FGF-2对人骨髓间充质干细胞增殖和向成骨细胞分化的影响

探讨体外培养条件下,成纤维细胞生长因子-2（FGF-2）和地塞米松（Dex）对第7代人骨髓间充质干细胞（MSCs）增殖和向成骨细胞分化的作用以及两者联合使用的效应。MSCs经含FGF-2或／和Dex的培养液作用后,于不同时间采用MTT法测定细胞增殖情况;对硝基苯磷酸（pNPP）法测定碱性磷酸酶（ALP）活性;ELISA法测定骨钙蛋白（OC）含量;茜素红S染色法对沉积的钙盐进行染色。发现：（1）FGF-2组细胞的生长速度为对照组的1．31倍,Dex／FGF-2组细胞的生长速度为FGF-2组的1．12倍。（2）Dex组的ALP活性、OC含量和细胞外基质钙盐沉积分别为对照组的17．0倍、2．12倍和10．56倍,并能形成成熟的羟基磷灰石（HA）结晶和骨结节;FGF-2组的ALP活性比对照组降低了76．7％,虽然OC含量、钙盐沉积增加,但不能形成成熟的HA结晶和骨结节;FGF-2对Dex诱导的ALP活性增加和HA结晶形成有拮抗作用。由此证明：（1）FGF-2可促进MSCs的增殖,Dex对MSCs的增殖无明显作用;Dex能增强FGF-2对MSCs的促增殖效应。（2）Dex可使MSCs分化为成熟的成骨细胞,是一个有效的成骨细胞分化诱导剂;FGF-2可使MSCs分化为未成熟的成骨细胞;FGF-2拮抗Dex诱导MSCs分化为成熟的成骨细胞。     

去卵巢对SD大鼠MSCs多向分化能力的影响

通过比较有、无诱导条件下正常与骨质疏松症（OP）大鼠骨髓间充质干细胞（MSCs）骨向、脂向及软骨向的分化情况,观察去卵巢对SD大鼠MSCs多向分化能力的影响。实验分为四组：正常组、正常诱导组、OP组、OP诱导组;分别在有或无成骨、成脂、成软骨诱导条件下,评价各组的分化情况。检测发现,无诱导条件下,正常大鼠MSCs骨向及软骨向分化的能力强于OP大鼠,而OP大鼠MSCs的脂向分化能力强于正常大鼠;诱导条件下,正常大鼠MSCs对骨向及软骨向诱导剂的反应能力强于OP大鼠,而OP大鼠MSCs对成脂诱导剂的反应能力强于正常大鼠。这些结果表明,去卵巢后大鼠MSCs骨向分化能力及对成骨诱导剂的反应力下降,脂向分化能力及对成脂诱导剂的反应力增强,对软骨诱导剂的反应力下降。     

在海藻酸钠凝胶上诱导骨髓间充质干细胞分化为成骨细胞

通过在海藻酸钠凝胶上诱导bMSCs向成骨细胞分化,探讨其对骨髓间充质干细胞（bone mesenchymal stem cells, bMSCs）的生物学效应。采用MTT、甲苯胺蓝染色、von Kossa染色和RT-PCR分别检测细胞的增殖、生长形态、诱导后细胞的钙化结节和成骨相关基因的表达。实验组bMSCs生长状况良好、细胞增殖迅速,与对照组的增殖无差异;bMSCs成集落样生长明显,集落中央细胞重叠生长形成钙化结节;培养至12d,实验组和对照组的成骨相关基因,包括碱性磷酸酶、I型胶原和骨钙素,均为阳性表达,但实验组的表达量高于对照组。海藻酸钠凝胶能够促进bMSCs向成骨细胞的分化,是良好的骨组织工程支架材料。     

