器官培养法保存角膜的内皮细胞活性研究及在基因转染和移植排斥研究中的应用
|
摘要
目的
1.对两种新型培养基—内皮细胞培养基(Endothelial Cell Medium,ECM)和无动物细胞成分培养基(Animal compound free medium,ACF)在无血清器官培养保存角膜内皮细胞活性方面的效果进行综合评估,对其在无血清角膜培养保存领域的应用前景进行初步探索。
2.观察新型人造胶体羟乙基淀粉(Hydroxyethyl Starch,HES)的最新剂型HES130/0.4对器官培养保存角膜片的脱水效果,并与葡聚糖T500相比较,探讨其成为新型角膜脱水剂的可行性。
3.为控制移植术后排斥反应发生,阻止内皮细胞损失,探索腺病毒载体对器官培养法保存的离体兔角膜片上内皮细胞的转染效率,以检测报告基因增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)的荧光表达来明确腺病毒的转染效率和动态过程。
4.建立不同类型器官培养法保存角膜植片的大鼠角膜移植模型,检测移植后数种参与两大类辅助性T细胞因子Th1因子和Th2因子平衡调节作用的细胞因子和T细胞亚群在眼局部和(或)全身的表达,分析其在器官培养保存角膜移植相关免疫排斥反应中的作用。
方法
1.将兔和大鼠角膜各80只平均分为5组,在5种含或不含胎牛血清(fetal calf serum,FBS)的培养基中(MEM+2%FBS、高糖DMEM+2%FBS、ECM+2%FBS/ECM、ACF)密闭保存3周后,进行角膜内皮细胞(corneal endothelial cells,CEC)计数和活性染色、兔角膜厚度测量、免疫组织化学染色法(immunohistochemistry,IHC)、免疫印迹法(western blotting,WB)和反转录-聚合酶链反应法(reverse transcription polymerase chain reagction,RT-PCR)定性或半定量检测兔角膜内皮层中肌动蛋白微丝(filament actin,F-actin)和大鼠角膜内皮层中紧密连接蛋白-1(zonula occludens-1,ZO-1)的表达来衡量CEC间连接的紧密程度以及透射电镜(Transmission electron microscopy,TEM)下CEC超微结构的观察。
2. 18对配对兔角膜片,一半于无血清ACF培养液中保存21d,葡聚糖T500脱水48h作为对照组;另一半于含10%HES 130/0.4的ACF培养液中保存21d作为实验组,不再另外行脱水程序。保存前后进行CEC计数及活性染色、角膜厚度和含水量的测定、角膜透明度和后弹力层皱褶程度的评估、IHC和WB法分别定性和半定量检测兔角膜内皮层中F-actin的表达以及TEM下CEC超微结构的观察。
3.去上皮兔角膜片24只随机分为4组,每组6只角膜,在添加2%FBS的ECM培养液中保存2~3周后与编码增强型绿色荧光蛋白(EGFP)的腺病毒载体Ad5(滴度范围5×106 vp/uL～5×109vp/uL)37℃下共同孵育3h进行基因转染,随后31℃下继续器官培养观察角膜2周。检测前剥离大部分植片基质,倒置荧光显微镜下观察不同时间点EGFP在CEC中的表达强度,比较不同滴度组腺病毒载体的转染效率、CEC计数,TEM观察CEC超微结构变化。
4.选用Wistar大鼠24只,SD大鼠54只,新西兰大白兔12只。将54只SD大鼠随机分为5组:①为正常角膜组成的对照组,②为新鲜大鼠角膜同种异体穿透性移植组(penetrating keratoplasty,PKP),③为去CEC同种异体大鼠角膜+器官保存兔角膜后弹力层和内皮细胞复合体(DM+CEC)移植组,④为器官培养法保存大鼠角膜同种异体PKP组,⑤为去CEC同基因大鼠角膜+器官保存兔角膜DM+CEC移植组。兔角膜保存时间2~3周不等。②～④组制备Wistar→SD大鼠PKP模型,⑤组制备SD→SD大鼠PKP模型。术后观察各组植片的排斥情况和存活时间,酶联免疫吸附实验(enzyme linked immunosorbent assay,ELISA)定量检测房水和血清中白细胞介素2(IL-2)、干扰素-gamma(IFN-γ)、白细胞介素4(IL-4)和肿瘤坏死因子α(TNF-α)的表达,IHC法定性检测术后植片内CD25+ T细胞的表达,RT-PCR半定量检测植片内TNF-α、IFN-γ、IL-4和CD25 mRNA的表达,流式细胞术检测外周血中CD25和CD28亚群的表达。
结果
1.结果显示,ECM和ACF保存组角膜的ECD最高,细胞超微结构与正常角膜间的区别最小,MEM组的ECD值则最低,降幅最大,细胞器等超微结构破坏较严重。F-actin和ZO-1在各组兔和大鼠角膜内皮层都有表达。WB显示F-actin蛋白在ECM和ACF组表达水平最高,DMEM组次之,MEM组最低, RT-PCR法证实ZO-1 mRNA在各组的表达趋势与兔F-actin的结果一致。
2.保存结束时含10%HES130/0.4的实验组植片较薄,但ECD值略低于对照组。对照组经葡聚糖T500脱水后,厚度和透明度与实验组差别变小,但ECD值明显降低。IHC和WB证实F-actin蛋白在两组内皮层都表达,实验组表达水平高于对照组。
3.在有效浓度范围内,EGFP的荧光在器官保存的兔角膜内皮层强烈表达,随时间延长强度逐渐降低。5×107~5×108vp/μL AdV是载体滴度最为适宜的转染组,转染组中EGFP在角膜内皮细胞中的表达面积为67～84%,相对表达强度为34～75。过高浓度AdV(5×109vp/μL)导致ECD值显著下降。
4.④和⑤组(即器官培养保存的同种异体全角膜和单纯异种异体内皮层移植组)的植片存活时间明显长于②和③手术组(即新鲜同种异体PKP组和联合器官培养保存的异种异体内皮层移植组),又以⑤组的存活期最长。术后各组血清和房水中IL-2、IFN-γ、IL-4和TNF-α的含量与排斥反应程度成正比,以②组最高,⑤组最低;各组植片内CD25的表达无显著差别;IFN-γ、TNF-α、IL-4和CD25mRNA在植片内的表达水平也与植片排斥程度成正比;外周血淋巴细胞中CD25亚群表达水平升高不明显,而CD28亚群的表达明显增强并与植片排斥程度成正比。
结论
1.无血清的ECM和ACF培养基在保持内皮细胞活力方面体现出了比常规含低浓度血清的MEM基础培养基更好的保护作用,在无血清器官培养保存角膜方面极具发展潜力。
2. HES能有效避免器官培养过程中角膜过度水肿,且细胞毒性小可以成为器官保存液中的持续添加组分。它的应用也简化了保存程序,在一定程度上减轻了感染风险,有希望成为角膜器官培养保存法中的新型脱水剂。
3.器官培养法保存后的角膜内皮细胞层可以同新鲜角膜一样被腺病毒载体高效率、特异性地转染成功并持续表达,转染效率只略低于新鲜角膜片。EGFP是监测这一转染过程的理想标记物。
4. Th1/Th2平衡调节机制和CD28+T细胞亚群在器官培养法保存角膜移植后的免疫排斥反应中发挥了重要作用;动态检测外周血CD28和IFN-γ等Th1因子和Th2因子的表达有助于临床了解局部免疫反应进程并预测排斥反应的发生。器官培养法保存的角膜,无论是以全层还是单纯DM+CEC复合体形式进行移植,术后植片的存活时间都得以延长。促进Th2因子表达,抑制Th1类因子表达可能有益于降低排斥反应。同基因大鼠去CEC角膜联合器官保存兔异种CEC移植取得了最佳效果,为深层角膜病患者及时有效复明提供了新思路。
Objective
1. Two specific media (ECM and ACF) were compared with the routinely used basic medium MEM and DMEM for their potentials in serum free culture of rabbit and rat corneas in the first part study.
2. To investigate the feasibility to use a new dilation agent, hydroxyethyl starch(HES), as an alternative to the standard deswelling additive dextran T500 in organ culture preservation media.
3. To modulate alloimmunity prior to keratoplasty so as to prevent corneal endothelial cell death,we optimized adenovirus-mediated gene transfer to organ-cultured donor corneal endothelium in vitro and to delineate the kinetics of reporter gene EGFP expression in rabbit corneal endothelial cells.
