【作者】 徐无忌；

【导师】 庄洪；

【作者基本信息】 广州中医药大学 ， 中医骨伤科学， 2008， 博士

【摘要】 实验研究目的:1.在成脂诱导的条件下,观察骨康含药血清对体外培养的大鼠骨髓基质干细胞的分化特性的影响;2.在与成骨细胞共培养的条件下,观察大鼠骨髓基质干细胞的成骨特性以及骨形态发生蛋白-2在其中的作用;3.在与成骨细胞共培养的条件下,采用中药血清学方法,观察骨康含药血清联合共培养对大鼠骨髓基质干细胞的成骨分化特性以及骨形态发生蛋白-2在其中的作用。方法:1.分离,鉴定并扩增MSCs,传至第四代时分为空白组与骨康组,均以成脂诱导液培养,骨康组中加入含药血清,对照组中加入空白兔血清,倒置相差显微镜下观察,培养第4天、7天、10天后行油红O染色,并计数脂肪细胞转化率。2.以淋巴细胞分离液自2月龄大鼠骨髓中分离骨髓基质干细胞,扩增后以免疫组化法鉴定其表型,成骨细胞取自1日龄乳鼠,酶消化法获得,扩增后用Gomori’s法行骨碱性磷酸酶染色以及茜素红染色钙结节进行鉴定。骨髓基质干细胞消化后分为A组:MSCs单独培养组,未进行共培养,培养液为完全培养基;B组:MSCs+雌激素含药血清组,培养液为完全培养基和雌激素含药血清;C组:MSCs+骨康含药血清组,培养液为完全培养基和骨康含药血清;D组:共培养组,培养液为完全培养基;E组:空白血清+共培养组,培养液为完全培养基和空白组兔血清;F组:雌激素含药血清+共培养组,培养液为完全培养基和雌激素含药血清;G组:骨康含药血清+共培养组,培养液为完全培养基和骨康含药血清。每3d换液1次。观察骨髓基质干细胞的形态学变化及矿化能力,酶联免疫吸附法检培养上清中骨形态发生蛋白-2、骨碱性磷酸酶以及骨钙素含量。3.所有数据均使用SPSS11.5进行统计处理。结果:1.随着诱导时间的延长,脂肪细胞转化率呈上升趋势;2.骨康组脂滴出现较空白组晚,第4天、7天、10天空白组中脂肪细胞转化率分别为24.31+12.53%、39.19±15.64%、50.13±15.13%,骨康组中则分别为11.73±9.09%、20.95±8.05%、30.73±9.52%,不同时相的两组之间比较差异有统计学意义(P＜0.05);3.上清中OC含量A组与C组无显著性差异(P=0.229),A组与B组比较有显著性差异(P＜0.05),D、E组与A组比较有非常显著性差异(P＜0.01),二者之间差异无统计学意义(P＞0.05),F组、G组与B、C、D组比较差异有统计学意义(P＜0.01),二者之间差异无统计学意义(P＞0.05),各组上清中B-ALP含量变化规律类似于OC,与之不同的是,A组与C组有显著性差异(P＜0.05);4.MSCs培养形成钙化结节数A组与B、C组比较以及B、C两组之间比较,有显著性差异(P＜0.05),B、D、E组与A组比较有非常显著性差异(P＜0.01),三者之间差异无统计学意义(P＞0.05),F组、G组与B、C、D、E组比较差异有统计学意义(P＜0.01),二者之间差异无统计学意义(P＞0.05)。经Spearman相关分析,上清中BMP-2浓度与钙化结节形成数之间存在正相关,相关系数γP=0.923,P=0.000。结论:1.随着诱导时间的延长,脂肪细胞转化率呈上升趋势;2.骨康含药血清可抑制成脂诱导剂对MSCs向脂肪细胞分化的促进作用,这可能是骨康治疗骨质疏松作用的机制之一;3.体外构建的共培养模型部分模拟了体内成骨环境;与成骨细胞共培养的骨髓基质干细胞成骨能力增强;4.骨康及雌激素含药血清联合共培养时,其成骨分化能力强于单一的干预因素施加;5.上清中BMP-2浓度与钙化结节形成数之间存在正相关,BMP-2浓度越高,钙化结节形成数量越多。临床研究目的:探讨绝经后骨质疏松症妇女血清BMP-2水平与腰椎侧位骨密度以及绝境年限的关系。方法:选择经骨密度测定及临床检查确诊为绝经后骨质疏松症患者88例,另外10名20-30岁未婚健康青年女性为对照组。对骨质疏松组的患者按照绝经年份进行分组,每5年为一个等级。测定血清中BMP-2水平,探讨绝经期后骨质疏松症患者绝经年限、骨量与BMP-2的相关性。结果:绝经后骨质疏松症妇女各组血清BMP-2水平与对照组相比较差异有统计学意义(P＜0.01),各绝经年限分组之间血清BMP-2水平差异无统计学意义(P＞0.05),但随着绝境年限增长呈现下降趋势。血清BMP-2水平与绝经年限呈负相关,相关系数γP=-0.570,P=0.000。绝经年限与腰椎骨密度之间存在负相关,相关系数γP=-0.471。腰椎骨密度与血清中BMP-2浓度二者之间存在正相关,γP=0.638,P=0.000。结论:1.绝经后骨质疏松症患者骨密度且随着绝经年限的延长而降低,二者存在负相关关系。绝经年限不同,腰椎骨量丢失速度不同,以绝经后2-10年间骨量丢失速度最快;2.绝经后骨质疏松症患者血清中BMP-2浓度明显较健康青年女性下降,与绝经年限存在负相关;3.腰椎骨密度与血清中BMP-2浓度存在正相关。随着妇女绝经的发生,BMP-2表达下调可能是绝经后骨质疏松症发生的原因之一。更多还原

【Abstract】 Experimental StudyObjective1.In order to study the effects of gukang on the differentiation of mesenchymal stem cells into adipocytes in vitro;2.To investigate osteogenic characteristics of rat bone marrow mesenchymal stem cells(MSCs)co-cultured with osteoblast(OB)indirectlyand effect of bone morphogenetic protein 2(BMP-2)in osteoblastic differentiation of MSCs;3.To observe the influence of pharmacal serum of GuKang and Estradiol-2(E-2) to osteogenic characteristics of rat bone MSCs under co-cultured with OB indirectly,and effect of bone morphogenetic protein 2(BMP-2)in osteoblastic differentiation of MSCs.Methods1.MSCs were isolated from 2 months old rats marrow,then separated by lymphocyte separating medium and proliferated in culture medium,detected in cell purity by flow cytometry analyzing cell phenotypes.The 4th passage MSCs were digested