转GmDREB1基因抗旱小麦T349的免疫毒理学评价

Immunotoxicologic assessment of genetically modified drought-resistant wheat T349 with GmDREB1

目的评价转CmDREB1基因抗旱小麦T349的免疫毒性作用。方法将250只6—8周龄、体重为18～22g的SPF级雌性BALB／c小鼠按体重分层随机分为5个大组，每大组50只，每大组再按体重分层随机分为5组，每组10只，分别为阴性对照组、普通小麦组、亲本小麦组、转基因小麦组和环磷酰胺阳性对照组。阴性对照组和环磷酰胺阳性对照组以AIN-93G饲料喂养，普通小麦组、亲本小麦组和转基因小麦组以掺入相应小麦的饲料喂养。饲喂30d后，检测小鼠的各项指标，包括体重、脏器系数、外周血淋巴细胞表型、血清细胞因子水平、血清免疫球蛋白水平、溶血空斑数、血清半数溶血值（HC50）、脾淋巴细胞增殖能力、迟发型超敏反应和吞噬细胞功能。结果阴性对照组、普通小麦组、亲本小麦组、转基因小麦组、阳性对照组小鼠饲喂30d后体重分别为（21．0±0．3）、（20．4±0．7）、（21．1±1．0）、（21．1±1．0）、（19．4±1．0）g（F=7．47，P〈0．01）；脾脏器系数分别为（0．407±0．047）％、（0．390±0．028）％、（0．402±0．042）％、（0．421±0．041）％、（0．304±0．048）％（F=12．41，P〈0．01）；胸腺脏器系数分别为（0．234±0．032）％、（0．246±0．028）％、（0．249±0．040）％、（0．234±0．034）％、（0．185±0．039）％（F=5．58，P〈0．01）；小鼠外周血T淋巴细胞百分比分别为（70．43±4．44）％、（68．33±5．37）％、（73．04±2．68）％、（74．42±2．86）％、（90．42±1．66）％（F=57．51，P〈0．01）；B淋巴细胞百分比分别为（13．89±3．19）％、（15．34±4．84）％、（13．06±4．22（12．93±2．36）％、（3．01±0．96）％（F=12．79，P〈0．01）；Th细胞百分比分别为（55．87±3．80（55．24±4．60）％、（57．92±3．70）％、（59．57±2．54）％、（77．37±2．31）％（F=68．58，P〈0．01％、％、；辅助性T细胞（Th）／抑制性T细胞（Ts）比值分别为4．16±0．29、4，73±0．96、4．19±0．78、4．52±0．40、6．34±0．73（F=17．57，P〈0．01）；小鼠血清IgG水平分别为（1046．38±210．67）、（1065．49±297．22）、（1517．73±299．52）、（1576．67±241．92）、（1155．88±167．05）ug／ml（F=10．53，P〈0．01）；小鼠血清IgM水平分别为（333．83±18．97）、（327．73±27．72）、（367．47±27．18）、（363．42±46．14）、（278．71±24．42）ug/ml（F=12．11，P〈0．01）；小鼠血清IgA水平分别为（51．69±10．10）、（42．40±8．35）、（32．11±4．22）、（37．12±4．90）、（41．45±8．89）ug/ml（F=8．25，P〈0．01）；小鼠溶血空斑数分别为（29．2±14．6）、（28．0±20．0）、（34．8±30．9）、（33．2±25．1）、（4．8±5．3）个／10^。脾细胞（F=3．33，P〈0．05）；HC50值分别为82．3±6．5、79．7±4．6、75．8±4．1、74．9±3．6、70．8±2．1（F=9．99，P〈0．01）；脂多糖诱导的脾细胞增殖能力分别为0．21±0．10、0．21±0．14、0．26±0．12、0．25±0．14、0．07±0．06（F=4．18，P〈0．05）。结论转GmDREB1基因抗旱小麦T349与亲本小麦对小鼠的免疫器官及免疫功能的影响具有实质等同性，无免疫毒性作用。

