【作者】 沈龙祥；

【导师】 陈峥嵘； Julie Glowacki； 周栓虎；

【作者基本信息】 复旦大学 ， 骨外科学， 2009， 博士

【摘要】 第一部分人骨髓基质干细胞体外培养与诱导分化鉴定【目的】利用实验室已建立的hMSCs的培养方案,建立hMSCs的培养体系并做鉴定,为后续的实验研究提供细胞来源。【方法】骨髓样本取自美国波士顿市Brigham and Women’s Hospital骨科因骨关节炎行全髋关节置换手术病人,从手术中分离的股骨头中分离获得。以密度梯度离心法获得单个核细胞,以贴壁分离方法获得hMSCs,在含有10%FBS的α-MEM的培养基中培养和传代扩增。在成骨、成脂肪、成软骨诱导下培养,分别用ALP活性检测、Alizarin Red S染色鉴定成骨细胞分化;利用Oil Red-O染色鉴定脂肪分化;利用Alcian blue染色鉴定软骨细胞分化。【结果】原代培养24h部分细胞开始贴壁伸展变形,48—72h后呈纺锤形或梭形,偶见形成集落,培养5—7d后见贴壁生长的细胞增殖迅速,可见多个细胞集落形成,当培养至第12—14d时,细胞已大部分融合,长满培养皿底,细胞成长梭形(成纤维样外观),有的细胞呈交叉重叠生长。一般5—10d可传一代,细胞可连续传4—10代。成骨细胞诱导分化后可检测到ALP活性,并见Alizarin RedS染色阳性细胞形成。成脂诱导分化后可见不同形状的Oil Red-O阳性染色细胞形成。成软骨细胞诱导分化可见Alcian blue阳性染色细胞形成。【结论】应用本实验方法从骨髓分离、培养和扩增获得hMSCs,具有向成骨细胞、脂肪细胞和软骨细胞分化的能力。第二部分年龄和性别对人骨髓基质干细胞WNT基因表达的影响【目的】已有的研究对于hMSCs的WNT基因表达的描述不相一致,作者推测年龄和性别和其它临床因素的差异可以解释过去的研究显示的WNT基因在hMSCs中表达的不同。利用不同年龄和性别来源的hMSCs,检测WNT基因的表达,建立WNT基因在hMSCs的表达谱。【方法】骨髓样本取自美国波士顿市Brigham and Women’s Hospital骨科行全髋关节置换手术的病人,从手术中分离的股骨头中分离获得。共19例样本来源的hMSCs被列入实验研究,来源包括12例女性和7例男性,年龄为36—85岁。其中9例为年轻样本(＜50岁,平均42.0±4.6岁),10例为老年样本(＞55岁,平均69.3±9.5岁)。12例样本来源女性为37—85岁,平均58.6±16.0岁,7例样本来源男性为36—82岁,平均52.6±16.0岁。将hMSCs在100mm组织培养皿中培养用传代至第2代(P2)和第4代(P4)的细胞进行实验。由于hMSCs来源于不同的个体,他们进行全髋关节置换的手术时间不同,为保证实验条件的一致性,所有的实验均采用标准的条件。hMSCs经体外培养、收集存储于Trizol试剂,冷冻于—80℃。所有19个样本收集齐全后同时进行RNA分离实验。最后采用RT-PCR检测所有WNT基因的表达。【结果】WNT2、3、4、5A、5B、6、7B、10B、11、13、14和16B共12个WNT基因被检测到在hMSCs中表达。相对于年老样本来源的hMSCs,年轻来源者表达WNT7B和WNT14显著升高。除此之外,WNT2和WNT13基因表达在年轻样本来源hMSCs组显示出表达显著升高的趋势。WNT基因表达和年龄相关性分析显示:WNT7B、WNT14和WNT13的表达强度与年龄呈显著反向相关。进一步分析显示,相对于女性来源的hMSCs样本,男性来源的hMSCs中WNT16B表达显著升高。WNT11在女性来源的hMSCs样本中表达呈现出显著性升高的趋势。在女性样本来源的hMSCs,WNT13基因表达与年龄呈现显著反向相关性。WNT4基因表达与年龄呈现显著正向相关性。在男性hMSCs,WNT7B基因表达与年龄呈现显著反向相关性,WNT14表达与年龄呈现反向相关性趋势。【结论】在hMSCs,大部分的老化相关的WNT基因属于经典WNT基因,WNT2、7B、13和14是年龄相关性WNT基因;除此之外,WNT4、7B、13、14和16的表达呈现性别特异性。第三部分WNT信号通路对于hMSCs向脂肪细胞、成骨细胞和软骨细胞分化的影响【目的】已有的采用小鼠脂肪前体细胞研究脂肪分化机制的结果表明,Wnt10b可以抑制脂肪分化并且促进成骨细胞分化,Wnt4和Wnt5可以促进脂肪细胞的分化,Wnt3a可以提高bmp2诱导的小鼠C3H10T1/2细胞向软骨细胞分化的能力。对于WNT信号通路在人脂肪细胞、成骨细胞和软骨细胞分化过程中的作用知之甚少。本实验设想WNT信号通路调节hMSCs向脂肪细胞分化、成骨细胞分化和软骨细胞分化,拟检测经典和非经典WNT信号通路在hMSCs向脂肪细胞分化中的作用,并且采用一个小分子的β-catenin激活剂(SB-216763)模拟经典WNT信号通路,阐述激活经典WNT信号通路对于hMSCs向脂肪细胞、成骨细胞和软骨细胞分化的影响。【方法】骨髓样本取自美国波士顿市Brigham and Women’s Hospital骨科因骨关节炎行全髋关节置换手术病人,经当地医学伦理委员会IRB允许,从手术中分离的股骨头中采用密度梯度离心分离获得。hMSCs在成脂、成骨、成软骨诱导培养基中培养,采用RT-PCR检测脂肪分化相关基因和WNT基因的表达,Western Immunoblotting检测β-catenin的表达,采用一个小分子的β-catenin激活剂SB-216763模拟经典WNT信号通路,检测激活经典WNT信号通路对于hMSCs向脂肪细胞、成骨细胞和软骨细胞分化的影响。【结果】对脂肪细胞的标志基因的检测结果显示,在hMSCs成脂肪诱导1d后,检测到PPARγ2和LPL的基因表达,并随着继续培养而升高。Adipsin的表达随着成脂诱导培养时间的延长亦呈进行性升高。经典WNT家族成员,包括WNT2、10B、13、14表达逐渐降低,同时,非经典WNT家族成员,包括WNT4和11表达逐渐升高。然而,WNT5A的表达基本保持不变。WNT11是唯一的一个在脂肪分化标志基因出现表达升高之前表达改变的WNT基因。SB-216763(5μM)可以升高β-catenin在hMSCs中的表达水平,并且通过阻断PPARγ2、LPL和adipsin的基因表达抑制脂肪分化。与分子水平的作用一致,SB-216763模拟的经典WNT信号通路抑制脂肪细胞分化呈现时间和剂量依赖性。激活经典WNT信号通路可以抑制成骨细胞标志基因ALP的表达,抑制ALP的活性,促进软骨细胞标志基因RUNX2和COLⅡ的表达,促进Ⅱ型胶原的分泌。【结论】在hMSCs向脂肪细胞分化的过程中,非经典WNT信号通路的WNT4和11而非WNT5A可能是主要促进因子,而激活经典WNT信号通路可以阻止hMSCs向脂肪细胞、成骨细胞分化,促进其向软骨细胞分化。本实验研究说明在不同物种间,Wnt信号通路在向脂肪细胞和成骨细胞分化中所起的作用存在不同。第四部分老化和性别对于hMSCs向脂肪细胞分化的影响【目的】本课题组前期的研究发现随着年龄的增加hMSCs发生一系列变化,向成骨细胞分化能力降低。