with 0.25%trypsin for 3-5 minutes and made into monoplast suspension.Cells were adjusted into 1×10~5 and then inoculated in 6 holes plates,divided into gukang group and control group.All groups were induced to differentiate into adipocytes in induction medium containing desamethasone,insulin and 1-methyl-3-isobutylxanthine.The induction medium of gukang contained the rabbits pharmacal serum of GuKang,and the control group’ s induction medium contained the normal rabbits serum.All groups were observed under microscopic lens.After 4days,7 days and 10days of adipocyte inducing,all groups were stained by Oil Red O;2.MSCs were isolated from normal 2 months old rats marrow,then separated by lymphocyte separating medium and proliferated in culture medium,detected in cell purity by immunohistochemistry method analyzing cell phenotypes. Osteoblast was got from 1 day old rat’ s skull by enzyme digestion method and the cells were observed through inverted microscope.ALP staining was made through Gomori’ s method and alizarin red staining of mineralized nodus to do the functional indentification.MSCs were adjusted into 1×10~5 and then inoculated in plates,divided into 7 groups:A group,MSCs group;B group, MSCs combine with pharmacal serum of E-2;C group,MSCs combine with pharmacal serum of GuKang;D group,MSCs co-cultured with OB group;E group, co-cultured combine with pharmacal serum of control rabit;F group, co-cultured combine with pharmacal serum of E-2;G group,co-cultured combine with pharmacal serum of GuKang;and control group.The change of morphology and mineralization ability of bone marrow MSCs were observed,and the contents of BMP-2 and bone alkaline phosphatase(BALP)and osteocalein(OC)in supernatant fluid was detected with enzyme-linked immunosorbent assay(ELISA) method.And the correlation between activity of mineralization ability and contents of BMP-2 in supernatant fluid was analyzed.3.All the data was processed and analyzed with SPSS11.5.Results1.The formation of lipid drop lets was in time-dependent manner;2.The formation of lipid drop lets in gukang groups were retarded,compared with control group.The adipocytes diferentiation rate of the control group at 4days,7 days and 10days were24.31±12.53%、39.19±15.64%、50.13±15.13%, and the rate of the gukang group were 11.73±9.09%、20.95±8.05%、30.73±9.52%,as detected by Red Oil O stain.There were significant difference between two groups in different time(P＜0.05).3.the contents of OC in supernatant fluid of A group and C group was no significant difference(P=0.229);A group and B group was significant difference(P＜0.05);D,E groups had very significant difference compared with A group(P＜0.01),and there had no significant difference between D,E groups(P＞0.05);F,G groups had very significant difference compared with B,C,D groups(P＜0.01),and there had no significant difference between F,G groups(P＞0.05).The change of contents of B-ALP in supernatant fluid of all groups liked OC,A group and C group was significant