4. To eatablish several rat corneal transplantion models using organ-cultured rat or rabbit corneas so ao to investigate the roles of several related cytokines and lymphocyte subsets in the corneal transplantions rejection after organ culture storage.
Methods
1. Eighty rabbit and eighty rat corneas were divided into 5 groups and cultured in five test organ culture media with or without serum content for 3 weeks and viability evaluation of corneal endothelial cells was performed. The evaluation parameters included: Endothelial cells densitie(ECD)before and after preservation and trypan blue vital staining of the endothelium after preservation; The mean central thickness of rabbit cornea before and after preservation;The expression of rabbit F-actin and rat ZO-1 in corneal endothelium using IHC,WB and RT-PCR;The transmission electron micronscopy observation of rabbit corneal endothelial cells.
2. A half of 18 pairs of rabbit corneas were cultured in serum free ACF culture medium for 21d and then were dehydrated in ACF with a concentration of 5% dextran T500 as acontrol.The other corneas were cultured in ACF medium with a supplement of HES 130/0.4 in concentrations of 10% from the beginning.The evaluation parameters included: The endothelial cells viability before and after storage; The mean central thickness of rabbit cornea before and after preservation;The mean water content after storage; The transparency and folding of rabbit corneas of two groups using two-level system; The expression of F-actin in corneal endothelium using IHC and WB;The electron micronscopy observation of rabbit corneal endothelial cells.
3. Twenty-four Newtherland rabbit corneas which had been stored in ECM+2%FBS mediun for 2~3weeks were incubated with replication-deficient adenovirus encoding enhanced green fluorescent protein (EGFP) or empty vector ex vivo at a dose of 5×106 to 5×109vp/ul at temperatures of 37°C. The epithelium had been removed before transduction.After ex vivo infection, and EGFP expression in the grafts was visualized in vitvo by inverted fluorescence microscope over 2 extended weeks.Finally,vital staining was performed to count endothelial cells desity in all grafts.
4. A total of 24 Wistar rats, 54 SD rats and 12 Newtheland rabbits were selected. The 54 SD rats were randomly divided into five groups:①normal control group(n=6),②allograft transplantions group with fresh rat cornea (n=12),③allograft transplantions group with fresh rat cornea without descemet membrane combined with organ-cultured rabbit endothelium(n=12),④allograft transplantions group with organ-cultured rat cornea (n=12),and⑤corneal transplantion group with fresh rat isograft without descemet membrane and organ-cultured rabbit endothelium.The former four groups received Wistar→SD keratoplasty and the latter 5 group SD→SD. After transplantion, immunological rejections were observed with a slit lamp microscope. Serum and aqueous humor levels of IL-2,IFN-γ,IL-4 and TNF-αwere measured by ELISA. IHC was performed to examine CD25+T expressions in grafts. Levels of TNF-αmRNA, IFN-γmRNA, IL-4mRNA and CD25mRNA in grafts were detected by using RT-PCR. The expression levels of CD25 and CD28 lymphocyte subsets in peripheral blood were also determined by Flow Cytometry(FCM).
Results
1. After storage,only corneas cultured in MEM medium with low serum content exhibited a higher endothelial cells loss and the highest densities were found in corneas cultured in ECM and ACF.F-actin and ZO-1 could be seen in all corneal endothelialiums using IHC.Further WB showed that F-actin protein expression of rabbit corneal endotheliums achieved the lowest level in MEM medium and the highest level in ECM and ACF medium.So did ZO-1mRNA expression levels using RT-PCR in rat corneas.
2. After 3 weeks storage, ECD of rabbit corneas cultured in ACF medium with 10% HES130/0.4 was slightly lower than that of the control group without dehydration. The experiment group also exhibited a thinner corneal thickness, better transparency and less folding compared with the control group. After dehydration for 48h using dextran T500, the control corneas became thin and transparent, however, the endothelial cell density decreased greatly at the same time. WB showed that F-actin protein expression in rabbit corneal endothelium achieved higher level in ACF medium with 10% HES130/0.4.
3. EGFP expression was found to be restricted to the corneal endothelium.Transduction of organ-cultured rat corneas with high doses (5×107~5×108 vp/μL) of AdV caused EGFP expression in 67～84% of corneal endothelial cells. Very high AdV dose (5×109 vp/μL) reduced endothelial cell densities to 1978(SD=281) nuclei/mm2.In vitro expression of EGFP in organ-cultured corneal grafts was demonstrated in 2 weeks observation period and weakened gradually in warm storage.
4.①The graft survival time were longer in the fourth and fifth groups than in the second and third groups which reached the longest 16.3d in the fifth group.②The serum and aqueous humor levels of IL-2,IFN-γ,IL-4 and TNF-αwere higher in all surgical groups than in the normal control group at days 6 ,13 and 24d after surgeries (P < 0.05),and the second and third groups had the highest expression level.③At day 13, CD25 was weakly expressed in all surgery groups.④The expressions of IFN-γmRNA,TNF-αmRNA,IL-4 mRNA and CD25mRNA in grafts were markedly increased (P < 0.05) at day 13 after surgeries,whereas there was no expression of these genes in normal corneas.⑤At day 13, the percentages of CD25 in lymphocytes of all groups didn't fluctuate greatly.However, the percentages of CD28 in lymphocytes of the second and third groups increased significantly and weakly expressed in the other two surgery groups.
Conclusion
1. ECM and ACF medium showed superiority over standard FCS medium in the ability to preserve endothelial cells viability.They have great potentials comparing with conventional MEM and DMEM medium and may play an important part in serum free organ cultured method in the future.
2. HES 130/0.4 keeps the corneas thin and transparent, is well torelated for endothelial cells and can be used as a continuous supplement during organ culture. It also simplifies the processing steps of corneas,makes further dehydration before transplantation unnecessary and reduces infection risk in a certain extent. HES 130/0.4 appears to be an alternative to the use of dextran as a deswelling additive in cornea organ culture media.
3. Organ-cultured rabbit corneal endothelial cells can be selectively and efficiently transduced by AdV vector following extended periods of warm storage, and reporter gene EGFP expression is retained in vitro during extended 2 weeks organ culture preservation. EGFP expression in fresh corneal grafts is more extensive than in organ-cultured grafts.
4. The cytokines involved in Th1/Th2-type immune response (IL-2,IFN-γ,IL-4 and TNF-α) and lymphocyte subsets CD28 play important roles during corneal allograft rejection. Monitoring the expression of them after transplantion surgeries can help to indicate the degree of local immune reaction and predict the corneal allograft rejection.It’s good for prolonging survival time to make use of organ-cultured corneal grafts.Increasing the expression of Th2 cytokines and decreasing the expression of Th1 cytokines and CD28 may help to inhibit corneal allograft rejection.
