【作者】 傅刘鹏；

【导师】 蔡绍晖；

【作者基本信息】 暨南大学 ， 药理学， 2010， 硕士

【摘要】 研究目的：本研究拟利用叶酸脂质体包封新型重组融合蛋白：TAT/54R/KDEL,以期既能有效增强TAT/54R/KDEL在体内的稳定性,还有助于TAT/54R/KDEL在肿瘤组织内靶向富集,促进其发挥靶向抗肿瘤效应。研究方法：1.将课题组前期构建的重组质粒TAT/54R/KDEL/pTAT-HA转化大肠菌株BL21(DE3),并通过IPTG诱导表达,镍柱亲和层析和HPLC纯化目的蛋白。纯化后的蛋白采用稀释、透析和超滤等方法进行复性。用流式细胞术考察TAT/54R/KDEL对高表达CXCR4的MOLT-4细胞的体外表型敲除活性。2.采用薄膜分散法结合冷冻超声法制备普通脂质体,并与叶酸臂共同孵育制备叶酸脂质体。通过测定叶酸脂质体对钙黄绿素的包封率和渗漏率,考察叶酸脂质体的稳定性。3.将表达CXCR4的小鼠乳腺癌4T1细胞,原位接种于BALB/c小鼠第2对乳房脂肪垫以建立小鼠乳腺癌移植模型,并利用该动物模型,考察所制备的包封钙黄绿素叶酸脂质体的体内靶向性。4.以叶酸脂质体对TAT/54R/KDEL的包封率为指标,考察磷脂和胆固醇用量、TAT/54R/KDEL用量、水相温度及超声时间对叶酸脂质体包封率的影响,利用正交设计方法对叶酸脂质体的制备工艺进行优化。5.利用4T1细胞构建的乳腺癌移植动物模型,考察所制备的包封TAT/54R/KDEL的叶酸脂质体在体内对乳腺癌细胞生长和转移的抑制效应。实验结果：1. SDS-PAGE电泳证实,TAT/54R/KDEL表达成功。采用镍柱亲和层析和HPLC纯化后TAT/54R/KDEL的纯度达到95%以上,其产率大约5 mg/L培养基。流式细胞术结果表明,TAT/54R/KDEL对MOLT-4细胞表面CXCR4具有较好的表型敲除活性,并且表型敲除活性大小与其浓度成正比。2.荧光显微镜和原子力显微镜观察结果证实,制备的叶酸脂质体分布均匀,大小一致,所包封钙黄绿素的荧光强度一致。叶酸脂质体对钙黄绿素的包封率为41.2%,而且叶酸脂质体在48h内是比较稳定的。3.病理学结果证实小鼠乳腺癌移植模型构建成功。荧光显微镜和荧光分光光度计结果显示,叶酸脂质体富集于原发性和转移性乳腺癌细胞的数量明显高于脂质体,表明叶酸脂质体具有肿瘤靶向性。4.正交设计方法表明,制备叶酸脂质体包封TAT/54R/KDEL的最佳实验方案为磷脂与胆固醇用量比例为3：1,磷脂与TAT/54R/KDEL用量比例为20：1,振荡水化时温度为4℃,超声时间为5min。叶酸脂质体对TAT/54R/KDEL的包封率为46.1%,而且叶酸脂质体在48h内是比较稳定的。5.乳腺癌动物移植模型证实,叶酸脂质体包封的TAT/54R/KDEL对乳腺癌细胞的生长和转移具有明显抑制效应。结论：1.成功表达和纯化出足量具有生物学活性的TAT/54R/KDEL,体外实验证实TAT/54R/KDEL对MOLT-4细胞膜表面的CXCR4具有表型敲除活性。2.成功制备包封钙黄绿素的叶酸脂质体,利用小鼠乳腺癌移植模型证实了叶酸脂质体对肿瘤的靶向性。3.初步证实,利用叶酸脂质体包封策略,能够增强TAT/54R/KDEL的体内稳定性,并能促进TAT/54R/KDEL对肿瘤组织的靶向作用,从而提高其对肿瘤细胞生长和转移的抑制效应。更多还原

