The roles of surfactant protein D during Aspergillus fumigatus infection in human corneal epithelial cells

AIM: To investigate roles of surfactant protein D (SP-D) and relative cytokines in human corneal epithelial (HCE) cells Exposed to aspergillus fumigatus (AF) antigens. METHODS: HCE cells cultured 47 in vitro with AF antigens and sampled at 0, 0.5, 1 hour, 2, 4, 6 and 8 hours. The Expression of SP-D mRNA was evaluated by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR).The expression of SP-D protein was shown by ELISA and immunocytochemistry SP methods. The expression of NF-kappa B and relative downstream cytokines such as TNF-alpha, IL-1 beta, IL-8 and IL-10 in supernatant fluid were measured by ELISA. RESULTS: SP-D mRNA and protein were detected in untreated HCE cells. The expression of SP-D and the relative downstream cytokines rose after being stimulated with AF antigens. SP-D mRNA began to rise at 0.5 hour and the most significantly peak was in 2 hours. The protein of SP-D in supernatant fluid had the same trend with mRNA. Immunocytochemistry of SP-D showed positive expression and gradually increased to 6 hours, and then the expression began to decline. NF-kappa B was activated after treated by AF antigens and the changes had correlation with SP-D. TNF-alpha, IL-1 beta, IL-8 and IL-10 began to rise after given AF antigens 1 hour and were 1.82, 1.43, 1.12 and 1.28 times higher than the untreated HCE cells separately. The expression of TNF-alpha and IL-1 beta reached the peak at 2 hours, separately 2.80 and 2.86 times than the untreated. The expression of IL-8 and IL-10 gradually increased with a time-dependent manner. ' CONCLUSION: HCE cells exists SP-D and it may play a significant role in pathogenesis of keratomycosis. AF may induce human corneal epithelial cells to express inflammatory cytokines via SP-D and NF-kappa B pathway. SP-D possibly mediates the recognition to AF mycelium.

Effects of exogenous recombinant human bone morphogenic protein-7 on the corneal epithelial mesenchymal transition and fibrosis

AIM：To evaluate the effect of exogenous recombinant human bone morphogenic protein-7（rhBMP-7）on transforming growth factor-β（TGF-β）-induced epithelial mesenchymal cell transition（EMT）and assessed its antifibrotic effect via topical application.METHODS：The cytotoxic effect of rhBMP-7 was evaluated and the EMT of human corneal epithelial cells（HECEs）was induced by TGF-β. HECEs were then cultured in the presence of rhBMP-7 and/or hyaluronic acid（HA）. EMT markers,fibronectin,E-cadherin,α-smooth muscle actin（α-SMA）,and matrix metaloproteinase-9（MMP-9）,were evaluated. The level of corneal fibrosis and the reepithelization rate were evaluated using a rabbit keratectomy model. Expression of α-SMA in keratocytes were quantified following treatment with different concentrations of rhBMP-7.RESULTS：Treatment with rhBMP-7 attenuated TGF-β-induced EMT in HECEs. It significantly attenuated fibronectin secretion（31.6%; P〈0.05）,the α-SMA protein level（72.2%; P〈0.01）,and MMP-9 expression（23.6%,P〈0.05）in HECEs compared with cells grown in the presence of TGF-β alone. E-cadherin expression was significantly enhanced（289.7%; P〈0.01）in the presence of rhBMP-7. Topical application of rhBMP-7 combined with 0.1% HA significantly reduced the amount of α-SMA~＋ cells by 43.18%（P〈0.05）at a concentration of 2.5 μg/mL and by 47.73%（P〈0.05）at 25 μg/mL,compared with the control group,without disturbing corneal reepithelization.CONCLUSION：rhBMP-7 attenuates TGF-β-induced EMT in vitro,and topical application of rhBMP-7 reduces keratocyte myodifferentiation during the early wound healing stages in vivo without hindering reepithelization. Topical rhBMP-7 application as biological eye drops seems to be feasible in diseases involving TGF-β-related corneal fibrosis with corneal reepithelization disorders.

