兔角膜缘干细胞两种培养方法的比较

目的对采用消化法和组织块法培养兔角膜缘干细胞进行对比,以期确定合理的角膜缘干细胞体外培养方法。方法采用3T3成纤维细胞滋养层培养体系,分别用消化法和组织块法培养兔角膜缘上皮细胞,用免疫组织化学技术检测培养的细胞中ΔNp63的表达。采用流式细胞技术分析分离的角膜缘上皮细胞悬液中间质细胞的含量,采用扫描电镜观察去除上皮的角膜缘基质表面。结果采用消化法培养,培养的细胞中ΔNp63表达较多,细胞最终可以分化为复层上皮结构。在分离的细胞悬液中,间质细胞含量小于5%,扫描电镜显示角膜缘上皮细胞能被完全消化。结论在角膜缘干细胞的培养中,在干细胞的含量方面,消化法培养要明显优于组织块法培养。     

角膜上皮干细胞的体外生长特征

目的　了解角膜上皮干细胞的生长特性。方法　用体外细胞培养的方法 ,从生长曲线、细胞倍增时间、细胞 DNA合成的活跃程度等几个方面 ,观察人角膜缘部、周边部及中央部角膜上皮细胞的生长特征。结果　角膜缘部细胞群体倍增时间最短 (4 9.79h±1.2 6 h) ,周边部次之 (6 4.89h± 2 .18h) ,中央部最长 (78.86 h± 1.38h) ,3组两两比较 P<0 .0 1;角膜缘部角膜上皮细胞 DNA合成最活跃 (CPM值 92 7.75± 47.94) ,周边部次之(CPM值 711.75± 2 9.47) ,中央部最弱 (CPM值 5 14.0 0± 72 .82 ) ,3组两两比较 P <0 .0 1。结论　结果进一部证实了角膜上皮干细胞存在于角膜缘的观点 ;也反映了角膜缘在角膜上皮创伤愈合 ,角膜上皮完整与稳定性维持过程中起着重要的作用     

EGF联合KGF促进人角膜上皮细胞生长

目的:寻找促进角膜上皮损伤修复,治疗持续性角膜上皮缺损的有效药物方法:用~3H—胸腺嘧啶核苷(~3H—TDR)掺入及液体闪烁技术,观察外源性表皮生长因子(EGF)联合角蛋白细胞生长因子(KGF)对体外培养的角膜上皮细胞DNA合成的影响,并计算细胞倍增时间。结果:10ng/mlEGF,10ng/mlKGF单独或联合应用均有明显促进人角膜上皮细胞DNA合成的作用(与对照组比较 P<0.01),联合用药,作用更强(P<0.05)。应用EGF与KGF明显缩短了细胞倍增时间。结论:外源性EGF与KGF对体外培养的人角膜上皮细胞有明显的促细胞增生作用,联合用药,效果更佳。表明EGF与KGF具有应用于临床,促进角膜上皮损伤修复的可能性。眼科学报1996; 12:107-109。     

不同体外方法培养兔角膜缘上皮细胞作用的研究

自从1986年Schermer等发现角膜缘干细胞位于角膜缘基底Vogt栅栏内后，对角膜缘干细胞缺失和角膜上皮病变治疗有了许多新的疗法，其中以角膜缘上皮细胞体外培养法治疗上述疾病比较有效。目前，多数学者采用组织块法进行角膜缘上皮细胞体外培养，但在培养系中干细胞能否从组织块中迁移出来尚有争议。另外，最近角膜缘上皮单细胞悬浮液超低温冷藏、复苏以及体外培养技术越来越受到人们的关注，成为角膜缘干细胞研究中的一项重要的课题。此种培养方法既可以将角膜缘干细胞和其他角膜上皮细胞长期保存在超低温状态下，减少细胞传代培养次数，[第一段]     

