肥大细胞缺陷大鼠经骨髓移植重建消化道肥大细胞

Reconstitution of mast cell population in gastrointestinal tract by bone marrow transplantation in mast cell deficient rats

目的探讨骨髓移植（BMT）是否能重建肥大细胞缺陷大鼠（Ws／Ws大鼠）的消化道肥大细胞，并了解新生肥大细胞的生长发育特性。方法42只Ws／Ws大鼠分成对照组及6种毁髓照射剂量组（6．0、7．0、8．0、9．0、10．0和11．0Gy），每组6只，以确定Co60 放射源的适宜毁髓照射剂量。16只Ws／Ws大鼠进行7．5Gy照射，于照射后第1、5、8、12天检测血象及骨髓象，每个时间点4只，另取4只无照射Ws／Ws大鼠的血象与骨髓象作为对照。将与Ws／Ws大鼠同基因背景的正常BrownNorway（BN）大鼠的骨髓细胞悬液，输注入经7．5Gy剂量照射后的Ws／Ws大鼠体内，构建BMT-Ws／Ws大鼠。4只雌性Ws／Ws大鼠接受雄性BN大鼠BMT后4周经Y染色体原位杂交（Y-FISH）鉴定BN大鼠骨髓在BMT-Ws／Ws大鼠体内的存活及分化迁移。另使用相同方法构建雄性BMT-Ws／Ws大鼠24只，于移植后5、8、13及23周（各6只）通过阿尔新蓝．番红O染色观察肥大细胞在全消化道的重建情况，对其进行计数，通过Western印迹法定量肥大细胞类胰蛋白酶和糜蛋白酶表达。结果Y-FISH显示雄性供体的骨髓可在雌性Ws／Ws大鼠体内存活并迁移分化为成熟的消化道肥大细胞。8周时BMT-Ws／Ws大鼠食管、胃及回肠远端肥大细胞数目最多，均明显高于BN对照大鼠[（103±6）比（35±4），（271±23）比（124±13），（200±13）比（103±7）个／mm2，均P〈0．05，n=6）；食管及回肠组织类胰蛋白酶含量也最高，明显高于BN对照大鼠（1．3±0．3比0．6±0．2，3．6±0．8比1．9±0．4，均P〈0．05，n=4）。13周之后肥大细胞数量及蛋白酶含量逐渐回落。结论骨髓移植可以重建Ws／Ws大鼠消化道肥大细胞，移植后8周至13周消化道肥大细胞及类胰蛋白酶含量最丰富，此期可作为BMT-Ws／Ws大鼠用于肥大细胞功能实验研究的最佳时间窗。

Objective To explore whether bone marrow transplantation （BMT） can restore gastrointestinal mast cells in mast eel1 deficient （Ws/Ws） rats and understand the features of these reconstituted mast cells. Methods Thirty-six Ws/Ws rats were subjected to Co60 radiation at 6 gradient doses （6. 0, 7.0, 8.0, 9.0, 10. 0 and 11.0 Gy, n =6） to confirm the appropriate dosage. And another 6 rats served as non-radiated controls. Sixteen Ws/Ws rats were exposed to a 7.5 Gy radiation and sacrificed at Day 1, 5, 8 and 12 （n =4） to obtain hemogram and myelogram. And 4 non-radiated Ws/Ws rats served as control. Ws/Ws rats received an intravenous injection of bone marrow ceils harvested from healthy cogenic Brown Norway （BN） rats after a dosage of 7. 5 Gy radiation. Y-chromosome fluorescence in situ hybridization （Y-FISH） was used to identify the survival and differentiation of bone marrow cells of male BN rats in female BMT-Ws/Ws rats （ n = 4 ）. Another set of 24 male BMT-Ws/Ws rats were sacrificed Weeks 5, 8, 13 and 23 post-BMT （n =6）. Tissues from esophagus, stomach, ileum and colon were harvested to perform mast cell quantification by Alcian blue staining. And mast cell derived proteinases （tryptase and chymase） were quantified by Western blotting. Results Y-FISH showed bone marrow cells from male BNrats survived in female BMT-Ws/Ws rats and mast cells were restored in gastrointestinal tract. Compared with control, the highest level of mast cell number （103± 6） vs （35± 4）/mm2 for esophagus, （271 ± 23 ） vs （ 124 ± 13 ）/mm2 for stomach, （ 200. 1 ± 13.3 ） vs （ 103.2 ± 6. 6 ）/mm2, all P 〈 0. 05 ） and mast cell proteinases （ 1.3 ± 0. 3 vs 0.6 ± 0. 2 for esophagus, 3.6 ± 0.8 vs 1.9 ±0. 4 for ileum, both P 〈 0.05 ） in BMT-Ws/Ws rats were observed at Week 8 post-BMT. The number of mast cell and proteinases decreased at Week 13 post-BMT. Conclusions BMT may restore mast cells in Ws/Ws rats. The best time period for using BMT-Ws/Ws rats in the study of mast cell function is between Weeks 8 - 13 post-BMT.