TGF—β与角膜伤口愈合

转化生长因子－β（ＴＧＦ－β）是一类具有多种生物学功能的多肽生长因子，能调节多种细胞内生长、分化和移行，参与调节细胞外基质成分的合成，在泪腺及角膜中均有分布。本文介绍了ＴＧＦ－β的生化特性，生物学作用以及在泪腺，角膜中的分布，着重就其与角膜伤口愈合的关系作简要综述。     

近视眼角膜上皮细胞EGFR、TGF—pRI及IL—1RI的表达

目的检测近视患眼角膜上皮细胞表皮生长因子受体(EGF R)、转化生长因子βⅠ型受体(TGF-βRⅠ)和白细胞介素1Ⅰ型受体(IL-1RⅠ)的表达.方法将PRK术中获取的角膜上皮细胞制成细胞涂片,ABC法检测.结果EGF R、TGF-βRⅠ和IL-1RⅠ在全部标本表达,强度依次为EGF R＞IL-1RⅠ＞TGF-βRI.结论近视患眼角膜上皮细胞表达此三种受体,推测PRK术后此三种受体参与角膜创面修复.     

慢病毒介导的TGFβ-R2shRNA抑制人LECs细胞TGFβ—R2的表达

目的：观察转化生长因子β-R2特异性短发夹 RNA （ shRNA ）对人晶状体上皮细胞TGFβ-R2表达的影响。 方法：根据小干扰RNA （ siRNA ）的设计原则,针对转化生长因子β-受体2基因序列特征构建特异性 shRNA （ RNAi ）载体,与TGFβ-R2过表达载体共转染293 T细胞,经Western blot筛选出有效RNAi载体后,进行扩增、纯化、滴度测定,并转染培养的人LECs , qPCR 检测TGFβ-R2 siRNA慢病毒载体对TGFβ-R2 mRNA表达的影响。 结果：经Western blot 筛选,TGFβ-R2/GV115RNAi＃1对目的基因的表达敲减效果最明显,慢病毒包装,滴度为8E＋8 TU/mL,感染人LECs细胞后,对TGFβ-R2基因有显著的敲减效果,达到78．1％（P＜0．05）。 结论：TGFβ-R2 RNAi载体构建成功,并且能抑制人晶状体上皮细胞TGFβ-R2 mRNA的表达。     

羊膜移植对兔角膜碱烧伤基质TGF—β1mRNA表达的影响

目的 观察兔角膜重度碱烧伤后羊膜移植对兔角膜碱烧伤基质TGF-β1mRNA表达的影响。方法 将18只新西兰白兔随机分成3组,每组6只,所有兔眼建立重度碱烧伤模型,左眼羊膜移植,右眼作为对照。分别观察1月、2月、3月。结果 1月时,羊膜移植组中基质TGF-β1mRNA的表达低于对照组。2月、3月时,两组中基质TGF-β1mRNA的表达无明显差异。结论 羊膜移植治疗兔角膜重度碱烧伤,早期可降低基质TGF-β1mRNA的表达,起到部分减轻基质浑浊的作用。     

羊膜对激光角膜切除术后TGF—β1表达调控的初步研究

目的 探讨羊膜对激光角膜光学切除术（Ｐｈｏｔｏｒｅｆｒａｃｔｉｖｅ ｋｅｒａｔｅｃｔｏｍｙ ＰＲＫ）后角膜组织ＴＧＦ－β１表达的影响。方法 对６只新西兰白兔双眼行ＰＲＫ,术后１眼立即行角膜表面羊膜移植术,只眼作为对照。术后４周用原位杂交方法检测角膜上皮和基质ＴＧＦ－β１ｍＲＮＡ的表达。结果 ＰＲＫ后４周对照组角膜上皮和基质有较浓度的ＴＧＦ－β１ｍＲＮＡ表达,羊膜移植组角膜上皮和基质亦有ＴＧＦ－β１     

