烧伤大鼠血清对不同来源间质干细胞的趋化作用

Chemotactic effects of burn rat serum on mesenchymal stem cells derived from different sources

目的了解间质干细胞（MSC）的来源，观察烧伤大鼠血清对不同来源MSC的趋化作用。方法将72只大鼠采用完全随机法分成烧伤组（36只，制作背部30％TBSAⅢ度烧伤模型）、假伤组（36只，37℃模拟致伤）。从两组大鼠骨髓、外周血中分离并培养MSC，在倒置显微镜下观察各组MSC的阳性率，比较其生长速度和细胞形态。同时观察不同血清对MSC的趋化作用及不同来源MSC的迁移能力。结果两组大鼠的骨髓内均培养出贴壁生长的MSC。烧伤组12只大鼠外周血中有7只培养出MSC，其阳性率（58％）明显低于骨髓培养（100％，P＜0．05）。假伤组大鼠的外周血内未见MSC（P＜0．05）。倒置显微镜下可见原代接种后24h有少量贴壁细胞，2～3d后见其生长，散在贴壁，大多呈梭形。各组MSC形态无明显差异，均呈纺锤形生长。烧伤大鼠血清处理的假伤组骨髓MSC的迁移数[（94±11）个／高倍视野]明显多于正常大鼠血清和胎牛血清处理者[（37±6）、（38±11）个／高倍视野，P＜0．01]，后两者处理的MSC迁移数相近（P＞0．05）。假伤组大鼠骨髓MSC被不同血清趋化时迁移的细胞数明显少于烧伤组大鼠骨髓和外周血MSC（P＜0．05或0．01）。烧伤组大鼠骨髓MSC与外周血MSC的迁移能力虽有差异，但无统计学意义（P＞0．05）。结论烧伤大鼠骨髓及外周血均能分离到MSC，而正常大鼠仅骨髓能分离到MSC。烧伤大鼠血清对MSC有较强的趋化作用。烧伤大鼠来源的MSC比正常大鼠来源的MSC具有更强的迁移能力。

Objective To isolate and culture mesenchymal stem cells （ MSC ） from different sources and to investigate the chemotactic effects of burn rat serum on MSC derived from different sources. Methods Seventy-two Wistar rats were randomly divided into burn （ n = 36, with 30% TBSA full-thickness burns on the back） and sham burn （n = 36, without burns）groups. Bone marrow and peripheral blood of the rats in both groups were collected to isolate and culture MSC. Ratio of MSC, growth speed and cell morphology were observed with inverted microscope. Effects of different serum （ fetal bovine serum, normal rat serum and burn rat serum） on chemotaxis of MSC derived from different sources and their migration ability were subsequently examined with a transwell system. Results MSC were obtained from bone marrow of the rats in both groups. MSC were successfully obtained from bone marrow of all burn rats（ 100% , P 〈 0.05 ） , but only from peripheral blood of 7 burn rat（ 58% ） , and no MSCs were obtained from peripheral blood of 12 rats in sham group.（ P 〈 0.05 ）. There was small amount of adherent cells 24 hrs after culture, and fusiform shaped adherent cells were sporadically observed in scattered distribution 2-3 days later with inverted microscope. There was no obvious difference in the cell morphology between the 2 groups, in the sham group, the number of MSC migrating to the lower surface of transwell after burn serum treatment [ （ 94 ± 11 ） cells/ highpower field] was significantly greater than that after the treatment with normal rat serum and fetal bovine serum [ （ 37 ± 6 ） , （ 38 ± 11 ） cells/highpower field , P 〈 0.001 ], while no difference in migration ability was found after normal serum treatment compared with that after fetal bovine serum treatment （ P 〉0.05 ）. The migra- tion rate of MSCs which were derived from bone marrow in sham group was obviously lower than those derived from bone marrow and peripheral blood from burn rats （ P 〈0.05 or 0.01 ）. Though some difference of the migration ability existed between MSC derived from bone marrow and peripheral blood, there was no statistieally significant difference （ P 〉 0.05 ）. Conclusion MSC can be isolated and cultured from bone marrow and peripheral blood of burn rat, but not from peripheral blood of normal rat. Burn rat serum has a stronger chemotactic effect on MSC. Moreover, the migration ability of MSC derived from burn rat is stronger than that of MSC derived from normal rat.