骨髓诱导成骨成脂分化机制研究进展

骨髓间充质干细胞(MSC)有分化为多种细胞的潜能,在无任何条件干预下可自然分化为成骨细胞系和脂肪细胞系,两者分化存在相互制约的平衡关系.细胞分化方向的调节通过一些复杂信号通路,如骨形态发生蛋白、Wnt蛋白、脂联素以及脂肪细胞、成骨细胞分化转录调节因子PPAR-γ、Runx2等之间相互作用实现,药物、年龄、微重力作用等因...     

骨髓诱导成骨成脂分化机制研究进展

骨髓间充质干细胞(MSC)有分化为多种细胞的潜能,在无任何条件干预下可自然分化为成骨细胞系和脂肪细胞系,两者分化存在相互制约的平衡关系。细胞分化方向的调节通过一些复杂信号通路,如骨形态发生蛋白、Wnt蛋白、脂联素以及脂肪细胞、成骨细胞分化转录调节因子PPAR-γ、Runx2等之间相互作用实现,药物、年龄、微重力作用等因素也可影响这些信号通路,调节MSC分化方向。该文就MSC成骨或成脂分化的影响因素研究进展作一综述。     

成骨细胞分化调控机制研究新进展

成骨细胞的分化是骨发生、骨形成的前提.成骨干细胞经过一步或几步分化为前成骨细胞(具有向成骨细胞和软骨细胞分化的双向分化潜能),再继续分化为有功能的成骨细胞和软骨细胞.成骨细胞在分化过程中受CTGF、FGF-2、BMP、Dlx-5、Runx2、Osx、Sox9、TGF-β1、TNF-α等多种因子的调控,调控过程相互联系,相互影响.     

兔成骨细胞和脂肪细胞横向分化的研究

目的 探讨兔成骨细胞和脂肪细胞两者横向分化的能力和方法.方法 选取3个月龄新西兰大白兔,获取、分离培养脂肪细胞,天花板贴壁法使其去分化,去分化脂肪细胞进行成骨诱导3周后行茜素红、碱性磷酸酶(ALP)和Ⅰ型胶原酶免疫组织化学染色.另选取出生7d内的乳兔,取颅骨骨块利用酶消-组织块培养法培养成骨细胞并传代培养,对其进行成脂诱导3周后行油红O染色、逆转录-聚合酶链反应(RT-PCR)检测过氧化物酶体增殖物激活受体γ(PPARγ) mRNA的表达.结果 提取的成熟脂肪细胞去分化后为长梭形成纤维细胞状;成骨诱导后,Ⅰ型胶原免疫组织化学染色显示实验组细胞内表达出Ⅰ型胶原,与对照组比较差异有统计学意义(P＜0.05);各组细胞第3天、第7天、第14天不同时段的ALP活性实验组较对照组高(P＜0.05),分别为0.165±0.007、0.253±0.005、0.345±0.007和0.067±0.004、0.076±0.006、0.082±0.003,且随着培养时间的延长实验组ALP活性逐渐增强(组内比较P＜0.05);提取的乳兔颅骨成骨细胞呈短梭形,成脂诱导3周后油红O染色阳性,PPARγmRNA表达阳性,对照组为阴性,实验组与对照组比较差异有统计学意义(P＜0.05).结论 成熟脂肪细胞可以通过体外培养实现去分化,脂肪细胞和成骨细胞在一定条件下可以相互转化.     

缝隙连接通讯对骨髓中成骨细胞与脂肪细胞横向分化调控的研究进展

骨质减少性疾病如骨质疏松症和骨坏死的主要病理基础是成骨细胞数量的减少及脂肪细胞数量的增多,两者发生的机制及如何减少脂肪细胞的数量或增加成骨细胞的活性一直是未解决的难题.国内外研究多从骨髓间质干细胞和细胞因子调控角度考虑,较少从两种细胞的横向分化和细胞间信号传导调控角度考虑.有研究结果表明分化成熟的成骨细胞和脂肪细胞具有相同的细胞表型[1],在一定条件下可以相互转化,即细胞的横向分化(transdifferentiation).     

