FK506抑制同种异体角膜缘移植术兔外周血T淋巴细胞CD25的表达

目的 探讨FK506滴眼液对同种异体角膜缘移植术后的免疫抑制作用。方法 新西兰兔48只48眼建立角膜缘缺陷症动物模型。随机分为FK506组、环孢霉素A(CsA)组及生理盐水对照组,每组16眼。1个月后实施同种异体角膜缘移植术,检测术前及术后1－8周兔外周血T淋巴细胞上CD25的表达。结果 各组表达CD25的外周血T淋巴细胞数均有升高。对照组较其他两组高;而CsA组显著高于FK506组(P＜0. 05)。 结论 FK506滴眼液可抑制同种异体角膜缘移植术后T淋巴细胞上CD25的表达,其免疫抑制效果优于CsA。     

FK506滴眼液抑制兔同种异体角膜缘移植排斥反应的免疫病理学研究

目的 :观察同种异体角膜缘移植术后排斥反应的免疫病理学变化 ,探讨FK5 0 6滴眼液的免疫抑制作用。方法 :新西兰兔 48只 48眼建立角膜缘缺陷症动物模型。随机分为PK 5 0 6组、环孢霉素A组 (CsA )及生理盐水对照组 ,每组16眼。 1月后实施同种异体角膜缘移植术。应用免疫病理学方法检测术后 4、 8、 10周角膜缘植片CD2 5的表达和淋巴细胞浸润。结果 :术后 4、 8、 10周三组角膜缘植片CD2 5的表达和淋巴细胞浸润均有升高 ,对照组较FK5 0 6组和CsA组高 (P <0 0 5 ) ,CsA组显著高于FK5 0 6组 (P <0 0 5 )。结论 :PK 5 0 6滴眼液可抑制同种异体角膜缘移植术后植片CD2 5的表达 ,其免疫抑制作用优于CsA。     

FK506眼液防治同种异体角膜缘移植免疫排斥反应的实验研究

目的　探索同种异体角膜缘移植排斥反应规律及FK5 0 6眼液的免疫抑制作用。方法4 8只新西兰白兔随机均分成FK5 0 6组、环孢素A组及非治疗组 ,建立右眼角膜缘缺陷症动物模型 ,1个月后行同种异体角膜缘移植术 ,术后分别用 0 5 %FK5 0 6眼液、1%环孢素A眼液及生理盐水滴眼4周。角膜缘移植术前 1d ,术后 2、4周分别进行角膜中央印迹细胞学检查 ;术前 1d及术后 1～ 8周动态监测外周血T淋巴细胞CD2 5的表达 ;移植术后每日观察植片存活、角膜新生血管和上皮再生情况 ,共 10周 ;分别取术后第 4、8及 10周的角膜缘植片观测淋巴细胞浸润及T淋巴细胞上CD2 5的表达。结果　角膜缘创伤后 1个月 ,所有兔眼角膜中央印迹细胞学检查均为结膜表型 ;同种异体角膜缘移植术后 4周 ,FK5 0 6组和环孢素A组角膜中央保持角膜细胞表型 ,非治疗组呈结膜细胞表型。非治疗组最早出现上皮排斥反应 ,FK5 0 6与环孢素A均可抑制排斥反应的发生 ,停药后环孢素A组先于FK5 0 6组出现上皮排斥反应 (P <0 0 5 )。FK5 0 6组外周血、角膜缘植片T淋巴细胞CD2 5表达及角膜缘植片中淋巴细胞浸润程度低于环孢素A组 ,两组均低于非治疗组 (P <0 0 5 )。结论　同种异体角膜缘移植术后早期应用 0 5 %FK5 0 6眼液滴眼可抑制外周血及植片T淋巴细胞CD2 5     

FK506抑制兔同种异体角膜缘移植免疫排斥反应的实验研究

目的 探讨FK506滴眼液对同种异体角膜缘移植术后排斥反应的免疫抑制作用。方法 新西兰兔48只48眼建立角膜缘缺陷症动物模型。随机分为FK506组、环孢霉素A组及生理盐水对照组3组，每组16眼。1个月后实施同种异体角膜缘移植术，眼表观察10周。结果 术后10周，对照组、环孢霉素A组、FK506组角膜新生血管指数平均秩分别为15．50、8．00、5．00。对照组角膜新生血管指数较其他2组高（P＝0．00）；而FK506组与环孢霉素A组间无显著差异（P＝0．12）；对照组上皮排斥反应发生最早，环孢霉素A组次之，FK506组最晚；术后10周，对照组、环孢霉素A组、FK506组移植排斥反应指数平均秩分别为13．67、8．07、4．58，对照组排斥反应指数较FK506组（P＝0．02）和环孢霉素A组（P＝0．00）高；环孢霉素A组移植排斥反应指数显著高于FK506组（P＝0．04）。同种异体角膜缘移植术后眼局部应用FK506滴眼液可延迟排斥反应发生，其免疫抑制效果优于环孢霉素A。     