Objective To assess the immunotoxicologic effects of genetically modified drought resistant wheat T349 with GmDREB1 gene. Methods A total of 250 female BALB/c mice（6 -8 week-old, weight 18- 22 g） were divided into five large groups （50 mice for each large group） by body weight randomly. In each large group, the mice were divided into five groups（ 10 mice for each group） by body weight randomly, which were set as negative control group, common wheat group, parental wheat group, genetically modified wheat group and cyelophosphamide positive control group, respectively. Mice in negative control and positive control group were fed with feedstuff AIN-93G, mice in common wheat group, non- genetically modified parental wheat group and genetically modified wheat group were fed with feedstuffs added corresponding wheat （ proportion up to 76% ） for 30 days, then body weight, organ coefficient of spleen and thymus, peripheral blood lymphocytes phenotyping, serum cytokine, serum immunoglobulin, antibody plaque-forming cell （PFC） , serum 50% hemolytic value （HC50） , mitogen-induced splenocyte proliferation, delayed-type hypersensitivity （DTH） reaction and phagocytic activities of phagocytes were detected respectively. Results After 30 days raise, among negative control group, common wheat group, non- genetically modified parental wheat group, genetically modified wheat group and cyclophosphamide positive control group,mice body weight were （21.0 ±0.3）, （20.4 ±0.7）, （21.1 ± 1.0）, （21.1 ± 1.0）, （ 19.4 ± 1.0） g, respectively （ F = 7.47, P 〈 0. 01 ） ; organ coefficient of spleen were （ 0. 407 ± 0. 047 ） % , （0. 390 ± 0. 028 ） %, （ 0. 402 ± 0. 042 ） %, （ 0. 421 ± 0.041 ） %, （ 0. 304 ± 0. 048 ） %, respectively （ F = 12.41, P 〈 0. 01 ） ; organ coefficient of thymus were （0. 234 ± 0. 032 ） %, （ 0. 246 ± 0. 028 ） %, （ 0. 249 ± 0. 040）%, （0. 234 ± 0. 034）%, （0. 185 ± 0. 039）%, respectively （F = 5.58, P 〈 0. 01 ） ;the percentage of T cell in peripheral blood were （ 70.43 ± 4.44） %, （68.33 ± 5.37 ） %, （ 73.04 ± 2. 68 ） %, （ 74. 42 ± 2. 86）%, （90.42 ± 1.66）%, respectively （F =57.51, P 〈0.01）; the percentage of B cell were （13.89±3.19）%, （15.34±4.84）%, （13.06 ±4.22）%, （12.93 ±2.36）%, （3.01 ±0.96）%, respectively （F= 12. 79, P 〈0.01）; the percentage of Th cell were （55.87 ±3.80）%, （55.24 ± 4.60）%, （57.92±3.70）%, （59.57 ±2.54）%, （77.37 ±2.31）%, respectively （F=68.58, P〈 0.01）;the Th/Ts ratio were 4.16 ±0.29, 4.73 ±0.96,4.19 ±0.78,4.52 ±0.40, 6.34 ±0.73, respectively （ F = 17.57, P 〈 0. 01 ） ; the serum IgG were （ 1046. 38 ± 210. 67 ）, （ 1065.49 ± 297. 22 ）, （ 1517.73 ± 299. 52）, （ 1576. 67 ± 241.92 ）, （ 1155.88 ± 167. 05 ） ug/ml, respectively （ F = 10. 53, P 〈 0.01）; the serum IgM were （333.83 ± 18. 97）,（327.73 ±27.72）,（367.47 ±27.18）,（363.42 ± 46. 14）, （278.71 ± 24. 42） ug/ml, respectively （F = 12. 11, P 〈 0. 01 ） ; the serum IgA were （51.69 ± 10. 10）, （42.40 ± 8.35 ）, （ 32. 11 ± 4. 22 ）, （ 37.12 ± 4. 90）, （41.45 ± 8.89 ） Ixg/ml, respectively （ F = 8.25, P 〈0.01） ; the PFC were （29. 2 ± 14. 6） ,（28. 0 ±20. 0） ,（34. 8 ±30. 9） ,（33.2 ±25.1）, （4. 8 ± 5.3） per 106 splenocyte, respectively （ F = 3.33, P 〈 0. 05 ） ; the HC50 were 82. 3 ± 6. 5, 79.7 ± 4. 6, 75.8 ±4. 1, 74. 9 ± 3.6, 70. 8 ± 2. 1, respectively （ F = 9. 99, P 〈 0. 01 ） ; the LPS-induced splenocyte proliferation were 0. 21 ± 0. 10, 0. 21 ± 0. 14,0. 26 ± 0. 12,0. 25 ± 0. 14,0. 07 ± 0.06, respectively （ F = 4. 18,P 〈 0. 05 ）. Conclusion The genetically modified drought-resistant wheat T349 was substantially equivalent to parental wheat in the effects on immune organs and immunologic functions of mice, and it didn＇t show immunotoxicity.