CLEC2B基因过表达的Jurkat细胞培养上清液对黑素瘤细胞B16的影响

Effects of Supernatant of CLEC2B Gene Overexpression in Jurkat Cells on the B16 Melanoma cell

目的将CLEC2B基因过表达的Jurkat细胞培养上清液作用于黑素瘤细胞,观察其上清液对黑素瘤细胞增殖、酪氨酸酶活性、黑素合成的影响,探讨CLEC2B基因在白癜风发病中的作用。方法培养人淋巴瘤细胞Jurkat细胞和小鼠黑素瘤细胞B16,采用脂质体介导的方法瞬时转染CLEC2B重组质粒入Jurkat细胞,半定量RT-PCR法鉴定CLEC2B基因过表达;将其细胞培养上清液作用于黑素瘤细胞48 h后,MTT法检测黑素瘤细胞的增殖情况,多巴氧化法检测酪氨酸酶活性,氢氧化钠裂解法检测黑素含量。结果 CLEC2B基因过表达的Jurkat细胞培养上清液作用后的黑素瘤细胞增殖比空载体组、正常对照组降低,差异有统计学意义(均P<0.05),CLEC2B过表达组黑素含量比空载体组、正常对照组减少,差异有统计学意义(均P<0.05),CLEC2B过表达组、空载体组、正常对照组之间的酪氨酸酶活性比较差异无统计学意义(均P>0.05),空载体组与正常对照组比较差异均无统计学意义(均P>0.05)。结论 CLEC2B基因过表达的Jurkat细胞培养上清液对黑素细胞的增殖和黑素合成有一定的抑制作用,对酪氨酸酶活性影响不明显。提示CLEC2B可能通过调节淋巴细胞功能间接影响黑素细胞增殖和黑素合成,从而参与白癜风发病。

Objective The melanoma cells were treated with culture supernataut of CLEC2B gene overexpression in Jurkat cells, to observe the impact of melanoma proliferation, the activity of tyrosinase and melanin synthesis, for searching CLEC2B gene participated in the vitiligo. Methods In this study, Jurkat ceils were transfected with recombinant plasmid pGCMV/EGFP/Neo-CLEC2B by DMRIE-C Reagent. RT-PCR identified CLEC2B gene overexpression. After 48 h transfection, the melanoma cells were treated with their culture supernatant for 48 h, then the proliferation of melanoma cells were detected by MTr, the activity of tyrosinase was detected by Dopa oxidation, melanin synthesis was detected by Sodium hydroxide lysis. Results The proliferation rate of melanoma cells in CLEC2B gene transfection group was significantly lower than the empty vector group and control group （P 〈0.05）, the activity of tyrosinase of melanoma cells with among CLEC2B gene transfection group, empty vector group and control group had no significant difference （P 〉0.05）, the melanin content of melanoma cells with in CLEC2B gene transfection group was significantly less than the empty vector group and control group （P 〈0.05）, the empty vector group had no significant difference with the control group （P〉0.05）. Conclusion The Jurkat cells culture supernatant with CLEC2B gene overexpression could inhibit the proliferation and melanin synthesis of melanocytes in some extent, it has no apparent effect on the activity of tyrosinase of melanocytes. CLEC2B could influence the proliferation and melanin synthesis of melanocytes by the way of affecting lymphocytes＇ function.