中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2015年
5期
440-445
,共6页
李斐%张琰%茹玉莎%刘会娟%赵少贞
李斐%張琰%茹玉莎%劉會娟%趙少貞
리비%장염%여옥사%류회연%조소정
间充质干细胞移植%角膜移植%移植物存活/免疫%T淋巴细胞%γ干扰素%白细胞介素%Lewis大鼠
間充質榦細胞移植%角膜移植%移植物存活/免疫%T淋巴細胞%γ榦擾素%白細胞介素%Lewis大鼠
간충질간세포이식%각막이식%이식물존활/면역%T림파세포%γ간우소%백세포개소%Lewis대서
Mesenchymal stem cell transplantation%Corneal transplantation%Graft survival/immunology%Tlmphocytes%Interferon-gamma%Interleukin%Rats,Lewis
背景 间充质干细胞(MSCs)已用于多种器官移植抗排斥反应的基础和临床研究.我们先前的研究表明,鼠尾静脉注射MSCs可延长大鼠角膜植片的存活时间,但治疗时MSCs的需求量较大且存在一定的全身不良反应.眼局部应用MSCs是否能够替代角膜移植排斥反应的全身治疗尚不清楚. 目的 探讨结膜下注射骨髓MSCs对大鼠角膜移植排斥反应的作用.方法 从清洁级Wistar大鼠股骨和胫骨骨髓中分离获得MSCs进行培养和传代,以体外成骨细胞分化和脂肪细胞分化法进行鉴定,取第3代MSCs用于实验.将20只Wistar大鼠双眼角膜作为角膜供体植片,40只Lewis大鼠右眼作为受体植床,行同种异体穿透角膜移植术.应用随机数字表法将40只受体大鼠分为PBS对照组和MSCs治疗组,另取6只正常Lewis大鼠作为正常对照组.MSCs治疗组大鼠分别于角膜移植术后即刻和第3天结膜下注射含2×106 MSCs的PBS 0.1 ml,PBS对照组大鼠以同样的方法仅给予等容积PBS.各组大鼠术后每天裂隙灯显微镜下观察角膜植片的混浊、水肿和新生血管情况,并按角膜植片排斥反应标准进行评分.于术后第10天分别处死PBS对照组和MSCs治疗组各12只大鼠,取出术眼角膜组织,采用实时荧光定量PCR法检测Th1细胞相关炎性因子γ干扰素(IFN-γ)mRNA和白细胞介素-2(IL-2)mRNA及Th2细胞相关炎性因子IL-4 mRNA和IL-10 mRNA在角膜中的相对表达量;采用ELISA法检测角膜组织中Th2细胞相关因子IL-4和IL-10蛋白的质量浓度. 结果 培养的细胞生长良好,成骨细胞分化的细胞茜素红染色呈橘黄色,脂肪细胞分化的细胞油红O染色呈红色,证实为MSCs.角膜移植术后MSCs治疗组角膜植片存活时间为(11.8±1.6)d,长于PBS对照组的(9.6±1.4)d,差异有统计学意义(P=0.004);MSCs治疗组大鼠角膜植片中IFN-γmRNA、IL-2 mRNA的相对表达量较PBS对照组下降,但2个组间差异均无统计学意义(均P＞0.05),但IL-4 mRNA和IL-10 mRNA相对表达量明显高于PBS对照组,差异均有统计学意义(均P=0.00).正常对照组、PBS对照组和MSCs治疗组大鼠角膜植片中IL-10蛋白质量浓度分别为(22.74±7.06)、(68.40±12.83)和(215.41±44.66) pg/ml,3个组间总体比较差异有统计学意义(F=55.06,P=0.00),且MSCs治疗组大鼠植片中IL-10蛋白质量浓度明显高于PBS对照组及正常对照组,差异均有统计学意义(均P＜0.05).结论 结膜下注射MSCs可以延长大鼠异体穿透角膜移植术后植片的存活时间,这一作用可能是通过调节Th 1/Th2平衡,特别是上调Th2细胞因子而介导的.
