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.

INTRODUCTION

Bone marrow mesenchymal stem cells (MSCs) are a kind of heterogenic cell mass. They are composed of cells, which are in the different stages of differentiation. How to perform in vitro proliferation and oriented differentiation of bone marrow MSCs, and then use them in various tissue repairs in clinic by tissue engineering technique has become a major problem in tissue engineering [1-5]. It has been confirmed that MSCs are the precursor cells of osteoblasts, chondroblasts and other many mesenchymal cells. After being in vitro proliferated, induced and differentiated, MSCs are used for autotransplantation without immunogenicity and the constraint of major histocompatibility complex (MHC).
This study was to investigate the effect of serum containing kidney-tonifying and blood-activating Chinese herbs on the in vitro proliferation of rat bone marrow mesenchymal stem cells.

MATERIALS AND METHODS

Materials
Experimental animals
This study was carried out in the Laboratory Animal Center, Guangzhou University of Traditional Chinese Medicine between January and March 2005. Forty healthy male SD rats of SPF grade, weighing 170-180 g, were provided by the Laboratory Animal Center, Guangzhou University of Traditional Chinese Medicine [Permission No. SCXK (yue) 2005-0001]. The rats were raised in the laboratory of SPF grade for 1 week and then used for the

experiment. The protocol was carried out in accordance with animal ethics guidelines for the use and care of animals.

Grouping
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.

Chinese herbs
Chinese herbs consisting of Shudi, Duzong, Buguzhi, Chuanniuxi, Mugua, Gouqizi, Danshen, Guangmuxiang, Honghua and Chuanxiong can tonify kidney to strength the bone, and dredge meridians and activate collaterals to relieve pain. So it was used for the treatment of osteoarthritis with kidney deficiency and blood stasis.

Main reagents and instruments
Percoll(Pharmacia, France, Lot No. 17089101), LDMEM(Gibco BRL Company, USA, Lot No. 1112422), fetal bovine serum (defined, Hyclone, USA, Lot No. AMC15819), Matrigel (BD Biosciences, USA, Lot No. 008092), 5-bromodeoxyuridineI (Boehringer Company, Germany, Lot No. 280879) were used in this study. Mouse anti-rat TH monoclonal antibody (Sigma Company), with working concentration of 1:1 000, diluted by antibody dilution, mouse anti-5 bromodeoxyuridine primary antibody (Sigma Company), mouse anti-CD34 primary antibody, mouse anti-c-kit primary antibody, ready-to-use SABC kit, DAB coloring kit (Boster Bioengineering Co., Ltd, Wuhan, China) were used in this study.
High-speed refrigerated centrifuge (SIGMA, 3K-15, Germany), CO2 incubator (Forma, USA), superclean bench (Suzhou Decontamination Equipment Factory) and an inverted microscope (Leica, Germany) were used.

Methods
Preparation of Chinese herbs
Shudi, Duzong, Buguzhi, Chuanniuxi, Mugua, Gouqizi, Danshen, Guangmuxiang, Honghua and Chuanxiong were added into 20 times of water and decocted twice, 1.5 hours once. Then, the mixture was filtered. Filtrate was evaporated on a thin film until extractum with the relative concentration of 1.30-1.40 (80 ℃) and made into particles with 6.32 g crude drug in each gram.

Isolation and purification of MSCs
Each rat was anesthetized by intraperitoneal injection of 40 mg/kg pentobarbital sodium. The parts below the waist were soaked in 75% ethanol. Five minutes later, the rat was fixed on the operation bench in dorsal position. The skin of unilateral thigh was dissected. Muscle layer was isolated to expose the distal femur. Under the aseptic condition, bone marrow was taken and anticoagulated by heparin, then diluted with LDMEM medium supplemented with 0.01 U/L heparin until 10 mL. The dissected skin of rat was sutured. The mixture was suspended into mononuclear cells, and centrifugated at 2 000 r/min for 20 minutes. Supernatant fluid was taken and added by 40%, 70% and 80% percoll test solution, separately. Each kind of mixture was centrifuged at 1 500 r/min for 10 minutes. 70%-80% percoll test solution was taken, diluted with 10% serum-α-MEM medium, suspended, counted and inoculated to 24-well plate at 1.5×108/L, 1 mL each well. Subsequently, the 24-well plate was incubated at 37 ℃ under 5% CO2-air and saturated humidity. When cells adhered to the wall 24 hours later, medium was changed for the first time, and then it was changed once every 7 days.
When approximately 80% confluence was reached, cells conducted passage. 2.5 g/L trypsase was used for digestion. When intracellular space was enlarged and cells were retracted, 10% serum-α-MEM medium was used to terminate the digestion. Under the microscope, cell solution was collected, centrifuged at 1 500 r/min for 10 minutes, made into cell suspension, and inoculated onto 24-well plate and Matrigel-coated 6-well plate with cover slip at 1×107/L for proliferation culture. Finally, cell growth was observed.

