恒河猴血管内皮细胞移植替代角膜内皮细胞的实验研究
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摘要
目的：探讨超声乳化破坏恒河猴角膜内皮细胞后角膜内表面的变化；通过观察移植到无内皮层的角膜内表面的恒河猴血管内皮细胞的位置、形态和功能改变,探索血管内皮细胞移植治疗角膜内皮细胞损伤的方法和前房注射法的可行性。
方法：体外培养增殖恒河猴脉络膜-视网膜血管内皮细胞株(RF\6A)并用5-溴脱氧尿嘧啶核苷(Brdu)标记。将恒河猴9只随机分为三组：实验组(A组,3只)、实验对照组(B组,2只),空白对照组(C组,4只)。A组：超声乳化角膜内表面后立即将体外培养增殖经Brdu标记的RF\6A通过角巩膜缘注射至前房,全麻下保持实验动物俯卧位3-4小时；B组：超声乳化术后一个月后用与A组相同的方法移植经Brdu标记的RF\6A；C组：行超声乳化术后不行细胞移植,其中一只术后立即摘眼行各项检测。术后观察各组角膜透明情况；术前、术后1、2、3、4、8、12周分别抽取房水,用ELISA法测定房水中血管内皮细胞生长因子(VEGF)浓度；使用全自动生化分析仪测定房水中Ca2+、Mg2+、P、Na+、K+、C1-六项离子的浓度,并行统计学分析。术后4、8、12周分别摘取术眼,行病理切片、抗Brdu单克隆抗体免疫组化染色、扫描电镜及透射电镜观察角膜内表面RF\6A分布和形态结构,虹膜及小梁网的形态结构。
结果：角膜透明度：在观察期内(3个月),A组、B组角膜比C组有较好的透明度,未出现新生血管及大泡性角膜病变。术眼前房深度均正常。病理切片：A组和B组的角膜内表面可见细胞层,抗Brdu单克隆抗体免疫组化染色阳性,示该细胞为体外培养经Brdu标记的RF\6A；后弹力层均基本完整。虹膜仅有轻度水肿；小梁网结构清晰,未见细胞碎片堵塞网孔。C组角膜内表面仅见周边残留角膜内皮细胞,免疫组化染色阴性；除术后立即摘眼的角膜后弹力层完整外,其他各时期后弹力层逐渐溶解消失。虹膜及小梁网结构清晰。扫描电镜：A组和B组角膜内表面可见RF\6A分布均匀,生长良好。虹膜上见散在RF\6A生长,但虹膜表面结构无明显改变；小梁网未见细胞碎片堵塞。C组角膜内表面仅见结构形态已破坏的CECs,术后后弹力层逐渐溶解,未见新的CECs生长。透射电镜：培养的RF\6A以及角膜内表面的RF\6A呈不规则扁圆形,其丰富的细胞器和特征性WP小体提示具备一定的活体血管内皮细胞的特征与活性。C组角膜内表面残存的CECs,胞质水肿,细胞器稀少。房水VEGF浓度：仅A组不同观察时间点的VEGF浓度差异有统计学意义。A、B和C组三组间Ca2+、Mg2+、P、Na+、K+、Cl-浓度无明显差异。术后3月时A组Na+浓度略低于C组。实验组内各时期Mg2+、P离子浓度有一定改变。
结论：前房注射法能够将恒河猴血管内皮细胞移植到角膜内表面,且细胞能在超声乳化破坏角膜内皮层的后弹力层上生长,并在一定程度上发挥屏障作用维持角膜的脱水状态和透明性。移植术后,对虹膜、前房角的组织结构形态在短期内不产生病理性的影响；对房水VEGF浓度和Ca2+、Mg2+、P、Na+、K+、Cl-的浓度有一定程度的影响。
Objective:It is to investigate the inner surface of the cornea changing process after the corneal endothelial cells (CECs) was destroyed by phacoemulsification surgery;It is to explore the method of the rhesus monkey vascular endothelial cell transplantation to substitute the allogeneic CECs,and the feasibility of anterior chamber injection transplantation method,through observe the position,morphologic and functional changes of the rhesus monkey vascular endothelial cell which was transplanted to the inner surface of cornea without CECs.