慢病毒介导的TGFβ-R2 shRNA抑制人LECs细胞TGFβ-R2的表达

目的：观察转化生长因子β-R2特异性短发夹RNA(shRNA)对人晶状体上皮细胞TGFβ-R2 表达的影响。

方法：根据小干扰RNA(siRNA)的设计原则,针对转化生长因子β-受体2基因序列特征构建特异性shRNA(RNAi)载体,与TGFβ-R2过表达载体共转染293T细胞,经Western blot筛选出有效RNAi载体后,进行扩增、纯化、滴度测定,并转染培养的人LECs,qPCR检测TGFβ-R2 siRNA慢病毒载体对TGFβ-R2 mRNA表达的影响。

结果：经Western blot筛选,TGFβ-R2/GV115RNAi#1对目的基因的表达敲减效果最明显,慢病毒包装,滴度为8E+8 TU/mL,感染人LECs细胞后,对TGFβ-R2基因有显著的敲减效果,达到78.1%(P<0.05)。

结论：TGFβ-R2 RNAi载体构建成功,并且能抑制人晶状体上皮细胞TGFβ-R2 mRNA的表达。     

PRK后兔角膜成纤维细胞分泌产生bFGF,TGF—β1及其对成纤维细？…

目的 探讨在准分子激光屈光手术后角膜伤口愈合过程,是否有生长因子ｂＦＧＦ及ＴＧＦ－β１参与,两种生长因子对成纤维细胞增殖的作用及其相应抗体对这一过程的影响。方法对ＰＲＫ后兔角膜成纤维细胞原代培养,观察有否内源性生长因子ｂＦＧＦ及ＴＧＦ－β１的作用,内源性及外源性生长因子ｂＦＧＦ及ＴＧＦ－β１对细胞增殖的影响及相应抗体对抗原的中和作用。结果 在ＰＲＫ后兔角膜伤口愈合过程中成纤维细胞分泌产生光屈光手术     

TGFβ1、iNOS在鸡FDM的表达及作用

目的 建立鸡形觉剥夺性近视眼(FDM)动物模型，研究视网膜和脉络膜TGF-β1和iNOS及其mRNA在FDM中的变化及作用。方法 出生一天的来亨鸡随机遮盖一眼，另眼作为对照。实验中分别行视网膜检影检测双眼屈光度，A超测量双眼眼轴长度。NO酶法试剂盒测定NO的含量；免疫组化分析TGF-β1和iNOS的表达；RT-PCR检测TGF—β1和iNOS mRNA的表达。结果 形觉剥夺使眼轴增长，近视屈光度增加，去遮盖后双眼轴长差异减小，近视度下降。形觉剥夺导致NO含量减少、TGF-β1和iNOS的免疫活性降低，TGF—β1和iNOS mRNA的表达下调；去遮盖后NO含量、TGF-β1和iNOS的免疫活性及TGF—β1mRNA的表达恢复正常。在去遮盖后1周，iNOS mRNA的表达高于对照组水平。结论 TGF-β1和iNOS可能参与调控FDM的形成，TGF-β1和iNOS之间可能有相互作用，也可能受其它因子的调控。     

异搏定对培养角膜基质细胞分泌转移生长因子—β1的影响

目的 探讨异搏定对体外培养兔角膜基质细胞分泌ＴＧＦ－β１的影响。为预防或减轻ＰＲＫ后混浊形成提供线索。方法 将传代细胞，在加药培养后４８ｈ，应用ＴＧＦ－β１Ｆｍａｘ＾ＴＭＩｍｍｕｎｏＡｓｓａｙＳｙｓｔｅｍ试剂盒和培养上清中ＴＧＦ－β１含量进行检测，根据标准曲线分别计算出各孔ＴＧＦ－β１的浓度。结果 Ｖｅｒａｐａｍｉｌ浓度５μｇ／ｍｌ和１０μｇ／ｍｌ，作用４８ｈ，培养上清中ＴＧＦ－μ１含量明显降低。     

