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目的 明确人肿瘤转移抑制基因TMSG- 1蛋白分子量大小和细胞定位,制备和鉴定TMSG- 1单克隆抗体,并探讨其在临床肿瘤标本检测的价值。方法 采用多肽固相合成法 (Fmoc法 )合成 15肽TMSG -1抗原片段,并与匙孔槭血蓝蛋白 (mcKLH)偶联,免疫BALB/C小鼠,采用细胞融合、ELISA法检测和快速有限稀释法筛选和制备单克隆抗体,并应用Western印迹和免疫组织化学ABC法染色进行抗体鉴定和肿瘤检测。结果 筛选出抗TMSG- 1抗体的单克隆杂交瘤细胞系C8,免疫球蛋白亚类测定为IgM。半抗原竞争抑制实验证实该抗体是特异的抗TMSG- 1抗体。Western印迹显示不转移癌细胞亚系 2B4和LH7有明显的相对分子质量为 45 .000蛋白条带,而高转移亚系 1E8和BE1则条带很弱。细胞免疫组织化学染色显示 2B4和LH7强阳性,为细胞膜和细胞质着色;而 1E8和BE1则为阴性。石蜡切片ABC法检测 52例乳腺癌和 41例结肠癌组织中TMSG- 1蛋白表达发现:未转移的癌组织多为中度阳性或强阳性,中度以上阳性率分别为 36% (乳腺癌 )和 52. 4% (结肠癌 );而转移的癌组织多为弱阳性或阴性, 中度以上阳性率分别为 7 .4% (乳腺癌 )和 35% (结肠癌 ),统计学分析表明TMSG 1在未转移癌组织中的表达显著高于转移性癌组织 (P<0 .05)。结论 成功制备了抗人肿瘤转移抑制基?
OBJECTIVE: To determine the size and location of TMSG-1 protein in human tumor metastasis suppressor gene TMSG-1 and to evaluate the value of TMSG-1 monoclonal antibody in the detection of clinical tumor samples. Methods Fifteen TMSG-1 antigen fragments were synthesized by Fmoc method and conjugated to keyhole limpet hemocyanin (mcKLH). BALB / C mice were immunized with the method of cell fusion, ELISA and rapid ELISA The monoclonal antibodies were screened and prepared by limiting dilution method. Antibody identification and tumor detection were performed by Western blotting and immunohistochemical ABC staining. Results The monoclonal hybridoma cell line C8 against TMSG-1 antibody was screened and the immunoglobulin subclass was determined to be IgM. Hapten competition inhibition assay confirmed that the antibody is a specific anti-TMSG-1 antibody. Western blotting showed that the non-metastatic cancer cell lines 2B4 and LH7 had a significant relative molecular mass of 45,000 protein bands whereas the highly metastatic sub-lines 1E8 and BE1 had weak bands. Immunohistochemical staining of 2B4 and LH7 showed strong positive staining for cell membrane and cytoplasm, while negative for 1E8 and BE1. Paraffin section ABC method detected 52 cases of breast cancer and 41 cases of colon cancer tissue TMSG-1 protein expression found that: non-metastatic cancer were more moderate or strong positive, moderate positive rates were 36% (breast cancer) And 52.4% (colon cancer). However, most of the metastatic cancerous tissues were weakly positive or negative, and the positive rates were 7.4% (breast cancer) and 35% (colon cancer) respectively. Statistical analysis showed that TMSG 1 in non-metastatic cancers was significantly higher than that in metastatic cancers (P <0.05). Conclusion The successful preparation of anti-human tumor metastasis-based?