组织块培养法及酶消化法培养人角膜缘上皮细胞

角膜上皮的完整和无血管状态对维持角膜的生理功能和透明性十分莆要。角膜上皮的完整性有赖于角膜缘干细胞（limbalstemcells,LSCs）的增生和分化,多种原因可造成LSCs的缺乏或功能障碍,使角膜丧失完整性和透明性。LSCs功能障碍有许多新疗法,其中角膜缘上皮细胞体外培养后移植成为研究热点。目前多数学者采用组织块法进行角膜缘上皮细胞的培养．培养体系巾干细胞能否从在组织块中迁移出来尚有争议,而酶消化法越来越受重视。     

不同移植手术治疗角膜缘干细胞缺损的实验研究

目的探讨以人羊膜为载体培养的角膜缘干细胞，自体及异体移植治疗全角膜缘干细胞缺损。方法制作兔眼角膜缘干细胞完全缺损3个月的模型。实验动物随机分为自体移植组和异体移植组，前者取对侧眼角膜缘组织，后者取异体兔眼角膜缘组织，均以去除上皮细胞的羊膜基底膜为载体，培养12d后行角膜缘干细胞羊膜移植术。术后观察3个月，以角膜上皮染色、角膜浑浊和新生血管3项指标进行临床疗效评定，通过病理检查评估术后角膜上皮修复情况，印迹细胞学检查移植前后角膜上皮的细胞表型。结果体外培养的兔角膜缘干细胞可在羊膜上粘附生长并增生，体外培养12d可形成复层。自体移植组和部分异体移植组术后角膜上皮逐渐愈合，透明度提高，基质细胞浸润减轻，新生血管减退或消失。印迹细胞学检查显示：移植前角膜上皮细胞PAS阳性，而移植后转为阴性；组织病理学显示：移植前角膜上皮大部分缺损，移植后呈现角膜上皮结构。部分异体移植组术后出现了免疫排斥反应。结论兔自体角膜缘干细胞羊膜移植术可重建眼表；免疫排斥反应仍是异体角膜缘干细胞羊膜移植术失败的主要原因。     

体外不同区域角膜上皮细胞对5—FU耐受性的研究

为体外培养角膜上皮移植选取细胞来源，本文应用溴代脱氧尿嘧啶（Ｂｒｄｕ，胸腺嘧啶的类似物）渗入ＤＮＡ合成，抗—Ｂｒｄｕ单克隆荧光抗体标记细胞法，在流式细胞仪（ＦＡＣＳｔａｒｐｌｕｓ）上检测离体情况下兔角膜不同区域上皮细胞对５－ＦＵ耐受情况，以确定长周期细胞的部位。结果显示：经５－ＦＵ处理后的角膜周边及角膜中央部上皮细胞增殖明显受到抑制，而角膜缘细胞对５－ＦＵ毒性则有相对耐受性。由此得出：角膜缘上皮细胞群中有长周期的角膜干细胞存在。因而，行体外培养角膜上皮时，应选取角膜缘部的细胞。     

羊膜培养兔角膜缘上皮细胞及自体移植--角膜缘干细胞缺损的治疗

目的　探讨以人羊膜为载体培养兔角膜缘上皮细胞及其自体移植治疗全角膜缘干细胞缺损。方法　在8只兔右眼用正庚醇脱上皮和角膜缘环切的方法构建全角膜缘干细胞缺损模型2月。其中6只兔为实验组,活体取左眼角膜缘浅层小块,置羊膜上常规和气_液培养42天后进行自体移植治疗右眼角膜缘干细胞缺损;2只兔为对照组,直接用解冻无细胞人羊膜移植治疗右眼角膜缘干细胞缺损。进行细胞和术眼活体观察、组织学观察和电镜观察。结果　角膜缘上皮细胞在羊膜上生长良好,形成复层,细胞间的联结结构存在,细胞与羊膜组织粘附牢固。实验组移植手术后角膜迅速上皮化,恢复角膜表面光滑和透明,组织学观察和电镜观察呈现生理角膜上皮层的结构特点。但眼睑闭合不全可导致手术失败。对照组术后出现角膜缘干细胞缺损导致的角膜病变。结论　以羊膜为载体培养角膜缘上皮细胞后自体移植可有效地治疗角膜缘干细胞缺损导致的角膜病变。     

