPs were evaluated by quantitative reverse transcription polymerase chain reaction(RT-q PCR) and the protein expression by enzyme-linked immunosorbent assay(ELISA).RESULTS:TJ proteins ZO-1 and Occludin were disrupted in primary HCECs exposed to hyperosmotic medium. The m RNA expression and protein production of TNF-α increased significantly in hyperosmotic media at 500 m Os M. TNF-α mediated the expression and production of MMP-1, MMP-13, MMP-9, and MMP-3 stimulated by hyperosmotic stress. The production of MMPs in hyperosmolar media were increased through the increase of TNF-α. GM-6001 prevent the destruction of ZO-1 and Occludin in hyperosmolar stress and rh-TNF-α treated medium. TNF-α induced activation of MMPs was involved in the TJ disruption by hyperosmolarity.CONCLUSION:TJ proteins ZO-1 and Occludin are disrupted by hyperosmolar stress and TNF-α, but protected by MMP inhibitor(GM-6001). It suggests that TNF-α/MMP pathway mediates the TJ disruption in primary HCECs exposed to hyperosmotic stress.

Evaluating alternative stem cell hypotheses for adult corneal epithelial maintenance

In this review we evaluate evidence for three different hypotheses that explain how the corneal epithelium is maintained. The limbal epithelial stem cell(LESC)hypothesis is most widely accepted. This proposes that stem cells in the basal layer of the limbal epithelium, at the periphery of the cornea, maintain themselves and also produce transient(or transit) amplifying cells(TACs). TACs then move centripetally to the centre of the cornea in the basal layer of the corneal epithelium and also replenish cells in the overlying suprabasal layers. The LESCs maintain the corneal epithelium during normal homeostasis and become more active to repair significant wounds. Second, the corneal epithelial stem cell(CESC) hypothesis postulates that, during normal homeostasis, stem cells distributed throughout the basal corneal epithelium, maintain the tissue. According to this hypothesis, LESCs are present in the limbus but are only active during wound healing. We also consider a third possibility, that the corneal epithelium is maintained during normal homeostasis by proliferation of basal corneal epithelial cells without any input from stem cells. After reviewing the published evidence, we conclude that the LESC and CESC hypotheses are consistent with more of the evidence than the third hypothesis, so we do not consider this further. The LESC and CESC hypotheses each have difficulty accounting for one main type of evidence so we evaluate the two key lines of evidence that discriminate between them. Finally, we discuss how lineage-tracing experiments have begun to resolve the debate in favour of the LESC hypothesis. Nevertheless, it also seems likely that some basal corneal epithelial cells can act as long-term progenitors if limbal stem cell function is compromised. Thus, this aspect of the CESC hypothesis may have a lasting impact on our understanding of corneal epithelial maintenance, even if it is eventually shown that stem cells are restricted to the limbus as proposed by the LESC hypothesis.

Effect of calcium on the proliferation and differen-tiation of murine corneal epithelial cells in vitro

AIM: To investigate the effect of calcium on the proliferation and differentiation of murine corneal epithelial cells in vitra METHODS: Mouse corneal epithelial cells were cultured in serum-free low-Ca(2+) medium (KSFM) and KSFM supplemented with 0.9mmol/L Ca(2+). Population doublings (PDs) were determined. The expression of corneal epithelial cell markets p63, keratin 19 (K19) and involucrin was investigated by RT-PCR analysis and semiquantitative analysis of Western blotting. RESULTS: Cells in KSFM were stably subcultured over 25 passages, however, none of the cell lines could pass P4 in KSFM with Ca(2+). In KSFM, the cells was were homogeneous and small cells with typical cobblestone appearance; and expressed p63, K19 and involucrin. After medium was supplemented with calcium, cells became a heterogeneous mix of small and large cells. Furthermore, semiquantitative analysis of Western blotting showed that the Expression of involucrin was increased significantly. CONCLUSION: Calcium has the effect of inhibiting proliferation and triggering differentiation on mouse corneal epithelial cells.

