新型VEGF靶向抗体FD006抑制角膜新生血管的实验研究

发布时间：2018-06-26 05:52

本文选题：血管内皮生长因子 + 单克隆抗体　；参考：《中国人民解放军医学院》2014年博士论文

【摘要】：目的：评价FD006的体外生物学活性。观察FD006抑制碱烧伤诱导的大鼠角膜新生血管（corneal neovascularization，CoNV）增殖的情况，初步探索FD006抑制大鼠CoNV生长的作用机制。评价FD006在眼部应用的安全性并初步探讨FD006的眼部药代动力学情况。材料与方法： 1．借助分子模拟、动态对接方法研究FD006与VEGF相互作用模式。蛋白-蛋白相互作用和ELISA检测FD006与VEGF的结合能力。CCK8检测FD006对VEGF诱导HUVEC增殖的影响。 2.建立碱烧伤诱导SD大鼠CoNV动物模型，碱烧伤术后第1天，随机分为：0.9%NaCl、溶剂、地塞米松组、贝伐单抗和FD006抗体5组，各组分别给予结膜下注射相应药物治疗；观察CoNV生长情况及角膜组织变化，并进行眼前节照相。分析CoNV的长度及面积。碱烧伤术后3、7、14、21和28天处死动物，取角膜组织行免疫组织化学法、Realtime PCR和Western Blot等方法检测VEGF、 VEGFR-1、 VEGFR-2、 MMP-9以及ICAM-1等基因表达水平。 3. CCK8法体外检测FD006抗体对角膜上皮细胞毒副作用。流式细胞仪检测FD006对角膜上皮细胞的凋亡影响。正常SD大鼠结膜下注射FD006抗体，观察眼部反应情况，通过裂隙灯显微镜检查、病理切片等，分析结膜和角膜病理改变。建立SD大鼠角膜上皮缺损模型，结膜下注射FD006抗体，观察角膜上皮愈合情况。 4.取8～12周龄健康成年新西兰白兔20只(40眼)，结膜下注射FD006，分别于给药后第5、7、14、21和28天取房水、玻璃体以及外周血，ELISA法检测各样本内的药物浓度，并使用WinNonlin药代动力学软件计算主要的药代动力学参数。结果： 1.FD006与VEGF结合能力优于贝伐单抗。实验条件下，FD006结合VEGF的EC50约为0.037μg/mL，贝伐单抗与VEGF结合的EC50为0.18μg/mL；结合动力学分析表明，FD006结合VEGF的亲和力是贝伐单抗的2倍，两者主要差别在于FD006解离速率慢于贝伐单抗。FD006抗体能显著抑制VEGF诱导的HUVEC的增殖，其IC50值0.031±0.0064μg/mL，优于贝伐单抗（0.047±0.0081μg/mL）。 2.结膜下注射地塞米松、贝伐单抗、FD006和对照组相比均能有效抑制CoNV的生长（P0.01）。溶剂组和0.9%NaCl组CoNV面积和长度没有显著差异（P0.05）。FD006治疗组和对照组相比能够有效降低角膜组织中VEGF,VEGFR-1, VEGFR-2, MMP-9and ICAM-1的表达。在治疗早期，FD006治疗组的CoNV长度和面积较贝伐单抗治疗组小(P 0.05)；与地塞米松相当(P0.05)；在治疗后期，FD006组和贝伐单抗组的CoNV长度和面积差异无统计学意义（P0.05）。 3. FD006不影响角膜上皮细胞增殖，，不促进角膜上皮细胞的凋亡。结膜下注射后，大鼠角结膜及眼部其他各组织形态正常，组织学检查显示结构正常，未见炎症细胞浸润。结膜下注射FD006不影响角膜上皮愈合。 4.结膜下注射FD006后，在血清和未注射的对侧眼的房水玻璃体内均检测到FD006。在各时间点，注射眼房水、玻璃体中的浓度都相应比未注射的对侧眼房水、玻璃体浓度更高。注射眼中玻璃体腔的FD006浓度高于房水。FD006结膜下注射后在注射眼的房水中半衰期约为10.7天，玻璃体内半衰期约为6.8天。结论： FD006抗体对能明显抑制CoNV，并可减轻碱烧伤引起的炎症反应。在治疗早期，FD006抗体的抗新生血管作用要优于贝伐单抗。FD006眼局部应用安全性较好，不影响角膜上皮细胞正常功能。FD006结膜下注射后可进入前房和玻璃体，并且可以通过血液循环，进入对侧眼。
[Abstract]:Objective: To evaluate the biological activity of FD006 in vitro. Observe the effect of FD006 on the proliferation of corneal neovascularization (corneal neovascularization (CoNV)) induced by alkali burn and preliminarily explore the mechanism of FD006 inhibiting the growth of CoNV in rats, evaluate the safety of FD006 in the eye application and preliminarily explore the pharmacokinetics of FD006 in the eye.
Materials and methods:
1. with the help of molecular simulation and dynamic docking, the interaction model of FD006 and VEGF was studied. Protein protein interaction and the binding ability of FD006 and VEGF by ELISA to detect the effect of FD006 on the proliferation of HUVEC induced by VEGF.
2. the CoNV animal model of SD rats was induced by alkali burn. First days after alkali burn, 5 groups were randomly divided into 5 groups: 0.9%NaCl, solvent, dexamethasone group, bevacizumab and FD006 antibody. Each group was given the corresponding treatment with subconjunctival injection; the growth of CoNV and the degeneration of corneal tissue were observed and the anterior segment was photographed. The length and surface of CoNV were analyzed. The animals were killed at 3,7,14,21 and 28 days after alkali burn. The expression levels of VEGF, VEGFR-1, VEGFR-2, MMP-9 and ICAM-1 were detected by immunohistochemistry, Realtime PCR and Western Blot.
3. CCK8 method was used to detect the side effects of FD006 antibody on corneal epithelial cells in vitro. Flow cytometry was used to detect the effect of FD006 on the apoptosis of corneal epithelial cells. FD006 antibody was injected under conjunctiva of normal SD rats, the eye reaction was observed, the slit lamp microscope examination, pathological section and so on were used to analyze the conjunctiva and pathological changes of cornea. A SD rat cornea was established. FD006 antibody was injected under conjunctiva to observe the healing of corneal epithelium.