difference(P＜0.05).4.The activity of mineralization ability of A group and B,C group was significant difference(P＜0.05),B,C group also had significant difference(P＜0.05);B,D,E groups had very significant difference compared with A group(P＜0.01),and there had no significant difference between B,D,E groups(P＞0.05);F,G groups had very significant difference compared with B,C,D,E groups(P＜0.01),and there had no significant difference between F,G groups(P＞0.05).The activity of mineralization ability was positively correlated with BMP-2(γP=0.923,P=0.000).Conclusions1.The formation of lipid drop lets was in time-dependent manner;2.The pharmacal serum of GuKang could inhibit differentiation of rat MSCs leading to differentiation into adipocytes,which maybe one of the mechanisms of GuKang in treatment of osteoporosis;3.The co-cultured model partly imitates osteogenic surroundings in vivo.Osteoblastic differentiation/proliferation of MSCs were improved when co-cultured with OB;4.The pharmacal serum of GuKang or E-2 combine with co-cultured increase the ability of osteoblastic differentiation/proliferation of MSCs.5.The activity of mineralization ability was positively correlated with BMP-2,The higher the contents of BMP-2 in supernatant fluid,the more of the activity of mineralization ability were.The Clinical StudyObjectiveTo observe the correlation between lumbar vertebrae(lateral position), menopausal years,serum BMP-2 in the postmenopausal osteoporosis(PMOP).Methods88 PMOP cases were taken part in the study,other 10 healthy young women whose age ranged from 20 to 30 years old were choosen as control group.88 PMOP cases were derided into 6 groups based on each 5 years of menopausal years.Bone mineral density(BMD)of lumbar vertebrae(lateral position)were tested through dual-photon sbsorptiometry(DXA),and the level of BMP-2 in serum were testd by ELISA method.The correlation between lumbar vertebrae(lateral position),menopausal years,serum BMP-2 in the PMOP who were difined as kidney-yang deficiency were observed. ResultsThe level of BMP-2 in serum of PMOP had significant difference compared with healthy control group(P＜0.01),and was negative correlated with menopause years(γP=-0.570,P=0.000).The level of BMP-2 of 6 groups of PMOP had no significant difference(P＞0.05).The level of BMP-2 was negative correlated with menopause years(γP=-0.471,P=0.000).There was positively correlated between BMD and BMP-2(γP=0.638,P=0.000).Conlusions1.BMD decreased as the durations of menopause increased,there was negative correlated between menopause years and BMD.The bone loss rate in lumbar varies among diferent groups of menopause years,and the rate was fastest in 2-10 years postmenopausal.2.the level of BMP-2 in serum of PMOP had significant difference compared with healthy young women,and was negative correlated with menopause years.3.BMD of lumbar vertebrae was positively correlated with BMP-2.the expression of BMP-2 decreased in the postmenopausal women may be one of the mechanisms of PMOP.更多还原