Effect of hypoxia on the proliferation of murine cornea limbal epithelial progenitor cells in vitro

AIM: To investigate the effect of hypoxia on the proliferation of mouse corneal epithelial cells in vitro. METHODS:Mouse corneal epithelial cells(MCEs) were cultured in normoxia (210mL/L O(2) and 50mL/L CO(2)) and hypoxia (20mL/L O(2) and 50mL/L CO(2)), respectively. Colony forming efficiency (CFE) and cell proliferation were determined. The expression of corneal epithelial progenitor cell marker p63 and K19 was Investigated by immunostaining. RESULTS: Normoxic colonies were smaller compared with colonies formed in hypoxia. CFE was (12.50 +/- 1.50)% in hypoxic cultures, which was similar compared with normoxia cultures [(11.13 +/- 1.86)%, P > 0.05)]. Cell proliferation was enhanced in hypoxia. Progenitor markers p63 and K19 were expressed in most cells under both normoxic and hypoxic conditions. O CONCLUSION: Murine limbal epithelial progenitor cells can be efficiently expanded in hypoxic conditions.

去上皮快速角膜胶原交联术对近视眼角膜屈光术后角膜扩张的疗效评估

目的探讨去上皮快速角膜胶原交联术(CXL)治疗近视角膜屈光手术后角膜扩张的有效性及安全性。方法采用系列病例观察研究方法,纳入2016年1月至2018年12月于陆军军医大学第一附属医院眼科确诊的近视角膜屈光手术后角膜扩张的患者12例22眼,患者均行去上皮快速CXL。分别于术前及术后1周、1个月、3个月、6个月、12个月,检测受试者裸眼视力(UCVA)、最佳矫正视力(BCVA);采用Topcon全自动综合验光仪测量球镜度、柱镜度、等效球镜度;采用Sirius眼前节分析仪测量角膜前表面最大曲率(Kmax)、前表面平均曲率(Km)、后表面Km、前表面对称指数(SIf)、角膜后表面对称指数(SIb)、最薄点角膜厚度(TCT)、总像差、总高阶像差、慧差、三叶草像差、球差;采用眼前节光学相干断层扫描仪测量角膜交联分界线深度;采用非接触眼压计测量眼压;采用角膜内皮细胞密度测量仪测量角膜内皮细胞密度;采用裂隙灯显微镜观察角膜炎性反应及角膜上皮下雾状混浊(haze)情况。结果所有受试者中接受小切口角膜基质透镜取出术者2例3眼,占13.64%,准分子激光角膜厚位磨镶术者10例19眼,占86.36%。去上皮CXL术后12个月UCVA(LogMAR)、BCVA(LogMAR)、柱镜度及等效球镜度分别为0.45±0.31、0.12±0.15、(-2.11±1.67)D和(-3.12±2.31)D,明显优于术前的0.61±0.42、0.24±0.23、(-2.83±2.39)D、(-3.60±2.66)D,差异均有统计学意义(t=4.054、4.956、-3.728、-2.742,均P<0.05)。术后12个月角膜前表面Kmax、前表面Km、SIf分别为(48.37±5.80)、(41.49±3.04)、(5.36±4.07)D,明显低于术前的(49.61±5.97)、(41.66±2.97)、(5.85±4.18)D,差异均有统计学意义(t=5.949、2.278、2.719,均P<0.05)。术前与术后12个月球镜度、后表面Km、SIb、TCT、总像差、总高阶像差、慧差、三叶草像差、球差、眼压、角膜内皮细胞密度比较,差异均无统计学意义(均P>0.05)。术后1个月4例8眼出现0.5~2级haze,经过醋酸泼尼松龙滴眼液点眼冲击治疗后,术后3个月复查haze均减退或消失,未出现UCVA及BCVA下降。术后1个月6例11眼在角膜基质平均深度(285.40±51.61)μm处查见明显角膜交联分界线。结论去上皮快速CXL能显著提高近视屈光术后角膜扩张患者术后视力,减少角膜散光,降低角膜曲率,有效阻止角膜扩张进展,具有良好的有效性及安全性。

