CAJ | 학술논문

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(50 mg/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1×109 L-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50 mg/支的注射用硝普钠1支,加入2.5 mL生理盐水混匀,从中取出1.0 mL液体加入到100 mL的生理盐水中,配成终浓度为200 mg/L,配置后4 h内使用.③各组小鼠细胞移植前均经5.0 Gy X射线全身照射4 h,吸收剂量率为1.45 Gy/min.照射完毕后,细胞移植组直接经尾静脉输注0.3 mL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200 mg/L硝普钠液体0.15 mL,1 min后立即输注骨髓间充质干细胞悬液0.3 mL(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60 d,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3 d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24 h内细胞完全贴壁,长梭形,增殖生长迅速,3 d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. 揹景:硝普鈉作為血管擴張劑加入骨髓間充質榦細胞培養過程中,對其分化潛能髮生何種影響?目的:觀察大鼠骨髓間充質榦細胞體內嚮造血細胞分化過程中加入硝普鈉後的變化,併與單純細胞移植的效果進行比較.設計:隨機區組,對照觀察.單位:廣東醫學院微生物免疫學教研室,廣州醫學院病理生理教研室.材料:實驗于2006-08在廣州醫學院完成.選取清潔級齣生七八週balb/c小鼠27隻作為受體,隨機數字錶法分為3組:細胞移植組、硝普鈉+細胞移植組、空白對照組,9隻/組.另選取4週齡SD大鼠1隻作為實驗用骨髓間充質榦細胞的供體來源.註射用硝普鈉(50 mg/支,北京雙鶴現代醫藥技術有限責任公司,國藥準字H11020907).方法:①無菌條件下取齣SD大鼠的股骨,進行骨髓間充質榦細胞的分離培養.傳至六七代作為供體細胞,消化離心,調整濃度為1×109 L-1.細胞懸液中加入熒光素標記的抗體,流式細胞術檢測錶型.②取50 mg/支的註射用硝普鈉1支,加入2.5 mL生理鹽水混勻,從中取齣1.0 mL液體加入到100 mL的生理鹽水中,配成終濃度為200 mg/L,配置後4 h內使用.③各組小鼠細胞移植前均經5.0 Gy X射線全身照射4 h,吸收劑量率為1.45 Gy/min.照射完畢後,細胞移植組直接經尾靜脈輸註0.3 mL骨髓間充質榦細胞懸液(含1.5×106箇細胞);硝普鈉+細胞移植組先註射已配好的200 mg/L硝普鈉液體0.15 mL,1 min後立即輸註骨髓間充質榦細胞懸液0.3 mL(含1.5×106箇細胞);空白對照組輸註等量無血清培養液.④移植後60 d,各組存活小鼠眼眶外週取血,處死後常規製備骨髓及脾髒單細胞懸液,流式細胞術檢測大鼠源性造血細胞CD11a與CD45的植入水平.主要觀察指標:①骨髓間充質榦細胞培養擴增情況.②不同組織大鼠源性造血細胞的植入水平檢測.結果:作為受體的27隻balb/c小鼠均存活至實驗結束.①骨髓間充質榦細胞培養擴增情況:培養3 d後細胞貼壁,形態比較均一,長梭形,至第6天細胞90%融閤,無重疊.傳代後24 h內細胞完全貼壁,長梭形,增殖生長迅速,3 d即達到完全融閤.②不同組織大鼠源性造血細胞的植入水平檢測:細胞移植組、硝普鈉+細胞移植組在外週血、骨髓、脾細胞懸液中均可檢測到低錶達的大鼠源性造血細胞CD11a與CD45,且硝普鈉+細胞移植組明顯彊于細胞移植組(t=2.619,P＜0.05);空白對照組CD11a與CD45呈陰性錶達.結論:大鼠骨髓間充質榦細胞具有嚮造血細胞分化的潛能,硝普鈉可促進其分化.
배경:초보납작위혈관확장제가입골수간충질간세포배양과정중,대기분화잠능발생하충영향?목적:관찰대서골수간충질간세포체내향조혈세포분화과정중가입초보납후적변화,병여단순세포이식적효과진행비교.설계:수궤구조,대조관찰.단위:엄동의학원미생물면역학교연실,엄주의학원병리생리교연실.재료:실험우2006-08재엄주의학원완성.선취청길급출생칠팔주balb/c소서27지작위수체,수궤수자표법분위3조:세포이식조、초보납+세포이식조、공백대조조,9지/조.령선취4주령SD대서1지작위실험용골수간충질간세포적공체래원.주사용초보납(50 mg/지,북경쌍학현대의약기술유한책임공사,국약준자H11020907).방법:①무균조건하취출SD대서적고골,진행골수간충질간세포적분리배양.전지륙칠대작위공체세포,소화리심,조정농도위1×109 L-1.세포현액중가입형광소표기적항체,류식세포술검측표형.②취50 mg/지적주사용초보납1지,가입2.5 mL생리염수혼균,종중취출1.0 mL액체가입도100 mL적생리염수중,배성종농도위200 mg/L,배치후4 h내사용.③각조소서세포이식전균경5.0 Gy X사선전신조사4 h,흡수제량솔위1.45 Gy/min.조사완필후,세포이식조직접경미정맥수주0.3 mL골수간충질간세포현액(함1.5×106개세포);초보납+세포이식조선주사이배호적200 mg/L초보납액체0.15 mL,1 min후립즉수주골수간충질간세포현액0.3 mL(함1.5×106개세포);공백대조조수주등량무혈청배양액.④이식후60 d,각조존활소서안광외주취혈,처사후상규제비골수급비장단세포현액,류식세포술검측대서원성조혈세포CD11a여CD45적식입수평.주요관찰지표:①골수간충질간세포배양확증정황.②불동조직대서원성조혈세포적식입수평검측.결과:작위수체적27지balb/c소서균존활지실험결속.①골수간충질간세포배양확증정황:배양3 d후세포첩벽,형태비교균일,장사형,지제6천세포90%융합,무중첩.전대후24 h내세포완전첩벽,장사형,증식생장신속,3 d즉체도완전융합.②불동조직대서원성조혈세포적식입수평검측:세포이식조、초보납+세포이식조재외주혈、골수、비세포현액중균가검측도저표체적대서원성조혈세포CD11a여CD45,차초보납+세포이식조명현강우세포이식조(t=2.619,P＜0.05);공백대조조CD11a여CD45정음성표체.결론:대서골수간충질간세포구유향조혈세포분화적잠능,초보납가촉진기분화.
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.