胎儿角膜上皮细胞分离方法的研究

目的:为了利用流产儿角膜组织体外培养获取角膜上皮细胞,做为构建组织工程化角膜上皮的种子细胞。方法:收集30例(60个角膜)人流产胎儿,分别采用酶消化法、酶消化法结合组织块法和组织块法3种方法分离并培养人胎儿角膜上皮细胞。结果:用Dispase和胰蛋白酶冷消化角膜上皮后,在获取细胞总数、细胞活力和原代培养成功率上没有明显差异,但用Dispase消化后原代细胞容易成活,传代可得到纯化的上皮细胞;酶消化法结合组织块法培养时加入角膜缘组织块可以缩短细胞形成单层的时间,增加传代后细胞的活力;组织块法培养时,在胎儿全角膜组织培养时可得到角膜上皮细胞,角膜组织切割后培养不能获得纯化的角膜上皮细胞。结论:体外构建组织工程化角膜上皮时,种子细胞的获取可以采用流产儿角膜组织为材料分离并培养角膜上皮细胞。     

角膜缘组织定位培养和冷冻后培养的实验研究

目的验证角膜缘干细胞的组织学定位,探讨低温冷冻保存对其增殖活性的影响。方法取新鲜角膜缘上皮组织和相应部位浅层巩膜组织各10块进行体外细胞培养,对比观察细胞生长情况。取冷冻保存的角膜缘上皮组织14例,观察体外培养后细胞生长情况。通过免疫组化方法检测冷冻保存的角膜缘上皮细胞的增殖活性和培养后单层细胞K3角蛋白的表达。结果10例新鲜角膜缘上皮组织培养后,7例有上皮细胞生长,1周形成细胞单层;10例浅层巩膜组织培养后未见细胞生长。14例冷冻角膜缘上皮组织培养后,4例有上皮细胞生长,9d形成细胞单层。5例冷冻角巩膜环组织冰冻切片中,3例可见角膜缘上皮基底细胞PCNA表达阳性。培养细胞对K3角蛋白特异性的AE-5单克隆抗体免疫反应阳性。结论角膜缘干细胞定位于角膜缘上皮基底部,低温冷冻保存的角膜缘干细胞组织可以保持增殖活性,体外培养后生长分化成为角膜上皮。     

深低温冻存及新鲜角膜用于穿透性角膜移植的对比研究

目的进一步探讨深低温保存角膜用于穿透性角膜移植术的价值及比较它与新鲜角膜各自的特点.方法选择36例属优良组的角膜白斑,随机分2组,分别采用新鲜及深低温保存角膜为供体,行穿透性角膜移植术,观察术后植片愈合及泪膜恢复情况,检测角膜厚度、内皮细胞密度及视力恢复情况等.结果采用新鲜及深低温保存角膜为供体,术后泪膜破裂时间(Break-up time,BUT)恢复正常的时间平均为(4.7±0.3)月(2～6月)及平均为(5.6±0.4)月(2～8月);泪液分泌试验(SchirmirTest)恢复正常的时间平均为(2.1±0.3)月(1～4月)及平均为(1.7±0.5)月(1～3.5月);移植片厚度平均为0.62 mm(0.56～0.68 mm)及平均为0.59 mm(0.54～0.62 mm);趋于稳定后的视力分别为0.46±0.03及0.44±0.05;排斥反应发生率分别为20%及19%;二组间均无显著性差异(P＞0.05).但前组术后植片上皮完整,植片始终透明,而后组术后植片上皮缺损,约3～5天才修复,植片出现2～3周暂时性水肿;前组植片内皮细胞密度平均为2 135个/mm2(2 043～2210),后组平均为1 672个/mm2(1 240～1 860个/mm2),二组间有显著性差异(P＜0.01).结论深低温保存角膜用于穿透性角膜移植术后在植片透明率、角膜厚度、视力恢复及排斥反应发生率等方面与应用新鲜角膜无差异,但上皮愈合相对延迟,内皮细胞密度相对较低.眼科学报2001;1768～71.     

