Objective: To explore the effects of allogeneic bone marrow mesenchymal stem cells (BMSCs) on polarization of peritoneal macrophages isolated from rats with sepsis induced by endotoxin/lipopolysaccharide (LPS). Methods: (1) BMSCs were isolated, cultured and purified from 5 SD rats with whole bone marrow adherent method. The third passage of cells were collected for morphologic observation, detection of expressions of stem cell surface markers CD29, CD44, CD45, and CD90 with flow cytometer, and identification of osteogenic and adipogenic differentiation. (2) Another 45 SD rats were divided into sham injury group (SI, n=5), LPS control group (LC, n=20), and BMSCs-treated group (BT, n=20) according to the random number table. Rats in groups LC and BT were injected with LPS (5 mg/kg) via tail vein to induce sepsis; rats in group SI were injected with the same amount of normal saline to simulate the damage. At post injury hour (PIH) 1, rats in group BT were given 1 mL BMSCs (2×10(6)/mL) via tail vein injection; rats in another two groups were injected with equal volume of phosphate buffer saline. Five rats in group SI at PIH 24 and in groups LC and BT at PIH 6, 12, 24, and 48 were sacrificed to harvest lung tissue for pathological observation with HE staining. In addition, rats in group SI at PIH 24 and in groups LC and BT at PIH 24 and 48 were simultaneously performed with intraperitoneal injection of low-glucose DMEM. Then peritoneal fluid was harvested to culture peritoneal macrophages. Flow cytometer was used to assess the positive expression of cell makers of macrophages including CD68 (making gate), CD11c, and CD206 in group SI at PIH 24 and in groups LC and BT at PIH 24 and 48. Data were processed with one-way analysis of variance and LSD test. Results: (1) The third passage of cells showed uniform fiber-like shape similar to fibroblasts. These cells showed positive expressions of CD29, CD44, CD90 and weak positive expression of CD45. They were able to differentiate into osteoblasts and adipocytes. These cells were identified as BMSCs. (2) At PIH 24, the structure of pulmonary alveoli of rats in group SI was clear and complete with no congestion or inflammatory cell infiltration. At PIH 6, the structure of pulmonary alveoli of rats in groups LC and BT was clear with a small amount of inflammatory cell infiltration, slight congestion and pulmonary interstitial thickening. At PIH 12, the inflammatory responses in lung tissue of rats in group LC were more severe than those in group BT with a large amount of inflammatory cell infiltration, serious congestion, and obvious pulmonary interstitial thickening. The pathological results of rats in group BT at PIH 12 was consistent with the results at PIH 6. At PIH 24, the pathological results of rats in groups LC and BT were similar to the results at PIH 12. At PIH 48, the structure of pulmonary alveoli tissue of rats in group LC was still severely disrupted, with a large number of inflammatory cell infiltration and congestion in lung tissue, but pulmonary interstitial thickening was slightly alleviated than before. The condition of rats in group BT nearly recovered to that in group SI. (3) At PIH 24, the positive expression rate of CD11c in peritoneal macrophages of rats in group LC [(83±10)%] was close to that in group BT [(87±7)%, P>0.05], and they were both significantly higher than the rate in group SI [(55±12)%, with P values below 0.01]. The positive expression rate of CD11c in peritoneal macrophages of rats in group LC [(59±11)%] at PIH 48 was close to that in group SI at PIH 24 (P>0.05), and they were both significantly higher than the rate in group BT [(20±11)%] at PIH 48 (with P values below 0.01). At PIH 24, the positive expression percentages of CD206 in peritoneal macrophages of rats were similar among the three groups (with P values above 0.05). The positive expression percentage of CD206 in peritoneal macrophages of rats in group SI at PIH 24 was close to that in group BT at PIH 48 (P>0.05), and they were both significantly lower than the percentage in group LC at PIH 48 (with P values below 0.01). Conclusions: BMSCs can reduce the pathological inflammatory responses in the lung of rats with sepsis and inhibit peritoneal macrophages from polarizing into M1 phenotype, whereas they can not promote macrophages to polarize into M2 phenotype.