本实验采用不同年龄和性别个体来源的hMSCs进行体外成脂诱导培养,观察其年龄和性别对hMSCs成脂肪细胞分化的影响,以期待揭示Ⅱ型骨质疏松患者骨髓腔脂肪容量增多的机制。【方法】15例hMSCs被列入实验研究,来源包括8例女性和7例男性,年龄为17—90岁,包括5例年轻样本和10例年老样本。在成脂诱导培养18d后检测不同年龄和性别组hMSCs形成脂肪细胞的差异。对于hMSCs脂肪细胞及成骨细胞相关基因表达相关实验研究,共有19例不同样本来源的hMSCs被纳入实验,包括12例女性和7例男性,年龄为36—85岁。其中9例为年轻样本,10例为老年样本,在基础培养条件下,检测脂肪细胞和成骨细胞标志基因在不同年龄和性别组之间表达的差异。【结果】年老来源的hMSCs在成脂诱导培养条件下,形成的脂肪细胞数目是年轻来源的hMSCs的2.35倍,但是统计学无显著性差异(P=0.30)。不同样本来源的hMSCs形成脂肪细胞数目和年龄的相关性分析显示,体外成脂诱导培养条件下,hMSCs分化成脂肪细胞的能力和年龄无显著相关性(Spearman,r=0.01899,P=0.95)。脂肪细胞的标志基因PPARγ2、LPL、Adipsin在年老来源的hMSCs中表达高于年轻来源的hMSCs,但是统计学均无显著性差异。成骨细胞标志基因RUNX2在年老来源的hMSCs中表达高于年轻来源者,而OC、ALP的表达在年老来源的hMSCs中高于年轻来源者,三者均无统计学差异。基因表达和年龄相关性分析显示,LPL基因表达随着年龄的升高显示出显著性升高的趋势。PPARγ2、Adipsin、Runx2、OC、ALP的表达与hMSCs来源个体的年龄均无显著相关性。在男性来源hMSCs,RUNX2的表达与年龄呈现反向相关性的显著趋势。【结论】体外诱导培养条件下,hMSCs脂肪细胞分化能力与年龄和性别无关,hMSCs向脂肪细胞分化能力与体内环境下表现不一致。结论由本研究,我们得到以下结论:1、通过本实验室建立的hMSCs的分离和培养方法,可获得足够数量的hMSCs用于实验,细胞培养体系稳定可靠;2、19个WNT基因中有12个在hMSCs中表达,并且基因表达呈现年龄和性别差异。3、非经典WNT信号通路中的WNT11和WNT4可能是hMSCs向脂肪细胞分化的促进因子,激活经典WNT信号通路可以抑制或阻断hMSCs向脂肪细胞、成骨细胞分化并且促进其向软骨细胞分化。4、体外培养条件下,hMSCs向脂肪细胞分化能力与hMSCs来源个体的年龄无关,男性和女性来源的hMSCs脂肪分化能力无明显差异。本课题创新点有三:1、采用不同年龄和性别来源的新鲜分离的hMSCs,研究了WNT基因在hMSCs中的表达谱,并且阐明了年龄和性别对于WNT基因表达的影响;2、、研究了WNT信号通路在hMSCs向脂肪细胞、成骨细胞、软骨细胞分化中的作用,发现WNT信号通路在人hMSCs向脂肪细胞、成骨细胞分化中与动物细胞中的差异;3、采用不同性别和年龄来源的hMSCs进行成脂诱导分化,阐明了体外诱导培养条件下年龄和性别对于hMSCs向脂肪细胞分化的影响。更多还原

【Abstract】 PartⅠThe in Vitro Culture and Identification of Human Bone Marrow Stromal Cells【Objective】To establish the culturing system of hMSCs according to the former established protocols of our lab,and provide cell source for following experiments.【Methods】Bone marrow samples were obtained with IRB approval from femoral tissue discarded during hip replacement surgery.Low-density mononuclear cells were isolated by density centrifugation on Ficoll/Histopaque 1077.Adherent hMSCs were isolated,hMSCs were cuLtured and passaged inα-MEM supplemented with 10%FBS.The cells were induced with adipocytogenic,osteogenic and chondrocyogenic medium separately.The analysis of ALP activity and Alizarin Red S staining were conducted for identification of osteogenesis,Oil Red-O for adipocytogenesis,and Alcian blue for chondrocytogenesis.【ResuLts】The primary cells attached after 24 hours cuLture.They exhibited the shape like spindle or shuttle,the clones of hMSCs formed after 48-72 hours cuLture.The cells proliferated rapidly after 5-7 days cuLture.The cells were almost confluence after 12-14 days cuLture with the appearance like fibroblasts. Some cells overlapped with others.The cells could be passaged after cuLturing for 5-10 days,and they can be passaged for 4-10 times.The ALP activity and Alizarin Red S positive area can be detected after the cells were cuLtured in osteogenic medium.The cells couLd differentiate to Oil Red-O positive cells after adipocytogenic cuLture.There were cells which were positive for Alcian blue after chondrocytogenic induction.