CAJ | 학술논문

补肾活血中药对大鼠骨髓间充质干细胞体外增殖的影响
보신활혈중약대대서골수간충질간세포체외증식적영향
Effect of serum containing kidney-tonifying and blood-activating Chinese herbs on the in vitro proliferation of rat bone marrow mesenchymal stem cells

万方数据

中国组织工程研究与临床康复中國組織工程研究與臨床康複 중국조직공정연구여림상강복
JOURNAL OF CLINICAL REHABILITATIVE TISSUE ENGINEERING RESEARCH
2008年 8期 1581-1585 ,共5页

曾意荣%樊粤光%刘红%夏雄智%范海蛟

증의영%번월광%류홍%하웅지%범해교

骨髓间充质干细胞%补肾活血中药%免疫细胞化学法骨髓間充質榦細胞%補腎活血中藥%免疫細胞化學法
골수간충질간세포%보신활혈중약%면역세포화학법

背景:骨髓间充质干细胞因其获取容易、创伤微小、具有无限扩增和多分化潜能、自体移植不存在免疫排斥反应而受到医学研究者的青睐.目的:体外分离纯化大鼠骨髓间充质干细胞,观察不同浓度补肾活血中药含药血清对其增殖的影响.设计:完全随机对照的动物实验.单位:广州中医药大学第一附属医院髋中心.材料:选用健康雄性SD大鼠40只,SPF级,体质量170～180 g,由广州中医药大学实验动物中心提供.实验过程中对动物的处置符合动物伦理学标准.将实验动物按随机数字表分为4组,正常对照组和高、中、低剂量给药组,每组10只.方法:实验于2005-01/03在广州中医药大学实验动物中心完成.采用Percoll密度梯度离心法分离纯化大鼠骨髓间充质干细胞,体外培养,建立大鼠骨髓间充质干细胞培养体系.高、中、低剂量给药组大鼠分别灌胃补肾活血中药4.4,2.2和1.1 g/kg,分别相当临床成人用量的20,10和5倍.正常对照组灌胃纯净水.连续给药1周.末次给药1 h后,无菌条件下,腹主动脉取血6 mL/只,2 000 r/min离心15 min,收集血清.高、中、低剂量给药组用10%大鼠含药血清代替10%的牛血清,正常对照组的培养基加入10%的血清.每天观察并记录细胞生长情况,绘制生长曲线.主要观察指标:①骨髓间充质干细胞的生长状况.②HE染色和吉姆萨染色观察骨髓间充质干细胞形态.③免疫细胞化学法检测骨髓间充质干细胞的抗原表达.④不同浓度含药血清对骨髓间充质干细胞生长情况的影响.结果:①骨髓间充质干细胞的生长状况:原代培养的骨髓细胞24 h后开始贴壁,7 d后可达80%融合.②骨髓间充质干细胞的一般形态:骨髓间充质干细胞的细胞多成长梭形或多角形,细胞体积小,胞核位于胞中或稍偏,核浆比略大.③髓间充质干细胞的抗原表达:单个核细胞的胞浆中有CD44表达,着蓝色,部分骨髓间充质干细胞表达c-Kit,在表型表达和细胞形态上具备骨髓间充质干细胞特征.④补肾活血中药对骨髓间充质干细胞生长曲线的影响:高、中、低剂量给药组在加入补肾活血中药血清3 d后,骨髓间充质干细胞数量比空白对照组明显增多,并与剂量呈正比.结论:补肾活血中药含药血清能促进骨髓间充质干细胞的体外增殖.
배경:골수간충질간세포인기획취용역、창상미소、구유무한확증화다분화잠능、자체이식불존재면역배척반응이수도의학연구자적청래.목적:체외분리순화대서골수간충질간세포,관찰불동농도보신활혈중약함약혈청대기증식적영향.설계:완전수궤대조적동물실험.단위:엄주중의약대학제일부속의원관중심.재료:선용건강웅성SD대서40지,SPF급,체질량170～180 g,유엄주중의약대학실험동물중심제공.실험과정중대동물적처치부합동물윤리학표준.장실험동물안수궤수자표분위4조,정상대조조화고、중、저제량급약조,매조10지.방법:실험우2005-01/03재엄주중의약대학실험동물중심완성.채용Percoll밀도제도리심법분리순화대서골수간충질간세포,체외배양,건립대서골수간충질간세포배양체계.고、중、저제량급약조대서분별관위보신활혈중약4.4,2.2화1.1 g/kg,분별상당림상성인용량적20,10화5배.정상대조조관위순정수.련속급약1주.말차급약1 h후,무균조건하,복주동맥취혈6 mL/지,2 000 r/min리심15 min,수집혈청.고、중、저제량급약조용10%대서함약혈청대체10%적우혈청,정상대조조적배양기가입10%적혈청.매천관찰병기록세포생장정황,회제생장곡선.주요관찰지표:①골수간충질간세포적생장상황.②HE염색화길모살염색관찰골수간충질간세포형태.③면역세포화학법검측골수간충질간세포적항원표체.④불동농도함약혈청대골수간충질간세포생장정황적영향.결과:①골수간충질간세포적생장상황:원대배양적골수세포24 h후개시첩벽,7 d후가체80%융합.②골수간충질간세포적일반형태:골수간충질간세포적세포다성장사형혹다각형,세포체적소,포핵위우포중혹초편,핵장비략대.③수간충질간세포적항원표체:단개핵세포적포장중유CD44표체,착람색,부분골수간충질간세포표체c-Kit,재표형표체화세포형태상구비골수간충질간세포특정.④보신활혈중약대골수간충질간세포생장곡선적영향:고、중、저제량급약조재가입보신활혈중약혈청3 d후,골수간충질간세포수량비공백대조조명현증다,병여제량정정비.결론:보신활혈중약함약혈청능촉진골수간충질간세포적체외증식.
BACKGROUND: Bone marrow mesenchymal stem cells (MSCs) have been widely accepted by medical investigators due to their advantages including easy obtaining, minimal invasion, with infinite proliferation and multi-differential potential, and without immunological rejection in the autologous transplantation. OBJECTIVE: The goal of this study is to isolate and purify rat bone marrow MSCs in vitro, so as to observe the effects of different concentrations of serum containing kidney-tonifying and blood-activating Chinese herbs on the in vitro proliferation of rat bone marrow MSCs.DESIGN: A randomized