骨髓基质干细胞成脂分化的调控及其对骨质疏松的影响

研究发现随着年龄的增长和绝经期后骨质疏松,骨髓腔内的脂肪细胞逐渐增多,取代了成骨细胞,因此抑制骨髓基质干细胞成脂分化是目前治疗骨质疏松的新途径。应用各种成脂诱导剂建立体外定向诱导骨髓基质干细胞成脂分化模型是研究新途径的基础。除了通过光学显微镜观察成脂分化的能力,还可运用分子生物学的方法定性、定量检测成脂分化的程度。过氧化物酶增殖子激活受体(PPARγ)是脂肪细胞的分化过程中起关键性作用的调节因子,组蛋白去乙酰化酶(SIRT1)被激活后能抑制PPARγ的表达,减少脂肪细胞的生成,起到预防和治疗骨质疏松的作用。笔者就以上方面和对减少骨髓脂肪分化的策略作了综述。     

骨康灵联合99Tc-MDP治疗糖皮质激素诱发骨质疏松细胞分子机制研究

目的观察骨康灵和99Tc-MDP联合治疗糖皮质激素诱发的骨质疏松优于单独治疗的细胞分子机制。方法用SD大鼠随机分为DEX(地塞米松)、DEX-MDP(地塞米松+99 Tc-MDP 2mg/kg)、DEX-Z(地塞米松+骨康灵2ml/kg)和DEX-MDP-Z(地塞米松+99 Tc-MDP 2mg/kg+骨康灵2ml/kg)组。给药后颈动脉放血,制备含药血清,用MTT和PNPP法检测其对成骨细胞增殖与分化的影响;用TRAP染色和骨片吸收陷窝法观察其对破骨细胞的数目和功能影响;取大鼠股骨骨髓细胞,用Real-time PCR方法检测骨髓细胞RUNX2和PPARγ的表达。结果显示与对照组及单独治疗组相比联合用药组对成骨细胞有微弱的促分化作用,对破骨细胞有较强的抑制其数量和骨吸收的功能;同时联合用药能上调RUNX2与PPARγ的比值。结论中药骨康灵联合99Tc-MDP能有效抑制破骨细胞的数目和功能,同时促进骨髓间充质干细胞向成骨细胞前体分化,这可能是其优于单独治疗的细胞分子机制之一。     

HDAC2转染对骨髓干细胞成骨细胞分化的影响

[目的]探讨组蛋白去乙酰化酶2 (histone deacetylase 2,HDAC2)转染对骨髓间充质干细胞(bone marrow mesenchy-mal stem cells,BMSCs)向成骨细胞分化的影响.[方法]HDAC2-siRNA重组序列、阴性随机对照序列分别转入大鼠BMSCs,命名为HDAC2-s...     

人骨髓间充质干细胞的生物学特性及成骨诱导分化的研究

[目的]探讨成人骨髓间充质干细胞分离、纯化、培养及鉴定的方法,观察其成骨分化过程中Runx2基因的动态表达以及生物学特性。[方法]取自人股骨近端骨髓标本,利用联合密度梯度离心和差异贴壁法分离骨髓间充质干细胞,体外扩增和传代培养,流式细胞仪检测细胞表面标记,诱导向成骨细胞分化,并采用RT-PCR和Western blot方法检测Runx2的动态表达。[结果]原代和传代细胞呈纺锤状外观,生长增殖能力良好,骨髓间充质干细胞的生长曲呈成"S"形,细胞表面标记物CD90阳性表达,CD34和CD45阴性表达。经定向诱导分化后,细胞分别呈现成骨细胞的表型特征,随着诱导时间的增加,Runx2的表达也明显增加,与对照组相比有统计学差异(P<0.05)。[结论]该方法能从人骨髓中高效分离和扩增MSCs,生物学性状稳定,具有成骨分化潜能,为骨组织工程提供理想的种子细胞,同时证实Runx2在成骨分化中起到重要的调控作用。     

