Abstract



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.

Hip Center, First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine, Guangzhou 510405, Guangdong Province, China

Zeng Yi-rong☆, Doctor, Associate chief physician, Hip Center, First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine, Guangzhou 510405, Guangdong Province, China
zeng6612@163.com

Supported by: the Grant from Guangdong Bureau of Traditional Chinese Medicine, No. 1040015*

Received:2007-08-16
Accepted:2007-09-18
(07-50-8-4446/SHM)

Zeng YR, Fan YG, Liu H, Xia XZ, Fan HJ.Effect of serum containing kidney-tonifying and blood-activating Chinese herbs on the in vitro proliferation of rat bone marrow mesenchymal stem cells.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(8):
1581-1585(China)

[www.zglckf.com/
zglckf/ejournal/
upfiles/08-8/
8k-1581(ps).pdf]

补肾活血中药对大鼠骨髓间充质干细胞
体外增殖的影响*☆

摘要

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。
关键词：骨髓间充质干细胞；补肾活血中药；免疫细胞化学法

曾意荣，樊粤光，刘 红，夏雄智，范海蛟
广州中医药大学第一附属医院髋中心，广东省广州市 510405
曾意荣☆，男，1966年生，江西省上饶市人，汉族，2005年广州中医药大学毕业，博士，副主任医师，主要从事髋膝关节疾病的临床研究。
2004年广东省中医药局基金项目（1040015）*

中图分类号: R394.2 文献标识码: A 文章编号: 1673-8225(2008)08-01581-05
曾意荣，樊粤光，刘红，夏雄智，范海蛟.补肾活血中药对大鼠骨髓间充质干细胞体外增殖的影响[J].中国组织工程研究与临床康复，2008，12(8):1581-1585
[www.zglckf.com/zglckf/ejournal/upfiles/08-8/8k-1581(ps).pdf]
（Edited by Yuan QL/Song LP/Wang L）