真皮成纤维细胞成骨分化的实验研究

目的比较不同诱导剂对真皮成纤维细胞成骨分化的不同影响,探讨成纤维细胞成骨分化机制。方法取新生大鼠皮肤进行组织块培养,真皮成纤维细胞分离培养及鉴定,并分别由地塞米松、1,25(OH)2D3以及地塞米松和1,25(OH)2D3进行成骨分化诱导。分别于诱导后14d行ALP含量测定,21d行茜素红染色,并进行TAZ表达检测。结果真皮成纤维细胞表达波形蛋白,不表达角蛋白;成纤维细胞诱导14d后,1,25(OH)2D3诱导组及地塞米松+1,25(OH)2D3诱导组ALP含量与对照组有显著差异;成骨诱导21d,地塞米松诱导组仅见少量散在红色钙结节形成,1,25(OH)2D3诱导组钙结节数量增高,地塞米松+1,25(OH)2D3诱导组钙结节数量明显增高,直径变大;1,25(OH)2D3诱导组及地塞米松+1,25(OH)2D3诱导组,经TAZ免疫荧光染色,可见部分细胞核表达TAZ。结论地塞米松可促进1,25(OH)2D3诱导真皮成纤维细胞成骨分化。     

川续断提取皂甙促进骨质疏松模型中骨髓基质细胞成骨分化的作用机制研究

摘要 目的：探讨川续断提取皂甙（ASA）促进骨质疏松模型中骨髓基质细胞（rBMSCs）成骨分化的作用机制。方法：选取3月龄的雌性SD大鼠60只,随机分为卵巢切除组和假手术组,每组30只。采取卵巢切除法构建骨质疏松模型。建模成功后采用微型计算机断层扫描获得其松质骨微观结构的三维图像并进行分析。采用CCK-8法测定ASA对卵巢切除组rBMSCs增殖的影响。分析碱性磷酸酶（ALP）活性;荧光定量PCR检测成骨基因ALP、骨桥蛋白（OPN）、Runt相关转录因子2（RUNX2）的表达情况;蛋白免疫印迹试验检测磷脂酰肌醇3激酶（PI3K）、磷酸化蛋白激酶B（p-AKT）蛋白表达情况。结果：与假手术组比较,卵巢切除组骨小梁数量、骨小梁厚度和骨体积分数下降,但骨小梁分离度升高,差异有统计学意义（P＜0.05）。第4天和第7天,ASA(10^-5 mol/L)组、ASA(10^-6 mol/L) 组、ASA(10^-7 mol/L) 组和ASA(10^-8 mol/L) 组rBMSC增殖均显著高于ASA(0学艺术mol/L)组,以ASA(10^-5 mol/L)最为显著;而ASA(10^-1 mol/L) 组、ASA(10^-2 mol/L) 组、ASA(10^-3 mol/L) 组和ASA(10^-4 mol/L) 组rBMSC增殖显著低于ASA(0 mol/L)组,以ASA(10^-4 mol/L)最为显著,差异均有统计学意义（P＜0.05）。第7天和第14天,ASA(10^-5 mol/L)组、ASA(10^-6 mol/L) 组、ASA(10 ^-7 mol/L) 组和ASA(10^-8 mol/L) 组ALP活性均显著高于ASA(0 mol/L)组,且第14天ALP活性高于第7天,差异均有统计学意义（P＜0.05）。与对照组比较,ASA组成骨相关基因ALP、OPN和RUNX2相对mRNA表达水平显著升高,差异有统计学意义（P＜0.05）;与ASA组比较,wortmannin组成骨相关基因ALP、OPN和RUNX2相对mRNA表达水平均显著降低,差异有统计学意义（P＜0.05）。与对照组比较,ASA组PI3K、p-AKT蛋白表达水平显著升高,差异有统计学意义（P＜0.05）;与ASA组比较,wortmannin组PI3K、p-AKT蛋白表达水平显著降低,差异均有统计学意义（P＜0.05）。结论：ASA能促进骨质疏松模型rBMSCs增殖,增强ALP活性,增强ALP、OPN和RUNX2的表达,而PI3K通路抑制剂wortmannin降低了这些成骨作用,并降低了ASA诱导的PI3K、p-AKT水平,表明ASA通过PI3K/AKT信号通路促进骨质疏松模型rBMSCs成骨分化。     

