鼠骨髓基质干细胞的特性及向成骨细胞的分化

目的分离纯化成年小鼠骨髓基质干细胞并进行定向诱导。方法利用Percoll液分离成年小鼠骨髓基质干细胞,Ter119、CD45磁珠纯化细胞,培养2-3代的细胞用矿化液进行定向诱导。对诱导前后的细胞进行免疫组化染色。结果 镜下见原代细胞呈多种形态。Ter119、CD45磁珠筛选可获得纯度达57.95％以上骨髓基质干细胞。碱性磷酸酶、Von Kossa染色阳性,油红染色弱阳性,矿化液诱导2周后,可形成钙结节。结论利用Percoll液结合磁珠分离法是离体条件下获得骨髓基质干细胞的一个简便有效的方法,纯化后定向诱导可得到较纯的成骨细胞。     

补骨脂素对大鼠骨髓间充质干细胞成骨分化的影响

目的研究不同浓度的补骨脂素对大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMMSCs)成骨分化的影响,为临床应用补骨脂素治疗骨质疏松症提供理论依据。方法选取8周龄健康雄性SD大鼠,取双侧股骨、胫骨,对大鼠的BMMSCs进行分离、原代培养和细胞表型鉴定。取第3代大鼠BMMSCs,体外利用成骨诱导培养基进行不同浓度补骨脂素的药物诱导,MTT法检测不同浓度的补骨脂素对大鼠BMMSCs生长的影响;通过碱性磷酸酶(ALP)活性测定、Western blot法和茜素红染色方法评价不同浓度补骨脂素对大鼠BMMSCs成骨分化能力。结果第3代大鼠BMMSCs表面抗原符合干细胞鉴定标准,成骨诱导后给予15μmol/L补骨脂素对大鼠BMMSCs的增殖作用最佳,ALP活性、Runx-2表达和钙化结节数目均明显高于经典成骨诱导组、5μmol/L和10μmol/L和20μmol/L补骨脂素组。结论15μmol/L补骨脂素成骨诱导促进BMMSCs向成骨分化,从而起到防治骨质疏松的作用。     

放射抑制小鼠骨髓间充质干细胞成骨潜能并导致骨损伤

目的 观察放射对小鼠骨髓间充质干细胞(BMMSCs)体外成骨潜能及体内骨组织的影响.方法 分离、培养正常的及接受4Gy放射后28d的小鼠BMMSCs,用碱性磷酸酶(ALP)和Von Kossa染色法鉴定BMMSCs体外成骨分化潜能的改变,并通过骨组织形态学和骨密度(BMD)检测放射后小鼠体内骨组织的相关变化.结果 4Gy照射28d后小鼠BMMSCs的成骨潜能明显降低,同时体内骨组织结构破坏,小鼠骨密度降低.结论 放射损伤后小鼠BMMSCs的成骨潜能显著降低,可能在干细胞水平参与了放射后骨损伤的发生.     

Perfusion culture enhances osteogenic differentiation of rat mesenchymal stem cells in collagen sponge reinforced with poly(glycolic Acid) fiber

The objective of this study was to obtain fundamental knowledge about in vitro culture systems to enhance the proliferation and differentiation of mesenchymal stem cells (MSCs) in collagen sponge reinforced by the incorporation of poly(glycolic acid) (PGA) fiber. A collagen solution with PGA fiber homogeneously localized at PGA:collagen weight ratios of 0.67, 1.25, 2.5, and 5 was freezedried, followed by cross-linking of combined dehydrothermal, glutaraldehyde, and ultraviolet treatment. Scanning electron microscopy revealed that collagen sponges exhibited homogeneous and interconnected pore structures with an average size of 180 microm, irrespective of PGA fiber incorporation. When rat MSCs were seeded into collagen sponge with or without PGA fiber incorporation, more attached cells were observed in collagen sponge incorporating PGA fiber than in collagen sponge without PGA fiber incorporation, irrespective of the PGA:collagen ratio. The proliferation and osteogenic differentiation of MSCs in PGA-reinforced sponge at a weight ratio of 5 were greatly influenced by the culture method and growth conditions. Alkaline phosphatase (ALP) activity and osteocalcin content of MSCs cultured in PGA-reinforced sponge by the perfusion method became maximum at a flow rate of 0.2 mL/min, although they increased with culture time period. It may be concluded that appropriate perfusion conditions enable MSCs to positively improve the extent of proliferation and differentiation.     