毛囊bulge细胞在角膜缘基质诱导下向角膜上皮细胞分化的初步研究

目的探讨体外培养的毛囊bulge细胞向角膜上皮细胞分化的可能性。方法体外分离培养毛囊bulge细胞,然后在transwell中与角膜缘基质细胞共培养诱导分化,观察毛囊bulge细胞的分化情况,免疫组化检测K12及K19表达的变化。结果体外培养的毛囊bulge细胞保持高增殖,低分化状态。经过2周左右的共培养,毛囊bulge细胞逐渐分化,部分细胞K12表达阳性。结论角膜缘基质细胞可以诱导毛囊bulge细胞向角膜上皮细胞分化。

光学相干断层扫描血管造影技术分析先天性上睑下垂角膜上皮厚度

目的利用光学相干断层扫描血管造影(OCTA)系统分析先天性上睑下垂患者角膜上皮各区域厚度和角膜全层厚度改变。方法对12例先天肌源性轻(≤2 mm)、中度(3～4 mm)上睑下垂患者(实验组)和随机选12位健康受试者的角膜上皮和角膜全层厚度的OCTA图谱进行分析,对双眼进行3次OCTA全层扫描,将所得的左眼数据翻转,并将双眼数据叠加,选取平均值,借助系统软件,用两种分区方式将角膜分别划分为25和17个区域。从测得的数值里选取最高信号强度指数的扫描图以进行分析。结果实验组角膜上皮的厚度最小值较薄,为(49.22±3.14)μm(P=0.032),最小值与最大值差值较大为(-10.40±1.63)μm(P=0.06),这一差异来源于角膜上皮上方(S)、鼻上(SN)、颞上(ST)角膜上皮变薄(P<0.05)。角膜全层厚度的最小值则与对照组差异无统计学意义(P>0.05)。结论上睑下垂对眼球表面的机械效应可能重塑角膜上皮,使得角膜上皮层和角膜全层部分区域变薄,且可利用OCTA技术检测出来。

不同培养方法对组织工程化小鼠角膜上皮构建影响的比较研究(英文)

目的:探讨滋养细胞在小鼠角膜上皮细胞复层化中的作用,并研究构建组织工程化角膜上皮的理想方法。方法:在Transwell气液界面培养系统中,分别采用接触滋养层培养法、分离滋养层培养法、复式滋养层培养法以及无滋养层培养法等4种方法进行组织工程化小鼠角膜上皮的重建。HE染色进行组织学观察,免疫荧光法检测p63、角蛋白19以及involucrin的表达。结果:接触滋养层培养法、分离滋养层培养法中,角膜上皮复层化为3-4层;复式滋养层培养法中,角膜上皮复层化达5-7层;而无滋养层培养法中,复层化仅为2-3层。复式滋养层培养法构建的小鼠角膜上皮,基底细胞层和基底细胞上层表达祖细胞标记p63和角蛋白19,角膜上皮全层均表达分化标记involucrin。结论:复式滋养层培养法为构建组织工程化小鼠角膜上皮的理想方法。

角膜酸烧伤后新生血管形成与上皮缺损和白细胞渗出关系的初步研究

目的 研究大鼠角膜酸烧伤后新生血管形成与角膜上皮缺损和白细胞渗出的关系。方法 建立轻、中、重度大鼠角膜酸烧伤新生血管模型,裂隙灯下动态观察角膜上皮缺损直径及新生血管形成情况,计算新生血管的面积与角膜面积的比例;各组分别于烧伤后不同时间点取眼球行HE染色,光镜下计算角膜白细胞浸润的数量。采用Pearson相关分析法分析新生血管形成与上皮缺损和白细胞浸润的关系。结果 角膜不同程度酸烧伤后上皮愈合速度不同,损害程度越重上皮愈合越慢。烧伤后可引起白细胞渗出,烧伤程度越重白细胞渗出越明显,有显著的差异(P <0 0 1)。轻度酸烧伤角膜未见新生血管形成,中重度酸烧伤在伤后第3天可观察到新生血管形成。不同程度酸烧伤新生血管形成有显著的差异(P <0 0 1)。酸烧伤后新生血管形成与上皮缺损(r =0 3 3 0 0 ,P <0 0 1)及白细胞渗出(r =0 1779,P <0 0 5 )有显著的相关性。结论 大鼠角膜酸烧伤后CNV的发生与烧伤程度与角膜上皮缺损和白细胞渗出有密切关系。