揹景 間充質榦細胞(MSCs)已用于多種器官移植抗排斥反應的基礎和臨床研究.我們先前的研究錶明,鼠尾靜脈註射MSCs可延長大鼠角膜植片的存活時間,但治療時MSCs的需求量較大且存在一定的全身不良反應.眼跼部應用MSCs是否能夠替代角膜移植排斥反應的全身治療尚不清楚. 目的 探討結膜下註射骨髓MSCs對大鼠角膜移植排斥反應的作用.方法 從清潔級Wistar大鼠股骨和脛骨骨髓中分離穫得MSCs進行培養和傳代,以體外成骨細胞分化和脂肪細胞分化法進行鑒定,取第3代MSCs用于實驗.將20隻Wistar大鼠雙眼角膜作為角膜供體植片,40隻Lewis大鼠右眼作為受體植床,行同種異體穿透角膜移植術.應用隨機數字錶法將40隻受體大鼠分為PBS對照組和MSCs治療組,另取6隻正常Lewis大鼠作為正常對照組.MSCs治療組大鼠分彆于角膜移植術後即刻和第3天結膜下註射含2×106 MSCs的PBS 0.1 ml,PBS對照組大鼠以同樣的方法僅給予等容積PBS.各組大鼠術後每天裂隙燈顯微鏡下觀察角膜植片的混濁、水腫和新生血管情況,併按角膜植片排斥反應標準進行評分.于術後第10天分彆處死PBS對照組和MSCs治療組各12隻大鼠,取齣術眼角膜組織,採用實時熒光定量PCR法檢測Th1細胞相關炎性因子γ榦擾素(IFN-γ)mRNA和白細胞介素-2(IL-2)mRNA及Th2細胞相關炎性因子IL-4 mRNA和IL-10 mRNA在角膜中的相對錶達量;採用ELISA法檢測角膜組織中Th2細胞相關因子IL-4和IL-10蛋白的質量濃度. 結果 培養的細胞生長良好,成骨細胞分化的細胞茜素紅染色呈橘黃色,脂肪細胞分化的細胞油紅O染色呈紅色,證實為MSCs.角膜移植術後MSCs治療組角膜植片存活時間為(11.8±1.6)d,長于PBS對照組的(9.6±1.4)d,差異有統計學意義(P=0.004);MSCs治療組大鼠角膜植片中IFN-γmRNA、IL-2 mRNA的相對錶達量較PBS對照組下降,但2箇組間差異均無統計學意義(均P＞0.05),但IL-4 mRNA和IL-10 mRNA相對錶達量明顯高于PBS對照組,差異均有統計學意義(均P=0.00).正常對照組、PBS對照組和MSCs治療組大鼠角膜植片中IL-10蛋白質量濃度分彆為(22.74±7.06)、(68.40±12.83)和(215.41±44.66) pg/ml,3箇組間總體比較差異有統計學意義(F=55.06,P=0.00),且MSCs治療組大鼠植片中IL-10蛋白質量濃度明顯高于PBS對照組及正常對照組,差異均有統計學意義(均P＜0.05).結論 結膜下註射MSCs可以延長大鼠異體穿透角膜移植術後植片的存活時間,這一作用可能是通過調節Th 1/Th2平衡,特彆是上調Th2細胞因子而介導的.
배경 간충질간세포(MSCs)이용우다충기관이식항배척반응적기출화림상연구.아문선전적연구표명,서미정맥주사MSCs가연장대서각막식편적존활시간,단치료시MSCs적수구량교대차존재일정적전신불량반응.안국부응용MSCs시부능구체대각막이식배척반응적전신치료상불청초. 목적 탐토결막하주사골수MSCs대대서각막이식배척반응적작용.방법 종청길급Wistar대서고골화경골골수중분리획득MSCs진행배양화전대,이체외성골세포분화화지방세포분화법진행감정,취제3대MSCs용우실험.장20지Wistar대서쌍안각막작위각막공체식편,40지Lewis대서우안작위수체식상,행동충이체천투각막이식술.응용수궤수자표법장40지수체대서분위PBS대조조화MSCs치료조,령취6지정상Lewis대서작위정상대조조.MSCs치료조대서분별우각막이식술후즉각화제3천결막하주사함2×106 MSCs적PBS 0.1 ml,PBS대조조대서이동양적방법부급여등용적PBS.각조대서술후매천렬극등현미경하관찰각막식편적혼탁、수종화신생혈관정황,병안각막식편배척반응표준진행평분.우술후제10천분별처사PBS대조조화MSCs치료조각12지대서,취출술안각막조직,채용실시형광정량PCR법검측Th1세포상관염성인자γ간우소(IFN-γ)mRNA화백세포개소-2(IL-2)mRNA급Th2세포상관염성인자IL-4 mRNA화IL-10 mRNA재각막중적상대표체량;채용ELISA법검측각막조직중Th2세포상관인자IL-4화IL-10단백적질량농도. 결과 배양적세포생장량호,성골세포분화적세포천소홍염색정귤황색,지방세포분화적세포유홍O염색정홍색,증실위MSCs.각막이식술후MSCs치료조각막식편존활시간위(11.8±1.6)d,장우PBS대조조적(9.6±1.4)d,차이유통계학의의(P=0.004);MSCs치료조대서각막식편중IFN-γmRNA、IL-2 mRNA적상대표체량교PBS대조조하강,단2개조간차이균무통계학의의(균P＞0.05),단IL-4 mRNA화IL-10 mRNA상대표체량명현고우PBS대조조,차이균유통계학의의(균P=0.00).정상대조조、PBS대조조화MSCs치료조대서각막식편중IL-10단백질량농도분별위(22.74±7.06)、(68.40±12.83)화(215.41±44.66) pg/ml,3개조간총체비교차이유통계학의의(F=55.06,P=0.00),차MSCs치료조대서식편중IL-10단백질량농도명현고우PBS대조조급정상대조조,차이균유통계학의의(균P＜0.05).결론 결막하주사MSCs가이연장대서이체천투각막이식술후식편적존활시간,저일작용가능시통과조절Th 1/Th2평형,특별시상조Th2세포인자이개도적.