Fungus induces the release of IL- 8 in human corneal epithelial cells, via Dectin-1-mediated protein kinase C pathways

AIM: To identify whether Aspergillus fumigatus(A.fumigatus) hyphae antigens induced the release of interleukin-8(IL-8) in anti-fungal innate immunity of cultured human corneal epithelial cells(HCECs) and determine the involvement of intracellular signalling pathways. METHODS: HCECs were treated with A. fumigatus hyphae antigens with different concentrations and time.The cytoplasmic calcium of HCECs were assessed by fluorescence imaging. Western blot was used to detect the expression of Ca2 +-dependent protein kinase C(PKC). The IL-8 levels were determined by specific human IL-8 enzyme-linked immunosorbent assay(ELISA) and reverse transcriptase polymerase chain reaction(RT-PCR). Using a series of pharmacological inhibitors, we examined the upstream signalling pathway responsible for IL-8 expression in response to A.fumigatus hyphae antigens. RESULTS: Cells exposed to A. fumigatus hyphae antigens showed higher level of IL-8 m RNA expression and protein production. We demonstrated here that stimulation of HCECs with A. fumigatus hyphae triggers an intracellular Ca2 +flux and results in the activation of Ca2 +-dependent PKC(α, βⅠ and βⅡ) which can be attenuated by pre-treatment of cells with laminarin,suggesting that Dectin-1 signals pathway induced cytoplasmic calcium and influence the activation of PKC in HCECs. Inhibitors of Ca2 +-dependent PKC(Ro-31-8220 and Go6976) significantly abolished hyphae-induced expression of IL-8.CONCLUSION: Our findings suggest that A. fumigatushyphae-induced IL-8 expression was regulated by the activation of Dectin-1-mediated Ca2 +-dependent PKC in HCECs.

SOX9通过转化生长因子β信号通路调节角膜内皮损伤后内皮-间质转化过程

目的探究性别决定区Y框蛋白9(sex-determining region Y-box protein 9,SOX9)是否会调控角膜内皮细胞损伤后的角膜上皮-间质转化(endothelial-to-mesenchymal transition,EndMT)过程及具体机制。方法转染小干扰RNA(small interfering RNA,siRNA)敲低人角膜内皮细胞B4G12中SOX9的表达,使用甲萘醌构建体外B4G12细胞损伤模型,设4个分组:si-NC组(转染siRNA-阴性对照)、si-SOX9组(转染siRNA-SOX9)、si-NC+甲萘醌组(转染siRNA-阴性对照后添加外源性甲萘醌做损伤处理)、si-SOX9+甲萘醌组(转染siRNA-SOX9后添加外源性甲萘醌做损伤处理)。通过实时荧光逆转录聚合酶链反应(real-time reverse transcription polymerase chain reaction,RT-PCR)和Western blot检测各组细胞中EndMT关键因子Snail家族转录抑制因子2(snail family transcriptional repressor 2,SNAIL2)及相关信号通路关键因子表达变化,阐明SOX9调控EndMT过程的功能和机制。结果转染siRNA-SOX9敲低细胞SOX9表达后,给予甲萘醌细胞损伤处理,观察到细胞中SNAIL2的表达会随SOX9的敲低而降低,同时观察到转化生长因子β(transforming growth factor beta,TGF-β)信号通路中SNAIL2的上游关键因子Smad2/Smad3的表达也随着SOX9的敲低而降低。结论SOX9通过TGF-β信号通路调控角膜内皮损伤后EndMT过程。