1.对两种新型培养基—内皮细胞培养基(Endothelial Cell Medium,ECM)和无动物细胞成分培养基(Animal compound free medium,ACF)在无血清器官培养保存角膜内皮细胞活性方面的效果进行综合评估,对其在无血清角膜培养保存领域的应用前景进行初步探索。
2.观察新型人造胶体羟乙基淀粉(Hydroxyethyl Starch,HES)的最新剂型HES130/0.4对器官培养保存角膜片的脱水效果,并与葡聚糖T500相比较,探讨其成为新型角膜脱水剂的可行性。
3.为控制移植术后排斥反应发生,阻止内皮细胞损失,探索腺病毒载体对器官培养法保存的离体兔角膜片上内皮细胞的转染效率,以检测报告基因增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)的荧光表达来明确腺病毒的转染效率和动态过程。
4.建立不同类型器官培养法保存角膜植片的大鼠角膜移植模型,检测移植后数种参与两大类辅助性T细胞因子Th1因子和Th2因子平衡调节作用的细胞因子和T细胞亚群在眼局部和(或)全身的表达,分析其在器官培养保存角膜移植相关免疫排斥反应中的作用。
方法
1.将兔和大鼠角膜各80只平均分为5组,在5种含或不含胎牛血清(fetal calf serum,FBS)的培养基中(MEM+2%FBS、高糖DMEM+2%FBS、ECM+2%FBS/ECM、ACF)密闭保存3周后,进行角膜内皮细胞(corneal endothelial cells,CEC)计数和活性染色、兔角膜厚度测量、免疫组织化学染色法(immunohistochemistry,IHC)、免疫印迹法(western blotting,WB)和反转录-聚合酶链反应法(reverse transcription polymerase chain reagction,RT-PCR)定性或半定量检测兔角膜内皮层中肌动蛋白微丝(filament actin,F-actin)和大鼠角膜内皮层中紧密连接蛋白-1(zonula occludens-1,ZO-1)的表达来衡量CEC间连接的紧密程度以及透射电镜(Transmission electron microscopy,TEM)下CEC超微结构的观察。
2. 18对配对兔角膜片,一半于无血清ACF培养液中保存21d,葡聚糖T500脱水48h作为对照组;另一半于含10%HES 130/0.4的ACF培养液中保存21d作为实验组,不再另外行脱水程序。保存前后进行CEC计数及活性染色、角膜厚度和含水量的测定、角膜透明度和后弹力层皱褶程度的评估、IHC和WB法分别定性和半定量检测兔角膜内皮层中F-actin的表达以及TEM下CEC超微结构的观察。
3.去上皮兔角膜片24只随机分为4组,每组6只角膜,在添加2%FBS的ECM培养液中保存2~3周后与编码增强型绿色荧光蛋白(EGFP)的腺病毒载体Ad5(滴度范围5×106 vp/uL～5×109vp/uL)37℃下共同孵育3h进行基因转染,随后31℃下继续器官培养观察角膜2周。检测前剥离大部分植片基质,倒置荧光显微镜下观察不同时间点EGFP在CEC中的表达强度,比较不同滴度组腺病毒载体的转染效率、CEC计数,TEM观察CEC超微结构变化。
4.选用Wistar大鼠24只,SD大鼠54只,新西兰大白兔12只。将54只SD大鼠随机分为5组:①为正常角膜组成的对照组,②为新鲜大鼠角膜同种异体穿透性移植组(penetrating keratoplasty,PKP),③为去CEC同种异体大鼠角膜+器官保存兔角膜后弹力层和内皮细胞复合体(DM+CEC)移植组,④为器官培养法保存大鼠角膜同种异体PKP组,⑤为去CEC同基因大鼠角膜+器官保存兔角膜DM+CEC移植组。兔角膜保存时间2~3周不等。②～④组制备Wistar→SD大鼠PKP模型,⑤组制备SD→SD大鼠PKP模型。术后观察各组植片的排斥情况和存活时间,酶联免疫吸附实验(enzyme linked immunosorbent assay,ELISA)定量检测房水和血清中白细胞介素2(IL-2)、干扰素-gamma(IFN-γ)、白细胞介素4(IL-4)和肿瘤坏死因子α(TNF-α)的表达,IHC法定性检测术后植片内CD25+ T细胞的表达,RT-PCR半定量检测植片内TNF-α、IFN-γ、IL-4和CD25 mRNA的表达,流式细胞术检测外周血中CD25和CD28亚群的表达。
结果
1.结果显示,ECM和ACF保存组角膜的ECD最高,细胞超微结构与正常角膜间的区别最小,MEM组的ECD值则最低,降幅最大,细胞器等超微结构破坏较严重。F-actin和ZO-1在各组兔和大鼠角膜内皮层都有表达。WB显示F-actin蛋白在ECM和ACF组表达水平最高,DMEM组次之,MEM组最低, RT-PCR法证实ZO-1 mRNA在各组的表达趋势与兔F-actin的结果一致。
2.保存结束时含10%HES130/0.4的实验组植片较薄,但ECD值略低于对照组。对照组经葡聚糖T500脱水后,厚度和透明度与实验组差别变小,但ECD值明显降低。IHC和WB证实F-actin蛋白在两组内皮层都表达,实验组表达水平高于对照组。
3.在有效浓度范围内,EGFP的荧光在器官保存的兔角膜内皮层强烈表达,随时间延长强度逐渐降低。5×107~5×108vp/μL AdV是载体滴度最为适宜的转染组,转染组中EGFP在角膜内皮细胞中的表达面积为67～84%,相对表达强度为34～75。过高浓度AdV(5×109vp/μL)导致ECD值显著下降。
4.④和⑤组(即器官培养保存的同种异体全角膜和单纯异种异体内皮层移植组)的植片存活时间明显长于②和③手术组(即新鲜同种异体PKP组和联合器官培养保存的异种异体内皮层移植组),又以⑤组的存活期最长。术后各组血清和房水中IL-2、IFN-γ、IL-4和TNF-α的含量与排斥反应程度成正比,以②组最高,⑤组最低;各组植片内CD25的表达无显著差别;IFN-γ、TNF-α、IL-4和CD25mRNA在植片内的表达水平也与植片排斥程度成正比;外周血淋巴细胞中CD25亚群表达水平升高不明显,而CD28亚群的表达明显增强并与植片排斥程度成正比。
结论
1.无血清的ECM和ACF培养基在保持内皮细胞活力方面体现出了比常规含低浓度血清的MEM基础培养基更好的保护作用,在无血清器官培养保存角膜方面极具发展潜力。
2. HES能有效避免器官培养过程中角膜过度水肿,且细胞毒性小可以成为器官保存液中的持续添加组分。它的应用也简化了保存程序,在一定程度上减轻了感染风险,有希望成为角膜器官培养保存法中的新型脱水剂。
3.器官培养法保存后的角膜内皮细胞层可以同新鲜角膜一样被腺病毒载体高效率、特异性地转染成功并持续表达,转染效率只略低于新鲜角膜片。EGFP是监测这一转染过程的理想标记物。
4. Th1/Th2平衡调节机制和CD28+T细胞亚群在器官培养法保存角膜移植后的免疫排斥反应中发挥了重要作用;动态检测外周血CD28和IFN-γ等Th1因子和Th2因子的表达有助于临床了解局部免疫反应进程并预测排斥反应的发生。器官培养法保存的角膜,无论是以全层还是单纯DM+CEC复合体形式进行移植,术后植片的存活时间都得以延长。促进Th2因子表达,抑制Th1类因子表达可能有益于降低排斥反应。同基因大鼠去CEC角膜联合器官保存兔异种CEC移植取得了最佳效果,为深层角膜病患者及时有效复明提供了新思路。
Objective
1. Two specific media (ECM and ACF) were compared with the routinely used basic medium MEM and DMEM for their potentials in serum free culture of rabbit and rat corneas in the first part study.
2. To investigate the feasibility to use a new dilation agent, hydroxyethyl starch(HES), as an alternative to the standard deswelling additive dextran T500 in organ culture preservation media.
3. To modulate alloimmunity prior to keratoplasty so as to prevent corneal endothelial cell death,we optimized adenovirus-mediated gene transfer to organ-cultured donor corneal endothelium in vitro and to delineate the kinetics of reporter gene EGFP expression in rabbit corneal endothelial cells.
4. To eatablish several rat corneal transplantion models using organ-cultured rat or rabbit corneas so ao to investigate the roles of several related cytokines and lymphocyte subsets in the corneal transplantions rejection after organ culture storage.
Methods
1. Eighty rabbit and eighty rat corneas were divided into 5 groups and cultured in five test organ culture media with or without serum content for 3 weeks and viability evaluation of corneal endothelial cells was performed. The evaluation parameters included: Endothelial cells densitie(ECD)before and after preservation and trypan blue vital staining of the endothelium after preservation; The mean central thickness of rabbit cornea before and after preservation;The expression of rabbit F-actin and rat ZO-1 in corneal endothelium using IHC,WB and RT-PCR;The transmission electron micronscopy observation of rabbit corneal endothelial cells.
2. A half of 18 pairs of rabbit corneas were cultured in serum free ACF culture medium for 21d and then were dehydrated in ACF with a concentration of 5% dextran T500 as acontrol.The other corneas were cultured in ACF medium with a supplement of HES 130/0.4 in concentrations of 10% from the beginning.The evaluation parameters included: The endothelial cells viability before and after storage; The mean central thickness of rabbit cornea before and after preservation;The mean water content after storage; The transparency and folding of rabbit corneas of two groups using two-level system; The expression of F-actin in corneal endothelium using IHC and WB;The electron micronscopy observation of rabbit corneal endothelial cells.