Determining MSC morphology by haematoxylin-
eosin (HE) staining and Giemsa staining
HE staining: Cover slip with cells was fixed with 100 g/L formalin for 30 minutes, washed with distilled water, stained with haematoxylin for 8 minutes, decolorized with hydrochloric acid-70% ethanol for 1 minute, soaked in 1 g/L NaHCO3, washed with water, stained with eosin for 1 minute, dehydrated, cleared and mounted with neutral balsam. Giemsa staining: Cover slip with cells was fixed with 100 g/L formalin for 10 minutes, placed in the dye vat containing Giemsa stain, washed with distilled water, dried, cleared and mounted with neutral balsam.

MSC antigen expression detected by immunocytochemical method
Cover slip with cells was stained by SP method, fixed with 40 g/L paraformaldehyde for 30 minutes, enveloped with 30 g/L H2O2 for 10 minutes and with goat serum for 10 minutes, performed CD34, CD44 and c-Kit staining separately according to the instruction from kit, developed with DAB and enveloped with neutral balsam.

Preparation of serum containing kidney-tonifying and blood-activating Chinese herbs
Rats in the high-, middle-, and low-concentration groups were respectively intragastrically administrated with kidney-tonifying and blood-activating Chinese herbs 4.4, 2.2 and 1.1 g/kg, which was 20, 10 and 5 times of adult dose, respectively. Rats in the normal control group were intragastrically administrated with purified water. Each rat was administrated for 1 week persistently. One hour after the last administration, under the aseptic condition, 6 mL blood was taken from abdominal aorta and centrifuged at 2 000 r/min for 15 minutes. Serum was collected, inactivated for 30 minutes at 56 ℃ and preserved at -80 ℃ for later use.

Effect of serum containing Chinese herbs on the growth of bone marrow MSCs
Twenty-four hours after the third generation of MSCs adhering to the wall, 10% serum containing Chinese herbs was used to replace 10% serum in the high-, middle-, and low-concentration groups. 10% serum added into the medium of normal control group. Cell growth was daily observed and recorded. Three wells were selected from each plate. 2.5 g/L trypsase was added in each well for cell count and drawing cell growth curve.

Statistical analysis
Statistical processing was performed by the Department of Statistics of Guangzhou University of Traditional Chinese Medicine with SAS 6.12 software. Chi square test was used for the comparison of difference of enumeration data, t test for measurement data, Ridit test for semi-quantitative data, and Grubbs for choosing questionable abnormal data.

RESULTS

Growth status of MSCs
MSCs cultured primarily began to adhere to the wall 24 hours later (Figure 1a). Adhered cells took spherical and different sizes of appearance. Nucleus could not be identified. Two days later, the adhered cells took polygon and short fusiform, with clear cytoplasm boundary and strong refraction. Generally, nucleus was located in the center of intumescent cell body. Most cells had one nucleolus. Irregular prominences appeared in the cell body. The adhered cells grew fast, and reached 80% confluence on about 7 days (Figure 1b).
 

General morphology of MSCs
Most MSCs took long fusiform or polygon, with small volume, nucleus being in or slightly partial away the center, and a higher karyoplasmic ratio (Figure 2).

