FSHR在肿瘤血管形成中作用的初步研究

发布时间：2018-09-19 10:57

【摘要】：卵泡刺激素受体(follicle-stimulating hormone receptor,FSHR)是一种跨膜糖蛋白,属于G蛋白偶联受体家族成员。在成年人和动物中,FSHR仅在睾丸支持细胞(testicular sertoli cells)和卵巢粒层细胞(ovarian granulosa cells)表面表达,在睾丸和卵巢血管内皮细胞低表达。促性腺激素卵泡刺激素(FSH)通过与睾丸支持细胞和卵巢粒层细胞表面的FSHR结合,在性腺发育过程中发挥重要作用。研究发现FSHR可能在肿瘤血管形成中发挥作用。本研究采用人脐静脉内皮细胞(Human Umbilical Vein Endothelial Cells,HUVEC)作为血管形成模型,分析FSH对HUVEC细胞增殖、迁移及成管作用的影响。另外,采用本研究组前期获的一株抗FSHR纳米抗体VHHFSHR-06与FSH拮抗剂苏拉明,研究阻断FSH-FSHR的结合对HUVEC细胞的增殖、迁移及成管作用的影响,为明确FSHR的作用和今后开展基于FSHR的抗血管治疗提供新的靶点。本研究包括两个方面:1、FSHR在肿瘤血管形成中的作用初探利用体外血管形成模型HUVEC细胞探讨FSH-FSHR在血管形成中的作用。采用RT-PCR、western blotting、细胞免疫化学和成管实验检测HUVEC细胞中FSHR的表达,结果表明,FSHR在HUVEC和卵巢腺癌细胞(Caov-3)均有表达,而在卵巢癌细胞(Skov-3)中不表达。为了更进一步分析FSHR在血管形成中的作用,以FSH(600 ng/m L)刺激HUVEC细胞,随后分别加入差异浓度的FSH阻断剂苏拉明[100,1000μg/mL],检测对细胞增殖、迁移及成管的影响。结果表明,FSH(600 ng/mL)作用于HUVEC细胞明显促进细胞的迁移,而不同浓度的苏拉明对由FSH刺激的迁移有抑制作用,呈剂量依赖性。FSH(600ng/mL)还能促成HUVEC细胞的增殖(P0.01),而苏拉明能够抑制这种FSH引起的细胞增殖,100μg/mL的苏拉明对细胞的增殖有明显抑制作用(P0.01),1000μg/mL苏拉明对细胞的抑制作用极显著(P0.001)。此外,苏拉明还能抑制由FSH刺激引起的HUVEC细胞成管现象。综上可以说明,FSH能够促进表达FSHR的HUVEC细胞的迁移、增殖及成管作用,苏拉明对FSHR阳性的HUVEC和Caov-3细胞的迁移和增殖具有一定抑制作用,但对FSHR阴性的CT-26细胞没有作用。初步提示FSH可能通过作用于其受体FSHR发挥以上作用。2、抗FSHR纳米抗体VHH-06亲和力成熟采用两种方法对前期从天然骆驼非免疫文库中筛选得到的抗FSHR纳米抗VHHFSHR-06的亲和力进行改进,以更好的利用该抗体开展FSH-FSHR的阻断实验和评价其抗血管形成效应。(1)采用定点饱和突变方式在VHH-06核酸CDR3序列处,进行除终止密码子外的氨基酸随机突变,形成由15个长度随机突变氨基酸组成的CDR3突变文库,连接到噬菌体载体pMECS中,电转化TG1感受态细胞,获得库容大小为7.36×108的突变文库(NNY)NL-06/ΔCDR3。采用FSHR234蛋白对VHH06突变库(NNY)NL-06/ΔCDR3进行6轮的反复亲和筛选,从输出噬菌体数量来看,克隆得到了富集。采用多克隆phage ELISA测定也表明,从第四轮开始,抗原特异的噬菌体得到了积累,但在随后对第六轮的100个克隆进行的单克隆phage ELISA检测中没有得到具有明显结合的克隆。原因可能是该突变文库的功能性多样性不足,文库质量存在问题。(2)将VHH-06与COMP48偶联来尝试提高纳米抗体与FSHR结合的能力。COMP48是源自人软骨寡聚基质蛋白(Cartilage oligomeric matrix protein)的一段具有螺旋卷曲结构的48肽。VHH-06羧基端与COMP48偶联,转化至BL21进行了表达和纯化,得到非还原条件下110 kD,还原条件下25kD大小的目的蛋白,但在随后的ELISA检测中,并没有显著提高与FSHR结合能力。本研究通过两种策略以期达到提高纳米抗体亲和力的效果,但并未取得预期目的。可能因为蛋白的不正确折叠或者是人工构建的突变库中有功能的序列太少等原因。细胞实验结果提示FSH可能通过作用于其受体FSHR对HUVEC细胞增殖、成管、迁移发挥作用。为FSHR作为新的抗肿瘤靶点提供支持,为抗血管形成治疗提供新思路、新依据。
[Abstract]:The follicle-stimulating hormone receptor (FSHR) is a transmembrane glycoprotein that is a member of the G protein-coupled receptor family. In adults and animals, FSHR is expressed only on the surface of testicular Sertoli cells and ovarian granulosa cells, and in the endothelial cells of testis and ovary. In this study, human Umbilical Vein Endothelial Cells (HU) were used to study the role of gonadotrophin follicle stimulating hormone (FSH) in gonadal development. VEC) was used as a model of angiogenesis to analyze the effects of FSH on proliferation, migration and tubulation of HUVEC cells. In addition, the effects of blocking the binding of FSH-FSHR on proliferation, migration and tubulation of HUVEC cells were studied by using VHHFSHR-06, an anti-FSHR nano-antibody, and suramin, an antagonist of FSH. This study included two aspects: 1. The role of FSHR in tumor angiogenesis was explored by using in vitro angiogenesis model HUVEC cells to explore the role of FSH-FSHR in angiogenesis. The FSHR in HUVEC cells was detected by RT-PCR, Western blotting, cytoimmunochemistry and vascular