联合rhBMP-2与成骨细胞诱导骨髓基质细胞成骨分化的实验研究

目的探讨rhBMP-2和成骨细胞联合诱导骨髓基质细胞(BMSCs)的成骨分化能力。方法应用全骨髓贴壁法分离培养4周龄SD大鼠的BMSCs,通过改进酶消化法结合反复贴壁法培养1日龄SD乳鼠成骨细胞。实验分为4组:rhBMP-2诱导组、成骨细胞诱导组、rhBMP-2+成骨细胞诱导组、单纯培养液组。倒置相差显微镜及扫描电镜观察细胞生长情况;分别收集第3、6、9天细胞培养液上清液定性及定量测定碱性磷酸酶并进行统计学分析。结果 rhBMP-2诱导组、成骨细胞诱导组、rhBMP-2+成骨细胞诱导组均检测出成骨细胞生成,其中,rhBMP-2+成骨细胞诱导组碱性磷酸酶含量明显大于前两组,差异有统计学意义(P0.05)。结论rhBMP-2、成骨细胞、rhBMP-2联合成骨细胞均能提供成骨微环境,并在体外诱导BMSCs向成骨细胞分化,rhBMP-2联合成骨细胞对BMSCs的成骨分化具有更优的诱导能力。     

人骨髓间充质干细胞向肝细胞诱导分化的进一步研究

目的：探索骨髓间充质干细胞（BM-MSCs）向肝细胞诱导的最有效方法,并对诱导后的细胞进行鉴定。方法：取健康成人的适量骨髓,采用梯度密度离心法分离BM-MSCs并行贴壁培养,扩增至第3代,取第3代细胞分3组分别进行诱导。A组：肝细胞生长因子（HGF）＋表皮生长因子（EGF）;B组：肝细胞生长因子（HGF）＋成纤维生长因子（FGF）;C组：肝细胞生长因子（HGF）＋表皮生长因子（HGF）＋成纤维生长因子（HGF）。分别进行培养,诱导前通过流式细胞仪分析细胞表面标志CD44、CD90的表达率对培养的BM-MSCs进行鉴定,诱导后通过RT-PCR、免疫组织化学法、糖原染色检测鉴定其诱导分化情况。结果：本文通过建立从人骨髓中分离、培养、扩增、传代、纯化BM-MSCs,定向诱导分化,使其成功向肝样细胞转化,图像分析表明C组鼠抗人白蛋白单克隆抗体（ALB）表达最为明显。结论：证明3种组合均可成功将骨髓间充质干细胞向肝样细胞诱导分化,均能分泌肝细胞特异性标记如鼠抗人甲胎蛋白单克隆抗体（AFP）、ALB、糖原,其中C组效果最为突出。     

槲皮素对人骨髓间充质干细胞增殖和成骨分化的影响及分子机制

目的 探讨槲皮素对人骨髓间充质干细胞（human bone marrow mesenchymal stem cells,hBMSCs）增殖和成骨分化以及对特异AT序列结合蛋白2（SATB2）基因表达的影响。方法 噻唑蓝（MTT）法检测不同浓度槲皮素对hBMSCs增殖活性的影响,碱性磷酸酶（alkaline phosphatase,ALP）试剂盒检测ALP活性,实时荧光定量PCR（qRT-PCR）检测成骨分化生物标志物Runt相关转录因子2（Runx2）、骨钙素（BGLAP）和分泌磷酸蛋白1（SPP1）mRNA的表达,qRT-PCR和蛋白免疫印迹（Western blot）检测SATB2 mRNA和蛋白表达变化,在hBMSCs转染SATB2特异性siRNA或SATB2过表达载体质粒,分析敲低或过表达SATB2对槲皮素干预的hBMSCs增殖和成骨分化的影响。结果 在一定范围内槲皮素呈浓度依赖性促进hBMSCs增殖,5 μmol/L时促进细胞增殖效果最佳,选择5 μmol/L的槲皮素用于后续研究。5 μmol/L的槲皮素能够有效促进ALP活性以及Runx2、BGLAP和SPP1 mRNA的表达,并且能够上调SATB2 mRNA和蛋白表达。敲低SATB2能够阻碍槲皮素对hBMSCs增殖和成骨分化的促进作用,而过表达SATB2能够增强槲皮素对hBMSCs增殖和成骨分化的促进作用。结论 槲皮素可通过上调SATB2基因的表达促进hBMSCs增殖和成骨分化。     