Laser scanning in vivo confocal microscopy of the normal human corneoscleral limbus

PURPOSE: To elucidate the structure of the human corneoscleral limbus by in vivo laser scanning confocal microscopy and to correlate limbal epithelial dimensions and density with the central epithelium and in relation to age. METHODS: Fifty adult subjects were recruited into one of two age groups: younger (age<45 years) and older (age>or=45 years). Fifty left eyes of these 50 healthy subjects were examined by laser scanning in vivo confocal microscopy, to assess the basal epithelium of the central cornea and inferior limbus. Mean epithelial cell diameter, area, and density were calculated for the central basal epithelium, limbus-corneal basal epithelium, and limbus-palisade epithelium. RESULTS: Data were analyzed in relation to the two age groups, group A, 30+/-6 years (n=25; mean+/-SD), and group B, 60+/-11 years (n=25; P<0.01). Mean epithelial density in the limbus-cornea and limbus-palisade regions decreased significantly with age: limbus-cornea group A=7253+/-1077 cells/mm2 group B=6614+/-987 cells/mm2, P=0.03; limbus palisade group A=5409+/-799 cells/mm2, group B=5055+/-722 cells/mm2, P=0.03). Central corneal epithelial density did not change with age: group A=6162+/-503 cells/mm2, group B=6362+/-614 cells/mm2, P=0.08. Mean epithelial density was greatest at the limbus-cornea (7010+/-1081 cells/mm2) and lowest at the limbus-palisades (5289+/-847 cells/mm2). The mean width of palisade ridges was 25.0+/-6.3 microm. CONCLUSIONS: This is the first study to image clearly the living human corneal limbus by laser scanning in vivo confocal microscopy and to demonstrate quantitative changes in the basal epithelium with age.     

小鼠角膜上皮细胞消化培养法和组织块培养法的比较研究

目的:比较小鼠角膜上皮细胞消化培养法和组织块培养法。方法:分别使用消化培养法和组织块培养法培养小鼠角膜上皮细胞。比较两种方法中小鼠角膜上皮细胞的克隆形成率(CFE)和群体倍增(PD)。通过Western blotting方法检测p63、角蛋白19以及角蛋白12的表达。结果:其中80%组织块培养法的原代培养可成功传代,而仅12%的消化培养法原代培养可成功传代;两者比较有显著性差异(P<0.05)。传代培养中,组织块培养法中55%的第一代(P1)细胞可以传代超过P10并继续稳定传代至少可传至P25。而消化培养法传代至P2即不能融合。在P1,组织块培养法细胞的CFE高于消化培养法(P=0·02);而组织块培养法P20细胞的CFE又显著高于其P1细胞(P=0.001)。免疫荧光染色显示消化培养法的P1细胞和组织块培养法的P1,P20细胞均表达p63和K19。K12仅在消化培养法的P1细胞和组织块培养法的P1中表达,而组织块培养法的P20细胞中,K12阴性表达。结论:小鼠角膜上皮细胞的培养,组织块培养法优于消化培养法。     

Adaptation of homeostatic ocular surface epithelium to chronic treatment with the opioid antagonist naltrexone

PURPOSE: To determine how ocular surface epithelium adjusts to an increase in cell replication after treatment with the opioid antagonist naltrexone (NTX). METHODS: Adult male rats were given twice daily injections of 30 mg/kg NTX or vehicle for 7 days. Outcomes of NTX administration included DNA synthesis (monitored with BrdU), mitosis (assayed using colchicine), number of cell layers and cell diameter, apoptosis and necrosis, and packing density for the peripheral corneal epithelium, limbus, and conjunctiva. Also, transit time from basal to surface epithelial layers in the peripheral cornea was assessed with [H]thymidine as a marker. RESULTS: DNA synthesis and mitosis in the basal layer of the peripheral corneal epithelium of NTX-treated rats were increased 69% and 85%, respectively, from control levels; no changes in either parameter were recorded in the limbal or conjunctival epithelium (stem cell region). Epithelial thicknesses in the NTX group were increased by 8% to 38% from control subjects, without more cell layers. Packing density in NTX-treated rats was increased from control values by 26% in the basal layer of the limbus and by 12% to 28% in the suprabasal layers of the corneal epithelium, limbus, and conjunctiva. Epithelial cell diameters from corneas of NTX-exposed rats were subnormal in the basal and suprabasal cells of the limbus and conjunctiva. Apoptosis and necrosis were negligible in the epithelium of NTX-treated and control rats. Transit times of peripheral corneal epithelial cells of animals in the NTX group were shortened by 63% from control levels. CONCLUSIONS: These data show that a 1-week treatment with NTX does not induce proliferative pathology or toxicity in ocular surface epithelium, has a minimal effect on stem cell proliferation, and accelerates normal homeostatic processes. Topical application of NTX for stimulation of corneal epithelial wound healing results in no adverse sequelae, thereby supporting the therapeutic role for this drug in the treatment of ocular surface abnormalities.     