3. Twenty-four Newtherland rabbit corneas which had been stored in ECM+2%FBS mediun for 2~3weeks were incubated with replication-deficient adenovirus encoding enhanced green fluorescent protein (EGFP) or empty vector ex vivo at a dose of 5×106 to 5×109vp/ul at temperatures of 37°C. The epithelium had been removed before transduction.After ex vivo infection, and EGFP expression in the grafts was visualized in vitvo by inverted fluorescence microscope over 2 extended weeks.Finally,vital staining was performed to count endothelial cells desity in all grafts.
4. A total of 24 Wistar rats, 54 SD rats and 12 Newtheland rabbits were selected. The 54 SD rats were randomly divided into five groups:①normal control group(n=6),②allograft transplantions group with fresh rat cornea (n=12),③allograft transplantions group with fresh rat cornea without descemet membrane combined with organ-cultured rabbit endothelium(n=12),④allograft transplantions group with organ-cultured rat cornea (n=12),and⑤corneal transplantion group with fresh rat isograft without descemet membrane and organ-cultured rabbit endothelium.The former four groups received Wistar→SD keratoplasty and the latter 5 group SD→SD. After transplantion, immunological rejections were observed with a slit lamp microscope. Serum and aqueous humor levels of IL-2,IFN-γ,IL-4 and TNF-αwere measured by ELISA. IHC was performed to examine CD25+T expressions in grafts. Levels of TNF-αmRNA, IFN-γmRNA, IL-4mRNA and CD25mRNA in grafts were detected by using RT-PCR. The expression levels of CD25 and CD28 lymphocyte subsets in peripheral blood were also determined by Flow Cytometry(FCM).
Results
1. After storage,only corneas cultured in MEM medium with low serum content exhibited a higher endothelial cells loss and the highest densities were found in corneas cultured in ECM and ACF.F-actin and ZO-1 could be seen in all corneal endothelialiums using IHC.Further WB showed that F-actin protein expression of rabbit corneal endotheliums achieved the lowest level in MEM medium and the highest level in ECM and ACF medium.So did ZO-1mRNA expression levels using RT-PCR in rat corneas.
2. After 3 weeks storage, ECD of rabbit corneas cultured in ACF medium with 10% HES130/0.4 was slightly lower than that of the control group without dehydration. The experiment group also exhibited a thinner corneal thickness, better transparency and less folding compared with the control group. After dehydration for 48h using dextran T500, the control corneas became thin and transparent, however, the endothelial cell density decreased greatly at the same time. WB showed that F-actin protein expression in rabbit corneal endothelium achieved higher level in ACF medium with 10% HES130/0.4.
3. EGFP expression was found to be restricted to the corneal endothelium.Transduction of organ-cultured rat corneas with high doses (5×107~5×108 vp/μL) of AdV caused EGFP expression in 67～84% of corneal endothelial cells. Very high AdV dose (5×109 vp/μL) reduced endothelial cell densities to 1978(SD=281) nuclei/mm2.In vitro expression of EGFP in organ-cultured corneal grafts was demonstrated in 2 weeks observation period and weakened gradually in warm storage.
4.①The graft survival time were longer in the fourth and fifth groups than in the second and third groups which reached the longest 16.3d in the fifth group.②The serum and aqueous humor levels of IL-2,IFN-γ,IL-4 and TNF-αwere higher in all surgical groups than in the normal control group at days 6 ,13 and 24d after surgeries (P < 0.05),and the second and third groups had the highest expression level.③At day 13, CD25 was weakly expressed in all surgery groups.④The expressions of IFN-γmRNA,TNF-αmRNA,IL-4 mRNA and CD25mRNA in grafts were markedly increased (P < 0.05) at day 13 after surgeries,whereas there was no expression of these genes in normal corneas.⑤At day 13, the percentages of CD25 in lymphocytes of all groups didn't fluctuate greatly.However, the percentages of CD28 in lymphocytes of the second and third groups increased significantly and weakly expressed in the other two surgery groups.
Conclusion
1. ECM and ACF medium showed superiority over standard FCS medium in the ability to preserve endothelial cells viability.They have great potentials comparing with conventional MEM and DMEM medium and may play an important part in serum free organ cultured method in the future.
2. HES 130/0.4 keeps the corneas thin and transparent, is well torelated for endothelial cells and can be used as a continuous supplement during organ culture. It also simplifies the processing steps of corneas,makes further dehydration before transplantation unnecessary and reduces infection risk in a certain extent. HES 130/0.4 appears to be an alternative to the use of dextran as a deswelling additive in cornea organ culture media.
3. Organ-cultured rabbit corneal endothelial cells can be selectively and efficiently transduced by AdV vector following extended periods of warm storage, and reporter gene EGFP expression is retained in vitro during extended 2 weeks organ culture preservation. EGFP expression in fresh corneal grafts is more extensive than in organ-cultured grafts.
4. The cytokines involved in Th1/Th2-type immune response (IL-2,IFN-γ,IL-4 and TNF-α) and lymphocyte subsets CD28 play important roles during corneal allograft rejection. Monitoring the expression of them after transplantion surgeries can help to indicate the degree of local immune reaction and predict the corneal allograft rejection.It’s good for prolonging survival time to make use of organ-cultured corneal grafts.Increasing the expression of Th2 cytokines and decreasing the expression of Th1 cytokines and CD28 may help to inhibit corneal allograft rejection.
引文
[1] Pels E, Schuchard Y. Organ-culture preservation of human corneas.Doc Ophthalmol. 1983;56:147–153.
[2]兔角膜器官培养保存与应用的研究.刘涛潘志强王立李纳.中华眼科杂志,2006,42(9):808-813.
[3] European Eye Bank Association Directory. 8th ed. ?rhus,Denmark, January 2000.
[4] Thuret G, Manissolle C, Campos-Guyotat L, Guyotat D, and Gain P.Animal Compound–Free Medium and Poloxamer for Human Corneal Organ Culture and Deswelling. Invest Ophthalmol Vis Sci. 2005;46:816–822.
[5] Bednarz J, Doubilei V, Wollnik PC, et al. Effect of three different media onserum free culture of donor corneas and isolated human corneal endothelial cells. Br J Ophthalmol. 2001;85:1416–1420.
[6] Rieck PW, Gigon M, Jaroszewski J, et al. Increased endothelial survival of organ-cultured corneas stored in FGF-2-supplemented serum-free medium. Invest Ophthalmol Vis Sci. 2003;44:3826–3832.
[7] Hempel B, Bednarz J, Engelmann K.Use of a serum-free medium for long-term storage of human corneas. Influence on endothelial cell density and corneal metabolism. Graefes Arch Clin Exp Ophthalmol. 2001 Oct;239(10):801-5.
[8] EEBA. European Eye Bank Association Directory. 12th ed. Amsterdam,The Netherlands; 2004.
[9] Ayoubi MG et al.Br J Ophthalmol,1996;80:740.
[10] Slettedal JK, Lyberg T, R?ger M,et al. Regeneration with proliferation of the endothelium of cultured human donor corneas with extended postmortem time.Cornea. 2008 Feb;27(2):212-9.
[11] Nusrat A,Parkos CA,Verkade P,Foley CS,Liang TW,Innis-Whitehouse W,et a1.Tight junctions ore membrane microdomains [M].J Cell Sci,2000,113(Pt 10):1771-1778.
[12] Stevenson B R,Siliciano J D,Mooseker M S,et a1.Identification of ZO-1:a high molecular weight polypeptide associated with the tight junction(zonula occludens)in a variety of epithelia[J].J Cell Biol,1986,103(3):755.
[13] Fanning A S,Jameson B J,Jesaitis L A,et a1.The tight junction protein ZO-1 establishes a link between the transnlenlbrane protein occludin and the actin cytoskeleton[J].J Biol Chem,1998,273:29745.
[14] Fanning A S,Ma T Y,Anderson J M.Isolation and functional characterization of the actin binding region in the tight junction protein ZO-1 [ J].FASEB J,2002,16(13):1835.
[15]鲍欢,包仕尧.缺氧后血脑屏障紧密连接变化的分子机制[J].圄外医学·脑血管疾病分册,2005,13(8):604.
[16]张文一,白海青,士峥华.视网膜色素上皮细胞中诱导型一氧化氮合酶、精氨酸代谢相关酶的表达及一氧化氮对细胞紧密连接的影响[J].中华眼底病杂志,2005,21(1):32.
[17] Crewe J M and John Armitage W.Integrity of Epithelium and Endothelium in Organ-Cultured Human Corneas. Invest Ophthalmol Vis Sci.2001;42:1757–1761.