test. The results showed that FSHR was expressed in both HUVEC and ovarian adenocarcinoma cells (Caov-3), but not in ovarian cancer cells (Skov-3). The results showed that FSH (600 ng/mL) significantly promoted the migration of HUVEC cells, while different concentrations of sulamine inhibited the migration stimulated by FSH in a dose-dependent manner. FSH (600 ng/mL) also promoted the proliferation of HUVEC cells (P 0.01), and sulamine inhibited the proliferation of HUVEC cells induced by FSH. In addition, suramin inhibited the tube formation of HUVEC cells stimulated by FSH. In conclusion, FSH could promote the migration, proliferation and tubulation of HUVEC cells expressing FSHR. Lamine inhibited the migration and proliferation of FSHR-positive HUVEC and Caov-3 cells, but had no effect on FSHR-negative CT-26 cells. It was preliminarily suggested that FSH might play the above role by acting on its receptor FSHR. 2. The affinity maturation of anti-FSHR nano-antibody VHH-06 from natural camel non-immune library was achieved by two methods. The affinity of the selected anti-FSHR nano-anti-VHHFSHR-06 was improved in order to make better use of the antibody to carry out the blocking test of FSH-FSHR and evaluate its anti-angiogenesis effect. (1) Site-directed saturation mutation was used to carry out random amino acid mutation at the CDR3 sequence of VHH-06 nucleic acid except termination codon, resulting in 15 random processes of length. CDR3 mutant library consisting of mutant amino acids was linked to the phage vector pMECS, and transformed into TG1 competent cells by electroporation. The mutant library (NNNY) NL-06/CDR3 with a capacity of 7.36*108 was obtained. The VH06 mutant library (NNNNY) NL-06/CDR3 was screened by using FSHR234 protein for six rounds of repeated affinity screening. Polyclonal phage ELISA assay also showed that antigen-specific phages were accumulated from the fourth round, but no clones were found to bind to each other in the subsequent phage ELISA assay of 100 clones from the sixth round. The reason may be due to the lack of functional diversity of the mutant library and the quality of the library. (2) Coupling VHH-06 with COMP48 is an attempt to improve the binding ability of nano-antibodies to FSHR. COMP48 is a 48-peptide with helical convolution structure derived from human cartilage oligomeric matrix protein. The carboxyl end of VHH-06 is coupled with COMP48 and transformed into BL21 for expression and purification under non-reduced conditions, 110. KD, the target protein of 25 kD size under reduction condition, did not significantly improve the binding ability to FSHR in subsequent ELISA detection. Two strategies were used to improve the affinity of antibodies, but the expected results were not achieved. These results suggest that FSH may play an important role in the proliferation, tube formation and migration of HUVEC cells by acting on its receptor FSHR.
【学位授予单位】：新疆大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R730.2

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