Phenotypic characterization of human corneal epithelial cells expanded ex vivo from limbal explant and single cell cultures

Cultivated human corneal epithelial cells have been successfully used for corneal reconstruction. Explant and single cell systems are currently used for human corneal epithelial cultivation. This study was conducted to characterize the phenotypes of human corneal epithelial cells expanded ex vivo by these two culture systems with regard to their growth potential, morphology and antigen expression patterns. Human corneal epithelial cells were expanded by limbal explant culture or limbal single cell suspension culture on a mitomycin C treated 3T3 fibroblast feeder layer. The phenotypes of primary cultured cells were evaluated by morphology and immunohistochemical staining with antibodies for proposed keratinocyte stem cell markers (p63, EGFR, K19 and integrin beta1) and differentiation markers (K3, involucrin and gap junction protein connexin 43). BrdU labeling was performed to identify the label-retaining cells. Human corneal epithelial cells were grown from limbal tissues preserved as long as 16 days by both culture systems. The growth rate depended on the tissue freshness, the time from death to preservation and the time from death to culture, but not on the donor age. Cell growth was observed in 96.2% (n = 43) of single cell suspension cultures and in 90.8% (n = 213) of explant cultures. The cell expansion was confluent in 10-14 days in single cell suspension cultures and 14-21 days in explant cultures. The cell morphology in single cell suspension culture was smaller, more compact and uniform than that in explant culture. Immunostaining showed a greater number of the small cells expressing p63, EGFR, K19 and integrin beta1, while more larger cells stained positively for K3, involucrin and connexin 43 in both culture systems. BrdU-label retaining cells were identified in 2.3+/-0.7% of explant cultures and 3.73+/-1.5% of single cell cultures chased for 21 days. In conclusion, the limbal rims are a great treasure for ex vivo expansion of human corneal epithelial cells. The phenotypes of corneal epithelial cells, ranging from basal cells to superficial differentiated cells, are well maintained in both culture systems. Slow-cycling BrdU-label retaining cells, that are characteristic of stem cells, were identified in the cultures.     

Preliminary study of the effect of FK506 nanospheric-suspension eye drops on rejection of penetrating keratoplasty.

OBJECTIVE: The aim of this study was to investigate the effect of a topical FK506 nanospheric suspension in a rat model of penetrating keratoplasty. METHODS: FK506 nanospheres were prepared by using a biodegradable poly (lactic-co-glycolic acid) copolymer (PLGA). Its distribution in the eye and blood after a single instillation was examined in rabbits. Sprague-Dawley (SD) rats received corneal heterografts and were topically treated with phosphate-buffered saline (PBS), PLGA, FK-506 0.01% (nanospheres), or dexamethasone 0.05% solutions twice a day for 28 days. Rejection index and graft-survival time were recorded and compared between the four groups. Three grafts were collected at different time points for immunohistochemical studies. RESULTS: In the cornea, the FK-506 concentration reached its peak within 1 h of a single eye-drop instillation and then decreased by half (1667.85 +/- 611.87 ng/g) at 8 h. FK-506 cannot be detected in rabbit blood. There were significant differences in the graft-survival time between the FK-506 nanosphere group (15.09 +/- 4.81 days) and the other three groups [PBS (7.90 +/- 1.20, t = -4.594, P < 0.001), PLGA (8.44 +/- 0.88, t = - 4.074, P = 0.001) and dexamethasone (10.44 +/- 1.42, t = -2.790, P = 0.012)]. The rejected corneas in the FK506 nanosphere group showed significantly fewer CD4, CD8, CD68, CD79, vascular endothelial growth factor, ICAM, and tumor growth factor-beta(1)-positive cells than those in the other groups. CONCLUSIONS: FK506 0.01% nanospheric-suspension eye drops delayed the occurrence of corneal allograft rejection and prolonged allograft survival time. The FK506 nanospheres may be valuable in suppressing corneal graft rejection.     

