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1、 原创性声明 本人声明， 所呈交的学位论文是本人在导师指导下进行的研究工作及取得的研究成果。尽我所知，除了论文中特别加以标注和致谢的地方外，论文中不包含其他人已经发表或撰写过的研究成果，也不包含为获得南华大学或其他单位的学位或证书而使用过的材料。 与我共同工作的同志对本研究所作的贡献均已在论文中作了明确的说明。 作者签名： 日期： 年 月 日 关于学位论文使用授权说明 本人同意南华大学有关保留、使用学位论文的规定，即：学校有权保留学位论文，允许学位论文被查阅和借阅；学校可以公布学位论文的全部或部分内容， 可以采用复印、 缩印或其它手段保留学位论文；学校可根据国家或湖南省有关部门规定送交学位论文
2、。 作者签名： 导师签名： 日期： 年 月 日3 中 文 摘 要 苦参碱对体外培养的大鼠视网膜微血管内皮细胞的增殖及 VEGF表达的影响苦参碱对体外培养的大鼠视网膜微血管内皮细胞的增殖及 VEGF表达的影响 硕士研究生： 蒋瑶祁 导 师： 彭辉灿 教授 目的：研究苦参碱（Matrine, Mat）对体外培养的大鼠视网膜微血管内皮细胞（rat retinal microvascular endothelial cells，RRMECs）的增殖及 VEGF表达的影响， 明确其可否抑制视网膜微血管内皮细胞的增殖及下调 VEGF 的表达，为视网膜新生血管的药物防治提供新的思路和理论依据。 方法：原代培
3、养 RRMECs，取第三代细胞用于实验。MTT 比色法检测不同剂量 Mat作用不同时间对 RRMECs 增殖的抑制作用；台盼蓝染色细胞计数法测定其对RRMECs 生长曲线的影响；AO/EB 荧光双染色法观察细胞凋亡形态学的改变。将RRMECs 按如下分组处理： （1）阴性对照组：用含 20%血清 DMEM 培养; （2）阳性对照组：含 20%血清 DMEM+血管抑素 130mg/L（3）Mat 组：含 20%血清 DMEM+Mat 40mg/L;（4）高糖组：含 20%血清高糖 DMEM（葡萄糖浓度 25mmol/L）;（5）Mat+高糖组。免疫细胞化学和 Western-Blotting 检
4、测不同分组处理 48 小时后细胞VEGF 的表达。 结果：1、MTT 比色法测定结果显示:用 5、10、20、40、80、160mg/L 的 Mat 处理 RRMECs，作用 24h 细胞增殖抑制率分别为 1.421.03%、11.501.98%、24.982.38%、40.272.49%、53.542.39%、63.883.12%;作用 48h 细胞增殖抑制率分别为 4.161.14%、14.401.78%、33.281.83%、53.611.32%、63.531.96%、73.791.12%;作用 72h 细胞增殖抑制率分别为 9.832.13%、22.174.29%、42.651.05%
5、、63.171.38%、74.352.60%、87.244.59%。在 5mg/L 以上各试验组与对照组相比均具有显著性差异（P0.05)，各试验组之间两两比较也具有显著性差异 （P0.05)。 随着浓度的增高， 作用时间的增加， Mat抑制细胞增殖的作用越明显，当浓度达到 40mg/L，作用 48 小时，细胞抑制率超4 过了 50%。细胞计数绘制生长曲线结果与 MTT 结果相一致。2、 AO/EB 荧光双染色法观察:在 Mat 浓度达到 40mg/L 后处理 RRMECs 呈现不同程度的细胞凋亡形态学特征。3、免疫细胞化学结果显示：VEGF 表达定位于胞膜和胞浆,阳性部分为棕黄色颗粒。血管抑
6、素（130mg/L）和 Mat(40mg/L)处理后其阳性表达明显降低，灰度值越少（P0.05）且两者作用无明显差别；高糖组的 VEGF 阳性表达最高，灰度值也最大，Mat(40mg/L)同样能下调其表达（P0.05）。4、Western-Blotting半定量检测 VEGF 蛋白的表达与免疫细胞化学的结果一致。 结论：1、 Mat 能有效地抑制体外培养的 RRMECs 的增殖，在一定的浓度和时间范围内其抑制作用呈明显的剂量和时间依赖性；2、Mat 能有效地诱导 RRMECs凋亡；3、通过免疫细胞化学和 Western-Blotting 证实其抑制主要是通过下调VEGF 表达而发生作用的，高糖
7、能上调 VEGF 的表达，Mat 对高糖条件下的 VEGF表达同样也有抑制作用， 提示 Mat 对糖尿病视网膜病变等引起的视网膜新生血管防治具有一定的应用前景。 关键词：苦参碱；视网膜微血管内皮细胞；血管内皮生长因子；视网膜新生血管；免疫印迹方法；免疫细胞化学 5 The effects of matrine on cell proliferation and expression of intercellular VEGF in microvascular endothelial cells from rat retina by cultured in vitro Abstract Obje
8、ctive ：To evaluate the effects of matrine on cell proliferation and expression of intercellular VEGF in RRMECs in vitro and to provide a new clue and theory basis for developing a drug of preventing and curing retinal neovascularization in the future. Methods ： RRMECs were primary cultured, the thir