miR-101-3p靶向TGFβR1/Smad抑制视网膜母细胞瘤细胞侵袭和迁移

目的　探索miR-101-3p对视网膜母细胞瘤细胞侵袭和迁移的影响及作用机制。方法　RT-PCR检测miR-101-3p及转化生长因子β受体1（transforming growth factor beta receptor 1，TGFβR1）表达水平；生物信息学预测miR-101-3p与TGFβR1的靶向关系并通过荧光素酶报告实验进行验证。将Y79细胞分为Control组、miR-101-3p mimic组（转染miR-101-3p mimic）、pcDNA-TGFβR1组（转染pc-TGFβR1）及miR-101-3p mimic+pc-TGFβR1组（共转染miR-101-3p mimic和pc-TGFβR1），MTT检测细胞增殖速率，流式细胞术检测细胞凋亡，Transwell实验检测细胞侵袭，划痕实验检测细胞迁移，Western blot检测TGFβR1、Ki67、Bcl-2、Bax、cleaved Caspase-9、基质金属蛋白酶（matrix metalloproteinase,MMP）-2、MMP-9、血管内皮生长因子、p-Smad2及p-Smad3的表达水平。结果　在视网膜母细胞瘤组织中,miR-101-3p的表达为0.35±0.05，明显低于正常视网膜组织（P<0.01）；视网膜母细胞瘤细胞株Y79中miR-101-3p的表达为0.24±0.04，明显低于视网膜上皮细胞株ARPE-19（P<0.01）；与Control组相比，miR-101-3p mimic组miR-101-3p mRNA水平显著升高、TGFβR1 mRNA水平显著降低（均为P<0.01）；miR-101-3p与TGFβR1之间存在连续结合片段。与Control组相比，miR-101-3p mimic组TGFβR1的表达水平显著降低，增殖速率及细胞中Ki67表达均显著降低，细胞凋亡率显著增加，Bax及cleaved Caspase-9表达显著升高、Bcl-2表达显著降低，侵袭细胞数目和划痕闭合率显著降低，MMP-2、MMP-9、血管内皮生长因子、TGFβR1、p-Smad2和p-Smad3的表达水平显著降低（均为P<0.01）。结论　miR-101-3p通过靶向下调TGFβR1抑制视网膜母细胞瘤细胞的侵袭和迁移。     

The Changes of TGF-α, TGF-β_1 and Basic FGF Messenger RNA Expression in Rabbit Cornea after Photorefractive Keratectomy

Objective : To study the mechanism of haze formation and investigate the expression changes of transforming growth factor-α(TGF-α), transforming growth factor-β1 (TGF-β1) and basic fibroblast growth factor (bFCF) mRNA in corneal epithelium and stroma after photorefractive keratectomy (PRK).Methods: Sixteen white rabbits were randomly divided into 4 groups, and PRK was performed on each eye of 12 rabbits. The haze formation was examined under a slit-lamp microscope at the 1st, 2nd and 3rd month after PRK, and the expressions of TGF-α , TGF-β1 and bFGF mRNA were detected with in situ hybridization.Results : The corneal haze formed at the 1" month after PRK. The most prominent haze formation was observed at the 2nd month, and declined gradually at the 3rd month after ablation. TGF-a mRNA expression was presented on the normal corneal epithelium and not on the corneal stroma. TGF-β1 and bGFG mRNA were expressed by both corneal epithelium and stroma. The capacities for cornea tissue expression of thre     

兔准分子激光角膜切削术后角膜血小板源性生长因子mRNA表达的变化

研究角膜上皮下混浊形成的发生机制,检测准分子激光屈光性角膜切削术后角膜上皮和基质血小板源性生长因子表达的变化。方法新西兰白兔施行ＰＲＫ后１,２,３月用裂隙灯显微镜观察ｈａｘｅ形成情况,并用原位酸分子杂交方法,检测角膜上皮和基质ＰＤＧＦ ｍＲＮＡ的表达。结果正常角膜上皮细胞有ＰＤＧＦ ｍＲＮＡ表达,基质层无表达;ＰＲＫ后角膜上皮细胞ＰＤＧＦｍＲＮＡ表达增加,术后２月表达最强,且基质中亦有轻微表达。上     

