不同方法诱导小鼠产生CD8^(+)记忆性T细胞的研究

Study on the effects of different methods to induce CD8^(+) memory T cells in mice

目的探讨不同方法诱导小鼠产生CD8^(+)记忆性T细胞的效果。方法将100只雌性Bal B/C小鼠作为受体小鼠,并将其中75只小鼠按随机数字表法分为移植组、1×10^(6)注射组、5×10^(6)注射组、1×10^(7)注射组和空白对照组,每组15只。取1只C57小鼠(供体小鼠)的脾脏淋巴细胞,按上述不同个数溶于0.2 ml PBS缓冲液后注射于各注射组小鼠腹腔中;颈椎脱臼处死后,取15块0.25 cm^(2)背部皮肤移植到移植组小鼠背部。处理后1、2、3个月时采用流式细胞术检测上述5组小鼠脾脏CD8^(+)记忆性T细胞百分比和CD3^(+)T细胞总数。剩余25只小鼠(每组5只)按上述方法分组和处理,然后纯化分选CD8^(+)记忆性T细胞,与经过辐照后的C57小鼠脾脏淋巴细胞进行共培养,1周后用流式细胞仪检测CD8^(+)记忆性T细胞增殖指数。结果前5组小鼠在处理后1个月时产生的CD8^(+)记忆性T细胞百分比差异无统计学意义(P>0.05),但在2、3个月时3个注射组高于空白对照组和移植组(均P<0.05),3个月时更明显(均P<0.05)。其中1×10^(6)注射组在2个月时CD8^(+)记忆性T细胞比例最高;在CD3^(+)T细胞总数方面,移植组及3个注射组组内比较均在2个月时最高,差异均有统计学意义(均P<0.05),但组间比较差异均无统计学意义(均P>0.05)。各组CD8^(+)记忆性T细胞的增殖指数差异无统计学意义(P>0.05)。结论通过体内注射脾脏淋巴细胞来诱导CD8^(+)记忆性T细胞是更高效的方法。

Objective To establish a new method for inducing CD8^(+)memory T cells to make the acquisition of CD8^(+)memory T cells easier and more efficient.Methods A total of 100 female Bal B/C mice were enrolled as recipient mice,and 75 of them were divided into 5 groups:skin transplantation group,1×10^(7) injection group,5×10^(6) injection group,1×10^(6) injection group and control group,with 15 mice in each group.Splenic lymphocytes from one C57 mouse(donor mouse)were extracted,dissolved in 0.2 ml PBS buffer and injected intraperitoneally into each injection group.C57 mice were sacrificed by cervical dislocation,and 0.25 cm2 of skin was transplanted to the back of recipient mice of transplantation group.At 1,2,or 3 months after treatment,the number of CD3^(+)T lymphocytes and the percentage of CD8^(+)memory T cells were measured by flow cytometry.The remaining 25 mice were grouped and treated as above,with 5 mice in each group.CD8^(+)memory T cells were purified and sorted,co-cultured with irradiated C57 mouse spleen lymphocytes,and proliferation index of CD8^(+)memory T cells was detected by flow cytometry after 1 week.Results There was no significant difference in memory T cells between the 5 groups of mice at 1 month(P>0.05),but the percentage of CD8^(+)memory T cells in the 3 injection groups was higher than that in the blank control and transplantation groups at months 2 and 3 after treatment.The 1×10^(6) injection group had the highest percentage of CD8^(+)memory T cells at month 2.At month 3 after treatment,the 3 injection groups had a higher proportion of CD8^(+)memory T cells than the transplantation group and the control group.Meanwhile,the difference in the proliferative capacity of CD8^(+)memory T cells among the groups was not statistically significant(P>0.05).Regarding the total number of CD3^(+)T cells,the intra-group comparison of each group in the transplantation group,1×10^(6) injection group,5×10^(6) injection group,and 1×10^(7) injection group,was the highest at the 2nd month after treatment,and the difference was statistically significant(P<0.05),but none of the differences were statistically significant in the inter-group comparison.Conclusion Injection of splenic lymphocytes in vivo is a more efficient method to induce CD8^(+)memory T cells.