Multidifferentiation potential of mesenchymal stem cells in three-dimensional collagen gel cultures

Mesenchymal stem cells (MSCs) have a great therapeutic potential resulting from their ability to differentiate into multiple tissues when cultured under specific conditions. However, it has not been clearly demonstrated whether or not MSCs exhibit a multidifferentiation potential in three-dimensional collagen gel cultures. This study was conducted to explore the multidifferentiation potential of MSCs cultured in three-dimensional collagen gels. Human MSCs were cultured in 0.3% collagen gel for 20 days in chondrogenic differentiation medium (CDM), and for 14 days in osteogenic differentiation medium (ODM). Increases in GAG deposits, intensity of toluidine blue staining, and mRNA expressions of chondrogenic markers (type II collagen and type X collagen) were found in human MSCs cultured in the collagen gel maintained in CDM. Positive staining for alkaline phosphatase (ALP) activity and alizarin red, and increases in mRNA expressions of osteogenic markers (type I collagen, bone sialoprotein and ALP) were noted in the MSCs maintained in ODM. These findings emphasize that human MSCs have an ability to differentiate into both bone and cartilaginous tissues in three-dimensional collagen gel cultures, indicating potential clinical applications of MSC transplant therapy with collagen gel as a scaffold for bone or cartilage regeneration in complicated tissue defects.     

Divergent expression and localization of aquaporin 5, an exocrine-type water channel,in the submandibular gland of Sprague-Dawley rats

By Western blot analysis, the expression level of aquaporin (AQP) 5 in the submandibular gland (SMG) was found to be different among individual rats of the Sprague-Dawley (SD) strain. Such differences were observed for AQP5 but not for AQP1 and consequently the SD strain was divided into two groups, one expressing a high level of AQP5 and the other a low one. The difference in average intensity of expression between the two groups was more than twofold. Immunohistochemical analysis of the SMG demonstrated that the AQP5 protein was localized in the basal and apical/lateral plasma membrane of acinar cells in rats expressing the high level of AQP5. In the rat expressing the low level, however, this channel protein was localized strongly in the apical/lateral plasma membrane, but only very weakly in the basal membrane of the acinar cells. Such a diverse localization of AQP5 was confirmed by Western blotting as well. Breeding between brother and sister was repeated for two times within high expressers and low expressers to obtain the third generation progenies (F2); the AQP5 level of the SMG in the third generation (F2 rats) from high expressers was significantly higher than the F2 from low expressers. Our present study suggests the existence of genetic variation in the expression of a water channel protein, AQP5, in rats.     

Modulation of differentiation and mineralization of marrow stromal cells cultured on biomimetic hydrogels modified with Arg-Gly-Asp containing peptides

We synthesized biomimetic hydrogels modified with an osteopontin-derived peptide (ODP) and used them as a substrate for in vitro culture of marrow stromal cells (MSCs) to investigate the effect of the biomimetic surface on differentiation of MSCs into osteoblasts. Proliferation and biological assays for 16 days proved that MSCs became differentiated into osteoblasts secreting osteogenic phenotypic markers such as alkaline phosphatase (ALP), osteopontin, and mineralized calcium. In addition, there was an additive effect of the cell-binding peptide on differentiation and mineralization of MSCs cultured in the presence of soluble osteogenic supplements in cell culture media. For example, calcium content at day 16 on peptide-modified hydrogels was significantly higher than on tissue culture polystyrene. Two general trends were observed: (1) proliferation of MSCs decreased as the amount of differentiation markers increased, and (2) higher peptide concentrations accelerated the differentiation of MSCs. On the hydrogel modified with ODP, ALP activity exhibited a maximum value of 36.7 +/- 4.2 pmol/cell/h at day 10 for the concentration of 2 micromol/g while the culture time needed for maximum ALP activity occurred on day 13 for the lower concentrations. On the same hydrogel, the calcium content at day 10 was 21.4 +/- 2.3 ng/cell for the peptide concentration of 2 micromol/g and 1.0 +/- 0.3 ng/cell for 1.0 micromol/g. We used Gly-Arg-Gly-Asp-Ser (GRGDS) for modification of the hydrogel as a comparison to the results with ODP. However, osteoblast development was not significantly affected by the nature of the binding peptide sequences. These results suggest that MSC function can be modulated by variation of the peptide concentration in biomimetic hydrogels used for scaffold-based bone tissue engineering.     