[18] Yonenmra S,hoh M,Nagafuchi A,et a1.Cel1-to-cell adherens junction formation and actin filament organization:similarities and diffrences between non-polarized fibroblasts and polarized epithelial cells.J Cell Sci,1995,108(Pt 1):127.
[19] Ando-Akatsuka Y, Yoflenlura S, Itoh M , et a1. Differential behavior of E-cadherln and occludin in their colocalization with ZO-1 during the establishment of epithelial cell polarity[J]. J Cell Physiol,1999,179 (2):115.
[1] Borderie VM, Baudrimont M, Lopez M, Carvajal S, Laroche L.Evaluation of the deswelling period in dextran-containing medium after corneal organ culture. Cornea. 1997;16:215–223.
[2] Thuret G, Manissolle C, Campos-Guyotat L, Guyotat D, Gain P.Animal compound-free medium and poloxamer for human corneal organ culture and deswelling. Invest Ophthalmol Vis Sci. 2005;46:816–822.
[3]兔角膜器官培养保存与应用的研究.刘涛潘志强王立李纳.中华眼科杂志,2006,42(9):808-813.
[4]熊加祥,黎海蒂,龚发云,等.严重烧伤影响大鼠脑内c—fos表达和Som—L1与脑水肿的关系[J].第三军医大学学报,2002,24(7):760—763. [5 ] Use of Poloxamers for Deswelling of Organ-Cultured Corneas. Zhao M, Thuret G, Piselli S,et al. Invest Ophthalmol Vis Sci. 2008;49:550–559.
[6] Sperling S. Human corneal endothelium in organ culture: the influence of temperature and medium of incubation. Acta Ophthalmol (Copenh). 1979;57:269–276.
[7] Pels E, Schuchard Y. The effects of high molecular weight dextranon the preservation of human corneas. Cornea. 1984;3:219–227.
[8] Reim M, Hesse R, Pietruschka G. The metabolism of organ cultures of cornea in TC 199 with added dextran 500 or hydroxyethyl starch 450.Klin Monatsbl Augenheilkd. 1990 Feb;196(2):76-80.
[9] van der Want HJ, Pels E, Schuchard Y, Olesen B, Sperling S.Electron microscopy of cultured human corneas: osmotic hydration and the use of a dextran fraction (dextran T 500) in organ culture. Arch Ophthalmol. 1983;101:1920–1926.
[10] Salla S, Redbrake C, Becker J, Reim M. Remarks on the vitality of the human cornea after organ culture. Cornea. 1995;14:502–508.
[11] Rieger R, Jaroszewski J, Jaeckel C, Hartmann C, Rieck P. Endothelial cell loss during storage of donor corneas in culture media containing dextrane. 15th conference of the European Eye Bank Association. Bruxelles, Belgium; 2003.
[12] Standl T, Burmeister MA, Schroeder F, et al. Hydroxyethyl starch (HES) 130/0.4 provides larger and faster increases in tissue oxygen tension in comparison with prehemodilution values than HES 70/0.5 or HES 200/0.5 in volunteers undergoing acute normovolemic hemodilution. Anesth Analg. 2003 Apr;96(4):936-943.
[13] Jungheinrich C, Sauermann W, Bepperling F, et al.Hes(130/0.4) can be safely administered to patients even with severe renal impairment. Anesth Analg.2002,95:544-551.
[14]赵晶,叶铁虎,徐庆,等.非心脏外科手术患者羟乙基淀粉130/0.4注射液容量治疗的安全性.中华麻醉学杂志, 2004,24(9):657-660.
[15] Reim M, Hesse R, Pietruschka G. The metabolism of organ cultures of cornea in TC 199 with added dextran 500 or hydroxyethyl starch 450. Klin Monatsbl Augenheilkd. 1990;196:76–80.
[16] Reim M, Pantenburg FJ, Ziegler CD. Effect of serum and osmotically active substances on metabolism in 262 tissue cultures of pig’s corneas: improved preservation of donor tissue for keratoplasty.Klin Monatsbl Augenheilkd. 2001;218:95–101.
[17] Redbrake C, Kompa S, Altmann G, Reim M, Arend O. HES 130 as a continuous supplement for organ culture of human corneas (in German)? Ophthalmologe. 2006;103:43–47.
[1] Teresa B. Recent developments in ocular gene therapy[J].Exp Eye Res,2003,76: 643-652
[2] Pleyer U, Bertelmann E, Rieck P, et al. Survival of corneal allografts following adenovirus-mediated gene transfer of interleukin-4. Graefes Arch Clin Exp Ophthalmol 2000;238:531–6.
[3] Klebe S, Sykes P, Coster D, et al. Prolongation of sheep corneal allograft survival by ex vivo transfer of the gene encoding interleukin-10. Transplantation 2001;71:1214–20.
[4] Comer RM, King WJ, Ardjomand N, et al. Effect of administration of CTLA4-Ig as protein or cDNA on corneal allograft survival. Invest Ophthalmol Vis Sci 2002;43:1095–103.
[5] Muller A, Zhang EP, Schroff M, et al. Influence of ballistic gene transfer on antigen-presenting cells in murine corneas. Graefes Arch Clin Exp Ophthalmol 2002;240:851–9.
[6] Rijneveld WJ, Remeijer L, van Rij G, Beekhuis H, Pels E. Prospective clinical evaluation of McCarey-Kaufman and organ culture cornea preservation media: 14-year follow-up. Cornea. 2008 Oct;27(9):996-1000.
[7]兔角膜器官培养保存与应用的研究.刘涛潘志强王立李纳.中华眼科杂志,2006,42(9):808-813.
[8] Chalfie M, Tu Y, Euskirchen G, et a1. Green fluorescent protein as a marker for gene expression[J]. Science,1994,263:802-805.
[9] Thuret G, Manissolle C, Campos-Guyotat L, Guyotat D, and Gain P.Animal Compound–Free Medium and Poloxamer for Human Corneal Organ Culture and Deswelling. Invest Ophthalmol Vis Sci. 2005;46:816–822.
[10] In vitro adenovirus mediated gene transfer to the human cornea Br J Ophthalmol. 2005 June; 89(6): 658–661. C F Jessup, H M Brereton, D J Coster, and K A Williams.
[11] Crystal RG. The gene as the drug. Nat Med 1995;1:15–7.
[12] Somia N, Verma IM. Gene therapy: trials and tribulations. Nat Rev Genet 2000;1:91–9.
[13] Shimomura O, Johnson FH, Saiga Y. Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea [J].J Cell Comp Physiol,1962,59:223-239.
[14] Tsien RY. The green fluorescent protein. Annu Rev Biochem[J], 1998, 67:509- 544.
[1] Remeijer L, van Rij G, Beekhuis H, Pels E, Rijneveld WJ. Prospective clinical evaluation of McCarey-Kaufman and organ culture cornea preservation media: 14-year follow-up.Cornea. 2008 Oct;27(9):996-1000
[2] Hitani K, Yokoo S, Honda N,et al. Transplantation of a sheet of human corneal endothelial cell in a rabbit model. Molecular Vision 2008; 14:1-9
[3] Niederkorn JY, Mellon J. Anterior chamber-associated immune deviation promotes corneal allograft survival. Invest Ophthalmol Vis Sci 1996;37(13):2700-2707
[4] Yamada J, Dana MR, Zhu SN, et al. Interleukin 1 receptor antagonist suppresses allosensitization in corneal transplantation. Arch Ophthalmol 1998;116(10):1351-1357
[5]何维.医学免疫学[M].北京:人民卫生出版社, 2005:250-256
[6] Mosmann TR, Cherwinski H, Bond MW, et al. Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J Immunol 1986;136(7):2348-2357
[7] Amirzargar A, Lessanpezeshki M, Fathi A, et al. TH1/TH2 cytokine analysis in Iranian renal transplant recipients. Transplant Proc 2005;37(7):2985-2987
[8] Williams KA, Coster DJ. Penetrating corneal transplantation in the inbred rat: a new model. Invest Ophthalmol Vis Sci 1985;26(1):23-30.
