复方倍他米松对异种角膜板层移植术后免疫排斥反应的影响

目的：观察结膜下注射复方倍他米松对鸵鸟-兔眼板层角膜移植术后免疫排斥反应的缓释治疗作用。

方法：16只6周龄健康新西兰白兔角膜(单眼)行板层角膜移植术,植片应用鸵鸟脱细胞的角膜基质,术后分成两组,实验组术毕及术后结膜下注射复方倍他米松注射液(每隔7d一次),对照组术毕及术后结膜下注射地塞米松磷酸钠注射液(每隔7d一次)。分别于术后1、2wk, 1、2mo取兔角膜组织做石蜡包埋切片,进行HE染色观察组织特点,同时进行间接免疫荧光法检测CD4⁺、CD8⁺ T淋巴细胞的变化。

结果：术后2mo,实验组角膜植片在位,并保持透明,新生血管极少,组织切片HE染色和间接免疫荧光染色结果显示,仅见少许中性粒细胞浸润,未见CD4⁺、CD8⁺ T淋巴细胞; 对照组炎症反应明显,角膜植片混浊,组织切片HE染色和间接免疫荧光染色结果显示,炎性反应以中性粒细胞浸润为主,可见CD4⁺、CD8⁺ T淋巴细胞。

结论：复方倍他米松作为长效缓释剂可有效抑制鸵鸟-兔板层角膜移植术后免疫排斥反应,延长植片的存活时间。     

沙利度胺对鸵鸟-兔异种板层角膜移植后新生血管生长的影响及其机制

目的探讨沙利度胺(thaolidiomide)对鸵鸟-兔异种板层角膜移植后新生血管生长的影响及其机制。方法选择新西兰白兔眼作为受体,鸵鸟角膜为供体,将其分为A组(兔-兔非处理组)、B组(鸵鸟-兔非处理组)、C组(鸵鸟-兔组,沙利度胺200mg/(kg·d)灌服,持续4wk)、和D组(鸵鸟-兔组,强的松1wk内0.1mg/(kg·d)灌服,之后每周减量0.005mg,持续4wk)共4组,每组6只兔。术后每天在裂隙灯下观察植片的反应,并按Holland标准给植片评分;术后4wk各组取全部的角膜植片行RT-PCR和免疫组化检测VEGF的表达,取角膜植片之前测量新生血管的长度、根数、钟点数及角膜的圆周半径。结果C组植片一直保持透明,无新生血管生长,存活时间长于异种移植其他各组(P<0.01)。C组植片VEGF的表达量显著低于异种移植的其他各组(P<0.01)。结论沙利度胺通过抑制VEGF的表达抑制异种板层角膜移植术后新生血管的生长,从而延长角膜植片的存活时间。     

鸵鸟角膜对兔板层角膜移植的排斥反应

目的：角膜穿孔属眼科急症，角膜外伤、感染和化学伤都可引起角膜穿孔，用角膜移植治疗穿孔效果较好，但角膜供体资源在我国目前仍较为缺乏。角膜移植排斥反应是造成移植失败的主要原因。为探索异种角膜移植材料，本实验研究鸵鸟对兔板层角膜移植的植片存活时间及排斥反应特点，以便评价鸵鸟角膜的应用前景。方法：将24只≤8周龄的健康新西兰白兔随机分成两组：A组12只兔为兔一兔同种异体板层移植组；B组12只兔为鸵鸟一兔异种角膜板层移植组，治疗及观察时间均为6wk。两组术后均予结膜下注射地塞米松。术后每日在裂隙灯显微镜下观察植片存活情况和排斥反应指数；术后每周测量新生血管面积；术后1，3，5wk各组取1只兔的术眼角膜行病理检查。结果：鸵鸟板层角膜植片的平均存活时间为23d。驼鸟一兔异种板层和同种板层角膜在1，2wk排斥反应指数及角膜新生血管面积无显著差异，第3wk起差异显著。结论：鸵鸟角膜植片在移植早期能保持透明，且有较好的生物相容性。鸵鸟角膜植片后期因新生血管生长和排斥反应加重而混浊，说明单独应用地塞米松不能完全控制异种植片的排斥反应。     