Germ cell differentiation of bone marrow mesenchymal stem cells

Bone marrow mesenchymal stem cells (BM‐MSCs) were first cultured under induction of retinoic acid (RA), Sertoli cells conditioned medium and RA + con (conditioned medium) as treatment groups. The presence of Sertoli cells was confirmed by immunocytochemistry of follicle‐stimulating hormone receptor in Sertoli cells and flow cytometry by anti‐Gata4 antibody. Cell viability and morphology of nucleus and cytoplasm of BM‐MSCs were evaluated by MTT test and DAPI staining respectively. The expression of Oct4, Plzf, Scp3, Caspases 8, 9 and 3 genes was evaluated by RT‐PCR. For increasing the accuracy of experiment, the expression of Vasa and SCP3 genes was investigated quantitatively by real‐time PCR after 0, 5, 10, 15 days of culture. The results showed that the number of apoptotic cells increased in RA group. The expression of apoptosis genes (Caspases 3, 8 and 9) was also observed in this group all days of culture. Measurement of Vasa and Scp3 genes by RT‐PCR confirmed the positive effects of retinoic acid on increasing of genes expression. So, in this study, a group with maximum expression of differentiation genes and minimum expression of apoptotic genes was RA + conditioned medium group. DNA fragmentation was not observed in all groups.     

Activation of Sirt1 decreases adipocyte formation during osteoblast differentiation of mesenchymal stem cells.

In vitro, mesenchymal stem cells differentiate to osteoblasts when exposed to bone-inducing medium. However, adipocytes are also formed. We showed that activation of the nuclear protein deacetylase Sirt1 reduces adipocyte formation and promotes osteoblast differentiation. INTRODUCTION: Mesenchymal stem cells (MSCs) can differentiate into osteoblasts, adipocytes, chondrocytes, and myoblasts. It has been suggested that a reciprocal relationship exists between the differentiation of MSCs into osteoblasts and adipocytes. Peroxisome proliferator-activated receptor gamma2 (PPARgamma2) is a key element for the differentiation into adipocytes. Activation of Sirt1 has recently been shown to decrease adipocyte development from preadipocytes through inhibition of PPARgamma2. MATERIALS AND METHODS: We used the mouse mesenchymal cell line C3H10T1/2 and primary rat bone marrow cells cultured in osteoblast differentiation medium with or without reagents affecting Sirt1 activity. Adipocyte levels were analyzed by light microscopy and flow cytometry (FACS) after staining with Oil red O and Nile red, respectively. Osteoblast and adipocyte markers were studied with quantitative real-time PCR. Mineralization in cultures of primary rat bone marrow stromal cells was studied by von Kossa and alizarin red staining. RESULTS: We found that Sirt1 is expressed in the mesenchymal cell line C3H10T1/2. Treatment with the plant polyphenol resveratrol as well as isonicotinamide, both of which activate Sirt1, blocked adipocyte development and increased the expression of osteoblast markers. Nicotinamide, which inhibits Sirt1, increased adipocyte number and increased expression of adipocyte markers. Furthermore, activation of Sirt1 prevented the increase in adipocytes caused by the PPARgamma-agonist troglitazone. Finally, activation of Sirt1 in rat primary bone marrow stromal cells increased expression of osteoblast markers and also mineralization. CONCLUSIONS: In this study, we targeted Sirt1 to control adipocyte development during differentiation of MSCs into osteoblasts. The finding that resveratrol and isonicotinamide markedly inhibited adipocyte and promoted osteoblast differentiation may be relevant in the search for new treatment regimens of osteoporosis but also important for the evolving field of cell-based tissue engineering.     