Culture of the corneal epithelium--comparison of the mitotic potential of limbal cells from living and cadaveric donors

PURPOSE: We analyse effectiveness of corneal limbal cells' culture prepared from heart-beating organ donors, that include living donors and from cadaver donors buttons following 3 days storage in 4 degrees C in Likorol. MATERIAL AND METHODS: For experiment 12 adults (living and heart-beating organ donors) aged 28-63 (mean 46.3) and 12 corneal buttons of cadaver (aged 18-51, mean 34.1) were qualified. Tissue samples (1 mm2) were taken from superior corneal limbus. Sample from living donor obtained during routine operation was sent immediately to laboratory, as well as from heart-beating organ donor. The limbal biopsy of preserved cornea was taken after 3 days storage in 4 degrees C (Likorol). Samples were treated with trypsin/EDTA solution before culture. Collected cells in similar density in 1 ml of medium laid on dishes inserts, covered with fibrin (Tissucol) and cultured in presence of feeder layer of fibroblasts (L929 line). Epithelial cells were cultured for 14 days at 37 degrees C in humidified 5% CO2 atmosphere in supplemented 2:1 mixture of DMEM and Ham's F12. On the 14th day cells were collected. Number of cells per 1 ml of medium was counted by cytometer. Immunostaining for epithelium type (Keratin 3) was performed. RESULTS: The number of cells obtained from cadaver donors reached 184.2+/-14.9% whereas from living donors revealed 1013.1+/-104.2%, increase in relation to number of delivered cells. We observed only 0.83+/-0.3 colonies per microscopic area in cultures from preserved tissue versus 6.67+/-0.6 colonies in cultures from living donor. CONCLUSIONS: The preservation in 4 degrees C in Likorol significantly decreases proliferative potential of the corneal limbus.     

角质细胞生长因子促进角膜上皮损伤修复的研究

目的寻找促进角膜上皮损伤修复，治疗持续性角膜上皮缺损的有效方法。方法用３Ｈ－胸腺嘧啶核苷（３Ｈ－ＴｄＲ）掺入及液体闪烁技术，观察不同浓度的外源性角质细胞生长因子（ｋｅｒａｔｉｎｏｃｙｔｅｇｒｏｗｔｈｆａｃｔｏｒ，ＫＧＦ）对体外培养的人角膜上皮细胞生长的影响，由此推算出有效滴眼液浓度并应用于兔眼。用计算机图形分析系统计算角膜上皮愈合速率；用光镜和电镜评估愈合的质量。结果０．１～１００ｎｇ／ｍｌＫＧＦ有明显促进体外培养的人角膜上皮细胞生长的作用（增长率为２７．６６％～７６．７３％），且呈剂量依赖性（ｒ＝０．９２３３，Ｐ＜０．００１）。１μｇ／ｍｌＫＧＦ滴兔眼，加速了角膜上皮损伤修复（愈合速率，ＫＧＦ组为１．７７±０．２３ｍｍ２／ｈ，与对照组１．４９±０．２４ｍｍ２／ｈ比较，Ｐ＜０．０５）。结论外源性ＫＧＦ对体外培养的人角膜上皮细胞有明显的促生长作用，其滴眼液有加速兔眼角膜上皮创伤修复的作用。     

Comparison of cell-suspension and explant culture of rabbit limbal epithelial cells

