靶向卵巢癌的纳米脂质微泡造影剂的制备及体外寻靶研究

发布时间：2018-11-03 14:43

【摘要】：目的：制备一种靶向卵巢癌的新型纳米脂质微泡造影剂，鉴定理化特性及其对卵巢癌细胞的靶向性，为实现肿瘤血管外显影研究提供了一个前期基础。方法： 1.将一定比例的二棕榈酰磷脂酰胆碱(DPPC)、生物素化二棕榈酰磷脂酰乙醇胺(DSPE-PEG2000-Biotin)及甘油、PBS等混合均匀，采用冷冻干燥法、机械振荡法制备非靶向脂质微泡造影剂。 2.光镜下观察微泡形态分散度、均一性，Zeta检测仪检测粒径大小范围。 3.将一定比例的二棕榈酰磷脂酰胆碱(DPPC)、生物素化二棕榈酰磷脂酰乙醇胺(DSPE-PEG2000-Biotin)及甘油、PBS等混合均匀，采用冷冻干燥法、机械振荡法、生物素-亲和素-生物素桥连法，制备非靶向纳米脂质微泡造影剂，将生物素化的促黄体生成素释放激素（luteinzing hormone-releasing hormone, LHRH）抗体与非靶向纳米脂质微泡造影剂连接，制备出卵巢癌靶向的纳米脂质微泡造影剂。 4.光镜下观察微泡形态分散度、均一性及浓度以及室温下保存时间，Zeta检测仪检测粒径大小范围、表面电位。 5.流式细胞术分别检测非靶向纳米脂质微泡造影剂、靶向纳米脂质微泡造影剂与二抗结合率。 6.光镜下观察靶向纳米脂质微泡造影剂与人卵巢癌OVCAR-3细胞结合情况。 7.预先用生物素化LHRH抗体与人卵巢癌OVCAR-3细胞饱和结合，再次观察靶向纳米脂质微泡造影剂与人卵巢癌OVCAR-3细胞结合情况。结果： 1.非靶向脂质微泡造影剂是一种乳白色混悬液，400倍光镜观察及Zeta检测仪检测机械振荡60s、90s、120s，微泡分布均匀，无团聚，单个微泡的外观圆整，粒径范围粒径范围分别为329～1008nm，295～468nm，369～618nm，157～268nm，400倍光镜及观察Zeta检测仪检测机械振荡150s，微泡分布不均匀，无团聚，单个微泡的外观圆整，粒径范围60～896nm。 2.非靶向纳米级脂质微泡造影剂和靶向纳米脂质微泡造影剂微泡成功制备，两种微泡的外观圆整，分布均匀。Zeta检测仪检测粒径范围分别为295～468nm和369～618nm，集中于360nm与508nm，两者粒径大小比较有统计学差异（P＜0.05 3.光镜下观察，微泡形状分散均匀和室温保存时间长。两种微泡电位均为-14.6mV。室温下保存7d时，，靶向纳米脂质微泡造影剂理化特性与刚制备时无统计学差异（P＞0.05）。 4.二抗与非靶向纳米脂质微泡造影剂、靶向纳米脂质微泡造影剂结合率分别为0.83%、75.6%。 5.光镜下OVCAR-3细胞周围可见靶向纳米脂质微泡造影剂围绕或黏附，呈花环样结构。 6.光镜下生物素化的LHRH抗体预先阻断OVCAR-3细胞不能与靶向纳米脂质微泡造影剂结合。结论： 1机械振荡90s为制作非靶向脂质微泡造影剂最佳时间，即为制备非靶向纳米脂质微泡机械振荡时间。 2.通过冷冻干燥法、机械振荡法和生物素-亲和素桥连法，可成功制备靶向卵巢癌的纳米脂质微泡造影剂此微泡粒径小、稳定性高。 3.靶向纳米脂质微泡造影剂体外能高效靶向人卵巢癌OVCAR-3细胞。 4.被生物素化的LHRH抗体阻断的饱和卵巢癌OVCAR-3细胞不能结合靶向纳米脂质微泡造影剂。 5.靶向纳米脂质微泡造影剂通过LHRH介导与卵巢癌OVCAR-3细胞结合。
[Abstract]:Purpose: preparing a novel nano lipid micro-bubble contrast agent for targeting ovarian cancer, identifying physicochemical properties and targeting of ovarian cancer cells, The foundation. Method: 1. A certain proportion of dipalmitate, choline (DPPC), streptavidin (DSPE-NHS-Bioin) and glycerol, PBS, etc. were mixed evenly, and the non-target was prepared by freeze drying and mechanical oscillation. The dispersion, homogeneity and Zeta of microvesicle were observed under light microscope. The size range of the particle size is detected by a detector. 3. A certain proportion of the dipalmitate, the phospholipid, the choline (DPPC), the Biotin dipalmitate and the ethanolamine (DSPE-NHS-Bioin) and the glycerol, the PBS and the like are uniformly mixed, the freeze drying method and the mechanical oscillation method are adopted, biotin-avidin-biotin bridging method is used to prepare non-targeted nano-lipid micro-bubble contrast agent, and biotin-induced luteinizing hormone releasing hormone (LAMP) antibody is connected with non-targeting nano lipid micro-bubble contrast agent to prepare egg The morphology, homogeneity and concentration of microvesicles and preservation time at room temperature were observed under light microscope. Zet a Detector detects particle size range, surface potential. 