ERK信号通路在檞皮素促大鼠MSCs成骨分化中的作用

目的:观察细胞外信号调节激酶(ERK)信号通路在檞皮素(QUE)促进SD大鼠骨髓间充质干细胞(MSCs)成骨分化过程中的作用。方法:(1)用0.01μmol/L、0.1μmol/L、1μmol/L、10μmol/L和100μmol/LQUE干预MSCs,MTT法检测各浓度QUE对MSCs增殖的影响,碱性磷酸酶(ALP)测定试剂盒检测各浓度QUE对MSCsALP表达的影响;(2)用ERK1/2抑制剂干预后,加入QUE,用ALP测定试剂盒检测ALP的表达,ELISA法检测Ⅰ型胶原(ColⅠ)和骨钙素(BGP)的表达,Westernblotting检测ERK1/2的表达,荧光定量PCR检测转化生长因子β₁(TGF-β₁)mRNA、骨形成蛋白2(BMP-2)mRNA和核心结合因子α1(Cbfα1)mRNA表达。结果:(1)0.1μmol/L、1μmol/L和10μmol/LQUE剂量依赖性地促进MSCsALP的表达,同时能促进MSCs的增殖;(2)与空白组相比,QUE组ALP、BGP和ColⅠ表达均增加(P<0.01),加入ERK1/2抑制剂后,磷酸化的ERK1/2表达减少(P<0.05),同时ALP、BGP和ColⅠ表达降低(P<0.01);(3)与空白组比较,QUE组TGF-β₁mRNA、BMP-2mRNA和Cbfα1mRNA的表达均增加(P<0.05),加入ERK1/2抑制剂后这3个基因的表达都下降(P<0.05)。结论:一定浓度的QUE能促进MSCs的增殖和成骨分化,ERK通路的激活在此过程中起到了重要的作用。     

力学应变对大鼠骨髓间充质干细胞增殖和成骨分化能力的影响

探讨周期性双轴力学应变对大鼠骨髓间充质干细胞(M esenchym a l stem ce lls,M SC s)增殖和成骨分化能力的影响。选用9月龄健康SD雌性大鼠,分离股骨、胫骨提取骨髓,采用密度梯度离心法分离M SC s。体外培养M SC s传至第3代,以1×105细胞浓度接种于双轴力学应变系统,选取4 000μstra in,频率为1hz的力学应变对M SC s加载。每天加载3次,每次2 h,间隔2 h。观察力学应变作用后1 d、3 d,M SC s增殖和成骨分化能力的变化,并与相应未加力学应变对照组比较。结果表明:(1)力学应变可增高M SC s的碱性磷酸酶(ALP)和骨桥蛋白(OPN)表达量;力学应变作用3 d后M SC s的ALP和OPN表达量明显高于力学应变作用1 d。I型胶原(COL I)仅在力学应变作用3 d增高;骨钙素(OCN)在各组无明显变化。(2)力学应变可促进M SC s增殖,但力学应变作用1 d和3 d对M SC s增殖的作用无明显差异。上述结果提示:力学应变可以促进M SC s的增殖和成骨分化能力。     

仿生活性人工软骨的体外构建及其异位成软骨分化实验研究

应用先进快速成形技术(RP)制备32枚粒度均匀(尺寸均为4mm×4mm×4mm)的聚乳酸-聚羟乙酸(PLGA)人工载体,该载体经I型胶原表面修饰后均分为A、B两组。A组载体复合人骨形态发生蛋白-2基因转染(rAAV-hBM P-2)的兔骨髓基质细胞(M SC s,2×104个细胞/枚);B组每枚载体复合等量、同代次、未基因转染M SC s。体外培养第5 d,从两组各取12枚细胞-载体复合物植入裸鼠皮下,术后30 d取材观察。结果发现rAAV-hBM P-2转染的M SC s成功表达目的基因。RP制备的PLGA载体具有良好的空间结构,大孔及材料表面微孔孔径分别为300μm和3~5μm。体外培养3~5 d,两组载体均复合生长着大量种子细胞。皮下埋植30 d,A组植入物形成较为典型的软骨细胞及基质,II型胶原蛋白表达阳性;同期B组植入物无软骨组织形成。A组聚酯材料面积百分率显著低于B组(P<0.01)。结果表明RP结合载体材料表面修饰,能制备出兼具理想孔隙结构和良好生物相容性的组织工程支架载体,该载体高效复合rAAV-hBM P-2转染的M SC s为组织工程软骨构建创造有利条件。     