阿司匹林对体外培养骨髓基质细胞成骨性分化的影响

目的：探讨阿司匹林对骨髓基质细胞成骨性分化的影响。方法：培养SD大鼠骨髓基质细胞（BMSCs）,传代3次后进行成骨诱导分化,诱导培养基中加入不同浓度阿司匹林（0.5、1、2、5、10mmol/L）,同时设立对照组。采用cck-8法分析细胞增殖情况。比较阿司匹林组与对照组在细胞碱性磷酸酶（ALP）活性、骨钙素（OC）分泌量、钙结节染色等方面的成骨性差异。结果：阿司匹林无促进细胞增殖活性,而高浓度阿司匹林能够强烈抑制细胞增殖。0.5、1、2mmol/L浓度阿司匹林可促进BMSCs的成骨性分化,中低浓度组碱性磷酸酶含量、骨钙素分泌量在不同阶段显著高于对照组。14天茜素红染色可见中低浓度组钙结节数量高于对照组。结论：中低浓度阿司匹林作用于骨髓基质细胞可促进其成骨细胞特性表达,这表明阿司匹林有促进骨代谢合成的作用。     

雌激素缺乏导致的骨质疏松发病过程中T淋巴细胞对骨髓间充质干细胞增殖和成骨分化的影响及与TNF-α的关系

探讨骨质疏松发病过程中T淋巴细胞对骨髓间充质干细胞（bonemarrow-derived mesenchymalstem cells,BMMSC）增殖分化的影响。选用健康雌性小鼠行双侧卵巢切除术（ovariectomy,OVX）,建立绝经后骨质疏松模型。选用同一批次健康小鼠行双侧卵巢脂肪组织部分切除,建立假手术组（sham）,Micro-CT确立模型成功建立。将sham组、OVX组、sham＋anti—TNFα组、OVX＋anti—TNFα组中T淋巴细胞与BMMSC共培养．ELISA检测sham组与OVX组T＇N-巴细胞上清液中TNF-α表达的差异,MTT法检测四组共培养体系中BMMSC生长曲线：成骨诱导后碱性磷酸酶和钙化结节茜素红染色法检测BMMsc成骨能力差异：ImPcR检测小鼠BMMSC成骨相关基因Runx2、碱性磷酸酶（alkaline phosphatase,ALP）的表达。结果显示,与sham组相比,OVX组中BMMsc的增殖受到了抑制,成骨分化减弱（P〈O．05）,OVXanti—TNF-α刺激组较OVX组增殖显著升高沪〈0．05）,成骨分化能力显著增强（P〈0．05）。以上结果证明,在雌激素缺乏下的T淋巴细胞能影响BMMSC增殖及成骨分化能力,这可能与T淋巴细胞表达TNF-α增强相关。     

软骨寡聚基质蛋白过表达对BMP-2诱导骨髓间充质干细胞分化的影响

目的：研究软骨寡聚基质蛋白（cartilage oligomeric matrix protein,COMP）过表达对BMP-2诱导骨髓间充质干细胞成骨及成软骨分化的影响。方法：BMP-2诱导骨髓间充质干细胞分化,通过脂质体转染含人COMP基因的质粒使骨髓间充质干细胞过表达COMP,采用实时定量PCR和Western blotting分析COMP基因过表达、成骨相关基因Ⅰ型胶原、RUNX2、骨钙蛋白以及成软骨相关基因Ⅱ型胶原、SOX9、蛋白聚糖、X型胶原的表达变化;通过茜素红染色观察成骨终末阶段矿化结节的生成情况,阿利新蓝染色观察细胞基质蛋白多糖的合成情况。结果：质粒转染后骨髓间充质干细胞COMP基因蛋白和mRNA表达水平显著提高（P<0.05）。COMP基因过表达后,成骨标记基因RUNX2、Ⅰ型胶原（Col1a1）mRNA水平均显著低于对照组（P<0.05）,RUNX2、骨钙蛋白（Osteocalcin）蛋白表达水平明显低于对照组（P<0.05）,而成软骨标记基因SOX9、蛋白聚糖（Aggrecan）mRNA水平均显著高于对照组（P<0.05）,SOX9、Ⅱ型胶原（Col2a1）蛋白表达均明显多于对照组（P<0.05）。细胞成骨茜素红染色弱于对照组,而阿利新蓝染色强于对照组。过表达组细胞X型胶原（Col10a1）基因表达显著低于对照组（P<0.05）,结论：骨髓间充质干细胞COMP基因过表达可抑制BMP-2诱导其成骨分化,促进骨髓间充质干细胞成软骨分化,并抑制软骨细胞的成熟肥大,为软骨组织工程研究提供新的方向。     

骨髓来源的间充质干细胞分化能力的鉴定

本文研究了人骨髓来源的间充质干细胞(MSCs)的成骨及成脂分化的潜能.通过加入诱导成骨的诱导剂,人的MSCs出现成骨分化的机箱,通过碱性磷酸酶活性测定,茜素红染色及主要调控基因BMP2和Runx2的表达,确定了MSCs具有成骨分化的潜能.对于成脂分化,通过油红O染色,及主要标志基因PPARγ的表达确定其具有成脂分化的潜能.所以,从骨髓分离的到的MSCs纯度达到标准,并且具有成骨成脂分化的多向潜能,是一种理想的实验模型细胞.     