controlled animal experiment.SETTING: Hip Center, First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine.MATERIALS: Forty healthy male SD rats of SPF grade, weighing 170 to 180 g, were provided by the Laboratory Animal Center, Guangzhou University of Traditional Chinese Medicine. The protocol was performed in accordance with ethics guidelines for the use and care of animals. The involved rats were divided into 4 groups by random digit table with 10 rats in each: normal control group, high-, middle-, and low-concentration groups. METHODS: This study was performed at the Laboratory Animal Center, Guangzhou University of Traditional Chinese Medicine between January and March 2005. Rat bone marrow MSCs were isolated and purified by Percoll density gradient centrifugation, and cultured in vitro to establish rat bone marrow MSCs culture system. Rats in the high-, middle-, and low-concentration groups were intragastrically administrated with 4.4, 2.2 and 1.1 g/kg serum containing kidney-tonifying and blood-activating Chinese herbs, which equaled to 20, 10 and 5 times of adult dosage, respectively. Rats in the normal control group were intragastrically administrated with purified water for 1 week. One hour after the last administration, 6 mL blood was taken from abdominal aorta of each rat under the aseptic condition. Then, it was centrifuged at 2 000 r/min for 15 minutes, and meanwhile serum was collected. 10% rat serum containing kidney-tonifying and blood-activating Chinese herbs was added to the medium in the high-, middle-, and low-concentration groups, while 10% fetal bovine serum was added in the normal control group. MAIN OUTCOME MEASURES: ① MSCs growth status; ② MSCs morphology was observed by HE staining and Giemsa's staining; ③ MSCs antigen expression was detected by an immunocytochemical method; ④ Effects of different concentrations of serum containing kidney-tonifying and blood-activating Chinese herbs on MSCs growth.RESULTS: ①The primarily cultured bone marrow MSCs began to adhere to the wall 24 hours later and 80% of them reached the confluence 7 days later. ② MSCs took appearance in long shuttle shape or polygon. These cells were little. Nuclei were located in the middle part of cells or a little deviation. The ratio of nucleus to cytoplasm was a little high. ③CD44 expression was found in the cytoplasm of mononuclear cells, and colored blue. Partial MSCs expressed c-Kit. Their cytomorphology and phenotypic expression have the characteristics of MSCs. ④Three days after serum containing kidney-tonifying and blood-activating Chinese herbal medicine being added to high-, middle-, and low-concentration groups, the number of bone marrow MSCs was dose-dependently increased as compared with that in the normal control group. CONCLUSION: Serum containing kidney-tonifying and blood-activating Chinese herbs promotes the in vitro proliferation of bone marrow MSCs.