Mice with cav-1 gene disruption have benign stromal lesions and compromised epithelial differentiation

Caveolin-1 (cav-1) is a major structural protein of caveolae, small invaginations of the plasma membrane that integrate and regulate signaling pathways involved in cell growth and differentiation. We previously generated a genetically engineered mice that are homozygous for a null mutation in exon 2 of cav-1 and documented increased incidence of urolithiasis in young male cav-1(-/-) mice. We attributed this, in part, to improper localization of plasma membrane calcium/calmodulin-dependent calcium ATPase in the distal convoluted tubules of the kidney. To document pathologies related to cav-1 function, we maintained cav-1(-/-) and control cav-1(+/+) mice for an extended time period. We report here that cav-1(-/-) mice demonstrate organ-specific growth-related disorders in stromal cells that normally have high levels of cav-1 expression. In many of these organs, epithelial cell growth/differentiation abnormalities were also observed, yet in most of these sites the epithelial cells normally express low to non-detectable levels of cav-1. We propose that loss of cav-1 function in stromal cells of various organs directly leads to a disorganized stromal compartment that, in turn, indirectly promotes abnormal growth and differentiation of adjacent epithelium.     

Modulation of mesenchymal stem cells behaviors by chitosan/gelatin/pectin network films

In this article, the chitosan/gelatin/pectin (CGP) network films were prepared to build appropriate physicochemical and mechanical microenvironment for attachment, proliferation, and differentiation of mesenchymal stem cells (MSCs). Results suggested that the hydrophilicity and mechanical character of CGP composites films could be modulated via adjusting the pectin content in the composites. The investigations of attachment and proliferation behaviors of mesenchymal stem cells (MSCs) on the CGP films were carried out. The morphology of cells was observed with hematoxylin/eosin staining (HE) and scanning electron microscope (SEM). The osteogenic differentiation of MSCs was investigated via ALP and polymerase chain reaction (PCR). Results suggested that the CGP films have excellent biocompatibility. MSCs seeded on CGP (0.1) film show higher proliferation capacity compared with other samples. Moreover, osteogenic differentiation of MSCs also depends on the properties of the substrate. The MSCs seeded on CGP (0.5) expressed the highest ALP activity, osteogenic gene expression and mineral formation capacity. These results suggest that the composition of the CGP network films could effectively modulate their physicochemical and mechanical properties and further regulate the cell behaviors of MSCs.     

The effect of glow discharge plasma surface modification of polymers on the osteogenic differentiation of committed human mesenchymal stem cells

Little is known of the effect of material surfaces on stem cell differentiation. The present study has addressed the hypothesis that the interaction of mesenchymal stem cells (MSCs) with material surfaces modified by glow discharge plasma is a major regulator of osteogenic differentiation. We found that biaxially oriented polypropylene (BOPP) plasma treated in ammonia significantly reduced up-regulation of expression of osteogenic marker genes, such as alkaline phosphatase (ALP), bone sialoprotein (BSP) and osteocalcin (OC). In contrast, ALP expression was up-regulated when cultured on treated Nylon-6 polyamide (Ny-t) but was substantially reduced when cultured on its pristine counterpart (Ny-p) on day 3. On day 7, ALP expression was down-regulated with MSCs cultured on Ny-t although its expression level was up again on day 14. BSP was expressed weakly on day 3, but was up-regulated when cultured on Ny-t and Ny-p. Its expression reached its maximum on day 14 when cultured on a polystyrene control, while it was cyclically up-regulated on Ny-t. Similarly, there was a slight increase in OC expression when MSCs were cultured on Ny-t and Ny-p on day 3, when compared to control. Thus, the nature of the surface can directly influence MSCs differentiation, ultimately affecting the quality of new tissue formation with BOPP-t suppressing osteogenic differentiation.     