【Conclusion】hMSCs can be isolated from bone marrow,and can be expanded after being cuLtured.The cells can differentiate into osteoblasts,chondrocytes, and adipocytes. PartⅡEffects of Age and Gender on WNT Gene Expression in Human Bone Marrow Stromal Cells【Objective】WNT signaling pathways play important roles in the behavior of human bone marrow stromal calls.Although WNT expression has been examined in human bone marrow stromal cells(hMSCs) with limited numbers of subjects or from commercial sources,there are conflicting resuLts on WNT gene expression in hMSCs.Furthermore,the effects of age and gender on WNT expression in hMSCs are largely unknown.【Methods】Bone marrow samples were obtained with IRB approval from femoral tissue discarded during hip replacement surgery.A total of 19 subjects,12 women and 7 men,age ranging from 36 years old to 85 years old,were included in this study. Of them,9 subjects were classified as young(≤50-years,mean 42.0±4.6 years) and 10 were classified as old(≥55-years,mean 69.3±9.5 years).There were 12 samples from women(37 to 85-years,mean 58.6±16.0 years) and 7 from men (36 to 82-years,mean 52.6±16.0 years).hMSCs were cuLtured in 100mm tissue cuLture dishes and Passage 2 or 4 cells were used.In each experiment,standardized conditions were used for all samples;cells were harvested and stored in Trizol reagent at -80℃for analysis at the same time to avoid technical differences between assays. WNT gene expression levels were measured by semi-quantitative RT-PCR.【ResuLts】Twelve of the 19 WNT genes,WNT2,3,4,5A,5B,6,7B,10B,11,13, 14,and16B were expressed in hMSC.WNT7B and 14 were expressed significantly higher in the younggroup.WNT2 and WNT13 showed a trend of higher expression in young group.WNT7B,13,and 14 were inversely correlated with age.Further analysis for gender-specific difference indicated that WNT16 was expressed significantly higher in men than in women.WNT11 showed a trend of higher expression in hMSCs from women.For the hMSCs from women,WNT13 was inversely correlated with age and WNT4 was positively correlated with age.For the hMSCs from men,WNT7B and WNT14 were inversely correlated with age.【Conclusion】These data indicated that most of the age-related WNT genes belong to the canonical WNT signaling pathway.Further,there are gender-specific differences in the expression of WNT4,7B,13,14,and 16 in hMSCs.Age and gender account for many of the sample-to-sample variations in WNT gene expression in human marrow stromal cells PartⅢEffects of WNT Signaling on Adipocytogenesis,osteogenesis,and chondrogenesis in Human Bone Marrow Stromal Cells【Objective】From the body of information available about mechanisms of adipocyte differentiation with murine preadipocytes,it is known that 1) Wnt10b inhibits adipogenesis and stimuLates osteoblastogenesis,2) Wnt4 and Wnt5a stimuLate adipocytogenesis and 3) Wnt3a can enhance the routine C3H10T1/2 cell differentiation into chondrocytes induced by bmp2.Because little is known about WNTs and human adipocytogenesis,osteogenesis,and chondrogenesis,we tested the hypothesis that WNT signaling reguLates adipocytogenesis,osteogenesis,and chondrogenesis of human bone marrow stromal cells(hMSCs);we assessed canonical and non-canonical WNT and effects of a small molecuLe stimuLator ofβ-catenin (SB-216763) during adipocytogenesis,osteogenesis,and chondrogenesis.