Inhibitory effect of CCR3 signal on alkali-induced corneal neovascularization

AIM: To investigate the effect of CC chemokine receptor 3 (CCR3) signal on corneal neovascularization (CRNV) induced by alkali burn and to explore its mechanism. METHODS: Specific pathogen-free male BALB/C mice (aged 6-8 weeks) were randomly divided into CCR3-antagonist treated group (experimental group) and control group. CRNV was induced by alkali burn in mice. The time kinetic CCR3 expression in injured corneas was examined by reverse transcription polymerase chain reaction (RT-PCR). CCR3-antagonist (SB-328437 at different concentration of 125 mu g/mL, 250 mu g/mL, and 500 mu g/mL) was locally administrated after alkali injury. The formation of CRNV was assessed by CD31 corneal whole mount staining at two weeks after injury. Monocyte chemotactic protein 1 (MCP-1), monocyte chemotactic protein 3 (MCP-3) expressions in the early phase after injury were quantified and compared by RT-PCR. Macrophage intracorneal accumulation in the early phase after injury was evaluated and compared by immunohistochemistry. RESULTS: Alkali injury induced the time kinetic intracorneal CCR3 expression. 500 mu g/mL of CCR3-antagonist treatment in the early phase but not the late phase resulted in significant impaired CRNV as compared to control group (P <0.05). CCR3-antagonist treatment in the early phase significantly reduced the intracorneal MCP-1 and MCP-3 enhancement compare to control group at day 2 and day 4 (P <0.05). Moreover, the number of intracorneal macrophage infiltration in the experimental group was reduced than those in control group at day 4 (P <0.05). CONCLUSION: CCR3 signal is involved in alkali-induced CRNV. CCR3-antagonist can inhibit alkali-induced CRNV by reducing the intracorneal MCP-1 and MCP-3 mRNA expression and the intracorneal macrophage infiltration.

TGFBI and CHST6 gene analysis in Chinese stromal corneal dystrophies

AIM:To investigate whether mutations in TGFBI gene or CHST6 gene correlated with stromal corneal dystrophies(CD) in 8 Chinese probands.· METHODS:Eight unrelated patients with stromal corneal dystrophies were recruited in this study;all affected members were assessed by completely ophthalmologic examinations.Genomic DNA was extracted from peripheral leukocytes,17 exons of TGFBI gene and the exon of CHST6 gene were amplified by polymerase chain reaction(PCR),sequenced directly and compared with the reference database.· RESULTS:Three heterozygous mutations in TGFBI gene were identified in six patients:c.370C>T(p.Arg124Cys) was found in exon 4 of TGFBI gene in three members,c.371G>A(p.Arg124His) was found in one patient;c.1663C>T(p.Arg555Trp) was found in exon 12 in other two members.In addition,four polymorphisms with the nucleotide changes rs1442,rs1054124,rs4669,and rs35151677 were found in TGFBI gene.Mutations were not identified in the rest of 2 affected individuals in TGFBI gene or CHST6 gene.· CONCLUSION:Within these patients,R124C,R124H and R555W mutations were co-segregated with the disease phenotypes and were specific mutations for lattice corneal dystrophy type I(LCD I),Avellino corneal dystrophy(ACD,GCDⅡ),granular corneal dystrophy type I(GCD I),respectively.Our study highlights the prevalence of codon 124 and codon 555 mutations in the TGFBI gene among the Chinese stromal corneal dystrophies patients.·

β-环糊精-透明质酸改性pHEMA角膜接触镜水凝胶的制备与性能

利用β-环糊精-透明质酸(β-CD-crHA)对聚甲基丙烯酸羟乙酯(pHEMA)水凝胶进行改性研究。借助核磁共振波谱仪、红外光谱仪、原子力显微镜、接触角仪、紫外-可见分光光度计、电子万能试验机等对水凝胶的组成、表面形貌、水接触角、透光率、拉伸性能、泪液蛋白吸附、双氯芬酸负载与释放等性能进行表征。结果表明,β-CD-crHA含量的增加显著提高了pHEMA水凝胶的表面亲水性,有效抑制了溶菌酶和牛血清白蛋白的吸附,同时增加了pHEMA水凝胶对双氯芬酸的负载量并减缓其释放速率,但不降低其透光率、拉伸应变等基本性能,是一种有前景的角膜接触镜材料。

