本文選題：血管內皮生長因子 + 單克隆抗體　； 參考：《中國人民解放軍醫(yī)學院》2014年博士論文

【摘要】：目的：評價FD006的體外生物學活性。觀察FD006抑制堿燒傷誘導的大鼠角膜新生血管（corneal neovascularization，CoNV）增殖的情況，初步探索FD006抑制大鼠CoNV生長的作用機制。評價FD006在眼部應用的安全性并初步探討FD006的眼部藥代動力學情況。 材料與方法： 1．借助分子模擬、動態(tài)對接方法研究FD006與VEGF相互作用模式。蛋白-蛋白相互作用和ELISA檢測FD006與VEGF的結合能力。CCK8檢測FD006對VEGF誘導HUVEC增殖的影響。 2.建立堿燒傷誘導SD大鼠CoNV動物模型，堿燒傷術后第1天，隨機分為：0.9%NaCl、溶劑、地塞米松組、貝伐單抗和FD006抗體5組，各組分別給予結膜下注射相應藥物治療；觀察CoNV生長情況及角膜組織變化，并進行眼前節(jié)照相。分析CoNV的長度及面積。堿燒傷術后3、7、14、21和28天處死動物，取角膜組織行免疫組織化學法、Realtime PCR和Western Blot等方法檢測VEGF、 VEGFR-1、 VEGFR-2、 MMP-9以及ICAM-1等基因表達水平。 3. CCK8法體外檢測FD006抗體對角膜上皮細胞毒副作用。流式細胞儀檢測FD006對角膜上皮細胞的凋亡影響。正常SD大鼠結膜下注射FD006抗體，觀察眼部反應情況，通過裂隙燈顯微鏡檢查、病理切片等，分析結膜和角膜病理改變。建立SD大鼠角膜上皮缺損模型，結膜下注射FD006抗體，觀察角膜上皮愈合情況。 4.取8～12周齡健康成年新西蘭白兔20只(40眼)，結膜下注射FD006，分別于給藥后第5、7、14、21和28天取房水、玻璃體以及外周血，ELISA法檢測各樣本內的藥物濃度，并使用WinNonlin藥代動力學軟件計算主要的藥代動力學參數(shù)。 結果： 1.FD006與VEGF結合能力優(yōu)于貝伐單抗。實驗條件下，F(xiàn)D006結合VEGF的EC50約為0.037μg/mL，貝伐單抗與VEGF結合的EC50為0.18μg/mL；結合動力學分析表明，F(xiàn)D006結合VEGF的親和力是貝伐單抗的2倍，兩者主要差別在于FD006解離速率慢于貝伐單抗。FD006抗體能顯著抑制VEGF誘導的HUVEC的增殖，其IC50值0.031±0.0064μg/mL，優(yōu)于貝伐單抗（0.047±0.0081μg/mL）。 2.結膜下注射地塞米松、貝伐單抗、FD006和對照組相比均能有效抑制CoNV的生長（P0.01）。溶劑組和0.9%NaCl組CoNV面積和長度沒有顯著差異（P0.05）。FD006治療組和對照組相比能夠有效降低角膜組織中VEGF,VEGFR-1, VEGFR-2, MMP-9and ICAM-1的表達。在治療早期，F(xiàn)D006治療組的CoNV長度和面積較貝伐單抗治療組小(P 0.05)；與地塞米松相當(P0.05)；在治療后期，F(xiàn)D006組和貝伐單抗組的CoNV長度和面積差異無統(tǒng)計學意義（P0.05）。 3. FD006不影響角膜上皮細胞增殖，，不促進角膜上皮細胞的凋亡。結膜下注射后，大鼠角結膜及眼部其他各組織形態(tài)正常，組織學檢查顯示結構正常，未見炎癥細胞浸潤。結膜下注射FD006不影響角膜上皮愈合。 4.結膜下注射FD006后，在血清和未注射的對側眼的房水玻璃體內均檢測到FD006。在各時間點，注射眼房水、玻璃體中的濃度都相應比未注射的對側眼房水、玻璃體濃度更高。注射眼中玻璃體腔的FD006濃度高于房水。FD006結膜下注射后在注射眼的房水中半衰期約為10.7天，玻璃體內半衰期約為6.8天。 結論： FD006抗體對能明顯抑制CoNV，并可減輕堿燒傷引起的炎癥反應。在治療早期，F(xiàn)D006抗體的抗新生血管作用要優(yōu)于貝伐單抗。FD006眼局部應用安全性較好，不影響角膜上皮細胞正常功能。FD006結膜下注射后可進入前房和玻璃體，并且可以通過血液循環(huán)，進入對側眼。
[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.
【學位授予單位】：中國人民解放軍醫(yī)學院
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R779.1

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