大鼠骨髓间充质干细胞体内造血分化过程中加入硝普钠的作用

背景:硝普钠作为血管扩张剂加入骨髓间充质干细胞培养过程中,对其分化潜能发生何种影响?目的:观察大鼠骨髓间充质干细胞体内向造血细胞分化过程中加入硝普钠后的变化,并与单纯细胞移植的效果进行比较。设计:随机区组,对照观察。单位:广东医学院微生物免疫学教研室,广州医学院病理生理教研室。材料:实验于2006-08在广州医学院完成。选取清洁级出生七八周balb/c小鼠27只作为受体,随机数字表法分为3组:细胞移植组、硝普钠 细胞移植组、空白对照组,9只/组。另选取4周龄SD大鼠1只作为实验用骨髓间充质干细胞的供体来源。注射用硝普钠(50mg/支,北京双鹤现代医药技术有限责任公司,国药准字H11020907)。方法:①无菌条件下取出SD大鼠的股骨,进行骨髓间充质干细胞的分离培养。传至六七代作为供体细胞,消化离心,调整浓度为1×109L-1。细胞悬液中加入荧光素标记的抗体,流式细胞术检测表型。②取50mg/支的注射用硝普钠1支,加入2.5mL生理盐水混匀,从中取出1.0mL液体加入到100mL的生理盐水中,配成终浓度为200mg/L,配置后4h内使用。③各组小鼠细胞移植前均经5.0GyX射线全身照射4h,吸收剂量率为1.45Gy/min。照射完毕后,细胞移植组直接经尾静脉输注0.3mL骨髓间充质干细胞悬液(含1.5×106个细胞);硝普钠 细胞移植组先注射已配好的200mg/L硝普钠液体0.15mL,1min后立即输注骨髓间充质干细胞悬液0.3mL(含1.5×106个细胞);空白对照组输注等量无血清培养液。④移植后60d,各组存活小鼠眼眶外周取血,处死后常规制备骨髓及脾脏单细胞悬液,流式细胞术检测大鼠源性造血细胞CD11a与CD45的植入水平。主要观察指标:①骨髓间充质干细胞培养扩增情况。②不同组织大鼠源性造血细胞的植入水平检测。结果:作为受体的27只balb/c小鼠均存活至实验结束。①骨髓间充质干细胞培养扩增情况:培养3d后细胞贴壁,形态比较均一,长梭形,至第6天细胞90%融合,无重叠。传代后24h内细胞完全贴壁,长梭形,增殖生长迅速,3d即达到完全融合。②不同组织大鼠源性造血细胞的植入水平检测:细胞移植组、硝普钠 细胞移植组在外周血、骨髓、脾细胞悬液中均可检测到低表达的大鼠源性造血细胞CD11a与CD45,且硝普钠 细胞移植组明显强于细胞移植组(t=2.619,P<0.05);空白对照组CD11a与CD45呈阴性表达。结论:大鼠骨髓间充质干细胞具有向造血细胞分化的潜能,硝普钠可促进其分化。

Sodium nitroprusside in the hematopoietic differentiation of rat bone marrow-derived mesenchymal stem cells in vivo

BACKGROUND: How dose sodium nitroprusside, as a vasodilatator, affect the potential of bone marrow-derived mesenchymal stem cells (MSCs) in hematopoietic differentiation?OBJECTIVE: To observe the changes during the differentiation of MSCs into hematopoietic cells after adding sodium nitroprusside, and compare the results with those of simple MSCs transplantation.DESIGN: A randomized grouping, controlled observation.SETTINGS: Department of Microbiology and Immunology; Department of Pathophysiology, Guangdong Medical College.MATERIALS: The experiments were carried out in Guangzhou Medical College in August 2006. Twenty-seven clean-degree balb/c mice of 7-8 weeks, were used as recipients, and were randomly divided into MSCs transplantation group (n =9), sodium nitroprusside+MSCs transplantation group (n =9) and blank control group (n =9). Another 4-week-old SD rat was selected as the MSCs donor. Sodium nitroprusside for injection (50 mg/piece) was provided by Beijing Double-Crane Modern Pharmaceutical Technology Co., Ltd (National drug approval: No. H11020907).METHODS: ① Under aseptic condition, the femur of SD rat was collected. MSCs in it were isolated for culture and amplifying in vitro. MSCs of passages 6-7 were digested and centrifugated, and the density was adjusted to 1×109 L-1.Monoclonal antibody with fluorescence labeled was added into cell suspension, and the phenotype was detected with flow cytometry. ② Sodium nitroprusside (50 mg/piece) was adjusted to the terminal concentration of 200 mg/L by adding with saline. It should be used within 4 hours. ③ Before transplantation, all the mice were exposed to 5.0 Gy. X-ray for 4 hours, and the absorbed dose was 1.45 Gy per minute. After irradiation, mice in the MSCs transplantation group were directly infused via caudal vein with 0.3 mL MSCs suspension (containing 1.5×106 cells); The mice in the sodium nitroprusside+MSCstransplantation group were firstly injected with the dispensed 200 mg/L sodium nitroprusside (0.15 mL), and immediately infused with 0.3 mL MSCs suspension (containing 1.5×106 cells) after 1 minute; The mice in the blank control group were infused with isovolume serum-free culture medium. ④ At 60 days after transplantation,peripheral blood was drawn from orbits of the survived mice in each group, single cell suspensions of bone marrow and spleen were prepared after the mice were killed, the levels of rat-derived hematopoietic cells CD11a and CD45 were detected with flow cytometry.MAIN OUTCOME MEASURES: ① MSCs culture and amplification; ② Levels of rat-derived hematopoietic cells in different tissues.RESULTS: All the 27 balb/c mice as recipients survived to the end of the experiment. ① MSCs isolation and amplification: The MSCs attached with uniform shapes of spindle and proliferated rapidly after culture for 3 days, and 90% of the cells were fused without overlapping at 6 days. The cells attached completely within 24 hours after passage,extended and became spindle again, rapidly proliferated and grew, and became fused completely at 3 days. ② Levels of rat-derived hematopoietic cells in different tissues: In the MSCs transplantation group and sodium nitroprusside+MSCs transplantation group, Iow expressions of rat-derived CD11a and CD45 hematopoietic cells could be detected in peripheral blood, bone marrow and spleen, and they were obviously higher in the sodium nitroprusside+MSCs transplantation group than in the MSCs transplantation group (t=2.619, P ＜ 0.05), while negative ones in the blank control group.CONCLUSION: MSCs have the ability to differentiate into hematopoietic stem cells, which can be promoted by sodium nitroprusside.