SOX9在角膜内皮损伤过程中的表达及功能

目的探究性别决定区Y框蛋白9(sex determining region Y box protein 9,SOX9)在角膜内皮损伤过程中的表达及功能。方法通过转录组数据库分析人角膜内皮损伤与SOX9表达的关联。冷冻法构建小鼠角膜内皮损伤模型,每只小鼠左眼造模、右眼空白对照,采用裂隙灯照相、眼前节光学相干断层扫描技术(optical coherence tomography,OCT)、免疫荧光技术评估动物模型质量,角膜铺片免疫荧光染色观察损伤后内皮细胞中SOX9的表达变化。分别用低浓度(30μmol/L)和高浓度(50μmol/L)的甲萘醌对人角膜内皮细胞(B4G12)处理3 h(短时间组)、6 h(中时间组)、12 h(长时间组)、24 h(超长时间组),构建细胞模型。采用显微镜照相、细胞计数试剂盒(cell counting kit-8,CCK-8)实验评估细胞模型质量,实时荧光定量聚合酶链式反应(real-time quantitative polymerase chain reaction,qPCR)、Western blot检测SOX9和介导角膜内皮-间质转化(endothelium-mesenchymal transition,EndMT)关键转录因子Snail家族转录抑制因子2(snail family transcriptional repressor 2,SNAIL2)的表达变化。结果数据库资料显示在角膜内皮损伤代表性疾病Fuchs角膜内皮营养不良(Fuchs′endothelial corneal dystrophy,FECD)患者中,FECD3期以上患者角膜内皮SOX9表达量比2期患者明显升高(P<0.05),且SOX9的下游基因富含半胱氨酸的酸性分泌蛋白(secreted protein acidic and rich in cysteine,SPARC)表达量比正常人明显升高(P<0.05)。在动物模型中,对照组角膜内皮铺片免疫荧光染色显示无SOX9荧光,损伤后短期出现大量SOX9荧光,损伤后中期、长期SOX9荧光回归极低水平。在甲萘醌处理的细胞模型中,SOX9表达随处理时间的延长呈现先升高后降低的趋势;同时发现SNAIL2的表达变化也会随甲萘醌处理时间的延长呈现先升高后降低的趋势。结论SOX9的表达随角膜内皮损伤进程产生变化,并与损伤修复中的EndMT紧密相关。

苯扎氯铵诱发小鼠角膜损伤模型的构建及损伤机制研究

目的 确定苯扎氯铵诱导小鼠角膜损伤模型的最适浓度,探索其损伤产生的相关分子机制。方法 C57BL/6雄性小鼠30只,适应性饲养1周,裂隙灯检查排除眼疾后随机分为对照组、苯扎氯铵组(浓度:0.2%、0.4%、0.6%、0.8%),每组6只鼠12只眼,对照组给予磷酸盐缓冲生理盐水(phosphate buffered saline,PBS),除对照组外,其他组连续给予对应浓度苯扎氯铵7 d后进行泪液分泌试验(schirmer I test,SIT),在裂隙灯下进行角膜浑浊度、角膜新生血管和荧光素染色评分。安乐死小鼠后取眼球并制备石蜡组织切片进行苏木精-伊红染色(hematoxylin-eosin staining,HE)和过典酸雪夫氏染色(periodic acid-schiff staining,PAS);Western bolt检测单核细胞趋化蛋白1(monocyte chemoattractant protein-1,MCP-1)表达评估炎症水平,检测细胞色素C(cytochrome C)表达评估凋亡水平。结果 与对照组相比,0.4%及以上浓度苯扎氯铵可导致小鼠泪液分泌量下降,角膜浑浊度和新生血管水平增加。HE染色结果证实0.4%及以上浓度苯扎氯铵处理后角膜上皮层变薄且细胞排列不规则,其基质层炎症细胞浸润明显。PAS染色显示0.4%及以上浓度苯扎氯铵处理可造成结膜杯状细胞数量降低,且呈浓度依赖性。MCP-1蛋白的表达随苯扎氯铵浓度的增加而升高,Cytochrome C的蛋白水平在0.4%苯扎氯铵组最高。结论 0.4%及以上浓度的苯扎氯铵可诱导小鼠严重的角膜损伤,且0.4%为最佳造模浓度,其损伤与炎症因子MCP-1以及凋亡因子Cytochrome C的表达增加有关。