Method:The RF\6A was cultivated to proliferation and marked by Brdu in vitro.The experimental monkeys are divided into3groups:experimental group (group A,3monkeys),control group(group B,2monkeys),blank control group(group C,4monkeys).In group A:the CECs was destroyed by phacoemulsification surgery;and the cultured RF\6A,marked by brdu,were transplanted onto the inside surface of cornea without CECs though anterior chamber injection transplantation method,then make the experimental animals facing down for3-4hours.In group B:one month after phacoemulsification surgery,using the methods the same as group A to transplant the cultured RF\6A which marked by Brdu.In group C:CECs was destroyed by phacoemulsification surgery but without cultured RMVECs transplantation.One of the group C rhesus monkeys was picked the experimental eye immediately for various testings afer the surgery.Corneal transparence of every monkey was frequently observed postoperation.Aqueous humor was collected in preoperation and1,2,3,4,8,12 postoperation weeks to measure the concentration of vascular endothelial growth factor(VEGF) in ELISA method. Six ions concentration were tested useing automatic biochemistry analyzer.Then all data proceed with data analysis.On4,8,12postoperative weeks,the experimental eyes were respectively detached to observe the distribution and morphological structure of the RF\6A in the inner surface of cornea.and morphological structure of both iris and trabecular meshwork by pathological section. anti Brdu monoclonal antibody immunohistochemistry,scanning electronic microscope (SEM) and transmission election microscopy(TEM).
Result:Corneal transpareney:In the experimental observation period(3months), group A and group B had better transparency than group C, with normal anterior chamber depth and without corneal neovascularization and bullous kerat-opathy.Pathological section:Cell layer with Brdu staining positive was found on the posterior surface of cornea in group A and group B,indicated the cells are RF\6A.The Descemet's membrane was basically complete.Mild edema was occurred in the iris.There wasn't cellular debris blocking trabecular meshwork and the angle of anterior chamber structure was clear.Only residual CECs with Brdu staining negative were foud on the peripheral posterior surface of cornea in group C. The Descemet's membrane was gradually dissolved and disappeared except the cornea which was immdiately picked off in postoperation.Both iris and trabecular meshwork had clear structure.SEM:Group A and group B showed that RF\6A with irregular shape uniformly distributed on the inner surface of cornea and growing well.Scattered RF\6A grown on the iris surface but the structure of iris surface had not significant changes.There was not cellular debris blocking trabecular meshwork.Group C showed with only residual CECs which structure and morphology were distroyed. The Descemet's membrane was gradually disappeared without new CECs growing on the inner surface of cornea.TEM:The cultured RF\6A and which in posterior surface of cornea were irregular oblateness.Abundant organelles and characteristic WBPs appeared in cytoplasm suggested the characteristic and vitality of vascular endothelial cells in vivo.Only residual CECs which cytoplasmic edema and scarce organelles had been fouded in posterior surface of cornea in group C.The concentration of VEGF in aqueous humor show no significant difference among these three groups,but significant difference among different time points in the group A.Among these three groups Ca2+,Mg2+,P,Na+,K+,C1-concentration show no significant difference.but the Na+concentration in group A is slightly lower than in the group C on the3postoperation months.And the Mg2+,P concentration in group A have some changes in some extent.
Conelusion:Anterior chamber injection transplantation method can transplant the rhesus monkey vascular endothelial cell into inner surface of cornea,and they can grow well on the Descemet's membrane which CECs were destroyed by phacoemulsification surgery.lt also can play a role of barrier to keep the corneal dehydration and transparency to a certain extent.There are not pathological influences to the morphological structure of iris and the anterior chamber angle in the short time after the transplantion surgery.But it shows some influence to the concentration of VEGF,Ca2+,Mg2+,P,Na+,K+,C1-in aqueous humor.