5. Flow cytometry respectively detects the non-targeted nano-lipid micro-bubble building Contrast ratio of contrast agent and nano-lipid micro-bubble contrast agent to two anti-binding agents: 6. Under light microscope, it is observed to target nano-lipid. Microbubble contrast agent binding to human ovarian cancer OVCAR-3 cells. 7. Pre-use the biotin-labeled antibody to bind to human ovarian cancer OVCAR-3 cells, and then observe the target again. rice lipoid Results: 1. Non-targeting lipid micro-bubble contrast agent is a milky white suspension liquid, 400 times light microscope observation and Zeta detector detect mechanical oscillation 60s, 90s, 120s, micro bubble distribution is uniform, no agglomeration, the appearance of single micro bubble is round, the particle size range is 329-1008, respectively. nm, 295 ~ 468nm, 369 ~ 618nm, 157 ~ 268nm, 400 times light microscope and observation of Zeta detector to detect mechanical oscillation 150s, micro bubble The distribution is uneven, no agglomeration, the appearance of a single micro bubble is round, the particle size range is 60-896nm. The shadow agent and the target nano lipid micro-bubble contrast agent microvesicle are successfully prepared, the appearance of the two micro bubbles is round, the distribution is uniform, and the detection particle size range of the Zeta detector is 295-468nm and 369-618nm, respectively, It is focused on the size of 360nm and 508nm. The difference in economics (P <0.05). Under light microscope, the micro-bubble shape was dispersed uniformly and the preservation time of room temperature was long. The two micro-bubble potentials were-14. 6mV. 7d, there was no statistical difference between the physical and chemical properties of the targeting Naomi microvesicles (P> 0.05). The combination rate of non-targeted nano-lipid micro-bubble contrast agent and target nano-lipid micro-bubble contrast agent was 0. 83%, 75.6%, respectively. 5. There is a rosette-like structure around OVCAR-3 cells under light microscope. 6 Under light microscope, the avidin preblocking OVCAR-3 cells could not be combined with the targeted nano-lipid micro-bubble contrast agent. Conclusion: 1 mechanical oscillation 90s is the best time for the preparation of non-target lipid micro-bubble contrast agent, that is, to prepare the non-targeting nano-lipid micro-bubble mechanical oscillation time. The targeted ovaries can be successfully prepared by freeze drying, mechanical oscillation and biotin-avidin bridging. The nano-lipid micro-bubble contrast agent of the cancer is small in particle size and high in stability. 3. Targeted nano-lipid micro-bubble contrast agent can target people in vitro Ovarian cancer OVCAR-3 cells. 4. Saturated ovaries blocked by Biotin Antibody
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.31

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