兔眼碱、酸、热烧伤后蛋白抗原性变化的研究

目的 了解角膜碱、酸、热烧伤后有无新的角膜变性蛋白产生并检测其抗原性。方法 利用SDS PAGE电泳技术和Western印迹实验分离角膜蛋白并确定其是否具有抗原性。结果 碱烧伤后 4周的角膜蛋白同正常角膜蛋白相比 ,可见在 2 3 5kda、2 5kda、3 5kda左右出现 3条新的蛋白带 ,其中 2 3 5kda和 3 5kda的蛋白能够刺激机体产生特异性的抗体并与之结合。热烧伤后 4周可见在 14 5kda左右出现l条新的蛋白带 ,不能刺激机体产生特异性的抗体。酸烧伤后 4周未出现新的蛋白带。结论 在角膜碱烧伤后的损伤机制中有变性抗原与抗体的病理性免疫反应参与 ,并持续较长时间存在。

春季角结膜炎活动期眼表和泪液蛋白的临床研究

目的对比观察春季角结膜炎(vernal keratoconjunctivitis,VKC)活动期患者眼表症状、泪膜、泪液蛋白以及角膜神经改变的特点。方法收集南昌大学第一附属医院2010年8月至2014年3月眼科门诊确诊为VKC活动期患者20例(40眼),分别对患者行眼表症状评分、泪膜四项检查、泪液蛋白测定、角膜知觉检查与中央角膜共聚焦显微镜检查,并与20例40眼健康对照组研究对象的检查结果进行对比分析。结果 VKC活动期患者左右两眼间和正常对照组研究对象的左右两眼间的眼表症状评分、泪膜四项、泪液蛋白、乳铁蛋白、溶菌酶、神经纤维密度及角膜敏感度差异均无统计学意义(均为P>0.05);VKC活动期患者右眼较对照组右眼干眼症状眼数增多,眼表疾病指数评分增高、泪膜破裂时间减短、基础泪液分泌试验结果增高、泪河高度降低、角膜荧光素染色增强,泪液总蛋白、乳铁蛋白、溶菌酶、角膜神经纤维密度及角膜敏感度值较对照组均明显降低,且差异均有统计学意义(均为P<0.05);VKC活动期患者左眼检查结果较对照组改变与右眼改变一致,差异也均有统计学意义(均为P<0.05)。共聚焦显微镜检查结果发现,VKC活动期患者中央角膜可见角膜上皮细胞部分结膜化,其上皮层存在大量炎症细胞浸润并伴有少量朗格汉斯细胞浸润,角膜上皮下有大量非成熟型朗格汉斯细胞浸润;角膜上皮下神经纤维纤细,呈盘旋、弯曲异常分布。结论 VKC活动期患者眼表及泪液改变明显,多表现为泪膜稳定性降低,泪液蛋白含量降低,角膜敏感度降低且角膜神经纤维密度下降、纤细及走形异常等特点。

应用GFP基因转染技术示踪组织工程技术构建角膜基质

目的 应用基因转染技术将绿色荧光蛋白 (GFP)标记角膜基质细胞 ,以此为种子细胞构建角膜基质并对构建过程进行示踪。方法 构建携带EGFP基因的重组逆转录病毒载体PLNCX2 EGFP ,转染兔角膜基质细胞 ,然后将该细胞接种于PGA ,形成细胞 -生物材料复合物 ,移植于母兔角膜基质板层内。术后 8周 ,组织学切片 ,HE染色 ,荧光显微镜下对绿色荧光蛋白 (GFP)进行示踪观察。结果 8周后 ,组织工程化角膜组织形成 ,组织学切片显示PGA完全降解 ,新生组织形成 ,组织排列规整 ,荧光显微镜下见新生组织呈绿色 ,提示EGFP表达。结论 组织工程角膜基质的组织学结构与角膜基质相类似。新生组织表达GFP 。

