穿孔素在重组白细胞介素-12治疗小鼠病毒性心肌炎过程中的表达

Perforin Expression in Course of Recombination Interleukin-12 Therapy for Viral Myocarditis in Mice

目的观察重组白细胞介素-12(rmIL-12)治疗小鼠病毒性心肌炎过程中自然杀伤细胞(NK细胞)活性、穿孔素表达水平及小鼠心肌病理变化等指标,探讨rmIL-12治疗病毒性心肌炎的机制。方法选取BALB/c雄性小鼠60只,随机分成6组,分别为空白对照组,病毒对照组,干扰素对照组,rmIL-12小、中、大治疗剂量组,每组10只小鼠。除空白对照组外,余每只小鼠均腹腔注射CVB30.2mL/d,连续3d。开始接种病毒日定为第0天,从第0天开始,于接种病毒4h,空白对照组和病毒对照组腹腔注射9g/L盐水0.1mL/d,干扰素对照组腹腔注射干扰素-α400 IU/d;rmIL-12小、中、大治疗组分别注射1、10、100ng/d rmIL-12,连续5d。第5天每组分别取5只鼠,颈椎脱臼处死,取心脏,留取心脏标本用于做心肌病理学检测。应用四甲基偶氮唑蓝法检测各组小鼠脾脏NK细胞活性。Trizol试剂提取心脏组织总RNA,应用RT-PCR法检测小鼠心脏穿孔素表达水平,应用HE染色法观察小鼠心肌病理改变。结果NK细胞活性病毒对照组与rmIL-12中剂量组及大剂量组比较有显著差异(Pa<0.05),与干扰素对照组及小剂量治疗组比较无明显差异(Pa>0.05)。NK细胞活性随着rmIL-12剂量的增加而增加。空白对照组无穿孔素表达,rmIL-12中剂量组和大剂量组表达量多于小剂量组和干扰素对照组。各组心肌炎性变化以rmIL-12中剂量治疗组心肌炎性反应浸润最轻,而rmIL-12大剂量组心肌细胞变性坏死最重。结论适当增加NK细胞的活性可以减轻心肌细胞的损伤,但NK细胞活性过度增加会加重心肌细胞损伤。rmIL-12通过增加NK细胞活性而增加了穿孔素的表达。穿孔素表达量随rmIL-12治疗剂量的增加而增加。

Objective To observe the NK cell activity, expression of perforin, mouse myocardial pathological changes in the course of rmlL- 12 treating mice viral myocarditis, and study the mechanism of recombination interleukin - 12 （rmIL - 12 ） therapy for mice viral myocarditis. Methods Sixty BALB/c male mice,were randomly divided into 6 groups（n = 10） as：the blank control group,virus control group, interferon control group,r raiL- 12 small, medium and large dose group. In addition to blank control group, each mouse were injected CVB3 0.2 mL/d in peritoneal for 3 d. The first day peritoneal injection CVB3 was defined as a day 0. After the virus injected 4 h ,blank control group and virus control group abdominal injection 9 g/L saline 0.1 mL/d,interferon control group abdominal injection of interferon - α 400 IU/d, rmlL - 12 small,medium and large dose group were injected rmIL - 12 for 1,10,100 ng/d,for 5 d. The first 5 days,5 rats were selected from each group, killed by cervical dislocation, raked the heart, collecting specimens for the heart to do cardiac pathology test observation the mice NK cell activity of splenocyte, detection the perforin expression in mouse heart using the methods of reverse transcription polymerase chain reaction and observation the mouse myocardial pathological changes. Results The results of the NK cell activity in different groups ： there were significant differences between virus control group and the medium dose rmlL- 12 group,and between virus control group and the large dose rmIL- 12 group （Pa 〈 0.05 ） ,there was no significant differences between virus control group and the interferon control group, and between virus control group and small dose group （P 〉 0.05 ）. NK cell activity was increased with the dose of rmIL - 12 increasing. Perforin expression：there was no perforin expression in blank control group. There were more expression in the rmIL- 12 medium dose and large dose group than in small dose group and interferon control group. The inflammation changes in different groups：tbe rmlL - 12 medium dose group with inflammation infiltration was minimum,and the rmIL - 12 large dose group was maximum. Conclusions Appropriately increase the activity of NK cell can be reduced myocardial injury. However,NK cell activity excessive increase will add to the myocardial injury, rmlL - 12 increases perforin expression by increasing the activity of NK cells. Perforin expression is increased with rmlL - 12 dose increasing.