阿司匹林对体外培养骨髓基质细胞成骨性分化的影响

目的:探讨阿司匹林对骨髓基质细胞成骨性分化的影响。方法:培养SD大鼠骨髓基质细胞(BMSCs),传代3次后进行成骨诱导分化,诱导培养基中加入不同浓度阿司匹林(0.5、1、2、5、10mmol/L),同时设立对照组。采用cck-8法分析细胞增殖情况。比较阿司匹林组与对照组在细胞碱性磷酸酶(ALP)活性、骨钙素(OC)分泌量、钙结节染色等方面的成骨性差异。结果:阿司匹林无促进细胞增殖活性,而高浓度阿司匹林能够强烈抑制细胞增殖。0.5、1、2mmol/L浓度阿司匹林可促进BMSCs的成骨性分化,中低浓度组碱性磷酸酶含量、骨钙素分泌量在不同阶段显著高于对照组。14天茜素红染色可见中低浓度组钙结节数量高于对照组。结论:中低浓度阿司匹林作用于骨髓基质细胞可促进其成骨细胞特性表达,这表明阿司匹林有促进骨代谢合成的作用。     

Differential activation of ERK1,2 MAP kinase signaling pathway in mesenchymal stem cell from control and osteoporotic postmenopausal women

Osteoblasts, the cells responsible for bone formation, derive from mesenchymal stem cells (MSCs) in bone marrow. To acquire a new cell phenotype, uncommitted MSCs must undergo several proliferation and differentiation changes. Although, it is known that extracellular signal-regulated protein kinases (ERKs) mitogen-activated protein (MAP) kinase pathway signaling is involved in the proliferation and differentiation processes, the role of ERKs in osteogenic differentiation it is controversial, at present. In addition, the function that ERK could play in MSCs derived from osteoporotic patients it is not well documented. In this study, we analyze whether previously observed differences in the dynamic response of MSCs from normal and osteoporotic postmenopausal women can be explained by changes in the activation of this signal transduction pathway. Levels of ERK phosphorylation and their correlation with osteogenic differentiation were evaluated in cultures of MSCs derived from osteoporotic postmenopausal women and "healthy" controls. The results show that, under basal conditions, MSCs derived from osteoporotic donors show a level of ERK phosphorylation 2.5 times higher than MSCs derived from control donors. The addition of the osteogenic stimulus only slightly increases the p-ERK level in cells derived from osteoporotic donors, and is higher in cells derived from control women. Important differences in the ability of PD98059 to inhibit phosphorylation of ERK in both types of cells were also observed, as well as the effect that this inhibition produced on calcium deposition. We conclude that the MAP kinase pathway signaling is differentially activated in MSCs derived from osteoporotic postmenopausal women. The high p-ERK levels in MSC derived from osteoporotic donors could determine the unresponsiveness of these cells to the osteogenic differentiation stimulus.     

Increased adipogenesis of osteoporotic human-mesenchymal stem cells (MSCs) characterizes by impaired leptin action

