热休克蛋白60诱导小鼠皮肤移植耐受的实验研究

目的探讨热休克蛋白60（heat shock protein60，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-2^k）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50gtgHSP60，2周后行BALB／C（H-2^d）小鼠皮肤移植：D组：受体小鼠背部皮下注射经0．1mL IFA乳化后的50μg HSP60，2周后行CBA／N（H-2^k）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（JP〈0．05）：A、B组间及C、D组间比较差异无统计学意义（JP〉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（interferonl，，IFN-γ）低于A、B组（JP〈0．05），A、B组间及C、D组间差异均无统计学意义（JP〉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。

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

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 x 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-2d) mice skin. In group C, C57BL/6 (H-2b) mice were administered HSP60 emulsified in IFA into the back 2 weeks before transplantation of BALB/C (H-2d) mice skin. In group D, C57BL/6 (H-2b) mice were treated with HSP60 emulsified in IFA into the back and followed by skin transplantation of CBA/N (H-2k) 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 mixed lymphocyte 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, 6581 +/- 573 and 6843 +/- 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 13286 +/- 1498, 12960 +/- 1376, 11936 +/- 1265 and 12374 +/- 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-gamma 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.