4. 8~12 weeks old healthy adult New Zealand white rabbits were taken (40 eyes), and FD006 was injected under conjunctiva. The aqueous humor, vitreous body and peripheral blood were taken on the first 5,7,14,21 and 28 days after the administration. The drug concentration in each sample was detected by ELISA method, and the pharmacokinetic parameters were calculated by the software of WinNonlin pharmacokinetics.
Result:
The combination of 1.FD006 and VEGF is superior to bevacizumab. Under experimental conditions, the EC50 of FD006 combined with VEGF is about 0.037 u g/mL, and the EC50 of bevacizumab and VEGF is 0.18 u g/mL, and the binding kinetics analysis shows that the affinity of FD006 to VEGF is 2 times that of bevacizumab, and the main difference is that the dissociation rate of FD006 is slower than the bevacizumab antibody. It can significantly inhibit the proliferation of HUVEC induced by VEGF, and its IC50 value is 0.031 + 0.0064 g/mL, which is better than bevacizumab (0.047 + 0.0081 g/mL).
2. subconjunctival injection of dexamethasone, bevacizumab, FD006 and the control group can effectively inhibit the growth of CoNV (P0.01). There is no significant difference in the area and length of CoNV between the solvent group and the 0.9%NaCl group (P0.05) the.FD006 treatment group and the control group can effectively reduce the expression of VEGF, VEGFR-1, VEGFR-2, MMP-9and ICAM-1 in the corneal tissue. The length and area of CoNV in the FD006 treatment group were smaller than that of the bevacizumab group (P 0.05), which was similar to that of dexamethasone (P0.05), and there was no significant difference in the length and area of CoNV between the FD006 group and the bevacizumab group (P0.05) in the later period of the treatment.
3. FD006 did not affect the proliferation of corneal epithelial cells and did not promote the apoptosis of corneal epithelial cells. After subconjunctival injection, the cornea conjunctiva and other tissues of the eyes were normal. Histological examination showed that the structure was normal and no inflammatory cells were infiltrated. Subconjunctival injection of FD006 did not affect the healing of the corneal skin.
After subconjunctival injection of FD006, FD006. was detected at all time points in the serum and uninjected contralateral aqueous humor glass. The concentration of the vitreous body in the vitreous body was higher than that in the uninjected contralateral eye. The concentration of the vitreous body in the vitreous body was higher than that in the uninjected eye. The concentration of FD006 in the vitreous cavity in the injection eyes was higher than that of the.FD006 conjunctiva injected into the injection eye. The half-life of the aqueous humor is about 10.7 days, and the half-life of the vitreous body is about 6.8 days.
Conclusion: FD006 antibody can obviously inhibit CoNV and reduce the inflammatory reaction caused by alkali burn. In the early stage of treatment, the anti neovascularization of FD006 antibody is better than the local application of bevacizumab.FD006 eye. It does not affect the normal function of corneal epithelial cells into the anterior chamber and vitreous body after the subconjunctival injection of.FD006. Through the blood circulation, enter the opposite eye.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R779.1

【参考文献】