The bone marrow contains mesenchymal stem cells (MSCs) that differentiate to the osteogenic and adipogenic lineages. The fact that the decrease in bone volume of age-related osteoporosis is accompanied by an increase in marrow adipose tissue implies the importance that the adipogenic process may have in bone loss. We previously observed that MSCs from control and osteoporotic women showed differences in their capacity to differentiate into the osteogenic and adipogenic pathways. In vitro studies indicate that bone marrow stromal cells are responsive to leptin, which increases their proliferation, differentiation to osteoblasts, and the number of mineralized nodules, but inhibits their differentiation to adipocytes. The aim of the present report was to study the direct effect of leptin on control and osteoporotic MSCs analyzing whether the protective effect of leptin against osteoporosis could be expressed by inhibition of adipocyte differentiation. MSCs from control, and osteoporotic donors were subjected to adipogenic conditions, in the absence or in the presence of 62.5 nM leptin. The number of adipocytes, the content of PPARgamma protein, and mRNA, and leptin mRNA were measured by flow cytometry, Western blot, and RT-PCR, respectively. Results indicate that control and osteoporotic MSCs differ in their adipogenic potential as shown by expression of active PPARgamma protein. Leptin exerted an antiadipogenic effect only on control MSCs increasing the proportion of inactive phosphorylated PPARgamma protein. Finally, results obtained during adipogenesis of osteoporotic cells suggest that this process is abnormal not only because of increased adipocyte number, but because of impaired leptin cells response.     

二甲双胍调控糖尿病大鼠骨髓间充质干细胞的增殖和分化

虽然二甲双胍广泛用于治疗2型糖尿病,但是其对骨骼的潜在影响知之甚少。因此,本研究评估了二甲双胍对培养的大鼠骨髓间充质干细胞(MSCs)和脂肪细胞两者的分化以及增殖的影响。首先随机组形成对照实验,其中对照组为在不经二甲双胍处理培养基中培养MSCs细胞21 d,而二甲双胍组则在用100μmol/L二甲双胍处理培养基中培养MSCs 21 d。结果表明,二甲双胍增强了大鼠MSCs的成骨细胞分化细胞中ALP的活性,抑制了培养中MSCs脂肪形成分化的过程,但是增强了MSCs细胞的增殖能力。     

Mesenchymal stem cells from osteoporotic patients produce a type I collagen-deficient extracellular matrix favoring adipogenic differentiation

Mesenchymal stem cells (MSCs), precursor cells resident in the bone marrow, have the capacity to differentiate into bone, cartilage, fat, and connective tissue. We have recently reported that MSCs from "healthy" donors differ from cells obtained from osteoporotic postmenopausal women in their proliferation rate, mitogenic response to osteogenic growth factors, and potential to mineralize. The purpose of this study was to examine the factors that explain the differential capacity of MSCs derived from "healthy" control and osteoporotic postmenopausal women to support mineralization. In addition, we examined the factors that regulate the differentiation of osteoporotic cells into adipocytes. For this purpose, we isolated MSCs from bone marrow of donors and analyzed the synthesis and deposition of type I collagen, the main component of bone extracellular matrix, the time course of gelatinolytic activity expression, the deposition of transforming growth factor beta (TGF-beta), and the ability of cells to differentiate into adipocytes. Our results indicate that cells derived from osteoporotic donors synthesized 50% less type I collagen than normal cells and maintained higher levels of gelatinolytic activity under differentiation conditions (70% versus 15% after 14 days in culture). MSCs derived from osteoporotic women produced 60-65% less TGF-beta and expressed higher adipogenic capacity. We conclude that the capacity of MSCs derived from osteoporotic postmenopausal women to generate and maintain type I collagen-rich extracellular matrix is decreased, favoring their adipogenic differentiation. These observations may explain the decreased mineralization previously observed in these types of cells.     

Tissue-engineered bone formation with cryopreserved human bone marrow mesenchymal stem cells

Bone marrow mesenchymal stem cells (MSCs) have become the main cell source for bone tissue engineering. It has been reported that cryopreserved human MSCs can maintain their potential for proliferation and osteogenic differentiation in vitro. There are, however, no reports on osteogenesis with cryopreserved human MSCs in vivo. The aim of this study was to determine whether cryopreservation had an effect on the proliferation capability and osteogenic differentiation of human MSCs on scaffolds in vitro and in vivo. MSCs were isolated from human bone marrow, cultured in vitro until passage 2, and then frozen and stored at −196 °C in liquid nitrogen with 10% Me₂SO as cryoprotectant for 24 h. The cryopreserved MSCs were then thawed rapidly, seeded onto partially demineralized bone matrix (pDBM) scaffolds and cultured in osteogenic media containing 10 mM sodium β-glycerophosphate, 50 μM l-ascorbic acid, and 10 nM dexamethasone. Non-cryopreserved MSCs seeded onto the pDBM scaffolds were used as control groups. Scanning electronic microscopy (SEM) observation, DNA content assays, and measurements of alkaline phosphatase (ALP) activity and osteocalcin (OCN) content were applied, and the results showed that the proliferation potential and osteogenic differentiation of MSCs on pDBM in vitro were not affected by cryopreservation. After 2 weeks of subculture, the MSCs/pDBM composites were subcutaneously implanted into the athymic mice. The constructs were harvested at 4 and 8 weeks postimplantation, and histological examination showed tissue-engineered bone formation in the pDBM pores in both groups. Based on these results, it can be concluded that cryopreservation allows human MSCs to be available for potential therapeutic use to tissue-engineer bone.     