PRK术后兔角膜表达bFGF mRNA的变化

目的 为研究ｂａｚｅ形成的发生机制,检测ＰＲＫ后角膜表达ｂＦＧＦ的变化。方法 新西兰白兔２０只,随机分成４组,其中１５只施行ＰＲＫ。手术后１、２、３月观察ｈａｚｅ形成的情况,并检测角膜ｂＦＧＦ ｍＲＮＡ的表达。结果 正常角膜上皮和基因均有ｂＦＧＦ ｍＲＮＡ轻微表达;ＰＲＫ后角膜组织ｂＦＧＦ ｍＲＮＡ持续高水平表达,且以术后１、２月最明显。ｂＦＧＦ ｍＲＮＡ表达水平与形成ｂａｚｅ的轻重有一定关系。结     

Relation between corneal haze and transforming growth factor-beta1 after photorefractive keratectomy and laser in situ keratomileusis.

PURPOSE: To investigate the relation between corneal haze formation and transforming growth factor-beta (TGF-beta) after photorefractive keratectomy (PRK) and laser in situ keratomileusis (LASIK). SETTING: Department of Ophthalmology, University of Tokyo Graduate School of Medicine, Tokyo, Japan. METHODS: White rabbits were divided into 4 groups, with each group receiving 1 of the following surgeries: manual epithelial abrasion, PRK, lamellar keratotomy, or LASIK. The degree of corneal haze was quantitatively analyzed by measuring the light scattering intensity of corneas before and 4 and 12 weeks after surgery. The expression of type IV collagen and TGF-beta1 in the corneas at baseline and at 4 weeks was examined immunohistochemically. RESULTS: The light scattering intensity was significantly greater 4 and 10 weeks after PRK. In contrast, epithelial abrasion, lamellar keratotomy, and LASIK did not influence the light scattering intensity of the corneas. Type IV collagen was detected in the basal lamina of the corneal epithelium and in Descement's membrane in the normal cornea. After epithelial abrasion, there was no change in the distribution of type IV collagen. Four weeks after PRK, the expression of type IV collagen was detected in the subepithelial layer of the laser-ablated area. Four weeks after lamellar keratotomy, type IV collagen was linearly and fragmentarily detected in the corneal stroma. Four weeks after LASIK, type IV collagen was linearly and continuously detected in the corneal stroma and was detected slightly in the subepithelial region of the laser-ablated area. In the normal corneas, the expression of TGF-beta1 was not detected in the keratocytes. Four weeks after PRK, the expression of TGF-beta1 increased in the keratocytes that proliferated in the subepithelial fibrous layer. In contrast, epithelial abrasion, lamellar keratotomy, and LASIK did not change the expression pattern of TGF-beta1 in the keratocytes. CONCLUSION: The multiplier effect of epithelial abrasion and excimer laser ablation in PRK may increase the expression of TGF-beta1 in keratocytes and induce corneal haze.     

准分子激光角膜切削术与准分子激光原位角膜磨镶术角膜创口愈合的对比实验研究

比较准分子激光角膜切削术(photorefractive keratectomy,PRK)与准分子激光原位角膜磨镶术(1aser in situ keratomileusis,LASIK)后激光对角膜组织的切削效应及角膜的愈合情况,从组织学角度探讨角膜雾状混浊(Haze)及屈光度数回退的成因。方法24只新西兰白兔按预矫屈光度数随机分为-4.00 D组和-8.00 D组,每只兔右眼行PRK,左眼行LASIK。术后10d,1、3及6个月观察Haze情况并验光,每组随机处死3只兔取角膜行光镜、电镜及免疫组化检查,检测胶原Ⅲ、胶原Ⅳ、纤维连结蛋白(fibronectine,FN)及转化生长因子-β(transforming growth factor-β1,TGF-β2)的含量。结果行PRK术的右眼术后出现不同程度的Haze及屈光度数回退,其程度与预矫正屈光度数成正比。行LASIK术的左眼术后除少数角膜瓣周围半环形混浊外,手术区域角膜透明,屈光度数回退较右眼轻。-4.00 D组右眼与左眼术后屈光度数均稳定,-8.00 D组右眼较左眼屈光度数回退明显。右眼术后角膜愈合反应重,恢复慢。6个月时角膜基质仍处于修复阶段。左眼术后除形成角膜上皮栓及对应处基质轻度增生外,手术区域角膜瓣与基质床间界面清晰,无明显增生,角膜基质愈合反应轻、恢复快。术后所有兔眼角膜均有TGF-β1表达及活化,持续时间与角膜愈合时间一致。Haze及屈光度数回退组织学改变为：角膜上皮细胞增生,基底膜不成熟,前基质角膜细胞活化、增殖,新生胶原Ⅲ合成、排列紊乱,细胞外基质FN在角膜上皮下沉积。结论LASIK矫正近视尤其是高度近视优于PRK;角膜伤口愈合特别是基质愈合的反应程度,是。Haze及屈光度数回退的关键;TGF-β1是角膜愈合过程中重要调节因子,可通过介导角膜上皮一基质相互作用,调节胶原Ⅲ及FN的含量,参与瘢痕形成。     

