Anti-GM1影响裸鼠NK细胞分化和杀伤活性研究

A study on the effects of Anti-GM1 on the differentiation and cytotoxic activity of NK cells in nude mice

目的研究抗单唾液酸神经节苷脂抗体(Anti-GM1)通过调节自然杀伤(NK)细胞分化,抑制裸鼠NK细胞的自然杀伤活性。方法多重免疫荧光染色鉴定人肾癌组织中NK细胞的分型特点。选择雄性BALB/c小鼠6只,GM1皮下注射每周1次,一共三周,以此来免疫小鼠制备Anti-GM1。选取6只BALB/c-Nude裸鼠,采用随机数字表法将裸鼠分为对照组(CON组)及实验组(Anti-GM1组),CON组用IgG连续腹腔注射7 d;Anti-GM1组用Anti-GM1连续腹腔注射7 d。7 d后经颈椎脱臼法处死裸鼠,取裸鼠脾脏并制成单细胞悬液,将脾细胞与YAC-1细胞共培养4 h。用LDH法检测NK细胞的杀伤活力。使用流式细胞术进行检测,根据CD27和CD11b表达来界定NK细胞亚群,同时检测CD107a的表达情况。ELISA检测NK细胞的γ干扰素(IFN-γ)及颗粒酶B(GzmB)分泌水平。结果人肾癌组织标本中有明显的NK细胞浸润现象,并且GzmB也呈现阳性表达。Anti-GM1可以显著抑制NK细胞的杀伤活力。与CON组相比,Anti-GM1组可以促进NK细胞向成熟亚群进行分化。Anti-GM1组NK细胞的CD107a水平显著下降。Anti-GM1组NK细胞的IFN-γ及GzmB分泌水平较CON组下降。结论NK细胞可以浸润在人肾癌组织中,并且能够分泌GzmB,发生自然杀伤作用。Anti-GM1可以促进NK细胞的成熟,但抑制NK细胞的杀伤活力,与其抑制NK细胞CD107a表达和抑制NK细胞的IFN-γ及GzmB分泌有关。

Objective To investigate that Anti-monosialoganglioside antibody(Anti-GM1)inhibits the natural killing activity of natural killer(NK)cells in nude mice by regulating NK cell differentiation.Methods Multiplex immunofluorescence staining was used to identify the NK cell typing characteristics in human renal cancer tissue.Six male BALB/c mice were selected,and GM1 was injected subcutaneously once a week for three weeks to immunize the mice for the preparation of Anti-GM1.Six BALB/c-Nude nude mice were randomly divided into the control group(CON)and the experimental group(Anti-GM1)using a random number table.The CON group was treated with IgG by continuous intraperitoneal injection for 7 days;the Anti-GM1 group was treated with Anti-GM1 by continuous intraperitoneal injection for 7 days.After 7 days,the nude mice were sacrificed by cervical dislocation,the spleens of the nude mice were taken and made into a single-cell suspension,and the splenocytes were co-cultivated in YAC-1 for 4 hours.Killing viability of the NK cells was detected by LDH assay.Detection was performed using flow cytometry,with NK cell subsets defined based on the expression of CD27 and CD11b,and the expression of CD107a was also assessed.ELISA was used to detect the secretion levels of NK cell interferon-gamma(IFN-γ)and granzyme B(GzmB).Results There was an obvious NK cell infiltration in the human renal cancer tissue specimens,and GzmB was also positively expressed.Anti-GM1 can significantly inhibit the killing activity of NK cells.Compared with the CON group,the Anti-GM1 group can promote the differentiation of NK cells into mature subsets.The level of CD107a in NK cells of the Anti-GM1 group was significantly decreased.The secretion levels of IFN-γand GzmB in NK cells of the Anti-GM1 group were decreased compared to the CON group.Conclusion NK cells can infiltrate into human renal cancer tissue and can secrete GzmB,exerting natural killing effects.Anti-GM1 can promote the maturation of NK cells but inhibit the killing activity of NK cells,which is related to its inhibition of NK cell CD107a expression and suppression of NK cell IFN-γand GzmB secretion.