胎牛血清对小鼠角膜上皮细胞生长和分化的影响(英文)

目的:探讨胎牛血清对小鼠角膜上皮细胞生长和分化的影响。方法:分别在无血清低钙培养基(KSFM)和含100mL/L胎牛血清的KSFM中培养小鼠角膜上皮细胞。比较两种培养基中角膜上皮细胞的群体倍增(PD)。通过RT-PCR和Western blotting方法检测p63、角蛋白19以及involucrin的表达。结果:在KSFM中,小鼠角膜上皮细胞可稳定传代超过25代,但在含胎牛血清的培养基中,细胞仅能存活3代。在KSFM中,培养细胞呈典型铺路石样外观,RT-PCR和Western blotting结果显示细胞表达p63、角蛋白19以及in-volucrin。当培养基中加入胎牛血清后,细胞形态明显增大,呈扁平状; involucrin表达显著增强。结论:胎牛血清可抑制小鼠角膜上皮细胞增生、诱导其分化。

诱导人脐带间充质干细胞分化为角膜上皮样细胞的研究

背景眼表疾病导致的角膜盲已成为全球致盲性角膜疾病中的主要原因之一。随着组织工程技术的发展和进步，组织工程角膜为眼表疾病的治疗开辟了新的途径。目的观察体外培养的人脐带间充质干细胞（UC—MSCs）移植到兔角膜基质后的分化发育情况，探讨人UC-MSCs分化为角膜上皮细胞以及治疗兔角膜损伤的可行性。方法获取人脐带组织，采用Ⅳ型胶原酶消化法分离纯化人UC—MSCs并传代，取第3代细胞用于扩增和实验。流式细胞仪检测细胞的免疫表型及诱导成骨分化鉴定。24只新西兰大白兔按随机数字表法随机分为2个组，将人UC—MSCs接种于去上皮的猪角膜基质上，培养4d后行实验组兔左眼板层角膜移植；对照组以相同的手术方法单纯移植去上皮猪角膜基质。术后对角膜定期行活体激光共焦显微镜检查，并分别于术后2、4、8周摘除各组实验眼行组织病理学和免疫荧光检查，评价移植到兔角膜基质的人UC-MSCs的存活、分化以及移植局部的反应等；应用免疫荧光技术检测移植后角膜上皮细胞中角蛋白3（CK3）、CKl2以及转运蛋白G超家族成员（ABCG2）的表达。结果消化培养的人UC—MSCs呈圆形，细胞胞体较大，贴壁后细胞呈长梭形。培养获得的人UC—MSCs的细胞表型CD105^＋／CD29^＋／CD44^＋／CD34^-／CD45^-，并可诱导分化为成骨细胞。实验组人ISC—MSCs接种到去上皮猪角膜基质后贴附良好、生长迅速，术后植片在植床上存活良好，种植了人UC-MSCs的去上皮猪角膜移植到兔眼，可见实验组受体角膜较对照组透明，未见明显新生血管，在活体共焦显微镜下可见新生的角膜上皮样细胞，未发生免疫排斥反应。免疫荧光检测可见在重建的角膜上皮层检测到CK3及CKl2的阳性表达，而未见ABCG2的表达。结论将种植了人UC—MSCs的猪角膜基质移植到损伤的兔角膜后，人UC—MSCs可以存活、增生并分化为角膜上皮样细胞，可用于修复甚至重建损伤的角膜表层。