1950 MHz射频电磁场对人脐带间充质干细胞增殖和成骨分化的影响研究

目的:间充质干细胞(Mesenchymal stem cells,MSCs)具有广阔的临床应用前景,但由于其体外增殖和定向分化等问题,制约了其进一步应用。本研究拟探讨1950MHz射频电磁场(Radio-frequency electromagnetic fields,RF-EMF)对人脐带间充质干细胞(Human umbilical cord mesenchymal stem cells,hUC-MSCs)增殖和成骨方向分化的影响,以期为MSCs的体外增殖和定向分化提供一条新途径。方法:华通氏胶组织块法分离培养人脐带间充质干细胞,流式细胞仪检测间充质干细胞特异性标志物。选择鉴定后的第3至第6代(P3-P6)hUC-MSCs用于实验。将hUC-MSCs细胞暴露或假暴露于频率为1950 MHz,比吸收率(Specific absorption rate,SAR)分别为0.5,1.0和2.0 W/kg的RF-EMF中,每天暴露1 h(5 min开,10 min关),连续暴露7 d。暴露结束后,流式细胞仪检测细胞周期,免疫荧光检测增殖相关蛋白Ki67表达,连续6天用CCK-8方法检测细胞数。在成骨分化研究中,将P3代的hUC-MSCs随机分为假暴露(sham)组,射频辐射暴露(RF)组,成骨诱导培养基组(Induction medium,OM)和成骨诱导培养基联合射频辐射暴露(OM+RF)组,暴露SAR值为2.0 W/kg,其它参数不变。暴露结束后立即检测细胞的碱性磷酸酶(Alkaline phosphatase,ALP)活性。结果:原代培养的细胞具有MSC典型外观,且表达MSCs特异性表面抗原。与sham组相比,不同SAR值RF暴露后,hUC-MSCs的增殖能力无明显变化,S期细胞比例及Ki67蛋白水平也无显著改变。此外,hUC-MSCs经SAR值为2.0W/kg的RF暴露7 d,与sham组相比其ALP活性无显著变化。与OM组相比,OM+RF组的ALP活性亦无显著改变。结论:华通氏胶组织块法能够培养出纯度较高的间充质干细胞,本实验条件下的1950 MHz射频电磁场对hUC-MSCs的增殖和成骨分化均无显著影响。     

Comparison of gingiva‐derived and bone marrow mesenchymal stem cells for osteogenesis

Presently, bone marrow is considered as a prime source of mesenchymal stem cells; however, there are some drawbacks and limitations. Compared with other mesenchymal stem cell (MSC) sources, gingiva‐derived mesenchymal stem cells (GMSCs) are abundant and easy to obtain through minimally invasive cell isolation techniques. In this study, MSCs derived from gingiva and bone marrow were isolated and cultured from mice. GMSCs were characterized by osteogenic, adipogenic and chondrogenic differentiation, and flow cytometry. Compared with bone marrow MSCs (BMSCs), the proliferation capacity was judged by CCK‐8 proliferation assay. Osteogenic differentiation was assessed by ALP staining, ALP assay and Alizarin red staining. RT‐qPCR was performed for ALP, OCN, OSX and Runx2. The results indicated that GMSCs showed higher proliferative capacity than BMSCs. GMSCs turned more positive for ALP and formed a more number of mineralized nodules than BMSCs after osteogenic induction. RT‐qPCR revealed that the expression of ALP, OCN, OSX and Runx2 was significantly increased in the GMSCs compared with that in BMSCs. Moreover, it was found that the number of CD90‐positive cells in GMSCs elevated more than that of BMSCs during osteogenic induction. Taking these results together, it was indicated that GMSCs might be a promising source in the future bone tissue engineering.