【Methods】Bone marrow samples were obtained with IRB approval as discarded femoral tissue from Orthopedic department,Brigham and Women’s Hospital.Low-density mononuclear cells were isolated by density centrifugation on Ficoll/Histopaque 1077.Upon near-confluence of hMSCs,medium was changed toα-MEM,with adipocytogenic,osteogenic,or chondrogenic supplements.The effect of adipocytogenic medium on the expression(RT-PCR) of adipocyte maker genes and WNT genes was performed at intervals to 10 days.The effects of SB-216763 on expression ofβ-catenin were tested by Western Immunoblotting.Eighteen days after treatment,adipocytes treated with and without SB-216763 were counted in cells positive for staining with 0.3%Oil Red-O.The effects of WNT signaling on osteogenesis and chondrogenesis of hMSCs were assayed by RT-PCR,ALP activity analysis,and Alcian blue staining.【ResuLts】Analysis of adipocyte marker genes indicated that the expression level of PPARγ2 and LPL was detectable after 1 day in adipocytogenic medium and increased thereafter.The expression of Adipsin also increased with time.Upon adipocytogenic differentiation of hMSCs(day 1),the expression of canonical WNT genes(2,10B,13,and 14) decreased,whereas non-canonical WNT genes (4 and 11),but not WNT5A increased.WNT11 was the only one to change prior to upreguLation of adipocyte signature genes.SB-216763(5μM),which increasedβ-catenin levels inhibited adipocytogenesis by blocking induction of PPARγ2,LPL,and Adipsin.Consistent with the molecuLar effects,SB-216763 inhibited generation of Oil Red-O adipocytes with duration- and dosage-dependence. Activation of canonical WNT signaling pathway in hMSCs by SB-216763 inhibited the expression of osteoblasts mark gene ALP,further confirmed by the ALP activity analysis.The chondrocytes mark gene,RUNX2 and COLⅡexpression were enhanced,accompanied by the collagenⅡproductivity in vitro confirmed by Alcian blue staining.【Conclusion】These study indicate that for human adipocytogenesis,non-canonical WNT11 and 4 may be major enhancers and that activation of canonical WNT signaling pathway prevents hMSCs from differentiating into adipocytes and osteoblasts,and stimuLates hMSCs differentiation into chondrocytes.Thus,there may be fundamental species differences in WNT signaling during adipocytogenesis and osteogenesis.PartⅣEffects of Age and Gender on Adipocyte Differentiation of Human Bone Marrow Stromal Cells【Objective】Our previous studies indicated an effect of age on many properties of hMSCs,including the decreased ability to differentiation into osteoblasts.In order to further identify the mechanism of the enhanced volume of fat tissue in the bone marrow of typeⅡosteoporosis patients,we surveyed the effects of age and gender on the adipocytogenesis of hMSCs,which were from different ages, both women and men.