培养的角膜缘上皮细胞增殖能力的动态研究

目的 探讨组织块培养角膜缘上皮细胞在不同时间以及第 2、3代培养的角膜缘上皮细胞早期 (6～ 8d)的增殖能力。方法 应用组织块培养法培养角膜缘上皮细胞 ,在d8、12、16、2 0、2 4用流式细胞仪检测其细胞周期 ,同时对组织块培养 16d的角膜缘上皮细胞进行传代 ,用同样方法检测第 2、3代细胞出现汇合时 (6～ 8d)的细胞周期。结果 组织块培养角膜缘上皮细胞增殖指数 (ProliferativeIndex ,PI)在高水平上呈下降趋势 (分别为 31 2 %、2 0 1%、17 6 %、15 6 %、13 4 % ) ;2、3代培养细胞早期的增殖指数较高 (分别为 30 3%、2 6 % )。结论 组织块培养的角膜缘上皮细胞增殖能力在 8～ 2 4d内在高水平上呈下降趋势 ,第 2、3代培养的细胞在培养早期的增殖能力也较高 。

复式滋养层培养法构建组织工程化小鼠角膜上皮的实验研究

目的探讨构建组织工程化小鼠角膜上皮的理想方法。方法在Transwell气液界面培养系统中,利用复式滋养层培养法构建组织工程化小鼠角膜上皮,HE染色进行组织学观察,分别利用RT-PCR、Western blot以及免疫荧光法检测上皮祖细胞标记p63、角蛋白19(K19)以及分化标记被膜素(involucrin)的表达。结果复式滋养层培养法构建可成功小鼠角膜上皮,上皮层数达5～7层。与RT-PCR和Western blot结果一致,免疫荧光结果显示上皮基底细胞层和基底细胞上层表达p63和K19,角膜上皮全层均表达involucrin。结论复式滋养层培养法可保持培养细胞的干细胞性质,是构建组织工程化小鼠角膜上皮的理想方法。

脱细胞猪角膜基质体外支持角膜上皮和基质细胞的生长(英文)

目的：探讨脱细胞猪角膜基质体外能否支持兔角膜细胞的生长。方法：体外培养兔角膜上皮细胞和基质细胞,并接种到制备的脱细胞猪角膜基质上,倒置相差显微镜和组织学观察细胞生长情况。结果：上皮细胞能在脱细胞猪角膜基质上贴附生长,10d时可形成2～3层的复层结构。基质细胞在脱细胞猪角膜基质上贴附生长后可向材料深层迁徙。结论：制备的脱细胞猪角膜基质体外可支持兔角膜上皮细胞和基质细胞的生长。

4％可卡因溶液对角膜毒性作用的实验研究

兔角膜表面置圆形直径９ｍｍ４％可卡因滤纸片１０、９０秒，擦去松解上皮，保留角膜缘内２ｍｍ完整上皮区。术后不同时间，光镜及电镜观察４％可卡因对角膜的毒性作用。结果：兔角膜上皮修复与滤纸片放置时间无关，角膜组织水肿恢复正常时间与滤纸片放置时间呈正相关。组织学上有明显差异，１０秒组角膜各层超微结构无明显改变；９０秒组角膜各层尤以内皮细胞层超微结构有明显改变。提示４％可卡因溶液长时间应用对角膜尤其是内皮细胞层具有毒性作用。