【Abstract】 ObjectiveIn this study, TAT/54R/KDEL, a novel recombinant protein, was enveloped by using folate liposomes, in order to effectively enhance the in vivo stability and targeting effect of anti-tumor.Methods1. The recombinant plasmid of TAT/54R/KDEL/pTAT-HA previously constructed was transformed into colorectal strain BL21 (DE3) and its expression was induced by IPTG. Then, the protein was purified through nickel affinity chromatography and HPLC. The purified protein needs deliquation, dislysis and ultrafiltration for renaturation. The phenotypic knockout activity of TAT/54R/KDEL in highly CXCR4-expressed cell (MOLT-4) was investigated by flow cytometry.2. Liposome was prepared by thin-film dispersion method and frozen ultrasound method, and incubated with FA’s arm to develop folate liposome. The envelopment and leakage rate of folate liposome to calcein were determined to evaluate the stability of folate liposome.3. The 4T1 mouse breast cancer cells was transplanted into BALB/c mouse vie the second breast fat pad to establish breast cancer model mouse where CXCR4 is highly expressed. Subsequently, the tumor targeting of wrapped calcein folate liposomes was investigated in this tumor model.4. Setting the envelopment rate of folate liposome to TAT/54R/KDEL as an index, the factors including the amount of phospholipid and cholesterol, the quantity of TAT/54R/KDEL, aqueous temperature and ultrasonic time were respectively investigated to know their effects on the envelopment rate of folate liposome to TAT/54R/KDEL. The systhesis process of folate liposomes was optimized through orthogonal design method.5. The activities of wrapped TAT/54R/KDEL folate liposomes on tumor growth as well as metastasis were evaluated through the 4T1 tumor-bearing model.Results1. SDS-PAGE electrophoresis showed that TAT/54R/KDEL was expressed successfully. After purification with nickel affinity chromatography and HPLC, the purity of TAT/54R/KDEL was more than 95%, with the yield rate of-5 mg/L in the medium. The results of flow cytometry demonstrated that, TAT/54R/KDEL has good knockout phenotype activity on the surface of CXCR4 MOLT-4 cells with concentration-depended activity.2. As the fluorescence microscope and atomic force microscopy showed, the prepared liposomes had uniform folate distribution and size, fluorescence intensity of the enveloped calcein was almost the same. the envelopment rate of folate liposomes to Calcein was 41.2%, and folate liposomes exhited relatively stablity within 48h.3. Pathological study confirmed that breast cancer mouse transplantation model was successfully constructed. Fluorescence microscopy and fluorescence spectrophotometer revealed that the accumulation of folate liposomes enriched in primary and metastatic breast cancer cells was significantly higher than that of liposomes, indicating that folate liposomes possessed favorable tumor targeting.4. Orthogonal design demonstrated that the most optimized experimental scheme for wrapped TAT/54R/KDEL folate liposomes was showed as followed:at a ratio of 3:1 (phospholipid: cholesterol), at the ratio of 20:1 (phospholipid:TAT/54R/KDEL), the temperature oscillation hydration at 4℃,5 min of ultrasond. Folate liposome encapsulation of TAT/54R/KDEL was at the radio of 46.1%, and folate liposomes was relatively stable within 48h.5. In vivo study indicated that the TAT/54R/KDEL enveloped by folate liposomes produced potent antitumor effect on breast cancer cell growth and also inhibited the metastasis.Conclusion1. TAT/54R/KDEL displayed potent phenotype-knockout activity on CXCR4 within the surface of MOLT-4 cell membrane in vitro.2. Folate liposomes exhibited favorable tumor targeting effect in the 4T1 tumor-bearing model mouse.3. TAT/54R/KDEL enveloped by Folate liposomes demonstrated enhanced tumor targeting, and produced potent inhibitory effect on tumor cell growth and metastasis in vivo.更多还原