TGF-β1 对角膜上皮移植后CD4+、CD8+、CD25+、CD71+的影响

目的 :角膜移植排斥反应是角膜移植失败的主要原因。方法 :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 ,我们观察了TGF β1对BALB/c小鼠角膜上皮移植术后外周血淋巴细胞亚群CD 4 、CD 8、CD 2 5、和CD 71活化的影响。研究中采用CD 4 、CD 8、CD 2 5、CD 71、免疫荧光标记及流式细胞仪检测技术 ,对BALB/c小鼠角膜上皮移植术后 12d的外周血中CD 4 、CD 8、CD 2 5、CD 71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CD 4 CD 2 5、CD 8CD 2 5、CD 4 CD 71、及CD 8CD 71双阳性的T淋巴细胞均有显著升高 ;术后经TGF β1治疗后 ,上述细胞的数量明显受到抑制。结论 :TGF β1可抑制特异性抗原介导的 ,以及非特异性炎症诱导的移植排斥反应     

沙利度胺对鸵鸟-兔异种角膜板层移植术后免疫排斥反应的影响

目的探讨沙利度胺(THD)对鸵鸟-兔异种角膜板层移植免疫排斥反应的治疗作用。方法选择新西兰白兔眼作为受体,新鲜的鸵鸟角膜为供体,将其分为4组,每组8只:A组(兔移兔对照组)、B组(鸵鸟移兔非处理组)、C组(鸵鸟移兔组,THD每日200mg/kg灌服,早晚各1次,持续4周)、D组(鸵鸟移兔组,结膜下注射地塞米松2mg,每日1次,持续4周)。于术后2、3、4周各组取角膜植片做病理组织学检查。分别于术前、术后1、2、3、4周各组取相同兔的外周血,采用流式细胞仪技术检测T淋巴细胞亚群CD4 、CD8 的动态变化。结果C组T淋巴细胞亚群CD4 /CD8 的比例在术后1周增高,之后随时间推移逐渐减低,而其余各组随时间推移逐渐增高。C组植片一直保持透明,无新生血管生长,仅见个别炎细胞。其余各组炎症均比C组重。结论THD能抑制异种角膜板层移植术后免疫排斥反应,延长异种移植物的存活。但有轻微的不良反应。     

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。     

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     

CD4+CD25+调节性T细胞参与大鼠角膜移植免疫耐受的研究

目的 探讨超抗原金黄色葡萄球菌肠毒素B(SEB)诱导大鼠高危角膜移植免疫耐受时CD4+CD25+调节性T细胞在眼局部及全身的表达情况.方法 本实验采用对照设计,以13只SD大鼠作为供体,26只Wistar大鼠作为受体建立高危穿透性角膜移植模型.所有受体大鼠抽签法随机分为2组,对照组和SEB组分别腹腔注射生理盐水和SEB(75 μg/kg体重),1次/4 d,共3次,末次注射后1 d行右眼穿透性角膜移植术.术后观察大鼠角膜植片的存活时间,并采用免疫荧光染色分析CD4+CD25+T细胞在大鼠角膜植片和虹膜的表达,使用流式细胞学方法分析外周血中的CD4+CD25+T细胞百分数.结果 对照组大鼠角膜植片平均存活时间为(11.63±2.83)d,SEB组为(14.13±0.99)d,两组间比较差异有统计学意义(P＜0.05).组织病理学检查可见发生排斥的角膜植片有大量炎性细胞浸润,炎性细胞数量与排斥反应程度呈正相关性.直接免疫荧光染色检查可见术前两组角膜组织均不表达CD4+CD25+T细胞,而术后20 d两组均有表达.虹膜铺片直接免疫荧光染色检查术前对照组未见CD4+CD25+T细胞表达,而SEB组(首次注射SEB后第10天)虹膜片可见CD4+CD25+T细胞表达,术后第20天两组大鼠虹膜铺片均可见CD4+CD25+T细胞表达,并且SEB组阳性细胞数更多.流式细胞学方法分析显示手术前后、对照组大鼠外周血中CD4+CD25+T细胞的百分数无变化,而SEB组呈先升高、后降低的变化趋势.结论 CD4+CD25+调节性T细胞参与超抗原SEB诱导大鼠高危角膜移植术后免疫耐受的形成.     

