骨髓干细胞动员对创伤失血犬免疫功能的调节作用

Mobilization of bone marrow stem cells can regulate immune functions following traumatic hemorrhage in dogs

目的观察创伤失血犬自体骨髓干细胞动员后外周血干细胞、免疫细胞亚群和细胞因子含量动态变化,为干细胞治疗战创伤提供依据。方法健康比格犬10只,采用手术方法切除左侧肾后,经肾动脉放血至血压60mmHg并维持30min。实验组(n=5)皮下注射GM-CSF5μg/kg,隔日1次,连续3次,对照组(n=5)同步注射等量生理盐水,分别于0、2、4、6、8、10d采集外周血,计数白细胞总数、淋巴细胞和中性粒细胞比例,测定CD3+、CD4+、CD8+、CD56+、CD34+、CD133+细胞比例和血清TNF-α、IL-1、IL-2含量。结果与0d比较,实验和对照犬在创伤失血后白细胞总数、中性粒细胞比例均逐渐升高,第4～6d达到高峰(P<0.05),然后逐渐下降,但实验组高于对照组(P<0.05);两组淋巴细胞的比例均下降(P<0.05),但实验组高于对照组(P<0.05);CD3+、CD4+、CD8+、CD56+细胞的比例下降(P<0.05),到第4～6d达到最低(P<0.05),随后逐渐升高,至第10d时接近正常,其中实验组高于对照组(P<0.05);两组CD34+、CD133+细胞比例均逐渐升高,至第2～4d达到高峰(P<0.05),随后逐渐下降,但实验组在整个实验过程中均高于对照组(P<0.05);实验组血浆TNF-α、IL-1、IL-2的含量均逐渐升高,至第6d达到高峰(P<0.05),随后下降至第10d时接近正常。结论骨髓干细胞动员可提高创伤失血犬外周血免疫亚群细胞比例和细胞因子含量,提示具有保护创伤失血犬免疫功能的作用,但也有促进炎症反应的作用。

Objective To observe the changes of peripheral blood stem cells, immune cell subsets and cytokine contents in dogs with traumatic hemorrhage after the mobilization of autologous bone marrow stem cells, and to provide a theoretical basis for the treatment of battle trauma with stem cells. Methods The left kidneys of 10 healthy beagles were resected and blood letting was performed via renal artery until the blood pressure declined to 60 mmHg and maintained for 30 min. The test group （ n = 5） was given the intramuscular injection of GM - CSF （5 μg/kg） once every other day for three times,while the control group （n =5 ） was given equal volume of normal saline. The peripheral blood samples were respectively gathered on day 0,2,4,6,8, 10,and then WBC and the proportion of lymphocyte and neutrophil were counted by blood cell analyzer. Flow eytometry was used to measure the proportion of CD3 ＋ , CD4 ＋ , CD8 ＋ , CD16 ＋ , CD56 ＋ , CD34 ＋ and CD133 ＋ cells. The plasma contents of TNF - or, IL - 1 and IL - 2 were determined by enzyme - linked immunosorbent assay （ELISA）. Results Compared with day 0, both WBC and the proportion of lymphocyte and neutrophil in the two groups gradually increased and reached the peak on day 4 - 6 （ P 〈 0.05 ）, and then declined gradually （ P 〈 0.05 ）, but they were higher in the test group than those in the control group （ P 〈 0.05 ）. The lymphocyte proportion in both groups declined （ P 〈 0.05 ）, and the test group was still higher. The proportion of CD3 ＋ , CD4 ＋ , CD8 ＋ , CD56 ＋ gradually decreased and reached the valley point on day 4 - 6 （ P 〈 0. 05 ）, and then raised gradually and approached to normal on day 10, and the test group was also higher（ P 〈 0.05 ）. The proportion of CD34 ＋ , CD133 ＋ cells both gradually raised to the peak on day 2 - 4 （ P 〈 0.05 ） , and then decreased gradually, but the test group was higher during the whole experiment （ P 〈 0.05 ）. In the test group, the plasma contents of TNF - α, IL - 1, IL - 2 gradually raised to the peak on day 6 （P 〈 0. 05 ）, and then decreased to nearly normal on day 10. Conclusion The mobilization of bone marrow stem cells can increase the proportion of immune cell subsets and cytokine contents in peripheral blood of dogs with traumatic hemorrhage. Stem cell mobilization can protect the immunologic functions of these dogs, but it may also promote inflammatory reactions.