糖尿病患者眼表及泪液蛋白改变的临床分析

目的分析2型糖尿病患者眼表及泪液蛋白成分,并探讨其变化的原因。方法 收集2型糖尿病患者65例(72眼),拟行白内障手术患者40例(40眼)作为对照组。根据其眼底改变将糖尿病患者分为:糖尿病无眼底病变(non-diabetic retinopa-thy,NDR)组、非增生性糖尿病视网膜病变(nonproliferative diabetic retinopathy,NPDR)组和增生性糖尿病视网膜病变(prolifera-tive diabetic retinopathy,PDR)组。对所有患者进行干眼症状的问卷调查,观察指标包括角膜知觉、Schirmer I试验、泪膜破裂时间(break-up time,BUT)、角膜荧光素染色等,分别测定各组泪液总蛋白和泪液中乳铁蛋白、溶菌酶的含量等,并与正常对照组的各项指标进行对比。结果 糖尿病组干眼症阳性的患者为46例(70.77%)明显高于对照组11例(27.50%);Schirmer I试验显示:PDR组(9.76±2.76)mm远小于对照组(14.60±4.53)mm,BUT检查发现PDR组(7.00±2.24)s远少于对照组(11.32±2.95)s,角膜荧光染色评分PDR组(1.14±0.91)分高于对照组(0.44±0.65)分(均为P<0.05);NPDR组、PDR组角膜知觉[39.25±10.92)mm、(29.52±15.48)mm]及角膜神经纤维密度[(1053±142)μm/视野、(890±175)μm/视野]与对照组[(49.20±9.43)mm、(1248±151)μm/视野]比较均明显下降(均为P<0.05)。泪液总蛋白各组间比较差异无显著性,但NP-DR与PDR组中乳铁蛋白、溶菌酶含量较对照组均明显降低(均为P<0.05)。糖尿病患者的病程及血糖控制情况与眼表改变各参数均有明显的相关性。结论 糖尿病患者,特别是PDR患者易发生眼表异常,定期进行相关检查及早发现并治疗眼表疾病意义重大。

毛果芸香碱滴眼对兔眼角膜内皮细胞凋亡作用的影响

目的探讨长期应用毛果芸香碱对兔眼角膜内皮细胞有无影响及影响方式。方法用2%的毛果芸香碱给兔滴眼1个月和3个月后,取下兔眼球迅速固定,分离角膜内皮组织,用蛋白印迹(Western-blot)法检测兔眼角膜内皮细胞中抗凋亡基因bcl-2蛋白及凋亡基因bax蛋白的表达,流式细胞仪检测角膜内皮细胞的凋亡率,透射电镜下观察各组角膜内皮细胞的超微结构变化。结果应用毛果芸香碱1个月及3个月后,均呈现兔眼角膜内皮细胞凋亡基因bax蛋白表达增多,抗凋亡基因bcl-2蛋白表达减少,3个月组兔眼角膜内皮细胞凋亡率高于对照组(P<0.05),透射电镜下观察可见,1个月及3个月组细胞内空泡形成,线粒体肿胀,内质网扩张,细胞核固缩,染色质固缩周边化等典型凋亡改变。结论长期应用2%的毛果芸香碱可以诱导兔眼角膜内皮细胞发生凋亡。

外源性小鼠干扰素诱导蛋白-10抑制实验性角膜新生血管的作用机制

背景干扰素诱导蛋白-10（IP-10）作为趋化因子调节免疫炎症反应方面的作用已被证实，但最近的研究发现其在抑制新生血管生成方面发挥一定的作用。角膜新生血管（CNV）的形成与多种血管新生相关因子有关，IP—10对CNV形成的作用及机制尚不明确。目的探讨外源性小鼠IP-10点眼对角膜碱烧伤诱导CNV形成的作用。方法选用SPF级BALB／c小鼠82只，并按随机数字表法分组。采用浸润1mol／LNaOH的滤纸贴附于左眼角膜中央40S的方法制作角膜碱烧伤小鼠模型，角膜碱烧伤后1d及7d开始局部应用IP-10共7d分别作早期点眼组10只眼和中晚期点眼组5只眼，各自的模型对照组均给予质量分数0．2％透明质酸钠（HA）点眼（分别为11只眼和6只眼）。早期点眼组于造模后2周，中晚期点眼组于造模后3周取角膜组织以CD31免疫荧光标记法比较模型对照组及实验组的CNV面积。收集早期IP-10碱烧伤后2d及4d小鼠的角膜组织，用逆转录聚合酶链反应（RT—PCR）法检测并比较各组小鼠角膜组织中趋化因子受体3（CXCR3）、血管内皮生长因子（VEGF）及转化生长因子β1（TGF—β1）mRNA的表达情况。所有实验动物的使用、饲养及处死均按照视觉及眼科学研究协会的有关规定及苏州大学的《实验动物管理及使用指南》进行。结果模型对照组小鼠CNV占角膜面积的比例为（88．67±10．22）％，IP-10早期点眼1周组小鼠CNV占角膜面积的（70．06±12．21）％，差异有统计学意义（t=3．77，P=0．00）。角膜碱烧伤后21d，模型对照组小鼠CNV占角膜面积的比例为（87．33±13．47）％，IP-10中晚期点眼1周组CNV占角膜面积的（86．56±12．47）％，差异无统计学意义（t=1．260，P〉0．05）。IP-10早期点眼2d和4d的小鼠角膜组织中CXCR3的表达较模型对照组I司期值明显升高（t=3．13、3．07，P〈0．05）、VEGF表达降低（t=5．99、6．27，P〈0．01）、TGF—β1表达降低（t=8．50，P〈0．01；t=4．53，P〈0．05）。结论角膜碱烧伤后外源性IP-10蛋白早期干预可通过上调CXCR3和下调VEGF及TGF—β1的表达抑制CNV的形成，中晚期干预不能减少角膜碱烧伤诱导的CNV。