骨形成过程中两条重要的信号传导通路

Wnt信号通路与BMP-2信号通路能够促进成骨细胞的分化和成骨细胞分泌的胞外基质的生物矿化,在这一过程中起着极其重要的作用。本文主要综述了,近年来对这两条信号通路研究的最新进展。     

体外诱导骨髓间质干细胞向软骨方向分化的研究

目的　探讨体外诱导骨髓间充质干细胞 (BMSCs)向软骨方向分化的条件和方法。方法　抽取兔股骨骨髓 ,用密度梯度离心和贴壁分离法分离BMSCs ,分别在高密度 (1× 10 6/ml)和低密度 (1× 10 4/ml)培养条件下用转化生长因子 β1(TGF β1)诱导BMSCs向软骨方向分化。倒置显微镜、透射及扫描电子显微镜观察细胞 ,用免疫组织化学、原位杂交、特殊染色方法检测Ⅱ型胶原及软骨基质成分的表达。结果　高密度诱导的BMSCsⅡ型胶原免疫组织化学阳性率为 89.2 %,Ⅱ型胶原原位杂交阳性率为 82 .6%,黏多糖特殊染色阳性 ,低密度诱导的BMSCsⅡ型胶原免疫组织化学阴性 ,Ⅱ型胶原原位杂交阳性率为 3 .8%,黏多糖特殊染色阴性。结论　TGF β1可以诱导BMSCs向软骨方向分化 ,细胞密度是BMSCs分化的重要影响因素。     

Rapamycin as an inhibitor of osteogenic differentiation in bone marrow-derived mesenchymal stem cells

Background An autograft of cultured bone marrow-derived mesenchymal stem cells has already been used in clinical practice. In those patients whose bone marrow cannot be used, a cell allograft with the use of immunosuppressant drugs will be an option in the future. However, little is known about the effects of immunosuppressant drugs on mesenchymal stem cells. This study assessed the effects of immunosuppressant drugs on osteogenic differentiation of mesenchymal stem cells and analyzed the manner in which immunosuppressant drugs modulate the osteogenic effect of dexamethasone. Methods Rat bone marrow cells were cultured with or without dexamethasone as an osteogenic supplement. In each experimental group, one of three immunosuppressants (rapamycin, cyclosporine A, or FK506) was added. As a control, cells were cultured without immunosuppressants. Histologically, mineralization was assessed by alizarin red S staining and phase-contrast microscopy. Biochemically, alkaline phosphatase activity, calcium content, and osteocalcin content were assessed. Results On histological analysis, no mineralized nodules were seen on alizarin red S staining or phase-contrast microscopy in the groups not treated with dexamethasone, except in the group that was treated with FK506. Mineralized nodules were seen in the groups treated with dexamethasone, except in the group that was treated with rapamycin. On biochemical analysis, it was found that, compared to the control group, rapamycin reduced alkaline phosphatase activity and the calcium content of mesenchymal stem cells; FK506 increased alkaline phosphatase activity, calcium content, and osteocalcin content; and cyclosporine A had negligible effects. Dexamethasone increased alkaline phosphatase activity, calcium content, and osteocalcin content, but these effects were decreased by rapamycin. Conclusions Rapamycin did not have an osteogenic effect on mesenchymal stem cells, but inhibited the effect of osteogenic differentiation induced by dexamethasone. In contrast, FK506 had an osteogenic effect on mesenchymal stem cells. Therefore, FK506 might be more useful than rapamycin in allogeneic transplantation of mesenchymal stem cells.     

Demonstration of the presence of independent pre-osteoblastic and pre-adipocytic cell populations in bone marrow-derived mesenchymal stem cells