【Methods】Bone marrow samples were obtained with IRB approval as discarded femoral tissue from Orthopedic department,Brigham and Women’s Hospital.A total of 15 subjects,8 women and 7 men,age ranging from 17 years old to 90 years old, were included in this study.Of them,5 subjects were classified as young and 10 were classified as old.The Oil Red-O positive cells were counted at after the hMSCs were cultured in adipocytogenic medium for 18 days.For the adipocyto marker gene and osteoblast marker gene expression experiment,a total of 19 subjects,12 women and 7 men,age ranging from 36 years old to 85 years old,were included in this study.Of them,9 subjects were classified as young and 10 were classified as old.There were 12 samples from women and 7 from men.RT-PCR was conducted to detect the gene expression.【ResuLts】Under the adipocytogenic induction,the number of adipocytes from old hMSCs was 2.35 folder as the ones from young group,but it was not statistically significant.The correlation analysis of the number of adipocytes form different subjects with age showed there was no significant correlation between them (Spearman,r=0.01899,P=0.95).There is no significant difference of the adipocytes number induced from hMSCs between women and men.The adipocyte marker genes,PPARγ2,LPL,and Adipsin were expressed higher in the hMSCs from old group than the young group,but there is no significant difference.For the osteoblast gene expression,the expression of RUNX2 was lower;OC and ALP were higher in hMSCs from old group as compared to young group.LPL expression showed a trend of positive correlation with age,while PPARγ2,Adipsin,RUNX2,OC, and ALP did not show significant correlations with age.【Conclusion】The in vitro adipocytogenic differentiation ability of hMSCs is not related to age and gender,there is difference of the adipocytogenic ability of hMSCs between in vivo and in vitro. Reflections and ConclusionsWe draw the following the theoretical considerations based on our resuLts:1.The cell isolation and cuLture system of our lab is stable and reliable,sufficient cells can be acquired for the following experiments.2.TweIve of 19 known WNT genes expressed in hMSCs,and there are differences of gene expression between young and old subjects,and between both genders.3.The non-canonical WNTs,WNT11 and WNT4 may be the enhancers of hMSCs differentiation into adipocytes.Activation of WNT/β-catenin signaling pathway can inhibit the adipocytogenic and osteogenic differentiation and enhance chondrogenic differentiation of hMSCs.The novelty of this study is threefold:1.These studies show variances in constitutive expression of many WNT genes in 19 samples of freshly isolated human bone marrow stromal cells from young and old subjects,both women and men.Further,they indicate effects of age and gender on expression of some WNT genes.2.Tested the effects of WNT signaling pathway during adipocytogenesis, osteogenesis,and chondrocytogenesis of hMSCs,and found there may be fundamental species differences in WNT signaling during adipocytogenesis and osteogenesis.3.Assessed the effects of age and gender on the adipocytogenic differentiation of hMSCs from young and old subjects both in women and men.更多还原