骨髓间充质干细胞在烟雾吸入性损伤家兔体内向功能细胞分化的观察

Observation on marrow-derived mesenchymal stem cells differentiating into functional cells in rabbit with smoke inhalation injury

目的 观察静脉移植的骨髓间充质干细胞（MSC）在烟雾吸入性损伤家兔体内＂归巢＂和分化情况.方法将32只新西兰大耳白兔按照随机数字表法分为正常对照组、吸入性损伤组、正常对照＋MSC处理组和MSC治疗组,每组8只.正常对照组家兔不致伤,经耳缘静脉注入10 mLPBS;正常对照＋MSC处理组家兔不致伤,经耳缘静脉注入内含1×107个溴脱氧尿苷（BrdU）标记的第3代兔MSC的PBS 10 mL.其余2组家兔造成重度烟雾吸入性损伤后,吸入性损伤组同正常对照组处理,MSC治疗组同正常对照＋MSC处理组处理.4组家兔分别于注射后7或28 d,取肺组织、气管组织行免疫组织化学染色观察MSC在受损组织中＂归巢＂情况;核标记物BrdU分别与肺或气管内特异性膜标记物水通道蛋白5（AQP-5）、碱性磷酸酶（AKP）、CD34以及角蛋白组合,行免疫组织化学双染色,观察MSC向功能细胞分化情况;HE染色观察肺及气管损伤情况.结果 （1）免疫组织化学染色显示,MSC治疗组可见MSC于注射后7 d在肺组织和气管组织中＂归巢＂;正常对照＋MSC处理组未见此现象.（2）免疫组织化学双染色显示,MSC治疗组于注射后28 d肺组织中可见AQP-5和AKP以及CD34表达阳性的MSC,气管组织中未见角蛋白表达阳性的MSC,正常对照＋MSC处理组未见上述阳性表达细胞.（3）HE染色显示,MSC治疗组肺损伤和气管损伤较吸入性损伤组明显减轻,Fb增殖显著减少.结论静脉移植的MSC能＂归巢＂至烟雾吸入性肺损伤炎症反应明显的肺组织和气管组织区域,并分化为肺泡Ⅰ型上皮细胞、肺泡Ⅱ型上皮细胞和肺血管内皮细胞,可能参与了烟雾吸入性损伤的组织修复过程.

Objective To observe the homing and differentiation of marrow-derived mesenchymal stem cells （MSC） transplanted intravenously in smoke inhalation injured rabbits. Methods Thirty-two New Zealand big ear rabbits were divided into normal control group （ NC ）, inhalation injury group （ Ⅱ）,normal control ＋ MSC treatment group （NM） , and MSC treatment group （MT） according to the random number table, with 8 rabbits in each group. Rabbits in NC group were injected with 10 mL phosphate buffered saline （PBS） via ear marginal vein. Rabbits in NM group were injected with 10 mL PBS containing the third generation MSC labeled by BrdU （ 1 × 107 per 10 mL PBS） via ear marginal vein. Severe smoke inhalation injury model was reproduced in the other two groups, among them rabbits in Ⅱ group were treated as rabbits in NC group, rabbits in MT group treated as rabbits in NM group. On the 7th and 28th day post treatment （PTD）, lung tissue and trachea tissue were harvested from four groups for observation on injury with HE staining. Homing of MSC in injured tissue was observed with immunohistochemistry staining. The differentiation of MSC into functional cells was observed with immunohistochemical double staining of combining nuclear marker BrdU with lung （trachea） membrane-specific marker aquaporin-5 （AQP-5） , alkaline phosphatase （AKP）, CD34, and cytokeratin respectively. Results （1） MSC homing in lung and trachea tissue was observed in MT group on PTD 7, which was not observed in NM group. （2） AQP-5, AKP, and CD34 positive MSC were observed in lung tissue in MT group on PTD 28, while cytokeratin positive MSC was not observed in trachea tissue. No positively marked MSC was observed in NM group. （3） Injury in lung and trachea was less severe in MT group than in Ⅱ group; and the proliferation of fibroblasts was less in MT group. Conclusions Intravenous injection of MSC to rabbits with smoke inhalation injury can migrate to lung and trachea tissue at obviously inflammatory site, and differentiate into alveolar epithelial cells type Ⅰ and Ⅱ , and pulmonary vascular endothelial cells, which may participate in the process of tissue repair in smoke inhalation injury.