方法：体外培养增殖恒河猴脉络膜-视网膜血管内皮细胞株(RF\6A)并用5-溴脱氧尿嘧啶核苷(Brdu)标记。将恒河猴9只随机分为三组：实验组(A组,3只)、实验对照组(B组,2只),空白对照组(C组,4只)。A组：超声乳化角膜内表面后立即将体外培养增殖经Brdu标记的RF\6A通过角巩膜缘注射至前房,全麻下保持实验动物俯卧位3-4小时；B组：超声乳化术后一个月后用与A组相同的方法移植经Brdu标记的RF\6A；C组：行超声乳化术后不行细胞移植,其中一只术后立即摘眼行各项检测。术后观察各组角膜透明情况；术前、术后1、2、3、4、8、12周分别抽取房水,用ELISA法测定房水中血管内皮细胞生长因子(VEGF)浓度；使用全自动生化分析仪测定房水中Ca2+、Mg2+、P、Na+、K+、C1-六项离子的浓度,并行统计学分析。术后4、8、12周分别摘取术眼,行病理切片、抗Brdu单克隆抗体免疫组化染色、扫描电镜及透射电镜观察角膜内表面RF\6A分布和形态结构,虹膜及小梁网的形态结构。
结果：角膜透明度：在观察期内(3个月),A组、B组角膜比C组有较好的透明度,未出现新生血管及大泡性角膜病变。术眼前房深度均正常。病理切片：A组和B组的角膜内表面可见细胞层,抗Brdu单克隆抗体免疫组化染色阳性,示该细胞为体外培养经Brdu标记的RF\6A；后弹力层均基本完整。虹膜仅有轻度水肿；小梁网结构清晰,未见细胞碎片堵塞网孔。C组角膜内表面仅见周边残留角膜内皮细胞,免疫组化染色阴性；除术后立即摘眼的角膜后弹力层完整外,其他各时期后弹力层逐渐溶解消失。虹膜及小梁网结构清晰。扫描电镜：A组和B组角膜内表面可见RF\6A分布均匀,生长良好。虹膜上见散在RF\6A生长,但虹膜表面结构无明显改变；小梁网未见细胞碎片堵塞。C组角膜内表面仅见结构形态已破坏的CECs,术后后弹力层逐渐溶解,未见新的CECs生长。透射电镜：培养的RF\6A以及角膜内表面的RF\6A呈不规则扁圆形,其丰富的细胞器和特征性WP小体提示具备一定的活体血管内皮细胞的特征与活性。C组角膜内表面残存的CECs,胞质水肿,细胞器稀少。房水VEGF浓度：仅A组不同观察时间点的VEGF浓度差异有统计学意义。A、B和C组三组间Ca2+、Mg2+、P、Na+、K+、Cl-浓度无明显差异。术后3月时A组Na+浓度略低于C组。实验组内各时期Mg2+、P离子浓度有一定改变。
结论：前房注射法能够将恒河猴血管内皮细胞移植到角膜内表面,且细胞能在超声乳化破坏角膜内皮层的后弹力层上生长,并在一定程度上发挥屏障作用维持角膜的脱水状态和透明性。移植术后,对虹膜、前房角的组织结构形态在短期内不产生病理性的影响；对房水VEGF浓度和Ca2+、Mg2+、P、Na+、K+、Cl-的浓度有一定程度的影响。
Objective:It is to investigate the inner surface of the cornea changing process after the corneal endothelial cells (CECs) was destroyed by phacoemulsification surgery;It is to explore the method of the rhesus monkey vascular endothelial cell transplantation to substitute the allogeneic CECs,and the feasibility of anterior chamber injection transplantation method,through observe the position,morphologic and functional changes of the rhesus monkey vascular endothelial cell which was transplanted to the inner surface of cornea without CECs.