MSC antigen expression
MSCs scarcely expressed hematopoietic cell surface marker antigen CD34 (Figure 3a). Blue-stained CD 44 expression was found in the cytoplasm of mononuclear cell (Figure 3b). Partial MSCs expressed c-Kit (Figure 3c), and blue-stained cells accounted for about 40%.

Effect of serum containing Chinese herbs on the growth of bone marrow MSCs (Figure 4)
Three days after serum containing Chinese herbs being added, bone marrow MSCs were obviously increased in dose-dependent manner in comparison with normal control group.

DISCUSSION

Cartilages are the natural seed cells for repairing cartilaginous tissue [6]. However, they have the disadvantages of rare source, progressive dedifferentiation occurred in the process of in vitro culture and proliferation, loss of cartilage exosyndrome, injury of donor site, etc. Bone marrow MSCs have a very strong capacity of self-duplication and can produce daughter cells with various phenotypes, and under different inducing conditions, they can form bone, cartilaginous tissue, fat, muscle, tendon connective tissue, nerve cells and so on [7-8]. Because of easy source, slight injury and easy-to-use in clinical practice, etc, bone marrow MSC is becoming a preferred seed cell in the cartilaginous tissue engineering[9]. There is only one bone marrow MSC in every 1×105 karyocytes from adult bone marrow. With aging, the number of bone marrow MSC is gradually decreased. Under the physiological state, 20% were quiescent cells.
MSCs can be cultured and proliferated in vitro, stably propagate and have the multi-directional differentiation potential. They are very ideal seed cells for cell transplantation. Nevertheless, they are very few in the bone marrow (one MSC in every about 1×105 karyocytes). Only by proliferation can they be used in the in vitro transplantation. Amplification condition should be controlled strictly in the in vitro culture of MSCs because of their multi-directional differentiation potential. It is reported that leukemia inhibitory factor (LIF), and other factors should be added in aforementioned in vitro culture[10]. In this study, we modified the method reported in the literature by omitting LIF and other expensive cytokines, and found that MSCs basically kept stem cell phenotype and in vitro propagated many times, without losing multi-potential transformation activity. After 15 generations of propagation, the number of MSCs was about 2×107, which meet the need for cell transplantation and in vitro induction and differentiation.
MSCs can express the surface markers of mesenchymal, endothelial and epidermal cells, and can differentiate into myocardial cells, hepatocytes, adipocytes, vascular endothelial cells, skin epithelial cells and so on [11]. It is generally accepted that integrin family member CD29, adhesion molecule CD44, CD166, CD105 and CD106 are the important markers of MSCs. MSCs do not express the surface antigens of hematopoietic cells, such as hematopoietic precursor cell marker antigen CD34, adult hematopoietic cell marker antigen CD38, leukocyte marker antigen CD45, lymphocyte surface antigen CD11a and monocyte/macrophage surface antigen CD14 [12-14]. MSCs can also express adhesion factors VLA-4 and P-selectin, which play important roles in the adhesion process of MSCs and endothelial cells [15-16]. Our study findings demonstrated that MSCs expressed important marker CD44, but not CD34. In this study, partial MSCs expressed c-kit. It has been reported in the literature that stem cells express c-kit, and the interaction of kit and stem cell factor as well as other cytokines are beneficial to the maintenance of stem cell phenotype [17-18]. It is demonstrated that the culture conditions used in this study contribute to the maintenance of stem cell phemotype of MSCs.
In this study, we isolated and purified rat bone marrow stromal cells. The harvested cells were given HE and Giemsa staining to determine the morphology of MSCs. Most MSCs took long fusiform or polygon, with small volume, nucleus being in or slightly partial away the center, and a higher karyoplasmic ratio. We detected the antigen expression of MSCs by immunocytochemical method. And we found that blue-stained CD 44 expression existed in the cytoplasm of mononuclear cells, partial MSCs expressed c-Kit, and blue-stained cells accounted for about 40%. Meanwhile, we also observed the effect of serum containing kidney-tonifying and blood-activating Chinese herbs on the growth of bone marrow mesenchymal stem cells. Experimental findings showed that MSCs could keep the features of stem cells and massively proliferate. Three days after serum containing Chinese herbs being added, bone marrow MSCs were obviously increased in dose-dependent manner in comparison with normal control group. Therefore, we could therefore assume that serum containing kidney-tonifying and blood-activating Chinese herbs possibly promoted the proliferation of rat bone marrow MSCs, and its action mechanism was possibly in relation to cytokine-like effects. This study seems a promising experiment for solving the problems of adult MSC amount, of development of Chinese herbs, and of Chinese herbal medicine combined with MSCs for the possible treatment of osteoarthritis.