大鼠异体角膜缘移植排斥的实验研究

目的通过建立大鼠角膜缘移植模型,对比观察同种异体角膜缘移植和自体角膜缘移植术后的表现及组织病理特点。方法32只SD大鼠随机分成两组,分别接受来自Wistar大鼠和自体对侧眼的角膜缘移植术。移植术后观察30d。依据裂隙灯下植片水肿、炎症反应、新生血管增加的情况,判断植片排斥反应的发生。PAS染色观察组织病理特点。结果根据眼表评分,异体角膜缘移植组中13只(81·3%)SD大鼠平均术后6d发生了移植排斥反应;自体移植组无一例发生排斥。两者比较具有统计学意义(P<0·01)。大鼠异体角膜缘移植排斥的主要表现为植片水肿、炎症、新生血管生成等。组织病理显示,在异体移植组有大量的淋巴细胞浸润;而自体角膜缘移植组仅有极少量炎症细胞浸润。结论通过尝试建立以大鼠为研究对象的角膜缘移植模型,观察异体角膜缘移植排斥反应的表现和组织病理特点,可以为异体角膜缘移植免疫排斥的研究提供新的平台。     

TGF—β1对角膜上皮移植后CD4^＋,CD^＋8,CD25^＋和CD71^＋的影响

目的：为有效控制角膜移植术后排斥反应的发生,提高角膜移植成功率,我们在小鼠（BALB／c)角膜上皮移植术后应用TCF－β1,并观察其对外周血淋巴细胞亚群CD4^ ,CD8^ ,CD25^ ,CD71^ 活化的影响,为今后临床应用TGF－β1抑制角膜移植术后免疫排斥反应奠定基础。方法：采用CD4^ ,CD8^ ,CD25^ ,CD71^ 免疫荧光标记及流式细胞仪检测技术。分别对设立的阴性对照组,阳性对照组,同基因组,异体角膜上皮组及TGF－β1治疗组的BALB/c小鼠角膜上皮移植术后12d的外周血中CD4^ ,CD25^ ,CD8^ ,CD25^ ,CD4^ ,CD71^ ,CD8^ CD71^ 的表达进行分析,结果：BALB/c小鼠角膜上皮移植术后12d的外周血中CD25^ CD4^ ,CFD25% CD8^ ,CD71^ CD4^ 和CD71^ CD8^ 双阳性的T淋巴细胞均有显升高,术后经TGF－β1治疗后,上述细胞的数量明显受到抑制。CD25^ CD8^ ,CD71^ CD4^ 和CD71^ CD8^ 双阳性的T淋巴细胞均有显升高,术后经GF－β1治疗后,上述细胞的数量明显受到抑制。结论：角膜上皮移植术后应用TGF－β1可抑制特异性抗原介导的,以及非特异性炎疗诱诱导的移植排斥反应。     

同种异体角膜缘移植术后免疫排斥反应的实验研究

目前导致异体角膜缘移植失败的主要原因是术后免疫排斥反应造成的干细胞永久性破坏。本研究通过建立大鼠同种异体角膜缘移植模型，分析了同种异体角膜缘移植术后免疫排斥反应的特征性表现，并初步探讨了其免疫排斥反应的机制。     

兔异体角膜缘移植的实验研究

目的：观察异体角膜缘移植反应的临床表现，初步探讨角膜缘移植反应的免疫机制。方法：20只健康白兔随机分为三组，分别行角膜缘环形切除＋角膜上皮剥离术（n=6），自体角膜缘移植术（n=7），异体角膜缘移植术(n＝7）；术后以1％荧光素钠染色观察角膜上皮化，移植排斥反应通过移植眼的临床表现和病理检查加以评估。结果：异体角膜缘移植术后早期可见角膜上皮化，术后10－14天（平均11．8天）发生角膜上皮炎症反应，反应可分为四个阶段：初发期、急性期、排斥期和慢性期；病理检查显示炎症反应的发生及其强度。结论：本试验模型中，移植反应的各个时期有明显的特征，对各期移植反应的说明将使我们在临床上更容易、准确的判断移植反应的发生及其强度。     