Currently, most investigators directly use limbal explants to culture corneal epithelial cells. However, it has not been identified that limbal stem cells do readily migrate from the limbal explants onto culture plate or amniotic membrane carrier. In this study a cell-suspension culture system for rabbit limbal stem cells was developed and compared with the direct explant method in the aspect of stem cells content in the culture system. Rabbit limbal epithelial cells were dissociated from rabbit eyes by dispase and single cell suspension was made for cell-suspension culture. DeltaNp63 expression of cultured rabbit limbal epithelial cells by cell-suspension technique and explant technique was detected. In cell-suspension culture, isolated cell-suspension was evaluated by flow cytometric analysis for vimentin expression and residual limbal tissue after dispase treatment was examined by scanning electron microscopy. In limbal epithelial cells suspension less than 5% cells were vimentin positive. Examination of residual limbal tissue confirmed that all the limbal epithelial cells had been removed. Histological examination revealed that with cell-suspension culture the cultured epithelial cells could differentiate better than with explant technique. In cells cultured with cell-suspension, there were much more cells expressing DeltaNp63 than in explant cultured cells. In cells cultured with explants, most of DeltaNp63 labelling cells mustered around the explants, and peripheral cells on the slides were DeltaNp63 negative. These results suggested that with pure limbal epithelial cells suspension including basal cells, which could directly enter into culture system, cell-suspension culture technique was significantly superior to explant culture technique in terms of stem cells content.     

Inhibitory effects of Triptolide on interferon-gamma-induced human leucocyte antigen-DR, intercellular adhesion molecule-1, CD40 expression on retro-ocular fibroblasts derived from patients with Graves' ophthalmopathy

PURPOSE: To explore the effects of Triptolide, the principal active diterpenoid from the Chinese Medicinal Herb Tripterygium Wilfordii Hook F that has immunosuppressive and anti-inflammatory properties, on cell proliferation, hyaluronic acid (HA) synthesis, and the expressions of human leucocyte antigen-DR (HLA-DR), intercellular adhesion molecule-1 (ICAM-1) and CD40 on cultured retro-ocular fibroblasts (RFs) from patients with Graves' ophthalmopathy. METHODS: After two to five passages, cultured RFs were incubated for 48 h within a medium alone or in the presence of recombinant human interferon-gamma (IFN-gamma) and various concentrations of Triptolide. Cell viability was assessed by MTT (3-[4.5-dimethylahiazol-2-yl]-2,5-diphenyltetrazolium Bromide). RFs proliferation was assessed by [(3)H]-thymidine incorporation assay. Flow cytometry was used to investigate the amount of HLA-DR, ICAM-1 and CD40. HA synthesis was measured by radioimmunoassay. RESULTS: Cell viability was not detrimentally affected when incubated with Triptolide from 0.01 microg/L to 10 microg/L for 48 h, and decreased with 20 microg/L Triptolide. The incorporation of [(3)H]-thymidine of RFs was 55 476 +/- 15 842 cpm incubated with medium alone or 18 352 +/- 3568 cpm with 10 microg/L Triptolide (t = 5.600, P < 0.01). Initially, the percentage of positive cells of HLA-DR, ICAM-1 and CD40 on RFs were 4.75 +/- 2.13%, 17.53 +/- 10.12% and 6.38 +/- 2.23%, respectively, and the synthesis of HA was 100 +/- 12%. Compared with basal values, 48-h incubation with IFN-gamma (100 U/mL) significantly enhanced the amount of HLA-DR, ICAM-1 and CD40, and HA synthesis. The values were 60.58 +/- 10.12% (t = 13.224, P < 0.01), 62.66 +/- 18.17% (t = 5.315, P < 0.01), 57.67 +/- 13.61% (t = 9.110, P < 0.01) and 164 +/- 22% (t = 9.238, P < 0.01), respectively. Triptolide 0.01 microg/L had little effect on IFN-gamma-induced HLA-DR, ICAM-1 and CD40 amounts, as well as HA synthesis. When the concentration ranged from 0.1 microg/L to 10 microg/L, Triptolide inhibited IFN-gamma-induced RFs activation in a dose-dependent manner. It was also found that Triptolide had the same inhibiting effects on IFN-gamma-induced RFs and skin fibroblasts from patients with normal individual conditions. CONCLUSIONS: Triptolide could inhibit IFN-gamma-induced activation of RFs derived from patients with Graves' ophthalmopathy.