The preliminary study of amniotic membrane on TGF-beta 1 expression after photorefractive keratectomy]

OBJECTIVE: To investigate the effect of amniotic membrane on TGF-beta 1 expression after photorefractive keratectomy(PRK). METHODS: PRK was performed bilaterally on 6 New Zealand white rabbits. One eye was randomly tranplanted with preserved human amniotic membrane, and the other eye served as the control. The expression of TGF-beta 1 mRNA was detected with in situ hybridization at the 4th weeks after PRK. RESULTS: TGF-beta 1 mRNA expression was present in the corneal epithelium and stroma at the 4th weeks after PRK. The cornea in amniotic membrane transplanted group had also TGF-beta 1 mRNA expression, and the expression extent was less than that in control group (P < 0.05). CONCLUSION: Amniotic membrane can reduce the expression of TGF-beta 1 mRNA in cornea after PRK.     

Effect of amniotic membrane on expressions of TGF-beta 1, collagens I, III and fibronectin in rabbit corneal healing after photorefractive keratectomy]

OBJECTIVE: To observed the effect of amniotic membrane transplantation (AMT) on expressions of transforming growth factor-beta 1(TGF-beta 1), collagens I, III and fibronectin (FN) in rabbit corneal healing after photorefractive keratectomy (PRK), and investigate the anti-scarring formation mechanism of AMT. METHODS: Ten rabbits underwent bilateral PRK to correct 8 diopters of myopia. One eye was randomly transplanted with preserved human amniotic membrane, and the other eye served as the control. The expressions of TGF-beta 1, collagens I, III and FN were studied by immunohistochemistry at 4 weeks after operation. RESULTS: The expression of TGF-beta 1 in corneal epithelium and keratocytes and collegans in anterior stroma was significantly less in AMT group than in the control group. The expressions of collagens III and FN in anterior stroma of ablation area were also significantly less in AMT group than in the control group. The staining of collagen I had no significant difference between the AMT group and the control group. CONCLUSION: The expressions of TGF-beta 1, collagens III and FN can be suppressed by amniotic membrane after PRK. These data suggest that part of the anti-scarring effect of AMT may be mediated through the suppression of TGF-beta 1, collagens III and FN expression.     