[9] Larkin DF, Calder VL, Lightman SL. Identification and characterization of cells infiltrating the graft and aqueous humour in rat corneal allograft rejection. Clin Exp Immunol 1997;107(2):381-391
[10] Skurkovich S, Kasparov A, Narbut N, et al. Treatment of corneal transplant rejection in humans with anti-interferon-gamma antibodies. Am J Ophthalmol 2002;133(6):829-830
[11] Jiang H, Liu C, Xu J, et al. Gene transfer of interleukin-4 delays acute rejection of splenic allografts in rats. Transplant Proc 2004;36(5):1600-1603
[12] Zhang EP, Franke J, Schroff M, et al. Ballistic CTLA4 and IL-4 gene transfer into the lower lid prolongs orthotopic corneal graft surviveal in mice. Graefes Arch Clin Exp Ophthalmol 2003;241(11):921-926
[13] De Karis,Zhu SN,Dana MR,et a1.TNF-alpha regulate corneal langerhans cell migration[J].J Immunol,1999,162(7):4235-4239.
[14] Watsky MA,Guna I,Ragsdate DN, et a1.Efect of tumor necrosis factor alpha on rabbit corneal endothelial pemeability [J].Invest Ophthalmol Vis Sci,1996,37:19
[15] Mathan A, Kuruvilla S, Abraham G. Interleukin 2 receptor expression in renal biopsies and the diagnosis of acute allograft rejection. Indian J Pathol Microbiol 2006;49(1):12-16.
[16] Modulation of Costimulation by CD28 and CD154 Alters the Kinetics and Cellular Characteristics of Corneal Allograft Rejection. Navid Ardjomand, James C. McAlister, Nicola J. Rogers, Peng H. Tan,Andrew J. T. George, and Daniel F. P. Larkin. Invest Ophthalmol VisSci. 2003;44:3899–3905
[17] Comer RM, King WJ, Ardjomand N, Theoharis S, George AJ,Larkin DF. Effect of administration of CTLA4-Ig as protein or cDNA on corneal allograft survival. Invest Ophthalmol Vis Sci 2002; 43:1095–103.
[18] Ardjomand N, McAlister JC, Rogers NJ, Tan PH, George AJ, Larkin DF. Modulation of costimulation by CD28 and CD154 alters the kinetics and cellular characteristics of corneal allograft rejection. Invest Ophthalmol Vis Sci 2003; 44:3899–905.
[1] Shimomura O, Johnson FH, Saiga Y. Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea [J].J Cell Comp Physiol,1962,59:223-239.
[2] Prasher DC, Eckenrode VK, Ward WW, et a1. Primary structure of the aequorea victoria green-fluorescent protein[J]. J Gene,1992,111:229-233.
[3] Chalfie M, Tu Y, Euskirchen G, et a1. Green fluorescent protein as a marker for gene expression[J]. Science,1994,263:802-805.
[4] Ormo M, Cubitt A B, Kallio K, et al. Crystal structure of the Aequorea victoria greenfluorescent protein[J]. Science,1996,273:1392-1395.
[5] Cody CW, Prasher DC, Westler WM, et a1. Chemical structure of the hexapeptide chromophore of the Aequorea green-fluorescent protein[J]. Biochemistry,1993,32: 1212-1218.
[6] Gubin AN, Reddy B, Njoroge JM, et a1. Long-term, stable expression of green fluorescent protein in mammalian cells[J]. Bioc hem Biophys Res Commun,1997, 236:347-350.
[7] Stadtfeld M, Varas F, Graf T. Fluorescent protein-cell labeling and its application in time-lapse analysis of hematopoietic differentiation[J]. Methods Mol Med,2005, 105:395-412.
[8] Tsien RY. The green fluorescent protein. Annu Rev Biochem[J], 1998, 67:509- 544.
[9] Tavare JM, Fletcher LM, Welsh GI, et al. Using green fluroscent protein to study intracelluar signaling[J].J Endocrinology,2001,170:297-306.
[10] Liu YP, Dovzhenko OV, Garthwaite MA, et al. Maintenance of pluripotency in human embryonic stem eelIs stably over-expressing enhanced green fluorescent protein[J].Stem Cells Dev,2004,l3:636-645.
[11] Kahn J, Byk T, Jansson-Sjostrand L, et al.Overexpression of CXCR4 on human CD34+ progenitors increases their proliferation,migration,and NOD/SClD repopulation[J]. Blood,2004,103:2942-2949.
[12] Teresa B. Recent developments in ocular gene therapy[J].Exp Eye Res,2003,76: 643-652
[13] Lai L, Lin K, Foulks G, et al. Highly efficient ex vivo gene delivery into human corneal endothelial cells by recombinant adeno-associated virus[J]. Curr Eye Res, 2005, 30:213-9.
[14]袁进,陈家祺,刘祖国,等.IL-1ra基因修饰角膜内皮细胞及其表达[J].眼科新进展,2006,26:2-6.
[15] Nguyen TH, Murakami A, Fujiki K, et al.Transferrin-polyethylenimine conjugate, FuGENE6 and TransIT-LT as nonviral vectors for gene transfer to the corneal endothelium[J]. Jpn J Ophthalmol,2002, 46:140-6.
[16] Choi J, Ko MK, Kay EP. Subcellular localization of the expressed 18 kDa FGF-2 isoform in corneal endothelial cells.Mol Vis, 2000, 6:222-31.
[17] Challa P, Luna C, Liton PB, et al. Lentiviral mediated gene delivery to the anterior chamber of rodent eyes[J]. Mol Vis,2005,11:425-30.
[18] Borrás T, Gabelt BT, Klintworth GK, et al. Non-invasive observation of repeated adenoviral GFP gene delivery to the anterior segment of the monkey eye in vivo [J]. J Gene Med, 2001,3:437-49.
[19] Qian Y, Leong FL, Kazlauskas A, et al. Ex vivo adenovirus-mediated gene transfer to corneal graft endothelial cells in mice[J]. Invest Ophthalmol Vis Sci,2004,45:2187-93
[20] Klebe S, Coster DJ, Sykes PJ, et al. Prolongation of sheep corneal allograft survival by transfer of the gene encoding ovine IL-12-p40 but not IL-4 to donor corneal endothelium[J]. J Immunol,2005,175:2219-26.
[21] Liu Y, Hamrah P, Zhang Q, et al. Draining lymph nodes of corneal transplant hosts exhibit evidence for donor major histocompatibility complex (MHC) class II-positive dendritic cells derived from MHC class II-negative grafts[J]. J Exp Med, 2002,195:259-68.
[22] Nakamura T, Ishikawa F, Sonoda KH, et al.Characterization and distribution of bone marrow-derived cells in mouse cornea[J]. Invest Ophthalmol Vis Sci,2005, 46:497-503.
[23] Brazelton TR, Blau HM. Optimizing techniques for tracking transplanted stem cells in vivo[J].Stem Cells,2005,23:125l-1265.
[24] Chinnery HR, Ruitenberg MJ, Plant GW, et al. The chemokine receptor CX3CR1 mediateshoming of MHC class II-positive cells to the normal mouse corneal epithelium.Invest Ophthalmol Vis Sci, 2007, 48:1568-74.
[2]兔角膜器官培养保存与应用的研究.刘涛潘志强王立李纳.中华眼科杂志,2006,42(9):808-813.
[3] European Eye Bank Association Directory. 8th ed. ?rhus,Denmark, January 2000.
[4] Thuret G, Manissolle C, Campos-Guyotat L, Guyotat D, and Gain P.Animal Compound–Free Medium and Poloxamer for Human Corneal Organ Culture and Deswelling. Invest Ophthalmol Vis Sci. 2005;46:816–822.
[5] Bednarz J, Doubilei V, Wollnik PC, et al. Effect of three different media onserum free culture of donor corneas and isolated human corneal endothelial cells. Br J Ophthalmol. 2001;85:1416–1420.
[6] Rieck PW, Gigon M, Jaroszewski J, et al. Increased endothelial survival of organ-cultured corneas stored in FGF-2-supplemented serum-free medium. Invest Ophthalmol Vis Sci. 2003;44:3826–3832.
[7] Hempel B, Bednarz J, Engelmann K.Use of a serum-free medium for long-term storage of human corneas. Influence on endothelial cell density and corneal metabolism. Graefes Arch Clin Exp Ophthalmol. 2001 Oct;239(10):801-5.
[8] EEBA. European Eye Bank Association Directory. 12th ed. Amsterdam,The Netherlands; 2004.
[9] Ayoubi MG et al.Br J Ophthalmol,1996;80:740.
[10] Slettedal JK, Lyberg T, R?ger M,et al. Regeneration with proliferation of the endothelium of cultured human donor corneas with extended postmortem time.Cornea. 2008 Feb;27(2):212-9.