Background Mesenchymal stem cells (MSCs) have been applied in basic and clinical researches of organ transplantation.Our previous study showed that intravenous injection of MSCs prolonged corneal allograft survival in rat.However,the effect of local administration of MSCs on corneal allograft rejection remains unclear.Objective The aim of this study was to investigate the effect of subconjunctival injection of MSCs on corneal allograft rejection in rat model of keratoplasty.Methods MSCs were isolated and cultured from femur and tibia bone marrow of clean Wistar rats,and then the cells were identified by induced differentiation of osteoblast and adipocyte.The third generation of MSCs was used in subsequent experiment.Allogenic penetrating keratoplasty was performed with the bilateral corneas of 20 Wistar rats as donor grafts and the right eyes of 40 Lewis rats as recipients.PBS 0.1 ml containing 2 × 106 MSCs and 0.1 ml PBS only was subconjunctivally injected immediately and postoperative 3 days respectively in randomized two groups,and another 6 normal Lewis rats served as the normal control group.Corneal rejection response was evaluated under the slit lamp after surgery based corneal opacity,edema and neovascularization,and the grafts were scored according to the criteria of Larkin.The corneal samples were extracted from 12 rats of the PBS control group and the MSCs group separately 10 days after surgery.The relative expressions of Th1 cytokines (interferon-γ [IFN-γ] mRNA and interleukin-2 [IL-2] mRNA) and Th2 cytokines (IL-4 mRNA and IL-10 mRNA) were detected by real-time quantitative PCR.Protein levels of IL-4 and IL-10 proteins in the corneas were assayed by ELISA.All experimental protocols involving rats were approved by the laboratory animal care and use committee of the Tianjin Medical University and treated with the ARVO statement for the use of animals in ophthalmic and vision research.Results The cells grew well with the orange stain for alizarin red in differentiated the osteoblasts and red stain for Oil red O in differentiated adipocytes.The survival time of corneal graft in the MSCs group was (11.8±1.6) days,it was significantly longer than (9.6±1.4) days in the PBS control group (P=0.004).The levels of IL-4 mRNA and IL-10 mRNA in the MSCs group were significantly higher than those in the PBS control group (both at P =0.00);while the levels of IFN-γ mRNA and IL-2 mRNA were not significantly different between the groups (both at P＞0.05).The IL-10 protein contents were (22.74 ±7.06),(68.40±12.83) and (215.41 ±44.66)pg/ml in the normal control group,PBS control group and MSCs group,showing significant difference among the three groups (F =55.06,P =0.00) and a significant increase in the MSCs group compared with the PBS control group and the normal control group (both at P ＜ 0.05).Conclusions Subconjunctival injection of MSCs prolongs the survival time of cornea allograft in penetrating keratoplasty probably by modulating the balance between Th1 and Th2 cytokines,especially by up-regulating Th2 cytokines.
