热休克蛋白60诱导小鼠皮肤移植耐受的实验研究

EXPERIMENTAL STUDY ON INDUCTION OF SKIN ALLOGRAFT TOLERANCE IN MICE BY HEAT SHOCK PROTEIN 60

目的探讨热休克蛋白60(heat shock protein 60,HSP60)对小鼠移植皮片成活的影响及其机制。方法选用60只C57BL/6(H-2b)小鼠为受体,45只BALB/C(H-2d)小鼠、15只CBA/N(H-2k)为供体,均为8～12周龄近交系雌性小鼠。受体小鼠剪下1cm×1cm全层皮肤,干纱布清创,即为植床;随机分为4组,每组15只。A组:无菌取供体BALB/C(H-2d)小鼠背部1cm×1cm全层皮片,刮去皮下组织后移植至受体小鼠背部;B组:受体小鼠背部皮下注射0.1mL不完全弗氏佐剂(imcompleted Freund’s adjuvant,IFA),2周后行BALB/C(H-2d)小鼠皮肤移植;C组:受体小鼠背部皮下注射经0.1mLIFA乳化后的50μgHSP60,2周后行BALB/C(H-2d)小鼠皮肤移植;D组:受体小鼠背部皮下注射经0.1mLIFA乳化后的50μgHSP60,2周后行CBA/N(H-2k)小鼠皮肤移植。B、C、D组供体皮肤移植方法同A组。术后观察移植皮片成活时间;移植术后7、25d行单向混合淋巴细胞反应及混合淋巴细胞培养上清液细胞因子检测;术后7d行迟发型超敏反应测定。结果A、B、C、D组移植皮片成活时间分别为(12.4±0.5)、(11.6±0.8)、(29.3±2.6)及(27.6±2.1)d;A、B组与C、D组比较,差异有统计学意义(P<0.05),A、B组间及C、D组间比较,差异无统计学意义(P>0.05)。皮肤移植术后7d,A、B、C、D组混合淋巴细胞反应每分钟放射脉冲数(counts of perminute impulse,cmp)值分别为12836±1357、11876±1265、6581±573及6843±612;A、B组与C、D组比较,差异有统计学意义(P<0.05);A、B组间及C、D组间比较差异无统计学意义(P>0.05);术后25d,各组cpm值分别为13286±1498、12960±1376、11936±1265及12374±1269,各组间差异无统计学意义(P>0.05)。皮肤移植术后7d,C、D组混合淋巴细胞培养上清液IL-10高于A、B组,IL-2、干扰素γ(interferon γ,IFN-γ)低于A、B组(P<0.05),A、B组间及C、D组间差异均无统计学意义(P>0.05);术后25d,各组IL-2、IL-10及IFN-γ差异无统计学意义(P>0.05)。皮肤移植术后7d,A、B、C、D组受体小鼠对供体小鼠脾细胞的迟发型超敏反应为(0.84±0.09)、(0.81±0.07)、(0.43±0.05)及(0.46±0.03)mm;A、B组与C、D组比较,差异有统计学意义(P<0.05),A、B组间及C、D组间差异无统计学意义(P>0.05)。结论HSP60对免疫耐受的诱导和维持有一定作用。

Objective To investigate the role and mechanism of heat shock protein 60 （HSP60） in induction of murine skin allograft tolerance. Methods At the age of 8-12 weeks, inbred female BALB/C （H-2d） mice （n=45） and CBA/N （H-2k）mice （n=15） were used as transplantation donors and C57BL/6 （H-2b） mice （n=60） as recipients. Recipients C57BL/6 （H-2b） mice were randomized into 4 groups （n=15）. In group A, 1 cm × 1 cm Wolfe-Krause skin graft was excised from the back of BALB/C （H-2d） mice and hypoderma was scraped off aseptically, and then transplanted to the back of C57BL/6 （H-2b） mice. The method of skin transplantation in the other 3 groups was the same as to group A. In group B, C57BL/6 （H-2b） mice were treated with imcompleted Freund＇s adjuvant （IFA） administration into the back 2 weeks before transplantation of BALB/C （H-2^d） mice skin. In group C, C57BL/6 （H-2^b） mice were administered HSP60 emulsified in IFA into the back 2 weeks before transplantation of BALB/C （H-2 ^d） mice skin. In group D, C57BL/6 （H-2^b） mice were treated with HSP60 emulsified in IFA into the back and followed by skin transplantation of CBA/N （H-2^k） mice 2 weeks later. The delayed type hypersensitivity was determined 7 days after transplantation. One-way mixed lymphocyte reaction, the concentration of cytokines in the mixedlymphocyte reaction culture supernatant was determined 7 days and 25 days after transplantation. The survival time of skin allograft was observed. Results The survival time of skin allograft in groups A, B, C and D was 12.4 ±0.5, 11.6 ± 0.8, 29.3 ± 2.6 and 27.6 ± 2.1 days, respectively. There was significant difference between groups A, B and groups C, D （P 〈 0.05）, while there was no significant difference between group A and group B as well as between group C and group D （P 〉 0.05）. The counts of per minute impulse （cpm） of mixed lymphocyte reaction 7 days after transplantation in groups A, B, C and D was 12 836 ± 1357, 11876 ± 1265, 6 581 ± 573 and 6 843 ± 612, respectively. There was significant difference between groups A, B and group C and group D （P 〈 0.05）, while there was no significant difference between group A and group B as well as between group C and group D （P 〉 0.05）. The cpm of mixed lymphocyte reaction at 25 days after transplantation in group A, B, C and D was 13 286 ±1 498, 12 960 ± 1 376, 11 936 ± 1 265 and 12 374± 1269, respectively. There was no significant difference among 4 groups （P 〉 0.05）.The concentration of IL-10 in the mixed lymphocyte reaction culture supernatant in groups C, D were higher than that in groups A, B, and IL-2 and IFN-y were lower than that in groups A, B 7 days after transplantation （P 〈 0.05）, while there was no significant difference between group A and group B as well as between group C and group D （P 〉 0.05）. There was no significant difference in cytokines among the 4 groups 25 days after transplantation （P 〉 0.05）. The delayed type hypersensitivity in groups A, B, C and D 7 days after transplantation was 0.84 ± 0.09, 0.81 ± 0.07, 0.43 ± 0.05 and 0.46 ± 0.03 mm, respectively. There was significant differences between groups A, B and groups C, D （P 〈 0.05）. While there was no significant difference between group A and group B as well as between group C and group D （P 〉 0.05）. Conclusion HSP60 may play a role in induction and maintenance of murine skin allograft tolerance.