REFERENCES

1 Schagemann JC, Mrosek EH, Landers R, et al. Morphology and function of ovine articular cartilage chondrocytes in 3-d hydrogel culture. Cells Tissues Organs 2006;182(2):89-97
2 Emans PJ, Hulsbosch M, Wetzels GM, et al. Repair of osteochondral defects in rabbits with ectopically produced cartilage. Tissue Eng 2005; 11(11-12):1789-1796
3 Song L, Baksh D, Tuan RS. Mesenchymal stem cell-based cartilage tissue engineering: cells, scaffold and biology. Cytotherapy 2004;6(6): 596-601
4 Naumann A, Dennis JE, Aigner J, et al. Tissue engineering of autologous cartilage grafts in three-dimensional in vitro macroaggregate culture system. Tissue Eng 2004;10(11-12):1695-1706
5 Bean AC, Almarza AJ, Athanasiou KA. Effects of ascorbic acid concentration on the tissue engineering of the temporomandibular joint disc. Proc Inst Mech Eng [H] 2006;220(3):439-447
6 Freed LE, Grande DA, Lingbin Z, et al. Joint resurfacing using allograft chondrocytes and synthetic biodegradable polymer scaffolds. J Biomed Mater Res 1994;28(8):891-899
7 Schwartz RE, Reyes M, Koodie L, et al. Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells. J Clin Invest 2002;109(10):1291-1302
8 Reyes M, Lund T, Lenvik T, et al. Purification and ex vivo expansion of postnatal human marrow mesodermal progenitor cells. Blood 2001; 98(9):2615-2625
9 Grande DA, Halberstadt C, Naughton G, et al. Evaluation of matrix scaffolds for tissue engineering of articular cartilage grafts. J Biomed Mater Res 1997;34(2):211-220
10 Bianco P, Riminucci M, Gronthos S, et al. Bone marrow stromal stem cells: nature, biology, and potential applications. Stem Cells 2001;19(3): 180-192
11 Baksh D, Song L, Tuan RS. Adult mesenchymal stem cells: characterization, differentiation, and application in cell and gene therapy. J Cell Mol Med 2004;8(3):301-316
12 Lazarus HM, Koc ON, Devine SM, et al. Cotransplantation of HLA-identical sibling culture-expanded mesenchymal stem cells and hematopoietic stem cells in hematologic malignancy patients. Biol Blood Marrow Transplant 2005;11(5):389-398
13 Zhao Y, Chen E, Li L, et al. Gene expression profiling in the inductive human hematopoietic microenvironment. Biochem Biophys Res Commun 2004;323(2):703-711
14 Monga SP, Tang Y, Candotti F, et al. Expansion of hepatic and hematopoietic stem cells utilizing mouse embryonic liver explants. Cell Transplant 2001;10(1):81-89
15 Segers VF, Van Riet I, Andries LJ, et al. Mesenchymal stem cell adhesion to cardiac microvascular endothelium: activators and mechanisms. Am J Physiol Heart Circ Physiol 2006;290(4): H1370-H1377
16 Rüster B, G ttig S, Ludwig RJ, et al. Mesenchymal stem cells display coordinated rolling and adhesion behavior on endothelial cells. Blood 2006;108(12):3938-3944
17 Crosby HA, Kelly DA, Strain AJ. Human hepatic stem-like cells isolated using c-kit or CD34 can differentiate into biliary epithelium. Gastroenterology 2001;120(2):534-544
18 Zhang JT. Modern Methodology in Pharmacological Experiment. Beijing: Associated Press of Peking Medical University and Peking Union Medical College 1998:1337-1340

补肾活血中药对大鼠骨髓间充质干细胞
体外增殖的影响*☆

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）*

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

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）