CD4和CD8基因敲除鼠行穿透性角膜移植术后免疫排斥特征的研究

目的　探讨CD4和CD8基因敲除小鼠行穿透性角膜移植术后免疫排斥反应发生的机制。方法　CD4、CD8基因敲除小鼠及C57BL/6小鼠各20只,分成3组,右眼接受穿透性角膜移植术,供体为BALB/c小鼠,术后裂隙灯显微镜评价角膜移植片情况,并详细记录免疫排斥的发生时间,在术后1、2、4周各取2只鼠术眼行免疫组织化学检查,观察眼前段CD+4 、CD+8 T细胞的变化。在术后2周, 3组小鼠各选其中10只接受皮肤移植,供体为BALB/c小鼠,监测皮肤移植后皮肤植片免疫排斥反应的时间和在皮肤发生排斥反应时角膜移植片的情况。结果　3组小鼠角膜移植术后免疫排斥发生时间明显不同,CD4基因敲除鼠角膜移植片保持透明,至少观察了90d未见免疫排斥反应发生;CD8基因敲除小鼠在(28±3)d时发生免疫排斥反应;C57BL/6小鼠发生免疫排斥反应的时间为(14±2)d(F=2034, P<0. 01)。移植皮肤后发生免疫排斥反应时间为:CD4基因敲除鼠(14±2)d,CD8基因敲除鼠(12±1)d,C57BL/6小鼠(10±1)d(F=42. 54, P<0. 01)。结论　小鼠行穿透性角膜移植术后免疫排斥反应可能是以T淋巴细胞,主要为CD+4 T细胞介导的免疫排斥反应,CD+8 T细胞参与了排斥反应过程。     

异种角膜基质异位植入后免疫病理学研究

目的 观察异种角膜基质异位植入后的免疫病理变化，评价其免疫相容性。方法将一定大小的新鲜的和甘油脱水的猪角膜基质植入到小鼠皮下组织内，同时以异种皮肤、假手术为阳性和阴性对照；术后1个月取材进行组织病理学观察，并通过免疫荧光细胞化学法检测植入材料内T淋巴细胞总数和亚群的变化。结果新鲜的和甘油脱水的猪角膜基质皮下植入后1个月，可见少量的淋巴细胞、巨噬细胞、CD3＋细胞、CD4＋细胞、CD8＋细胞浸润．且CD4＋细胞多于CD8＋细胞，但未达到阳性对照水平（P〈0．05）。结论异种角膜基质异位植入后早期局部未引发免疫排斥反应，免疫相容性好。     

Mechanism of immune tolerance induced by donor derived immature dendritic cells in rat high-risk corneal transplantation

AIM: To study the role of immature dendritic cells (imDCs) on immune tolerance in rat penetrating keratoplasty (PKP) in high-risk eyes and to investigate the mechanism of immune hyporesponsiveness induced by donor-derived imDCs.METHODS:Seventy-five SD rats (recipient) and 39 Wistar rats (donor) were randomly divided into 3 groups:control, imDC and mature dendritic cell (mDC) group respectively. Using a model of orthotopic corneal transplantation in which allografts were placed in neovascularized high-risk eyes of recipient rat. Corneal neovascularization was induced by alkaline burn in the central cornea of recipient rat. Recipients in imDC group or mDC group were injected donor bone marrow-derived imDCs or mDCs of 1×10⁶ respectively 1 week before corneal transplantation via tail vein. Control rat received the same volume of PBS. In each group, 16 recipients were kept for determination of survival time and other 9 recipients were executed on day 3, 7 and 14 after transplantation. Cornea was harvested for hematoxylin-eosin staining and acute rejection evaluation, Western blot was used to detect the expression level of Foxp3.RESULTS: The mean survival time of imDC group was significantly longer than that of control and mDC groups (all P<0.05). The expression level of Foxp3 on CD4⁺CD25⁺T cells of imDC group (2.24±0.18) was significantly higher than that in the control (1.68±0.09) and mDC groups (1.46±0.13) (all P<0.05).CONCLUSION: Donor-derived imDC is an effective treatment in inducing immune hyporesponsiveness in rat PKP. The mechanism of immune tolerance induced by imDC might be inhibit T lymphocytes responsiveness by regulatory T cells.     