9、d generation of RRMECs were treated. The effect of Mat on the proliferation of RRMECs was measured by MTT assay. The influence of Mat on the growth of RRMECs was determined by cell counting method in trypan blue staining. AO/EB double fluorescence staining method was used to observe the morphologica
10、l changes of RRMECs apoptosis after treatment with Mat. RRMECs were divided into 5 groups: the control group, angiostatin (130mg/L) group, Mat (40mg/L) group, high glucose (25mmol/L)group, and Mat(40mg/L)+high glucose(25mmol/L) group. After 48 hour, the localization of VEGF protein was examined by i
11、mmunocytochemistry. Western-blotting was used to measure the protein level of VEGF. Results ：1.We observed the inhibitory effects of Mat on RRMECs through MTT assay, which were exposed to 5mg/L,10mg/L,20mg/L,40mg/L,80mg/L and 160mg/L concentrations for 24, 48, 72 hours respectively. The inhibitory r
12、ate were 1.421.03%,11.501.98%,24.982.38%,40.272.49%,53.542.39%,63.883.12% after they were incubated with Mat for 24 hours, they are 4.161.14%, 14.401.78%, 33.281.83%,53.611.32%,63.531.96%, 73.791.12% for 48 hours and 9.832.13%, 22.174.29%, 42.651.05%, 63.171.38%, 74.352.60%, 87.244.59%.for 72 hours.
13、 When Mat concentrations exceed 5mg/L, there were significant differences between experimental groups in which the RRMECs were disposed with different concentrations and incubation time of Mat and control group disposed with DMEM 6 (P0.05), as well as among experimental groups (P0.05). The inhibitor
14、y rates of RRMECs increased with both the raise of the Mat concentrations and the prolongation of time. These results were consistent with the results of cell counting method drawing growth curve in RRMECs. 2. AO/EB double fluorescence staining method indicated that we could observe typical apoptoti
15、c cells with fluorescence microscope when Mat whose concentration exceed 5mg/L. 3. Immunohistochemical techniques indicated that the expression of VEGF is in cell membranes and cytoplasm. Compared with the control group, An and Mat decreased the expression of VEGF (P0.05); Compared with the control
16、group, high glucose increased the expression of VEGF (P0.05). Mat could decrease the expression as well (P0.05). These results were consistent with the results of Western-blotting. Conclusions ：1. Mat can effectively inhibit the proliferation of RRMECs, the inhibitory rates are increased in both time-dependent and dose-dependent ways by Mat in vitro. 2. Mat can effectively induce