FK-506抑制同种异体角膜缘移植术后免疫排斥反应的研究

目的：探讨ＦＫ－５０６抑制角膜缘干细胞移植术后免疫排斥反应的临床可行性与有效性。方法：应用前瞻性评估研究方法,将角膜缘移植术后病例６４只眼按随机原则分投药组及对照组各半,投药组应用０．５％ＦＫ－５０６滴眼液,对照组应用１％ＣｓＡ滴眼液。平均随访期半年,以术后角膜缘植片新生血管、水肿、混浊及溶解程度为临床主要评估指标。结果：随访期内在新生血管指数、上皮排斥发生率、移植排斥指数及假性胬肉指数四项指标投     

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。     

Efficacy of systemic cyclosporine A and amniotic membrane on rabbit conjunctival limbal allograft rejection

PURPOSE: To evaluate the effect of intramuscular cyclosporine A (CsA) and amniotic membrane (AM) on conjunctival limbal allograft survival in a rabbit model. METHODS: Eighty-two female rabbits (59 New Zealand white rabbits, 23 Dutch pigmented rabbits) were used. The New Zealand white rabbits were divided into 4 treatment groups: group 1 (n=13), conjunctival limbal autograft transplantation; group 2 (n=12), conjunctival limbal allograft transplantation without additional treatment; group 3 (n=18), conjunctival limbal allograft transplantation and human AM; and group 4 (n=16), conjunctival limbal allograft transplantation and systemic CsA (10 mg/kg/day intramuscularly). The 23 Dutch pigmented rabbits were used as limbal stem cell allograft donors. The rejection index, the mean survival time, and the rejection rates were calculated for each group. RESULTS: After 28 days of follow-up, there were no episodes of limbal rejection in groups 1 and 4, whereas the rejection rate was 100% in groups 2 and 3. There was no significant difference in mean survival time of the rejected grafts between groups 2 and 3. CONCLUSIONS: A model of rejection of conjunctival limbal transplantation was developed in the rabbit. Intramuscularly injected CsA effectively prevents limbal allograft rejection. Human AM is not useful for this purpose.     

Survival of rabbit limbal stem cell allografts after administration of cyclosporin A.

OBJECTIVES: To evaluate the efficacy of systemic or topical administration of cyclosporin A (CsA) after limbal transplantation of stem cell allografts in rabbits. METHODS: Thirty-six rabbits underwent corneal epithelial debridement and limbal ablation to induce ocular surface disease and were then treated by limbal allograft transplantation. Animals received either systemic CsA (10 mg/kg per day, intramuscularly), 1% CsA eyedrops, or vehicle eyedrops immediately after transplantation and 28 days thereafter. Concentration of CsA in plasma and aqueous humor was determined by fluorescence polarization immunoassay after 4 weeks of therapy. Graft survival was inspected clinically. RESULTS: Both systemic and topical administration of CsA resulted in a significant prolongation of graft survival. In addition, one of seven of the limbal allografts in either group of systemic and topical CsA survived >60 days on cessation of CsA. There was no significant difference in mean survival time between systemic and topical application, although plasma levels of CsA were significantly higher after systemic administration. However, a significant higher aqueous concentration was found in topical treatment. CONCLUSIONS: Limbal allografts were stable in maintaining the reconstructed ocular surface under attentive postoperative immunosuppression. Topically administered CsA was as effective as systemic use.     

Long-term survival of allogeneic donor cell-derived corneal epithelium in limbal deficient rabbits