ROCK抑制剂Y-27632在角膜保存时对角膜缘干细胞活性的促进作用

背景 角膜缘干细胞（LSCs）对维持角膜上皮的稳定和角膜组织透明性有重要作用.Rho关联卷曲螺旋蛋白激酶（ROCK）抑制剂Y-27632能够促进人胚胎干细胞、角质上皮细胞等的增生,减少细胞凋亡.目的 探讨Y-27632对离体兔角膜保存和体外LSCs扩增能力的影响. 方法 在细胞培养基MEM中加入质量分数12.5％硫酸软骨素、质量分数10.0％低分子右旋糖酐、20.0 mg/L地塞米松、100 mg/L妥布霉素注射液、9.5 g/L Hepes,使用时添加0.375 mg/L L-谷氨酰胺,制备成角膜活性保存液.将新西兰大白兔的角膜组织片分别置于含或不含Y-27632的角膜活性保存液中保存4、7、14d,采用质量分数0.25％锥虫蓝和质量分数0.2％茜素红染色法观察角膜内皮细胞密度及形态,采用Giemsa染色法并使用Image J图像分析软件计数克隆球数量和LSCs活性,计算LSCs存活率和克隆形成效率. 结果 角膜片保存4d时,Y-27632保存液组和单纯角膜中期保存液组角膜内皮细胞形态无明显差别,角膜片保存7d时,Y-27632保存液组角膜内皮细胞形态仍保持规则的六边形,而单纯角膜中期保存液组角膜内皮细胞的细胞膜轻微皱缩,少数细胞体积变大.角膜片保存14 d时单纯角膜中期保存液组角膜内皮细胞可见较多的茜素红斑.单纯角膜中期保存液组角膜内皮细胞计数为（2 262±75）/mm2,Y-27632保存液组角膜内皮细胞计数为（2 425 ±95）/mm2,差异有统计学意义（P＜0.001）.新鲜角膜片和角膜片保存4d时,Y-27632保存液组和单纯角膜中期保存液组LSCs的克隆球均较大,其内细胞数较多,而保存7d和14 d时,与Y-27632保存液组比较,单纯角膜中期保存液组LSCs克隆球直径明显缩小.新鲜分离的角膜片和角膜片保存4d时,Y-27632保存液组与单纯角膜中期保存液组LSCs的克隆形成率和角膜上皮细胞存活率的差异均无统计学意义（均P＞0.05）;角膜片保存7d和14d时,角膜缘上皮细胞的活性率分别为（73.00±2.12）％和（56.00±0.71）％,明显高于单纯角膜中期培养液组的（66.00±4.00）％和（49.00±0.71）％,差异均有统计学意义（t=3.098,P=0.018;t=9.798,P=0.000）;角膜片保存7d和14 d时,Y-27632保存液组与单纯角膜中期保存液组LSCs的克隆形成效率分别为（11.05±0.21）％和（3.10±1.97）％,明显高于单纯角膜中期保存液组中的（2.05±1.20）％和（0.40±0.14）％,差异均有统计学意义（t=18.107,P=0.000;t=3.184,P=0.017）.结论角膜保存液中添加Y-27632可明显提高离体角膜保存的效果,同时可维持LSCs的活性及克隆形成能力.Y-27632可作为角膜保存液的有效添加成分。

丝素蛋白和乳酸-己内酯共聚物纳米纤维支架构建组织工程化角膜上皮的实验研究

目的评估兔角膜缘上皮细胞在丝素蛋白(SF)和乳酸-己内酯共聚物(PLCL)复合纳米纤维薄膜上的黏附、增殖和分化情况,探讨SF-PLCL复合纳米纤维薄膜作为组织工程角膜上皮支架材料的可行性。方法利用六氟异丙醇(HFIP)作为溶剂,配制浓度为8%的SF和PLCL共混物(质量比25∶75)的纺丝溶液。通过静电纺丝技术制备纳米纤维膜。将体外培养的第一代兔角膜缘上皮细胞种植于SF-PLCL纳米纤维薄膜上进行复合培养。利用扫描电镜观察SF-PLCL纳米纤维薄膜的表面形态及细胞材料复合物的表面形貌;利用免疫荧光染色分析角膜上皮细胞标记物K3的表达情况;通过细胞活力检测观察细胞在材料上的生长分布规律。结果 SF-PLCL纳米纤维薄膜的平均纤维直径为(715±186)nm,材料具有较好的透明度。扫描电镜显示角膜缘上皮细胞在材料上保持正常上皮细胞的铺路石样形态,黏附生长良好,细胞表面布满微绒毛,细胞间紧密联接;免疫荧光染色表明角膜缘上皮细胞在材料上可分化为成熟的角膜上皮细胞。细胞活力检测证实角膜缘上皮细胞在材料上生长良好,均匀分布,形成多层层化结构。结论角膜上皮细胞与SF-PLCL纳米纤维膜的生物相容性良好,SF-PLCL纳米纤维膜是一种潜在、合适的组织工程角膜上皮支架材料。