Method:The RF\6A was cultivated to proliferation and marked by Brdu in vitro.The experimental monkeys are divided into3groups:experimental group (group A,3monkeys),control group(group B,2monkeys),blank control group(group C,4monkeys).In group A:the CECs was destroyed by phacoemulsification surgery;and the cultured RF\6A,marked by brdu,were transplanted onto the inside surface of cornea without CECs though anterior chamber injection transplantation method,then make the experimental animals facing down for3-4hours.In group B:one month after phacoemulsification surgery,using the methods the same as group A to transplant the cultured RF\6A which marked by Brdu.In group C:CECs was destroyed by phacoemulsification surgery but without cultured RMVECs transplantation.One of the group C rhesus monkeys was picked the experimental eye immediately for various testings afer the surgery.Corneal transparence of every monkey was frequently observed postoperation.Aqueous humor was collected in preoperation and1,2,3,4,8,12 postoperation weeks to measure the concentration of vascular endothelial growth factor(VEGF) in ELISA method. Six ions concentration were tested useing automatic biochemistry analyzer.Then all data proceed with data analysis.On4,8,12postoperative weeks,the experimental eyes were respectively detached to observe the distribution and morphological structure of the RF\6A in the inner surface of cornea.and morphological structure of both iris and trabecular meshwork by pathological section. anti Brdu monoclonal antibody immunohistochemistry,scanning electronic microscope (SEM) and transmission election microscopy(TEM).
Result:Corneal transpareney:In the experimental observation period(3months), group A and group B had better transparency than group C, with normal anterior chamber depth and without corneal neovascularization and bullous kerat-opathy.Pathological section:Cell layer with Brdu staining positive was found on the posterior surface of cornea in group A and group B,indicated the cells are RF\6A.The Descemet's membrane was basically complete.Mild edema was occurred in the iris.There wasn't cellular debris blocking trabecular meshwork and the angle of anterior chamber structure was clear.Only residual CECs with Brdu staining negative were foud on the peripheral posterior surface of cornea in group C. The Descemet's membrane was gradually dissolved and disappeared except the cornea which was immdiately picked off in postoperation.Both iris and trabecular meshwork had clear structure.SEM:Group A and group B showed that RF\6A with irregular shape uniformly distributed on the inner surface of cornea and growing well.Scattered RF\6A grown on the iris surface but the structure of iris surface had not significant changes.There was not cellular debris blocking trabecular meshwork.Group C showed with only residual CECs which structure and morphology were distroyed. The Descemet's membrane was gradually disappeared without new CECs growing on the inner surface of cornea.TEM:The cultured RF\6A and which in posterior surface of cornea were irregular oblateness.Abundant organelles and characteristic WBPs appeared in cytoplasm suggested the characteristic and vitality of vascular endothelial cells in vivo.Only residual CECs which cytoplasmic edema and scarce organelles had been fouded in posterior surface of cornea in group C.The concentration of VEGF in aqueous humor show no significant difference among these three groups,but significant difference among different time points in the group A.Among these three groups Ca2+,Mg2+,P,Na+,K+,C1-concentration show no significant difference.but the Na+concentration in group A is slightly lower than in the group C on the3postoperation months.And the Mg2+,P concentration in group A have some changes in some extent.
Conelusion:Anterior chamber injection transplantation method can transplant the rhesus monkey vascular endothelial cell into inner surface of cornea,and they can grow well on the Descemet's membrane which CECs were destroyed by phacoemulsification surgery.lt also can play a role of barrier to keep the corneal dehydration and transparency to a certain extent.There are not pathological influences to the morphological structure of iris and the anterior chamber angle in the short time after the transplantion surgery.But it shows some influence to the concentration of VEGF,Ca2+,Mg2+,P,Na+,K+,C1-in aqueous humor.
引文
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