Comparative study of osteogenic differentiation potential of mesenchymal stem cells derived from bone marrow and adipose tissue of osteoporotic female rats

Osteoporosis causes reduction of osteogenic differentiation of mesenchymal stem cells (MSCs) from bone marrow and adipose tissue. This study was designed to compare the osteogenic potential of bone marrow mesenchymal stem cells (BMMSCs) and adipose-derived stem cells (ADSCs) of ovariectomized (OVX) rats. MSC were harvested from bone marrow and inguinal fat pads of six OVX rats. The limitations of this report are that cells from different animals were pooled for the purpose of the experiments that were carried out in this study. At 7, 14 and 21?d of osteogenic differentiation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) conversion, alkaline phosphatase activity and gene expression for collagen I, osteocalcin, bone sialoprotein, osteopontin and bone morphogenetic protein-2 bone morphogenetic protein-2 (BMP-2) were analyzed. At 21?d, percentage of cells per field and percentage of mineralized nodule were analyzed. The data were subjected to analysis of variance, and the means were compared by Student–Newman–Keuls test. The cells, regardless of group, showed phenotypic characteristics consistent with stem cells. MTT conversion, alkaline phosphatase activity, percentage of mineralized nodule and expression of collagen I, osteocalcin and BMP-2 of ADSCs from OVX rats were higher when compared to BMMSCs from OVX rats in at least one of the evaluated periods (p?p?相似文献   

Role of aquaporin 3 in development, subtypes and activation of dendritic cells

Dendritic cells (DCs) uptake soluble antigens and large volumes of fluid through macropinocytosis and migrate for antigen presentation. Aquaporin 3 (AQP3), a water and glycerol transporting protein, is highly expressed in immature DCs. To elucidate the role of AQP3 in DC function, we investigated subtype and activation of DCs in AQP3 knock-out (AQP3(-/-)) mice. Depletion of AQP3 did not affect the development of bone marrow-derived DCs (BM-DCs) by GM-CSF or the Flt3 ligand and the level of expression of CD86 on unstimulated and LPS-stimulated BM-DCs. In addition, the percentage of CD86(+) cells among splenic cDCs after LPS treatment in both in vitro and in vivo conditions was similar in wild type and AQP3(-/-) mice. However, the frequency of CD4(+) cDCs in the spleen of AQP3(-/-) mice was significantly lower than that of wild type mice. There was higher expression of CD103 in the CD8(+) subpopulation of splenic cDCs from AQP3(-/-) mice than wild type mice. In the dermis, more CD103-expressing cells were detected in AQP3(-/-) mice than in wild type mice and the LPS-induced decrease of CD103(+) dermal DCs was impaired in AQP3(-/-) mice. AQP3 depletion did not affect the uptake of either albumin or dextran by CD11c(+) splenic DCs. However, HgCl(2), which is an AQP inhibitor, significantly inhibited the uptake of albumin but not dextran by CD11c(+) splenic DCs. These results suggest that AQP3 may play a role in modulating DC population and migration.     

Aquaporin-3 facilitates epidermal cell migration and proliferation during wound healing

Healing of skin wounds is a multi-step process involving the migration and proliferation of basal keratinocytes in epidermis, which strongly express the water/glycerol-transporting protein aquaporin-3 (AQP3). In this study, we show impaired skin wound healing in AQP3-deficient mice, which results from distinct defects in epidermal cell migration and proliferation. In vivo wound healing was approximately 80% complete in wild-type mice at 5 days vs approximately 50% complete in AQP3 null mice, with remarkably fewer proliferating, BrdU-positive keratinocytes. After AQP3 knock-down in keratinocyte cell cultures, which reduced cell membrane water and glycerol permeabilities, cell migration was slowed by more than twofold, with reduced lamellipodia formation at the leading edge of migrating cells. Proliferation of AQP3 knock-down keratinocytes was significantly impaired during wound repair. Mitogen-induced cell proliferation was also impaired in AQP3 deficient keratinocytes, with greatly reduced p38 MAPK activity. In mice, oral glycerol supplementation largely corrected defective wound healing and epidermal cell proliferation. Our results provide evidence for involvement of AQP3-facilitated water transport in epidermal cell migration and for AQP3-facilitated glycerol transport in epidermal cell proliferation.