角膜移植排斥反应的铺片免疫组化研究

目的 探讨角膜移植排斥反应的发生机制及其参与细胞的表型。方法 制备正常大鼠和穿透性角膜移植鼠的角膜和虹膜睫状体铺片,用８种单克隆抗体,于铺片上进行免疫组化染色。结果 正常周边角膜及角膜缘可见少量的Ｔ细胞（ＣＤ３）、辅助／诱导性Ｔ细胞（ＣＤ４）、抑制／细胞毒Ｔ细胞（ＣＤ８）、巨噬细胞、树突细胞、主要组织相容性复合体Ⅱ类抗原阳性细胞及β转化生长因子阳性细胞;同种角膜移植术后７及１２天角膜和虹膜睫状体中     

组织工程角膜基质的体外构建及移植的实验研究

目的探讨利用组织工程技术体外构建角膜基质进行板层角膜移植的可行性和有效性。方法将猪角膜基质去细胞处理后制备成组织工程角膜基质载体;取幼兔角膜基质细胞体外培养,将其种植在载体上,体外构建成组织工程角膜基质,用PKH26荧光标记兔角膜基质细胞示踪角膜基质的构建;将16只兔的角膜基质内植入壳聚糖膜使之形成无菌性角膜溃疡,随机从16只兔中选8只,进行组织工程角膜基质移植;另外8只作为对照组,进行新鲜的同种异体兔板层角膜移植。术后对角膜进行裂隙灯、光学显微镜、透射电镜观察。结果体外构建的组织工程角膜的基质细胞具有活性,其结构与正常角膜基质相近。移植治疗无菌性角膜溃疡术后,1~2周有新生血管侵入组织工程角膜基质植片边缘,植片为灰白色半透明状;3~4周随着新生血管减退,组织工程角膜基质植片局部开始透明变薄;术后8~10周角膜溃疡完全修复,角膜恢复透明性,角膜神经可再生;观察最长达10月,角膜仍保持透明,无免疫排斥发生,与对照组疗效相同。结论体外构建的组织工程角膜基质无免疫原性、具有良好的生物相容性,可作为临床治疗角膜溃疡的移植材料。     

Dynamic Expression of Chemokines and the Infiltration of Inflammatory Cells in the HSV-Infected Cornea and its Associated Tissues

Background: The chemotactic signals regulating cell trafficking in the herpes simplex virus type 1 (HSV-1) infected cornea are well documented, however, those in the cornea-associated tissues, such as the trigeminal ganglion (TG) and draining lymph nodes (LNs), are largely unknown. Objectives: To examine chemokine expression and subsequent cell infiltration in the HSV-1 infected cornea and its associated tissues. Study design: Eight-week-old female BALB/c mice were infected with 10 μ l HSV-1 (CHR3 strain: 5 × 10⁶ PFU/ml) by corneal scarification. Total RNAs were extracted from the corneas, TGs, and LNs at pre-inoculation, 3 days post-inoculation (P.I.) and 7 days P.I. The mRNA for 28 different chemokines in the extracts was amplified by RT-PCR. Infiltrating cells were identified by immunohistochemistry. Result: After the HSV-1 infection, the corneal stroma became edematous by infiltrated cells under the eroded epithelium. The TG and LNs were markedly swollen. The cornea was infiltrated with granulocytes and CD11b⁺ cells at 3 days P.I., followed by CD4⁺ and CD8⁺ T cells at 12 days P.I. In the TG, CD11b⁺ cells, but no granulocytes, infiltrated throughout the observation period. T cells migrated into the TG earlier than into the cornea. Gene expressions of neutrophil-attracting chemokines (CXCL1, 2, 3, and 5) increased in the cornea, but they did not enhance in the TG or LNs. On the other hand, gene expressions of chemokines which attract CD11b⁺ cells such as CCL2, 8, 7, 12, CCL3, 4, and CCL5, increased in the cornea and TG with its peak at 3 days P.I. Gene expressions of chemokines those work on T cells and B cells, such as CCL19, CCL21, CXCL9, CXCL13, CXCL10, XCL1, and CXCL16, were up-regulated and peaked at 3 days P.I. in the cornea and in the TG. Thus, pattern of chemokine gene expression was similar in the cornea and in the TG. On the contrary, gene expressions of chemokines in the draining LNs affecting CD11b⁺ cells and T cells were temporarily down-regulated. Conclusion: Upon HSV-1 infection, dynamic gene expression of chemokines was observed not only in the inoculated cornea but also in its associated tissues.     

