摘要
背景：研究发现骨质疏松症与骨髓间充质干细胞的分化关系密切，随着年龄的增加，骨髓间充质干细胞绝对含量下降，向成骨分化能力减弱，向成脂分化能力增强。目的：在成脂诱导条件下，观察中药骨康含药血清对体外培养的大鼠骨髓间充质干细胞分化特性的影响。设计、时间及地点：细胞学体外观察，于2007—01／08在广州中医药大学附属骨伤科医院细胞室完成。材料：清洁级6月龄新西兰大白兔4只，SPF级2月龄SD大鼠5只，购自广州中医药大学动物实验中心。中药骨康方的主要成分为丹参、补骨脂、淫羊藿等，含生药量1．43g／mL，由广州中医药大学附属骨伤科医院制剂室提供。方法：2只兔按4．8g／kg给予生药灌胃制备含药血清，相当于临床剂量的10倍，2次／d，每次间隔5h，连续灌胃7d；另2只兔给予等体积生理盐水灌胃。分别于最后1次给药后2h行心脏采血，离心后取上清，-20℃保存备用。密度梯度离心＋贴壁法分离培养大鼠骨髓间充质干细胞，膑蛋白酶扩增纯化，将传至第4代的骨髓间充质千细胞按2×10^8L^-1接种，加入成脂诱导液的同时，骨康组加入上述含药血清，对照组加入空白血清，均调整浓度为10％。主要观察指标：流式细胞仪检测细胞表面标志，油红O染色观察脂滴形成情况，计数脂肪细胞转化率。结果：第2代骨髓间充质干细胞均一性〉90％，CD44呈阳性表达，CD34、CD45呈阴性表达。培养第4，7，10天后，两组细胞中均含大小不等的橙红色脂肪滴，细胞核被脂滴挤于细胞一侧，骨康组脂肪细胞转化率均明显低于空白组（F=0．025—0，037，P〈0．05）。结论：中药骨康含药血清可抑制成脂诱导剂对骨髓间充质干细胞向脂肪细胞分化的促进作用。
BACKGROUND： Studies have demonstrated that osteoporosis is closely related to bone marrow mesenchymal stem cell （BMSC） differentiation. Absolute content of BMSCs decreases with aging, and osteogenic ability decreases but adipogenic ability increases. OBJECTIVE： To explore the effects of Gukang-conditioned serum on BMSC differentiation under adipogenic induction. DESIGN, TIME AND SETTING： In vitro observation of cytology was performed at the Cell Laboratory, Orthopedics and Trauma Hospital, Guangzhou University of Traditional Chinese Medicine from January to August 2007. MATERIALS： Four 6-month-old New Zealand rabbits and five 2-month-old SD rats were provided by the Animal Experimental Center of Guangzhou University of Traditional Chinese Medicine. Chinese herb Gukang included Danshen, Buguzhi, and Finyanghuo with crude drug of 1.43 g/mL and was provided by Manufacturing Laboratory of Orthopedics and Trauma Hospital, Guangzhou University of Traditional Chinese Medicine. METHODS： Two rabbits were infused with crude drug at dose of 4.8 g/kg （10 times of clinical dose） to prepare drug-conditioned serum, twice a day, 5 hours interval for 7 days. The other rabbits were infused with the same volume of normal saline. Blood was harvested from the heart 2 hours following the last medication. The superuatant was obtained after centrifugalization, and stored at -20 ℃. MSCs were isolated from normal rats marrow using density gradient centrifugation and attachment method, purified by trypsin, The 4th passage of MSCs were incubated in adipogenic induction at density of 2×10^8 L^-1. Then the Gukang group was added drug-conditioned serum, while the control group added serum at concentration of 10%. MAIN OUTCOME MEASURES： Cell surface marker was detected using flow cytometry; lipid droplet formation was observed using Oil Red O staining to count adipocyte transformation rate. RESULT： The homogenicity of the second passage of MSCs was 〉 90%, and cells were CD44 positive, and CD34 and CD45 negative. Formation of lipid droplets was found in two groups, and nucleus moved to lateral of the cells. Adipocyte transformation rate in Gukang group was significantly lower than the control group （F=0.025 0.037, P 〈 0.05）. CONCLUSION： Chinese herb Gukang-conditioned serum could inhibit the induction of adipocytes from MSCs.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2008年第43期8434-8438,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
