趋化因子CCL5与糖尿病相关的弥漫大B细胞淋巴瘤关系的实验研究

Relationship between Chemotactic Factor CCL5 and Diabet-associated Diffuse Large B Cell Lymphoma

目的:探讨趋化因子CCL5是否是糖尿病并发弥漫大B细胞淋巴瘤(DLBCL)的重要因子。方法:体外培养人正常B细胞和DLBCL细胞,应用RT-PCR分别检测其CCL5 mRNA表达;通过5、30 mmol/L两种糖浓度体外培养人DLBCL细胞株和鼠源性DLBCL细胞株A20,使用RT-PCR分别检测其CCL5 mRNA表达。给BALB/c小鼠腹腔注射小剂量链脲佐菌素(STZ)构建糖尿病鼠模型,通过慢病毒转染技术建立稳定的低表达CCL5和高表达CCL5细胞株;将低表达CCL5和高表达CCL5二种细胞株皮下注入糖尿病BALB/c小鼠和正常血糖BALB/c小鼠体内。根据血糖水平将实验小鼠分为2组:糖尿病组(A组)和血糖正常组(B组);然后根据CCL5的表达水平A组和B组又分为高表达组(A1组和B1组)和低表达组(A2组和B2组),分别观察成瘤率、成瘤时间、肿块大小和质地;对瘤组织常规HE染色切片,免疫组织化学检测各组CCL5的表达。结果:体外培养人DLBCL细胞株CCL5mRNA表达高于正常B细胞(P<0.05);高糖培养人DLBCL细胞株和鼠DLBCL细胞株中CCL5 mRNA表达高于低糖培养株(P<0.05)。糖尿病小鼠体内注射高表达CCL5、低表达CCL5的小鼠A20细胞株的糖尿病小鼠中A1组成瘤率为93.3%、A2组为60%,成瘤时间A1组中为7.0±0.85 d、A2组为9.5±2.8 d。正常血糖鼠成瘤率在B1组中为20%、B2组中为20%,成瘤时间B1组为12±1.3 d、B2组为14±2.5 d;免疫组织化学检测糖尿病小鼠成瘤组织内CCL5表达高于正常血糖小鼠瘤组织。结论:高血糖可致患DLBCL的风险增加,并加快肿瘤的生长,趋化因子CCL5可能在糖尿病致DLBCL生长、转移中发挥作用。

Objective: To explore whether the chemotactic factor CCL5 is the major factor of diffuse large B cell lymphoma( DLBCL) induced by diabetes or not. Methods: The normal human B cells and DLBCL cells were cultured in vitro; the RT-PCR was used to detect their CCL5 mRNA expression,the human DLBCL cell line and mouse-derived DLBCL cell line A20 were cultured in vitro by using glucose at 5 and 30 mmol /L,respectively,then their CCL5 mRNA expression was detected by PT-PCR; the diabetic mouse model was constructed through peritoneal injection of streptozotocin at low dose in the BALB /c mice; the cell lines with stably high and low expression of CCL5 were established via lentiviral transfection and the cell lines with low and high expression of CCL5 were subcutaneously injected into diabetic mice and mice with normal blood sugar level. According to blood sugar level,the experimantal mice were divided into 2 groups: diabetic group( A group) and normal blood sugar group as control( B group); then according to CCL5 expression level,the A group and B group were divided as well into high expression group( A1 group and B1 group) and low expression group( A2 group and B2 group),respectively,the tumor-formation rate,tumor-formation time,tumor size and texture were analyzed,respectively; the CCL5 expression was detected by using HE staining of tumor tissue and immunohistochemical method. Results: The expression of CCL5 mRNA in human DLBCL cell line cultured in vitro was higher than that in normal B cells( P < 0. 05); the expressions of CCL5 mRNA in human DLBCL cells cultured in high sugar concentration in vitro and mouse DLBCL cells were higher than those in cells cultured in low sugar concentration( P < 0. 05). The tumor-formation rates in diabetic mice injected with high and low expression of CCL5 cell line A20 were 93. 3% in A1 group and 60% in A2 group; the their tumor-formation time was7. 0 ± 0. 85 days in A1 group and 9. 5 ± 2. 8 days in A2 group,while the tumor formation rates in mice with normal blood sugar level were 20% in B1 group and 20% in B2 group,and their tumor-formation time was 12 ± 1. 3 days and14 ± 2. 5 days respectively; the CCL5 expression level in tumor tissue of diabetic mice was higher than that in tumor tissue of mice with normal blood sugar level. Conclusion: The high blood glucose level can casase increase of DLBCL risk and promote the tumor growth; the chemotactic factor CCL5 may play an important role in the growth and migration of DLBCL caused by diabetes mellitus.