[11] Nusrat A,Parkos CA,Verkade P,Foley CS,Liang TW,Innis-Whitehouse W,et a1.Tight junctions ore membrane microdomains [M].J Cell Sci,2000,113(Pt 10):1771-1778.
[12] Stevenson B R,Siliciano J D,Mooseker M S,et a1.Identification of ZO-1:a high molecular weight polypeptide associated with the tight junction(zonula occludens)in a variety of epithelia[J].J Cell Biol,1986,103(3):755.
[13] Fanning A S,Jameson B J,Jesaitis L A,et a1.The tight junction protein ZO-1 establishes a link between the transnlenlbrane protein occludin and the actin cytoskeleton[J].J Biol Chem,1998,273:29745.
[14] Fanning A S,Ma T Y,Anderson J M.Isolation and functional characterization of the actin binding region in the tight junction protein ZO-1 [ J].FASEB J,2002,16(13):1835.
[15]鲍欢,包仕尧.缺氧后血脑屏障紧密连接变化的分子机制[J].圄外医学·脑血管疾病分册,2005,13(8):604.
[16]张文一,白海青,士峥华.视网膜色素上皮细胞中诱导型一氧化氮合酶、精氨酸代谢相关酶的表达及一氧化氮对细胞紧密连接的影响[J].中华眼底病杂志,2005,21(1):32.
[17] Crewe J M and John Armitage W.Integrity of Epithelium and Endothelium in Organ-Cultured Human Corneas. Invest Ophthalmol Vis Sci.2001;42:1757–1761.
[18] Yonenmra S,hoh M,Nagafuchi A,et a1.Cel1-to-cell adherens junction formation and actin filament organization:similarities and diffrences between non-polarized fibroblasts and polarized epithelial cells.J Cell Sci,1995,108(Pt 1):127.
[19] Ando-Akatsuka Y, Yoflenlura S, Itoh M , et a1. Differential behavior of E-cadherln and occludin in their colocalization with ZO-1 during the establishment of epithelial cell polarity[J]. J Cell Physiol,1999,179 (2):115.
[1] Borderie VM, Baudrimont M, Lopez M, Carvajal S, Laroche L.Evaluation of the deswelling period in dextran-containing medium after corneal organ culture. Cornea. 1997;16:215–223.
[2] Thuret G, Manissolle C, Campos-Guyotat L, Guyotat D, Gain P.Animal compound-free medium and poloxamer for human corneal organ culture and deswelling. Invest Ophthalmol Vis Sci. 2005;46:816–822.
[3]兔角膜器官培养保存与应用的研究.刘涛潘志强王立李纳.中华眼科杂志,2006,42(9):808-813.
[4]熊加祥,黎海蒂,龚发云,等.严重烧伤影响大鼠脑内c—fos表达和Som—L1与脑水肿的关系[J].第三军医大学学报,2002,24(7):760—763. [5 ] Use of Poloxamers for Deswelling of Organ-Cultured Corneas. Zhao M, Thuret G, Piselli S,et al. Invest Ophthalmol Vis Sci. 2008;49:550–559.
[6] Sperling S. Human corneal endothelium in organ culture: the influence of temperature and medium of incubation. Acta Ophthalmol (Copenh). 1979;57:269–276.
[7] Pels E, Schuchard Y. The effects of high molecular weight dextranon the preservation of human corneas. Cornea. 1984;3:219–227.
[8] Reim M, Hesse R, Pietruschka G. The metabolism of organ cultures of cornea in TC 199 with added dextran 500 or hydroxyethyl starch 450.Klin Monatsbl Augenheilkd. 1990 Feb;196(2):76-80.
[9] van der Want HJ, Pels E, Schuchard Y, Olesen B, Sperling S.Electron microscopy of cultured human corneas: osmotic hydration and the use of a dextran fraction (dextran T 500) in organ culture. Arch Ophthalmol. 1983;101:1920–1926.
[10] Salla S, Redbrake C, Becker J, Reim M. Remarks on the vitality of the human cornea after organ culture. Cornea. 1995;14:502–508.
[11] Rieger R, Jaroszewski J, Jaeckel C, Hartmann C, Rieck P. Endothelial cell loss during storage of donor corneas in culture media containing dextrane. 15th conference of the European Eye Bank Association. Bruxelles, Belgium; 2003.
[12] Standl T, Burmeister MA, Schroeder F, et al. Hydroxyethyl starch (HES) 130/0.4 provides larger and faster increases in tissue oxygen tension in comparison with prehemodilution values than HES 70/0.5 or HES 200/0.5 in volunteers undergoing acute normovolemic hemodilution. Anesth Analg. 2003 Apr;96(4):936-943.
[13] Jungheinrich C, Sauermann W, Bepperling F, et al.Hes(130/0.4) can be safely administered to patients even with severe renal impairment. Anesth Analg.2002,95:544-551.
[14]赵晶,叶铁虎,徐庆,等.非心脏外科手术患者羟乙基淀粉130/0.4注射液容量治疗的安全性.中华麻醉学杂志, 2004,24(9):657-660.
[15] Reim M, Hesse R, Pietruschka G. The metabolism of organ cultures of cornea in TC 199 with added dextran 500 or hydroxyethyl starch 450. Klin Monatsbl Augenheilkd. 1990;196:76–80.
[16] Reim M, Pantenburg FJ, Ziegler CD. Effect of serum and osmotically active substances on metabolism in 262 tissue cultures of pig’s corneas: improved preservation of donor tissue for keratoplasty.Klin Monatsbl Augenheilkd. 2001;218:95–101.
[17] Redbrake C, Kompa S, Altmann G, Reim M, Arend O. HES 130 as a continuous supplement for organ culture of human corneas (in German)? Ophthalmologe. 2006;103:43–47.
[1] Teresa B. Recent developments in ocular gene therapy[J].Exp Eye Res,2003,76: 643-652
[2] Pleyer U, Bertelmann E, Rieck P, et al. Survival of corneal allografts following adenovirus-mediated gene transfer of interleukin-4. Graefes Arch Clin Exp Ophthalmol 2000;238:531–6.
[3] Klebe S, Sykes P, Coster D, et al. Prolongation of sheep corneal allograft survival by ex vivo transfer of the gene encoding interleukin-10. Transplantation 2001;71:1214–20.
[4] Comer RM, King WJ, Ardjomand N, et al. Effect of administration of CTLA4-Ig as protein or cDNA on corneal allograft survival. Invest Ophthalmol Vis Sci 2002;43:1095–103.
[5] Muller A, Zhang EP, Schroff M, et al. Influence of ballistic gene transfer on antigen-presenting cells in murine corneas. Graefes Arch Clin Exp Ophthalmol 2002;240:851–9.
[6] Rijneveld WJ, Remeijer L, van Rij G, Beekhuis H, Pels E. Prospective clinical evaluation of McCarey-Kaufman and organ culture cornea preservation media: 14-year follow-up. Cornea. 2008 Oct;27(9):996-1000.
[7]兔角膜器官培养保存与应用的研究.刘涛潘志强王立李纳.中华眼科杂志,2006,42(9):808-813.
[8] Chalfie M, Tu Y, Euskirchen G, et a1. Green fluorescent protein as a marker for gene expression[J]. Science,1994,263:802-805.
[9] Thuret G, Manissolle C, Campos-Guyotat L, Guyotat D, and Gain P.Animal Compound–Free Medium and Poloxamer for Human Corneal Organ Culture and Deswelling. Invest Ophthalmol Vis Sci. 2005;46:816–822.
[10] In vitro adenovirus mediated gene transfer to the human cornea Br J Ophthalmol. 2005 June; 89(6): 658–661. C F Jessup, H M Brereton, D J Coster, and K A Williams.
[11] Crystal RG. The gene as the drug. Nat Med 1995;1:15–7.
[12] Somia N, Verma IM. Gene therapy: trials and tribulations. Nat Rev Genet 2000;1:91–9.
[13] Shimomura O, Johnson FH, Saiga Y. Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea [J].J Cell Comp Physiol,1962,59:223-239.
[14] Tsien RY. The green fluorescent protein. Annu Rev Biochem[J], 1998, 67:509- 544.