PURPOSE: To investigate the capability of cultivated allogeneic epithelial stem cells to restore a functional ocular surface in a limbal deficient cornea; to verify the long term survival of epithelial allograft; and to examine the host immune response to heterologous cell transplant in a rabbit model. METHODS: Limbal deficiency was established by performing limbectomy on rabbits (n = 100). Corneal epithelial stem cells were obtained from the limbus and replicated in vitro without a supporting layer. The cell (3 x 10(5)) suspension was then transplanted via topical application as eye drops. Animals were divided into allograft, autograft, and control groups. Females were used as recipients and males as donors for the allograft. Corneas were collected at 7, 14, 21, 40 days as well as 2, 3, 7 and 8 months after cell transplantation. Experimental corneas were evaluated by histology, immunofluorescence, immunohistochemistry and Y chromosome analysis. RESULTS: A well-differentiated corneal epithelium was recognized at 14 to 40 days after cell transfer overlying an infiltrated corneal stroma. Corneal re-epitheliazation was confirmed in 31 of 36 allograft corneas. No significant immune rejection was noted. Stromal abnormality caused by previous limbal deficiency was mostly resolved three months after the regeneration of corneal epithelium. CONCLUSIONS: Transplanted corneal epithelial stem cells were able to differentiate into normal corneal epithelium in vivo without the use of membrane scaffolding. This non-autologous donor cell-derived corneal epithelium survived up to 8 months without immunosuppression and was able to reverse the stromal scarring. Thus, cultivated epithelial stem cells have great potential as an alternative to multiple-surgical procedures in the treatment of limbal deficiency states.     

Effect of Immunosuppression on Survival of Allograft Limbal Stem Cells

Purpose To examine whether the application of immunosuppression enhances the survival of limbal allograft stem cells.Methods Wistar (allograft) or Fischer 344 (isograft) rat limbal tissue was transplanted into superior lamellar excision sites in Fischer 344 rats. Allograft-recipient rats received an immunosuppressive agent or vehicle for 8 weeks. Graft-recipient rats were examined by slit-lamp microscopy for clinical signs of rejection, and some recipients were killed for immunohistochemical analysis during rejection. One month from the time of transplantation, other (five in each group) recipients received daily injections of 5-bromo-2-deoxyuridine (BrdU) at a dose of 5mg/100g for 2 weeks, followed by a 1-month BrdU-free period before death. After the rats were killed, limbal allograft eyes were removed for BrdU staining to identify label-retaining cells. The labeling index of transplanted limbal basal epithelial cells was determined.Results In nonimmunosuppressed allograft recipients, clinical rejection occurred between days 5 and 10 after transplantation (median, 7 days). In contrast, rejection was suppressed for more than 60 days (median value) when the immunosuppressive was administered. Numerous MHC class II⁺, CD4⁺, and CD8⁺ T cells were identified with acute rejection. Immunosuppressive-treated allografts had significantly less inflammation compared with untreated controls. Furthermore, immunosuppressive-treated allografts showed more label-retaining basal cells in transplanted limbal epithelium compared with untreated allograft controls (P < 0.01).Conclusions The survival of limbal allograft stem cells can be improved by immunosuppression. The limbal allograft procedure described here provides a useful model for evaluating a suitable alternative means of sustaining the survival of corneal stem cells. Jpn J Ophthalmol 2004;48:440–447 © Japanese Ophthalmological Society 2004     

角膜移植患者外周血中T细胞CD28分子的表达及其意义

目的　探讨角膜移植患者外周血T细胞及亚群CD2 8分子表达的变化及其意义。方法　于术前1d、术后第1、2、3、4周,应用流式细胞仪检测2 5例角膜移植患者外周血中CD2 8分子表达水平的变化。结果　角膜高血管化植床组患者术后外周血T细胞CD2 8分子表达比角膜无血管化植床组患者明显增高,也比术前明显增高(P <0 .0 1) ;6例发生排斥反应的患者均出于高血管化植床组;术后早期外周血T细胞中以CD4 + 细胞为主。结论　外周血T细胞CD2 8分子表达与角膜移植排斥反应关系密切,检测角膜移植患者外周血中的CD2 8分子表达可更早监测免疫排斥反应的发生     

兔同种异体角膜缘移植角膜印迹细胞学检测

目的 研究兔角膜缘干细胞缺乏和同种异体角膜缘移植后临床和角膜上皮表型的改变。方法 建立兔角膜缘干细胞缺乏模型,1个月后对治疗组进行同种异体角膜缘移植,术后联合使用免疫抑制剂。比较治疗组与非治疗组的临床表现和角膜表型的改变。结果 兔角膜缘干细胞缺乏后,角膜混浊,新生血管化,持续性上皮缺损;角膜上皮为结膜细胞表型。移植术后角膜上皮完整,新生血管减少,角膜透明度增加;上皮恢复角膜表型。结论 兔同种异体角膜缘移植联合术后使用免疫抑制剂是治疗角膜缘干细胞缺乏症的有效方法。印迹细胞学检查是角膜缘干细胞缺乏症诊断和角膜缘移植术后的评价手段。