目的:比较脐血与成人外周血粒细胞Toll样受体(TLRs)的表达情况,为新生儿相关疾病的临床治疗积累实验资料.方法:取脐血和成人外周血,用密度梯度离心结合红细胞裂解法分离、收集粒细胞,流式细胞术鉴定粒细胞纯度,RT-qPCR检测TLRs 10个成员的mRNA表达水平,流式细胞术测定部分TLRs的蛋白荧光强度.结果:(1)流式细胞术检测结果:采用密度梯度离心结合红细胞裂解法分离收集的脐血粒细胞CD19-CD24+为(95.66±1.73)％,CD3+为(4.27±1.22)％,成人外周血粒细胞CD19-CD24+为(95.48±2.13)％,CD3+为(4.82±1.07)％.(2) RT-qPCR结果显示:脐血粒细胞和成人外周血粒细胞TLRl(0.141 ±0.091 vs 0.691 ±0.447)、TLR2(0.388 ±0.337vs0.901 ±0.508)、TLR4 (0.093 ±0.071 vs 0.254 ±0.147)、TLR6(0.056±0.045 vs 0.202±0.034)、TLR7(0.001±0.001 vs 0.004±0.003)和TLR8(0.046±0.040 vs 0.211±0.146)的表达差异有统计学意义(P＜0.01),TLR3、TLR5、TLR9和TLR10的表达差异无统计学意义(P＞0.05)；其中TLR3、TLR7和TLR9在脐血和成人外周血粒细胞的相对表达水平均较低.(3)流式分析显示脐血与成人外周血粒细胞TLR2平均蛋白荧光强度(21.40±3.09 vs 30.50±5.69)差异有统计学意义(P＜0.05)；而TLR4平均蛋白荧光强度差异无统计学意义(P＞0.05).结论:脐血粒细胞TLR1、TLR2、TLR4和TLR6 mRNA及TLR2蛋白表达低于成人外周血粒细胞,提示新生儿粒细胞识别细菌性病原微生物感染的能力可能存在缺陷或尚未完全成熟.这是否与新生儿急性细菌性毒血症发病及病死率较高有直接联系,有待进一步研究.%AIM: To compare the expression levels of Toll-like receptors (TLRs) in the granulocytes isolated from umbilical cord blood and adult periphERαl blood. METHODS: The granulocytes in umbilical blood and adult periphERαl blood were isolated by the method of density gradient centrifugation combined with red blood cell splitting. The purity of the cells was evaluated by flow cytometry. The mRNA expression levels of 10 TLRs were detected by RT-qPCR, and the protein levels of some TLRs were also tested by flow cytometry. RESULTS: The populations of CD19 CD24 + cells and CD3 + cells were (95.66 ±3.15)% and(4.19±1.54)% in neonatal granulocytes, respectively, and were (95. 36 ± 1.74)% and (4.30 ±0.96)% in adult granulocytes, respectively. The relative mRNA expression levels of TLRs in the granulocytes isolated from umbilical cord blood and adult periphERαl blood were as follows: TLR1 0. 141 ±0.091 and 0. 691 ±0.447, TLR2 0.388 ±0.337 and 0.901 ±0.508, TLR4 0.093 ±0.071 and 0. 254 ±0. 147, TLR6 0.056 ±0.045 and 0. 202 ± 0. 034, TLR7 0. 001 ± 0. 001 and 0. 004 ± 0. 003 , and TLR8 0. 046 ± 0. 040 and 0. 211 ± 0. 146, and the difference had statistical significance (P<0.01). However, no difference in the expression levels of TLR3, TLR5, TLR9 and TLR10 between the neonatal and adult gra-nulocytes was observed (P > 0. 05). Among them, the mRNA expression ofrnTLR3, TLR7 and TLR9 was at a low level in both neonatal and adult granulocytes. The protein level of TLR2 in adult gra-nulocytes (30.50+5.69) was higher than that in neonatal granulocytes (21.40+3.09, P<0.05). CONCLUSION: Low mRNA expression of TLR1, TLR2, TLR4 and TLR6, and low protein level of TLR2 in neonatal granulocytes indicate that the ability of recognizing bacterial pathogen by neonatal granulocytes may be defective or not yet fully mature.
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