[1] Remeijer L, van Rij G, Beekhuis H, Pels E, Rijneveld WJ. Prospective clinical evaluation of McCarey-Kaufman and organ culture cornea preservation media: 14-year follow-up.Cornea. 2008 Oct;27(9):996-1000
[2] Hitani K, Yokoo S, Honda N,et al. Transplantation of a sheet of human corneal endothelial cell in a rabbit model. Molecular Vision 2008; 14:1-9
[3] Niederkorn JY, Mellon J. Anterior chamber-associated immune deviation promotes corneal allograft survival. Invest Ophthalmol Vis Sci 1996;37(13):2700-2707
[4] Yamada J, Dana MR, Zhu SN, et al. Interleukin 1 receptor antagonist suppresses allosensitization in corneal transplantation. Arch Ophthalmol 1998;116(10):1351-1357
[5]何维.医学免疫学[M].北京:人民卫生出版社, 2005:250-256
[6] Mosmann TR, Cherwinski H, Bond MW, et al. Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J Immunol 1986;136(7):2348-2357
[7] Amirzargar A, Lessanpezeshki M, Fathi A, et al. TH1/TH2 cytokine analysis in Iranian renal transplant recipients. Transplant Proc 2005;37(7):2985-2987
[8] Williams KA, Coster DJ. Penetrating corneal transplantation in the inbred rat: a new model. Invest Ophthalmol Vis Sci 1985;26(1):23-30.
[9] Larkin DF, Calder VL, Lightman SL. Identification and characterization of cells infiltrating the graft and aqueous humour in rat corneal allograft rejection. Clin Exp Immunol 1997;107(2):381-391
[10] Skurkovich S, Kasparov A, Narbut N, et al. Treatment of corneal transplant rejection in humans with anti-interferon-gamma antibodies. Am J Ophthalmol 2002;133(6):829-830
[11] Jiang H, Liu C, Xu J, et al. Gene transfer of interleukin-4 delays acute rejection of splenic allografts in rats. Transplant Proc 2004;36(5):1600-1603
[12] Zhang EP, Franke J, Schroff M, et al. Ballistic CTLA4 and IL-4 gene transfer into the lower lid prolongs orthotopic corneal graft surviveal in mice. Graefes Arch Clin Exp Ophthalmol 2003;241(11):921-926
[13] De Karis,Zhu SN,Dana MR,et a1.TNF-alpha regulate corneal langerhans cell migration[J].J Immunol,1999,162(7):4235-4239.
[14] Watsky MA,Guna I,Ragsdate DN, et a1.Efect of tumor necrosis factor alpha on rabbit corneal endothelial pemeability [J].Invest Ophthalmol Vis Sci,1996,37:19
[15] Mathan A, Kuruvilla S, Abraham G. Interleukin 2 receptor expression in renal biopsies and the diagnosis of acute allograft rejection. Indian J Pathol Microbiol 2006;49(1):12-16.
[16] Modulation of Costimulation by CD28 and CD154 Alters the Kinetics and Cellular Characteristics of Corneal Allograft Rejection. Navid Ardjomand, James C. McAlister, Nicola J. Rogers, Peng H. Tan,Andrew J. T. George, and Daniel F. P. Larkin. Invest Ophthalmol VisSci. 2003;44:3899–3905
[17] Comer RM, King WJ, Ardjomand N, Theoharis S, George AJ,Larkin DF. Effect of administration of CTLA4-Ig as protein or cDNA on corneal allograft survival. Invest Ophthalmol Vis Sci 2002; 43:1095–103.
[18] Ardjomand N, McAlister JC, Rogers NJ, Tan PH, George AJ, Larkin DF. Modulation of costimulation by CD28 and CD154 alters the kinetics and cellular characteristics of corneal allograft rejection. Invest Ophthalmol Vis Sci 2003; 44:3899–905.
[1] Shimomura O, Johnson FH, Saiga Y. Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea [J].J Cell Comp Physiol,1962,59:223-239.
[2] Prasher DC, Eckenrode VK, Ward WW, et a1. Primary structure of the aequorea victoria green-fluorescent protein[J]. J Gene,1992,111:229-233.
[3] Chalfie M, Tu Y, Euskirchen G, et a1. Green fluorescent protein as a marker for gene expression[J]. Science,1994,263:802-805.
[4] Ormo M, Cubitt A B, Kallio K, et al. Crystal structure of the Aequorea victoria greenfluorescent protein[J]. Science,1996,273:1392-1395.
[5] Cody CW, Prasher DC, Westler WM, et a1. Chemical structure of the hexapeptide chromophore of the Aequorea green-fluorescent protein[J]. Biochemistry,1993,32: 1212-1218.
[6] Gubin AN, Reddy B, Njoroge JM, et a1. Long-term, stable expression of green fluorescent protein in mammalian cells[J]. Bioc hem Biophys Res Commun,1997, 236:347-350.
[7] Stadtfeld M, Varas F, Graf T. Fluorescent protein-cell labeling and its application in time-lapse analysis of hematopoietic differentiation[J]. Methods Mol Med,2005, 105:395-412.
[8] Tsien RY. The green fluorescent protein. Annu Rev Biochem[J], 1998, 67:509- 544.
[9] Tavare JM, Fletcher LM, Welsh GI, et al. Using green fluroscent protein to study intracelluar signaling[J].J Endocrinology,2001,170:297-306.
[10] Liu YP, Dovzhenko OV, Garthwaite MA, et al. Maintenance of pluripotency in human embryonic stem eelIs stably over-expressing enhanced green fluorescent protein[J].Stem Cells Dev,2004,l3:636-645.
[11] Kahn J, Byk T, Jansson-Sjostrand L, et al.Overexpression of CXCR4 on human CD34+ progenitors increases their proliferation,migration,and NOD/SClD repopulation[J]. Blood,2004,103:2942-2949.
[12] Teresa B. Recent developments in ocular gene therapy[J].Exp Eye Res,2003,76: 643-652
[13] Lai L, Lin K, Foulks G, et al. Highly efficient ex vivo gene delivery into human corneal endothelial cells by recombinant adeno-associated virus[J]. Curr Eye Res, 2005, 30:213-9.
[14]袁进,陈家祺,刘祖国,等.IL-1ra基因修饰角膜内皮细胞及其表达[J].眼科新进展,2006,26:2-6.
[15] Nguyen TH, Murakami A, Fujiki K, et al.Transferrin-polyethylenimine conjugate, FuGENE6 and TransIT-LT as nonviral vectors for gene transfer to the corneal endothelium[J]. Jpn J Ophthalmol,2002, 46:140-6.
[16] Choi J, Ko MK, Kay EP. Subcellular localization of the expressed 18 kDa FGF-2 isoform in corneal endothelial cells.Mol Vis, 2000, 6:222-31.
[17] Challa P, Luna C, Liton PB, et al. Lentiviral mediated gene delivery to the anterior chamber of rodent eyes[J]. Mol Vis,2005,11:425-30.
[18] Borrás T, Gabelt BT, Klintworth GK, et al. Non-invasive observation of repeated adenoviral GFP gene delivery to the anterior segment of the monkey eye in vivo [J]. J Gene Med, 2001,3:437-49.
[19] Qian Y, Leong FL, Kazlauskas A, et al. Ex vivo adenovirus-mediated gene transfer to corneal graft endothelial cells in mice[J]. Invest Ophthalmol Vis Sci,2004,45:2187-93
[20] Klebe S, Coster DJ, Sykes PJ, et al. Prolongation of sheep corneal allograft survival by transfer of the gene encoding ovine IL-12-p40 but not IL-4 to donor corneal endothelium[J]. J Immunol,2005,175:2219-26.
[21] Liu Y, Hamrah P, Zhang Q, et al. Draining lymph nodes of corneal transplant hosts exhibit evidence for donor major histocompatibility complex (MHC) class II-positive dendritic cells derived from MHC class II-negative grafts[J]. J Exp Med, 2002,195:259-68.
[22] Nakamura T, Ishikawa F, Sonoda KH, et al.Characterization and distribution of bone marrow-derived cells in mouse cornea[J]. Invest Ophthalmol Vis Sci,2005, 46:497-503.
[23] Brazelton TR, Blau HM. Optimizing techniques for tracking transplanted stem cells in vivo[J].Stem Cells,2005,23:125l-1265.
[24] Chinnery HR, Ruitenberg MJ, Plant GW, et al. The chemokine receptor CX3CR1 mediateshoming of MHC class II-positive cells to the normal mouse corneal epithelium.Invest Ophthalmol Vis Sci, 2007, 48:1568-74.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |