Maintenance of hepatocyte functions by coculture with bone marrow stromal cells

Rat hepatocytes and bone marrow stromal cells (BMSCs) were cocultured with the aim of maintaining differentiated hepatocyte functions. After BMSCs were expanded to a confluent monolayer, freshly isolated hepatocytes were cultured with them, separated by a semipermeable membrane. The BMSCs significantly increased the urea synthesis and albumin secretion activities of the hepatocytes. Conditioned medium prepared from a BMSC monoculture had the same effect. Further study showed that interleukin-6 was involved in the maintenance of urea synthesis and another factor in the maintenance of albumin secretion.     

Mag-seeding of rat bone marrow stromal cells into porous hydroxyapatite scaffolds for bone tissue engineering

Bone tissue engineering has been investigated as an alternative strategy for autograft transplantation. In the process of tissue engineering, cell seeding into three-dimensional (3-D) scaffolds is the first step for constructing 3-D tissues. We have proposed a methodology of cell seeding into 3-D porous scaffolds using magnetic force and magnetite nanoparticles, which we term Mag-seeding. In this study, we applied this Mag-seeding technique to bone tissue engineering using bone marrow stromal cells (BMSCs) and 3-D hydroxyapatite (HA) scaffolds. BMSCs were magnetically labeled with our original magnetite cationic liposomes (MCLs) having a positive surface charge to improve adsorption to cell surface. Magnetically labeled BMSCs were seeded onto a scaffold, and a 1-T magnet was placed under the scaffold. By using Mag-seeding, the cells were successfully seeded into the internal space of scaffolds with a high cell density. The cell seeding efficiency into HA scaffolds by Mag-seeding was approximately threefold larger than that by static-seeding (conventional method, without a magnet). After a 14-d cultivation period using the osteogenic induction medium by Mag-seeding, the level of two representative osteogenic markers (alkaline phosphatase and osteocalcin) were significantly higher than those by static-seeding. These results indicated that Mag-seeding of BMSCs into HA scaffolds is an effective approach to bone tissue engineering.     

Protective effects of lupeol against benzo[a]pyrene induced clastogenicity in mouse bone marrow cells

Fruits and vegetables contain a variety of ingredients that exhibit chemopreventive effects against an array of xenobiotics. In the present study, the antigenotoxic potential of lupeol, a triterpene, and mango pulp extract (MPE) was evaluated in Swiss albino mice. Benzo[a]pyrene (B[a]P), a well-known mutagen, was given at a single dose of 100 mg/kg body weight intraperitoneally. Pretreatment with lupeol (1 mg/animal) and MPE (1 mL, 20%) was given through oral intubation for 7 days prior to B[a]P administration. Animals from all the groups were killed at sampling time of 24 h and their bone marrow tissue was analyzed for chromosomal damage and micronuclei induction. In B[a]P-treated animals a significant induction of chromosomal aberration and micronuclei was recorded, with a decrease in mitotic index. In lupeol- or MPE-supplemented groups, a significant decrease in B[a]P-induced clastogenicity was recorded. The incidence of aberrant cells and micronuclei was found to be reduced by both lupeol and MPE when compared to the B[a]P-treated group. The anti-cytotoxic effects of lupeol or MPE were also evident, as observed by significant increase in mitotic index. Thus, results of the present investigation revealed that lupeol and MPE have protective effects against B[a]P-induced clastogenic changes in Swiss albino mice.     

Ex vivo expansion of human cord blood hematopoietic progenitor cells using glutaraldehyde-fixed human bone marrow stromal cells

Human stromal cells were immortalized and fixed with glutaraldehyde to support an ex vivo expansion of human cord blood hematopoietic progenitor cells. In addition, this enabled glutaraldehyde-fixed stromal cells to be stored at 4 degrees C. Although freeze-dried glutaraldehyde-fixed stromal cells did not increase the number of the progenitor cells, the percent decrease in the number of CD34(+) cells in the presence of freeze-dried glutaraldehyde-fixed stromal cells was less than that in the absence of the stromal cells. Thus, glutaraldehyde-fixed stromal cells can serve as a stabilizing device for hematopoietic cell expansion.     

Availability of bone marrow stromal cells in three-dimensional coculture with hepatocytes and transplantation into liver-damaged mice

Rat bone marrow stromal cells (BMSCs) were cultured in porous hydroxyapatite (HA) disks for 2 weeks to form a cell layer on the surface. Freshly isolated hepatocytes were then inoculated into both BMSC-cultured and non-treated HA disks. Hepatocytes cocultured with BMSCs secreted significantly more albumin than those in monoculture in vitro. The cell-packed HA disks were implanted into the peritoneal cavity of Nagase analbuminemia rats (NARs), and 4 weeks later, blood samples were collected to measure the albumin concentration. The cotransplantation of BMSCs with hepatocytes significantly increased the serum albumin concentration in NARs. The HA disks coculturing mice hepatocytes and BMSCs were also implanted into mice, in which liver damage had been induced using carbon tetrachloride and phenobarbital. The decreased serum albumin level in liver-damaged mice was completely recovered by the transplantation of hepatocytes and BMSCs. The serum level of IL-6 in liver-damaged mice was also increased by the cotransplantation of BMSCs and hepatocytes. Thus, the transplantation of BMSCs appears to have a systemic effect on recipients through the increase in the serum cytokine level as well as a local effect on cotransplanted hepatocytes.     

Effect of temperature on cell population balance in Dexter's culture of murine bone marrow hematopoietic cells with stromal cells

The effect of temperature in the range from 25 to 37 degrees C on the population balance of stromal and multiple-lineage hematopoietic cells from murine bone marrow at various stages of differentiation in Dexter's culture was investigated. The length of time required for stromal cells to reach confluence after inoculation of both cell types harvested from murine bone marrow was shorter at higher temperatures. On the other hand, the hematopoietic cell concentration initially decreased more rapidly at higher temperatures until the confluence of stromal cells, which should then support the proliferation of hematopoietic cells. The concentration of hematopoietic cells began to increase after 2 weeks incubation at 33 and 37 degrees C and after 4 weeks at 29 degrees C. However, the growth of hematopoietic cells was not stable at 37 degrees C, and neither stromal nor hematopoietic cells showed significant growth at 25 degrees C. The specific growth rate of hematopoietic cells at 29 degrees C after 4 weeks was comparable or higher than that at 33 degrees C, while the final concentration of hematopoietic cells was maximal at 33 degrees C. Using a cultivation method in which hematopoietic cells were recharged onto a confluent layer of stromal cells prepared at 33 degrees C, the maintenance and growth of hematopoietic cells were better at 29 degrees C than at higher temperatures. The content of progenitor cells among the hematopoietic cells increased prior to the increase in the hematopoietic cell concentration, and the progenitor cell was greater content at the lower temperature. These results suggest that cultivation at 29 degrees C might be superior to 33 degrees C for the maintenance and growth of hematopoietic cells with a prepared confluent layer of stromal cells.     

The mixed coculture effect of primary rat hepatocytes and bone marrow cells is caused by soluble factors derived from bone marrow cells

Heterospheroids consisting of hepatocytes and bone marrow cells (BMCs) are formed by the mixed coculture of these cells and enhance the expression and maintenance of the liver-specific functions of hepatocytes. Not only the soluble factors derived from these cells, but also functional organoid (heterospheroid) formation, are considered to underlie this coculture effect. Therefore, in the present study, we aimed to clarify the mechanism of this co-culture effect. We performed hepatocyte monoculture with conditioned media prepared from hepatocyte cultures, BMC cultures and a coculture of hepatocytes and BMCs. When using any type of conditioned medium, no hepatocyte spheroids formed, and the hepatocytes formed a monolayer. In addition, an effect for these conditioned media was shown in terms of the albumin production and ammonia metabolism activities of the hepatocytes; conditioned medium from BMCs showed the strongest effect. The monocultured hepatocytes in the conditioned medium derived from BMCs showed equivalent albumin production and ammonia metabolism activities to the cocultured spheroids of hepatocytes and BMCs. Therefore, it was determined that the effect of the coculture of hepatocytes and BMCs was caused by soluble factors derived from BMCs.     

The effects of acrylamide on the frequency of megakaryocytic emperipolesis and the mitotic activity of rat bone marrow cells

BACKGROUND: Although the pathophysiological importance of emperipolesis is not known exactly, it has been reported to increase significantly in cases of various cancer types, different tumours and thrombosis disorders. In this study the effects of acrylamide on the frequency of megakaryocytic emperipolesis and the mitotic activity in rat bone marrow cells were determined. For this purpose, two separate experiments were performed with Sprague‐Dawley rats gavaged with 0, 30, 45 and 60 mg acrylamide kg^?1 body weight (BW) for five consecutive days. In the second experiment, 3 mg colchicine kg^?1 BW was injected intraperitoneally 2 h before cervical dislocation. Bone marrow samples were taken 24 h after the last application in both experiments. RESULTS: It was found that only the highest dose of acrylamide significantly decreased the incidence of megakaryocytic emperipolesis and that the types of bone marrow cells engulfed by megakaryocytes were mostly neutrophil granulocytes. Neither megakaryocytes nor engulfed cells showed any morphological degeneration. In the mitotic activity experiment, doses of 45 and 60 mg acrylamide kg^?1 BW decreased the mitotic activity of bone marrow cells in comparison with the control group. CONCLUSION: It was concluded that the decrease in megakaryocytic emperipolesis frequency might be a consequence of the decrease in mitotic activity in bone marrow cells. Copyright © 2011 Society of Chemical Industry     

植物乳杆菌胞外多糖对小鼠树突状细胞分泌的调控

采用细胞因子诱导法,以加入重组小鼠粒细胞巨噬细胞集落刺激因子(rmGM-CSF)、重组小鼠白细胞介素-4(rmIL-4)及10%胎牛血清的RPMI1640为完全培养基培养树突状细胞(DCs)。实验组加入植物乳杆菌胞外多糖,脂多糖LPS和RPMI1640分别作为阳性和空白对照。采用Griess法和双抗体夹心ELISA检测DCs分泌NO,促炎症因子IL-12p70,抗炎症因子IL-10和趋化因子RANTES的浓度。结果表明:植物乳杆菌胞外多糖刺激DCs后,DCs分泌NO、IL-12p70、IL-10和RANTES的浓度分别是空白组的110%、194%、76%和133%,且呈剂量依赖关系。植物乳杆菌胞外多糖能促进小鼠骨髓来源的DCs分泌能力。     

Identification of genes regulated by interleukin-1beta in human endometrial stromal cells

Interleukin-1beta (IL-1b) is an important immune regulatory factor that in human endometrium plays a role in both menstruation and implantation in the event of pregnancy. It promotes inflammatory-like processes and also stimulates tissue remodelling. We present a cDNA microarray study documenting the major effects of IL-1beta on gene expression in stromal cells from human endometrium. Endometrial stromal cells from five normal healthy women at the mid secretory phase were cultured with or without IL-1beta at 50 and 500 pg/ml for 48 h. cDNA microarrays were used to compare the levels of gene expression in total RNA isolated from cells stimulated with IL-1beta. These cDNA arrays were produced containing 15 164 sequence-verified clones, which included genes known to be important in angiogenesis, immune modulators, apoptosis, cell signalling, extra-cellular matrix (ECM) remodelling and cell cycle regulation. Genes which were regulated by IL-1beta were identified by analysis of the microarray data using the Significance Analysis of Microarrays software package. Upregulated (n = 23) and downregulated (n = 6) different genes were observed, which changed at least 3-fold, at a false discovery rate of less than 2% (P < 0.02). Our results have identified genes regulated by IL-1beta, which are involved in leukocyte recruitment, ECM remodelling and other cellular functions. Changes in three genes, IL-8, colony-stimulating factor 2 and aldoketo reductase family 1 member 1, which were upregulated by IL-1beta, were verified using real-time PCR. Novel functions regulated by IL-1beta in endometrium, including genes involved in free radical protection, and fatty acid metabolism were also identified. These results also provide new insights into the role of IL-1beta in disorders of the endometrium, especially in implantation-related infertility and endometriosis, in which this cytokine plays a major role.     

Testosterone inhibits matrix metalloproteinase-1 production in human endometrial stromal cells in vitro

Androgen receptor (AR) is reported to be expressed in human uterine endometrium, but not much information is available on the role of androgens in human endometrium. The purpose of this study is to investigate the role of androgens in the regulation of matrix metalloproteinase (MMP)-1, which is one of the important MMPs for menstruation and embryo implantation in human endometrium. Human endometrial stromal cells (HESCs) were obtained from human endometrium by enzymatic dissociation method. Purified HESCs were incubated with 17beta-estradiol (E2), testosterone, or E2 + testosterone. Progestins (natural progesterone or medroxyprogesterone acetate) or vehicle (dimethyl sulfoxide) were also added to the media instead of testosterone. Furthermore, hydroxyflutamide (FLU),a specific AR antagonist, was also supplemented to cultured media. The amounts of MMP-1 in cultured media and in HESC lysates were examined by ELISA measurements and western blotting analysis respectively. The expression of ARmRNA in HESCs RNA was analyzed by RT-PCR. Testosterone significantly inhibited MMP-1 in both cultured media and cell lysates in a dose-dependent manner. Progestins also inhibited MMP-1. Furthermore, FLU completely recovered the decrease of MMP-1 induced by testosterone. ARmRNA was detected in all HESCs RNA. The present study demonstrated that the secretion and production of MMP-1 in HESCs in vitro were inhibited by testosterone through androgen receptors in a manner similar to that seen for progesterone. These findings indicate that androgen may play an important role in morphological and functional changes of human endometrium.     

High inoculation cell density could accelerate the differentiation of human bone marrow mesenchymal stem cells to chondrocyte cells

The effects of the density of human mesenchymal stem cells (MSCs) on their differentiation to chondrocytes in a differentiation medium supplemented with dexamethasone, TGFbeta3, and IGF-1 were investigated for the regenerative therapy of cartilage. The increase in the initial density of MSCs from 0.05 x 10(4) to 0.9 x 10(4) cells/cm(2) accelerated the increase in the expression level of aggrecan mRNA during the differentiation culture for 7 d. The conditioned medium harvested at 7 d from the differentiation culture with an initial MSC density of 0.3 x 10(4) cells/cm(2) accelerated the initial increase in the expression level for 3 d in the differentiation culture with an initial MSC density of 0.3 x 10(4) cells/cm(2), whereas the conditioned medium harvested at 7 d in the differentiation culture with an initial MSC density of 0.05 x 10(4) cells/cm(2) did not. The differentiation culture after 14 d with an initial MSC concentration of 0.3 x 10(4) cells/cm(2) showed an expression level 1.7-fold that in the case of the culture with an initial MSC concentration of 0.05 x 10(4) cells/cm(2). Thus, a high MSC inoculum density might be appropriate for the rapid differentiation of MSCs to chondrocytes.     

Shrinkage-free preparation of scaffold-free cartilage-like disk-shaped cell sheet using human bone marrow mesenchymal stem cells

Aiming for the clinical application of cartilage regeneration, a culture method for mesenchymal stem cells (MSCs) derived from human bone marrow to obtain scaffold-free cartilage-like disk-shaped sheet of uniform sizes without the shrinkage was investigated. A disk-shaped cell sheet having the same diameter as that of the membrane without the shrinkage was formed after the cultivation of MSCs (18.6 × 10(5)cells/well) for 3 weeks in a cell culture insert (CCI) containing a flat membrane whose porosity was 12%, while 6.2 and 31.0 × 10(5)MSCs/well, respectively, resulted in the shrinkage of the aggregate and the hole formation in the center part of the sheet. Cell aggregates shrunk also in a 96-well plate and CCIs having lower porosity. The disk-shaped cell sheet showed the comparable thickness (1.2mm) and sulfated glycosaminoglycan (sGAG) density to those of the pellet formed in a pellet culture. The gene expression levels of aggrecan and type II collagen in the disk-shaped cell sheet were not lower than those in the pellet. In conclusion, the usage of CCI having 12% porosity and 18.6 × 10(5)MSCs/well could avoid the shrinkage from the formation of the scaffold-free cartilage-like disk-shaped cell sheet.     

Antioxidant activities and inhibitory effects of dietary plants against sodium nitroprusside induced lipid peroxidation in the mouse brain and liver

The antioxidant properties of aqueous extracts of 6 medicinal plants, Phyllanthus emblica, Terminalia chebula (black and yellow), Terminalia arjuna, Balsamodendron Mukul, and Alium sativum against lipid peroxidation in mouse tissues were investigated. Extracts showed inhibition against thiobarbituric acid reactive species (TBARS) induced by pro-oxidant (5 μM sodium nitroprusside) in the mouse brain and liver. Extracts displayed high free radical scavenging activities against DPPH (IC₅₀, 23.23±1.2 μg/mL, P. emblica), 20.24±0.9 μg/mL (T. chebula yellow), 17.33±1.1 μg/mL (T. chebula black), 19.44±0.45 μg/mL (T. arjuna), 56.59±2.1 μg/mL (Balsamo-dendron Mukul), and higher than 200 μg/mL (A. sativum). Higher antioxidant and inhibitory effects of T. chebula black are attributed to a higher phenolic content, Fe(II) chelating ability, reducing ability, nitric oxide radical scavenging, and free radical scavenging activity. Oxidative stress in the brain and liver could potentially be managed/prevented by dietary intake of these plants.     