Mesenchymal stem cells (MSC) are defined as plastic-adherent, clonal cells that are common progenitors for osteoblasts and adipocytes. An inverse relationship between bone and fat has been observed in several clinical conditions and has been suggested to be caused by re-directing MSC differentiation into one particular lineage. However, this inverse relationship between bone and fat is not consistent and under certain in vivo conditions, bone and fat can change independently suggesting separate precursor cell populations. In order to test for this hypothesis, we extensively characterized two plastic-adherent clonal MSC lines (mMSC1 and mMSC2) derived from murine bone marrow. The two cell lines grew readily in culture and have undergone more than 100 population doublings with no apparent differences in their growth rates. Both cell lines were positive for the murine MSC marker Sca-1 and mMSC1 was also positive for CD13. Both cell lines were exposed to in vitro culture induction of osteogenesis and adipogenesis. mMSC1 and not mMSC2 were only able to differentiate to adipocytes evidenced by the expression of adipocyte markers (aP2, adiponectin, adipsin, PPARgamma2 and C/EBPa) and the presence of mature adipocytes visualized by Oil Red O staining. On the other hand, mMSC2 and not mMSC1 differentiated to osteoblast lineage as demonstrated by up-regulation of osteoblastic makers (CBFA1/RUNX2, Osterix, alkaline phosphatase, bone sialoprotein and osteopontin) and formation of alizarin red stained mineralized matrix in vitro. Consistent with the in vitro results, mMSC2 and not mMSC1, were able to form bone in vivo after subcutaneous implantation in immune-deficient (NOD/SCID) mice. Our data suggest that contrary to the current belief, bone marrow contains clonal subpopulations of cells that are committed to either osteoblast or adipocyte lineage. These cell populations may undergo independent changes during aging and in bone diseases and thus represent important targets for therapy.     

Inhibition of osteoblast differentiation but not adipocyte differentiation of mesenchymal stem cells by sera obtained from aged females

Aging is associated with decreased osteoblast-mediated bone formation leading to bone loss and increased risk for osteoporotic fractures. However, the cellular mechanisms responsible for impaired osteoblast functions are poorly understood. In the present study, we hypothesized that changes in bone microenvironment composition with aging are responsible for impaired osteoprogenitor cell recruitment and differentiation. As a model for bone microenvironment, we examined the effects of sera obtained from young (age 20-30 year old [yo], n=20) and old (70-84 yo, n=19) healthy female donors on cell proliferation and differentiation capacity into osteoblasts and adipocytes of human mesenchymal stem cells (hMSC). Cell proliferation rate determined by counting cell number was similar when the cells were cultured in the presence of media containing 5% sera from old or from young donors. Similarly, the number of adipocytes and levels of adipocytic gene expression was similar in cultures incubated with sera from young or old donors. We observed decreased osteoblastic gene expression in hMSC cultured either in pooled or individual sera of old donors compared to sera from young donors: core binding factor/runt-related binding factor 2 (Cbfa1/Runx2) 46%+/-2% (P<0.05), alkaline phosphatase (ALP) 45%+/-2% (P<0.05), collagen type I (Col-I) 50%+/-1% (P<0.05), and osteocalcin 65%+/-3% (P<0.05). This down-regulation of the mRNA was accompanied by reduced ALP enzyme activity by 25%+/-1% (P<0.01), immunocytochemical staining for osteoblastic markers: ALP, Col-I, and bone sialoprotein (BSP) as well as reduced in vitro mineralization as determined by Alizarin red staining. In conclusion, age-related changes in the serum composition and possibly hMSC microenvironment may contribute to the impaired osteoblast functions with aging. The factors mediating these changes remain to be determined.     

应用聚集培养技术进行兔骨髓间充质干细胞体外成软骨诱导培养

目的观察离心管诱导培养条件下,兔骨髓间充质干细胞(MSCs)的成软骨分化,从而为应用该技术提供实验依据。方法分离扩增兔骨髓MSCs和关节软骨细胞,采用离心管内聚集培养技术诱导培养:MSCs,用含转化生长因子-β1的DMEM培养液换液,以相同培养条件下的软骨细胞为阳性对照组,以常规培养液换液的MSCs为阴性对照组;分别于培养1、2、3、4周后,收集培养物行苏木素-伊红(HE)染色、Ⅱ型胶原免疫组织化学染色和图像分析。结果MSCs诱导培养1周后开始表达Ⅱ型胶原,随时间延长而表达增强,并逐渐产生细胞外基质,但4周内表达强度始终弱于软骨细胞组(P<0.01)。阴性对照组中部分MSCs死亡,培养物崩解。结论采用离心管诱导培养技术可以促进MSCs向软骨细胞表型分化;该技术方法操作简单、诱导确切,适宜于鉴定干细胞的软骨分化潜能。