In vitro tissue engineering of lamellar cornea using human amniotic epithelial cells and rabbit cornea stroma

AIM:To reconstruct the lamellar cornea using human amniotic epithelial (HAE) cells and rabbit cornea stroma in vitro using tissue engineering technology.METHODS: Human amnia taken from uncomplicated caesarean sections were digested by collagenase to obtain HAE cells, and the cells were cultured to proliferate. Rabbit corneal epithelial cells were removed by n-heptanol to make lamellar matrix sheets. The second passage of HAE cells were cultured on the corneal stroma sheets for 1 or 2 days, then transferred to an air-liquid interface environment to culture for 2 weeks. Tissue engineered lamellar cornea (TELC) morphology was observed by Hematoxylin-eosin (HE) staining; its ultrastructure was observed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM); corneal epithelial cell-specific keratin 3 and keratin 12 were detected with immunofluorescence microscopy.RESULTS:HAE cells grew on the rabbit corneal stroma, forming a monolayer after 1-2 days. About 4-5 layers of epithelial cells developed after 2 weeks of air-liquid interface cultivation, a result similar to normal corneal epithelium. Rabbit corneal stromal cells were significantly reduced after one week, then almost completely disappeared after 2 weeks. TEM showed desmosomes between the epithelial cells; hemidesmosomes formed between the epithelial cells and the basement membrane. SEM revealed that the HAE cells which grew on the lamellar cornea had abundant microvilli. The tissue-engineered cornea expressed keratin 3 and keratin 12, as detected by immunofluorescence assay.CONCLUSION: Functional tissue-engineered lamellar corneal grafts can be constructed in vitro using HAE cells and rabbit corneal stroma.     

异体角膜对人外周血T细胞及其亚群CD69分子表达的体外调节

目的 观察异体角膜体外对人外周血T细胞及其亚群CD69分子表达的调节。方法 应用异体角膜与外周血淋巴细胞体外共同培养和流式细胞分析技术(FACS)。结果 对照组T细胞CD69表达为10．3％；受角膜或佛波醇脂(PDB)激活后，T细胞CD69表达分别为31.6％和53.8％；受角膜激活后，CD4和CD8T淋巴细胞CD69表达分别为47.3％和30．3％。结论 角膜组织体外能够刺激人外周血T细胞CD69的表达和T细胞活化，早期以CD4T细胞活化更明显。     

PDR患者外周血CD4+CD25+T细胞和相关基因FOXP3的变化及临床意义

目的：探讨增殖性糖尿病视网膜病（ proliferative diabetic retinopathy,PDR）患者外周血CD4＋CD25＋T细胞及相关基因FOXP3表达变化意义,观察其在PDR发病中的临床意义。
　　方法：采用流式细胞术检测PDR患者及正常对照外周血CD4＋CD25＋T细胞比例,Q－PCR测PDR患者、糖尿病和正常人中 FOXP3, IL－17和CTLA－4的 mRNA 表达,并用Wilcoxon秩和检验和直线相关分析法分析PDR患者糖化血红蛋白、年龄、血脂、肾功能的临床资料及它们之间相关度。
　　结果：PDR患者CD4＋T细胞降低,CD4＋CD25＋T无显著差异,FOXP3和CTLA－4在PDR中下降, IL－17增高。 CD4＋T细胞与年龄相关,CD4＋CD25＋T与患者的血肌酐存在正相关性,CD4＋CD25＋T细胞水平与年龄、糖化血红蛋白、血脂、血肌酐（ Cr）、尿素氮（ BUN）均无明显相关性。 PDR患者糖化血红蛋白、甘油三酯、总胆固醇、低密度脂蛋白均高于正常值。
　　结论：CD4＋CD25＋T细胞可能参与PDR的发病机制,并且血脂异常提示PDR可能与血脂相关。