Dietary hydroxycinnamates prevent oxidative damages to liver,spleen, and bone marrow cells in irradiation-exposed mice

Food Science and Biotechnology - Dietary hydroxycinnamates are considered as attractive materials for radioprotection. This study explores whether hydroxycinnamates protect against...     

菜籽多糖WPS-1对小鼠脾淋巴细胞的免疫作用

采用尼龙毛柱法从脾淋巴细胞分离T、B淋巴细胞。通过MTT法检测,WPS-1对T淋巴细胞具有明显的增殖活性,并表现出明显的剂量-效果关系,增殖的最佳浓度为40μg/mL,并且WPS-1比APS-2作用效果更好。采用半定量RT-PCR进一步研究WPS-1对T淋巴细胞分泌的细胞因子白细胞介素2（IL-2）、IL-4、IL-6、干扰素γ（IFN-γ）mRNA表达的影响。首先研究了各细胞因子mRNA随WPS-1作用时间的变化,WPS-1促进IL-2、IL-4、IL-6mRNA表达最佳作用时间都为24h,而促进IFN-γmRNA表达的最佳作用时间为12h。采用各细胞因子mRNA表达的最佳作用时间,进一步考察WPS-1浓度的影响,得到其对促进IL-2、IL-6、IFN-γmRNA表达的最佳浓度都为40μg/mL,而对IL-4则为20μg/mL。WPS-1可能对淋巴细胞的免疫功能具有调节作用。     

The inhibitory effects of 5-hydroxy-3,6,7,8,3',4'-hexamethoxyflavone on human colon cancer cells

Scope: Previously, we reported that 5‐hydroxy‐3,6,7,8,3′,4′‐hexamethoxyflavone (5HHMF), a polymethoxyflavone found in citrus peels, potently inhibited the growth of multiple human colon cancer cells. Herein, we further investigated the anti‐cancer mechanisms of 5HHMF in human colon cancer cells. Methods and results: Colony formation assay revealed that 5HHMF dose dependently inhibited colony formation of multiple colon cancer cells. Western blot analysis demonstrated 5HHMF decreased nuclear β‐catenin levels and increased the E‐cadherin level in a dose‐dependent manner. 5HHMF also modified plasma membrane‐associated proteins, such as K‐Ras, EGFR, and their downstream effectors, such as Akt. Moreover, treatments with 5HHMF inhibited nuclear translocation of NF‐κB, which may contribute to its anti‐cancer effects. Add‐back study showed that the inhibitory effect of 5HHMF was not associated with the production of reactive oxygen species (ROS). In addition, 5HHMF treatment inhibited the capillary tube formation of human umbilical vein endothelial cells (HUVECs) on matrigel, suggesting a potential anti‐tumor angiogenesis effect. Conclusion: Our results demonstrated that 5HHMF suppressed multiple oncogenic molecular events in colon cancer cells.     

IGFs对小鼠乳腺IGFBP-5和cyclin D1表达的影响

运用荧光显微镜技术，采用小鼠乳腺组织培养及免疫荧光组化方法，对IGFs在乳腺发育和乳腺功能调控中的作用进行研究。结果表明：①IGFs能抑制各时期小鼠乳腺中IGFBP-5的表达，并且IGF—Ⅰ对小鼠乳腺中IGFBP-5表达的抑制作用强于IGF—Ⅱ，说明IGFs抑制小鼠乳腺细胞凋亡，延缓小鼠乳腺退化过程；②IGFs能促进各时期小鼠乳腺cyclin D1的表达，并且IGF—Ⅰ对小鼠乳腺cyclin D1表达的促进作用强于IGF—Ⅱ．证明IGFs可促进小鼠乳腺腺泡的发育。