目的： 探讨同种异体骨髓间充质干细胞(BMSC)对LPS诱导的脓毒症大鼠腹腔巨噬细胞极化改变的影响。 方法： (1)取5只SD大鼠，采用全骨髓贴壁法分离纯化培养BMSC，取第3代细胞行形态学观察，流式细胞仪检测干细胞表面标志物CD29、CD44、CD45、CD90的表达，并进行成骨细胞、成脂肪细胞诱导分化鉴定。(2)取45只SD大鼠，按随机数字表法分为假伤组5只、LPS对照组20只、BMSC治疗组20只。对LPS对照组、BMSC治疗组大鼠予尾静脉按5 mg/kg注射LPS致脓毒症，假伤组大鼠注射等量的生理盐水模拟致伤。伤后1 h，BMSC治疗组大鼠另予尾静脉注射1 mL BMSC(2×10(6)个/mL)，另2组大鼠注射等量PBS。假伤组大鼠于伤后24 h，LPS对照组、BMSC治疗组于伤后6、12、24、48 h各取5只大鼠处死后取肺组织行HE染色观察病理变化。假伤组伤后24 h的大鼠与LPS对照组、BMSC治疗组伤后24、48 h的大鼠另同时通过腹腔注射低糖DMEM培养液后取腹腔液培养巨噬细胞。流式细胞仪检测假伤组伤后24 h，LPS对照组、BMSC治疗组伤后24、48 h大鼠腹腔巨噬细胞标志物CD68(用于设门)、CD11c、CD206阳性表达情况。对数据行单因素方差分析、LSD检验。 结果： (1)分离培养的第3代贴壁细胞形态呈均一纤维样，与Fb相似；CD29、CD44、CD90呈阳性表达，CD45呈弱阳性表达；诱导后细胞可分化为成骨细胞和成脂肪细胞，鉴定为BMSC。(2)伤后24 h，假伤组大鼠肺泡结构清晰、完整，无充血或炎性细胞浸润。伤后6 h，LPS对照组、BMSC治疗组大鼠肺泡结构清晰，少量炎性细胞浸润，肺组织轻微充血，肺间质稍增厚。伤后12 h，LPS对照组大鼠肺组织较BMSC治疗组炎症反应明显，可见大量炎性细胞浸润，肺组织充血，肺间质明显增厚；而BMSC治疗组大鼠与伤后6 h情况基本一致。伤后24 h，LPS对照组、BMSC治疗组大鼠肺组织病理改变较伤后12 h无明显变化。伤后48 h，LPS对照组大鼠肺泡组织结构仍严重破坏，大量炎性细胞浸润，肺组织充血，肺间质增厚情况较前稍有减轻；而BMSC治疗组大鼠基本恢复至假伤组水平。(3)伤后24 h，LPS对照组、BMSC治疗组大鼠腹腔巨噬细胞CD11c阳性表达率分别为(83±10)%和(87±7)%，二者相近(P>0.05)，且均明显高于假伤组的(55±12)%(P值均小于0.01)。LPS对照组伤后48 h大鼠腹腔巨噬细胞CD11c阳性表达率[(59±11)%]与假伤组伤后24 h相近(P>0.05)，且均高于BMSC治疗组伤后48 h的(20±11)%(P值均小于0.01)。伤后24 h，3组大鼠腹腔巨噬细胞CD206阳性表达比相近(P值均大于0.05)。假伤组伤后24 h和BMSC治疗组伤后48 h大鼠腹腔巨噬细胞CD206阳性表达比相近(P>0.05)，且均低于LPS对照组伤后48 h(P值均小于0.01)。 结论： BMSC可减轻脓毒症大鼠肺组织的炎症反应，并且能够抑制腹腔巨噬细胞向Ⅰ型巨噬细胞极化，尚不能促使巨噬细胞向Ⅱ型巨噬细胞极化。.
Keywords: Bone marrow mesenchymal stem cells; Macrophages; Sepsis.