纤维介素蛋白2(FGL-2)在大鼠角膜移植排斥反应中的作用

目的研究纤维介素蛋白2(fibrinogen-like protein 2,FGL-2)在大鼠角膜移植排斥反应发生发展过程中的作用及糖皮质激素对其表达的影响。方法 60只成年雌性Wistar大鼠随机分为正常对照组、自体角膜移植组、同种异体角膜移植组和糖皮质激素组。正常对照组大鼠未行任何手术,自体角膜移植组、同种异体角膜移植组及糖皮质激素组均行穿透性角膜移植术。术后裂隙灯显微镜下观察术眼角膜炎性反应情况,按照Larkin的标准进行排斥反应评分,计算排斥反应指数,并在术后9 d和14 d分别取眼球制备角膜组织切片,行组织病理学检查和免疫组织化学检测,观察角膜组织的炎性反应和FGL-2在角膜组织中的表达;采用实时荧光定量PCR法检测FGL-2 mRNA在大鼠角膜植片中的相对表达量。结果术后9 d内各组角膜植片均出现不同程度的水肿、混浊及新生血管生长。同种异体角膜移植组大鼠平均在角膜移植术后9.8 d发生排斥反应并获病理学支持;自体角膜移植组和糖皮质激素组大鼠术后排斥反应计分均未达到诊断标准。免疫组织化学结果显示,不同时间点同种异体角膜移植组炎性细胞明显多于自体角膜移植组和糖皮质激素组。术后3组手术干预组角膜FGL-2 mRNA的表达均增加,在术后9 d和14 d时同种异体角膜移植组均较自体角膜移植组和糖皮质激素组高,差异均有统计学意义(均为P<0.05)。结论 FGL-2可能参与角膜移植免疫排斥反应,糖皮质激素可能通过降低其表达发挥抗排斥作用。

水通道蛋白1在人角膜内皮细胞的表达

目的 研究水通道蛋白 1(water channel protein1,AQP1)抗体在人角膜中的表达及水转运机制。方法 运用免疫组织化学法测定人角膜中 AQP1的表达。结果 在角膜内皮细胞和基质细胞中有 AQP1的表达 ,而角膜上皮细胞不表达 AQP1。结论 本实验证实了 AQP1在角膜内皮的表达 。

