摘要
目的观察GSB中药血清对大鼠MSCs定向诱导分化成骨的影响。方法将培养的rMSCs分为对照组（C组）[含10%胎牛血清,100U/ml青霉素和100μg/ml链霉素的完全培养液]、诱导组（O组）[完全培养液,加诱导培养液（0.1μmol/L地塞米松、10mmol/Lβ-甘油磷酸钠及50μg/ml维生素C）,加20%空白血清]和含药血清诱导组（G组）[完全培养液加诱导培养液诱导培养液中加入20%含药血清）。显微镜下观察细胞形态变化,生化法测定上清中Ca^2＋浓度,Gomori改良钙钴法进行ALP染色,Von Kossa法进行矿化结节染色,RT-PCR法测定Ⅰ型胶原（COLL1）和脂蛋白脂酶（LPL）的表达,放免法测定上清中骨钙素含量。结果O组和G组在加入诱导培养液3-4天后梭形细胞比例减少,细胞变形成多角形、乃至方形,部分区域细胞成多层生长;改良Gomori钙钻法碱性磷酸酶染色阳性细胞,Von Kassa染色发现钙盐沉积;培养9d后细胞Ca^2＋浓度开始升高,并随时间持续显著增长,且G组明显高于O组。自第15d后,O组和G组细胞ALP活性随时间延长而明显增高,且G组显著高于O组;培养的第14d,G组骨钙素分泌量明显高于O组。成骨诱导剂作用MSCs12d后,COLLⅠ mRNA表达阳性,LPL mRNA表达阴性,且G组比O组COLLⅠ mRNA表达更强。结论补肾填精中药GSB促进骨钙素分泌、增强ALP活性、促钙盐沉积,具有增强成骨诱导剂诱导rMSCs向成骨细胞分化,并促进成骨细胞成熟作用。
Objective To study the tonifying kidney and benefiting essence of life by the traditional Chinese medicine, we investigate the effect of serum containing GSB-extract（GSB serum） on proliferation and differentiation of rat mesenchymal stem cells （rMSCs）. Methods rMSCs derived from the fourth passages were incubated in LDMEM supplemented with different concentration of GSB serum（5%, 10% and 20% ）. Expanded cells were detected by MTT, to confirm appropriate serum concentration to be used. Subsequently, cultured cells at passage 4 were nontreated in LDMEM medium with 10% FBS（as a control, C group） or treated in LDMEM medium with 20% rat serum with（G group） or without（O group） experimental recipe-GSB extracted. The mRNA levels of type Ⅰ collagen and lipoprotein lipase were measured by RT-PCR. Results rMSCs changed from a spindle shape to a polygonal appearance, ALP staining and von Kossa staining were positive in both O group and G group while the controls were not. The ALP activity in every group of every period of time had significant difference. The ALP activity of O group and G group were higher than that of C group on fifth day; there＇re significant differences between O group and C group, G and C group on fifth and tenth day, no difference between G and O group. There＇re significant differences between C group and each other group only in the first twenty days, and there＇re significant differences between O group and G group in every period of time. The concentration of Ca^2＋ was significantly higher in GSB serum group than that of control group after rMSCs were incubated for 9 days. There＇re no clear differences between the three groups on third and sixth day. There＇re significant differences between C group and each other group only in the first twenty one days. However there＇re significant differences between O group and G group after fifteen days. There＇re significant differences between the three groups on the fourteenth and twenty first day on the content of osteocalcin. The OCN content of O and G groups are higher than C group. The OCN content of G group is higher than that of O group on the fourteenth and twenty first day with significant difference. The mRNA level of type Ⅰ collage was measured after rMSCs were induced for 12 days, higher level in G group than in C group. The expression of lipoprotein lipase was not measured. Conclusions GSB serum may promote proliferation and osteogenic differentiation of rMSCs. Experimental recipe-GSB possesses to manufacture the essence.
出处
《中国骨质疏松杂志》
CAS
CSCD
2007年第7期480-485,共6页
Chinese Journal of Osteoporosis
关键词
含药血清
间充质干细胞
成骨细胞
分化
Serum containing GSB-extract （GSB serum ）
Marrow mesenchymal stem cells
Osteoblast
Differentiation