应用共焦显微镜评估bFGF对LASIK术后角膜胶原和神经修复的影响

背景角膜损伤后分泌的碱性成纤维细胞生长因子（bFGF）对细胞再生、胶原纤维重建、神经轴突再生具有重要作用,然而局部分泌的bFGF并不能满足机体修复需要。目的研究应用活体共焦显微镜观察外源性bFGF即重组bFGF（rbFGF）对兔准分子激光原位角膜磨镶术（LASIK）术后角膜胶原、神经纤维的修复作用及安全性。方法建立34只成年新西兰大白兔双眼LASIK模型。于术后第1天双眼用妥布霉素地塞米松滴眼液点眼共10d,右眼加用rbFGF点眼3个月,左眼同法用玻璃酸钠点眼。术后1周、2周,2、3、5个月时共焦显微镜下观察前部基质细胞密度、上皮基底膜下神经纤维密度、前基质神经纤维密度、角膜haze分级、灰度差、角膜缘新生血管等指标,分析角膜胶原、神经纤维修复及前基质细胞情况。结果共19只兔符合LASIK模型标准纳入研究。术后2个月时前基质细胞密度最高,haze改变与之相反。术后各时间点2组问平均前基质细胞密度、haze评分、灰度差值的差异均无统计学意义（P〉O．05）。LASIK术后2组的神经纤维密度均呈递增趋势,上皮基底膜下神经纤维更明显;术后2周时,rbFGF组前部基质神经纤维密度明显高于玻璃酸钠组,差异有统计学意义（Z=-2．070,P：0．038）,5个月时上皮基底膜下神经纤维密度明显高于对照组（Z=-2．060,P=0．039）。实验期间2组均无角膜新生血管（CNV）发生。2组组内术后灰度值与haze分级值呈正相关（rbFGF组：b=22．687,F=37．975,P=0．000,玻璃酸钠组：b=20．410,F=18．516,P=0．000）,而haze程度与基质细胞密度无明显相关（rbFGF组：b=0．001,F=0．164,P=0．668;玻璃酸钠组：b=-0．002,F=1．896,P=0．178）。结论外源性bFGF可安全、有效地促进LASIK术后角膜神经修复及基质胶原细胞规则再生,有利于术后角膜胶原的规则重建。     

准分子激光屈光性角膜切削术后兔眼角膜组织细胞外基质成分的变化

目的 动态观察准分子激光屈光性角膜切削术（photorefractive keratectomy,PRK) 后角膜组织细胞外基质（extracellular matrix,ECM)成分的变化,并探讨其与角膜雾状混浊（haze）的关系。方法 24只兔按术后即刻、24h、1周、2周、1个月、3个月、6个月及12个月不同处死时间分为8个实验组,每组3只,按设计矫正度数－10．00D行右眼PRK术,术后裂隙灯显微镜检查各组角膜的haze分级,并取角膜组织以及免疫组织化学方法检测ECM成分,包括Ⅰ型胶原蛋白、Ⅲ型胶原蛋白、Ⅳ型胶原蛋白、细胞纤维连接蛋白（cellular fibronectin,cFN)、细胞黏合素（tenascin,TN)以层黏连蛋白（laminin,LN)。结果 PRK术后haze的发生率为100％;角膜基质浅层内沉积新合成的Ⅰ、Ⅲ、Ⅳ型胶原蛋白及cFN、TN、LN等ECM成分,其表达量变化与haze分级变化基本一致。结论 PRK术后切削区角膜基质浅层ECM成分的沉积与角膜haze的出现密切相关。     

Growth factor mRNA and protein in preserved human amniotic membrane

PURPOSE: To investigate the expression of growth factor mRNA and the level of growth factor protein in preserved human amniotic membrane (AM). METHODS: RT-PCR was used to examine the expression of mRNA for eight growth factors (EGF, TGF-alpha, KGF, HGF, bFGF, TGF-beta1, -beta2, -beta3) and two growth factor receptors (KGFR and HGFR) in human AM preserved at -80 degrees C for one month. In addition, ELISAs were used to measure the protein concentrations of seven growth factors (EGF, TGF-alpha, KGF, HGF, bFGF, TGF-beta1, -beta2) in preserved human corneas and in AM both with and without amniotic epithelium. RESULTS: RT-PCR revealed that human AM expresses mRNA for EGF, TGF-alpha, KGF, HGF, bFGF, TGF-beta1, -beta2, -beta3, KGFR and HGFR, while ELISAs showed that it contains EGF, TGF-alpha, KGF, HGF, bFGF, TGF-beta1, -beta2. AM without amniotic epithelium also contains all seven growth factors examined, however, in this tissue the protein levels of EGF, KGF, HGF and bFGF were found to be significantly lower than in native AM. CONCLUSIONS: Preserved human AM expresses mRNAs for a number of growth factors and contains several growth factor proteins that might benefit epithelialization after AM transplantation. High levels of EGF, KGF, HGF and bFGF in AM with amniotic epithelium as compared to AM without amniotic epithelium suggest an epithelial origin for these growth factors. We feel that EGF, KGF and HGF in particular might play important roles in ocular surface wound healing after AM transplantation.