不同切口超声乳化术对糖尿病合并白内障患者角膜神经和泪液蛋白的影响研究

目的探讨年龄相关性白内障糖尿病患者超声乳化术后眼表症状,角膜神经和泪液蛋白的特点。方法年龄相关性白内障糖尿病患者64例(64眼,均为右眼),随机分为A、B组。A组采用上方透明角膜切口行超声乳化白内障吸除联合人工晶状体(IOL)植入术,B组在颞侧透明角膜切口行超声乳化白内障吸除联合IOL植入术,分别于术前、术后1周、2周、1个月及3个月对患者行眼表症状评分、泪液蛋白测定、角膜知觉计检查和共聚焦显微镜检查。结果术前两组干眼各亚症状评分、泪液蛋白、乳铁蛋白、溶菌酶、神经纤维密度及角膜敏感度差异无显著性(P>0.05)。术后2周,两组患者各症状中干涩感、异物感、畏光、疼痛、眼红和流泪比较,差异均有显著性(P<0.05);术后3个月,两组患者各症状间差异无显著性(P>0.05)。术后3个月,两组患者泪液总蛋白量,乳铁蛋白,溶菌酶及神经纤维密度差异无显著性(P>0.05),但角膜敏感度、角膜切口连接型神经比较差异有显著性(P均<0.05)。结论年龄相关性白内障糖尿病患者行上方切口超声乳化联合IOL植入术可以明显改善角膜知觉,减轻角膜切口神经损伤,提高患者的视觉质量。

腺病毒与慢病毒载体转染离体兔角膜基质细胞的有效性对比研究

目的观察比较腺病毒载体(adenovirus vector,AV)与慢病毒载体(lentivirus vector,LV)分别介导增强型绿色荧光蛋白(enhanced green fluorescent proteins,EGFP)对体外培养的兔角膜基质细胞(rabbit corneal stroma cells,RCSC)的转染效率,探讨较优一种病毒载体作为角膜基因治疗载体的可行性。方法行离体RCSC原代、传代培养及鉴定;实验分为转染组(AV-EGFP转染组和LV-EGFP转染组)和对照组,观察转染组在不同感染复数(multiplicity of infection,MOI)及感染后24h、48h、72h在倒置荧光显微镜下EGFP的表达情况,流式细胞技术检测转染效率。结果 AV-EGFP转染组感染RCSC后从24～48h就开始可见明显的绿色荧光,起始时间早于LV-EGFP转染组(48h)。LV-EGFP转染组在MOI≤1000时,随着MOI值增大,检测到的细胞转染率逐渐增大,两两比较差异均有统计学意义(均为P<0.05);AV-EGFP转染组在MOI>10时,随着MOI值增大,检测到的细胞转染率逐渐增大,两两比较差异均有统计学意义(均为P<0.05);而在同一MOI值下(MOI>1),LV-EGFP较AV-EGFP转染效率更高,差异均有统计学意义(均为P<0.05)。结论 AV与LV均可有效转染离体RCSCs同一MOI值下(MOI>1),LV转染效率更高。

小牛血去蛋白提取物眼用凝胶与卡波姆眼用凝胶对SBK后角膜上皮修复疗效的比较

目的探讨小牛血去蛋白提取物眼用凝胶与卡波姆眼用凝胶在准分子前弹力层下角膜磨镶术(sub-Bow-man’s keratomileusis,SBK)后对角膜上皮损伤修复作用的情况,为指导术后用药提供依据。方法连续选取2012-05～06月在广州军区武汉总医院准分子激光中心拟行SBK的近视患者23例(46眼),采用随机双盲方法,将患者左右眼随机分别纳入小牛血去蛋白提取物眼用凝胶组和卡波姆眼用凝胶组。术前查双眼最佳矫正视力等准分子手术常规术前检查,术后即刻随机点两种眼用凝胶,术后当日依据患者主诉症状制定的评分标准记录评分,术后第一天查双眼裸眼视力(uncorrectedvisual acuity,UCVA)、泪膜破裂时间(breakup time,BUT)、角膜荧光素钠(fluorescein sodium,FS)染色及依据患者主诉症状制定的评分标准记录评分,对比两种药物对SBK术后角膜上皮损伤修复的差异。结果所有患者术后均感双眼异物感明显,小牛血去蛋白提取物眼用凝胶组术后舒适度优于卡波姆眼用凝胶组;术后第一天UCVA、BUT、角膜FS染色,两组差异无统计学意义;依据患者主诉症状制定的评分标准进行评分,术后当日、术后第一天,两组差异无统计学意义(P>0.05),但两组术后第一天反应均较术后当日轻。结论 SBK术后短期应用小牛血去蛋白提取物眼用凝胶及卡波姆眼用凝胶在改善术后刺激症状,促进角膜上皮愈合,改善患者术后眼干不适症状方面未见明显差异。