Comparison Study on the Effect of Mesenchymal Stem Cells-Conditioned Medium Derived from Adipose and Wharton’s Jelly on Versican Gene Expression in Hypoxia

Background:Mesenchymal stem cells enhance tissue repair through paracrine effects following transplantation. The versican protein is one of the important factors contributing to this repair mechanism. Using MSC conditioned medium for cultivating monocytes may increase versican protein production and could be a good alternative for transplantation of MSCs. This study investigates the effect of culture medium conditioned by human MSCs on the expression of the versican gene in PBMCs under hypoxia-mimetic and normoxic conditions. Methods:The conditioned media used were derived from either adipose tissue or from WJ. Flow cytometry for surface markers (CD105, CD73, and CD90) was used to confirm MSCs. The PBMCs were isolated and cultured with the culture media of the MSC derived from either the adipose tissue or WJ. Desferrioxamine and cobalt chloride (200 and 300 µM final concentrations, respectively) were added to monocytes media to induce hypoxia-mimetic conditions. Western blotting was applied to detect HIF-1α protein and confirm hypoxia-mimetic conditions in PBMC. Versican gene expression was assessed in PBMC using RT-PCR. Western blotting showed that the expression of HIF-1α in PBMC increased significantly (p < 0.01). Results:RT-PCR results demonstrated that the expression of the versican and VEGF genes in PBMC increased significantly (p < 0.01) in hypoxia-mimetic conditions as compared to normoxia. Conclusion:Based on the findings in the present study, the secreted factors of MSCs can be replaced by direct use of MSCs to improve damaged tissues.Key Words: Adipose tissue, Hypoxia, Mesenchymal stem cells, Wharton’s jelly     

Effects of Nanofiber Scaffolds Coated with Nanoparticulate and Microparticulate Freeze Dried Bone Allograft on the Morphology,Adhesion, and Proliferation of Human Mesenchymal Stem Cells

Background:Freeze dried bone allograft nanoparticles on a nanofiber membrane may serve as an ideal scaffold for bone regeneration. This study aimed to assess the biological behavior of human MSCs in terms of proliferation and adhesion to nanoparticulate and microparticulate FDBA scaffolds on PLLA nanofiber membrane. Methods:In this experimental study, PLLA nanofiber scaffolds were synthesized by the electrospinning method. The FDBA nanoparticles were synthesized mechanically. The FDBA nanoparticles and microparticles were loaded on the surface of PLLA nanofiber membrane. A total of 64 scaffold samples in four groups of n-FDBA/PLLA, FDBA/PLLA, PLLA and control were placed in 24-well polystyrene tissue culture plates; 16 wells were allocated to each group. Data were analyzed using one-way ANOVA and Bonferroni test. Results:The proliferation rate of MSCs was significantly higher in the nanoparticulate group compared to the microparticulate group at five days (p = 0.034). Assessment of cell morphology at 24 hours revealed spindle-shaped cells with a higher number of appendages in the nanoparticulate group compared to other groups. Conclusion:MSCs on n-FDBA/PLLA scaffold were morphologically more active and flatter with a higher number of cellular appendages, as compared to FDBA/PLLA. It seems that the nanoparticulate scaffold is superior to the microparticulate scaffold in terms of proliferation, attachment, and morphology of MSCs in vitro.Key Words: Allografts, Bone regeneration, Mesenchymal stem cells, Nanofibers     

Characteristics of Human Endometrial Stem Cells in Tissue and Isolated Cultured Cells: An Immunohistochemical Aspect

Background:

The aim of this study was to investigate the percentage of the stem cells population in human endometrial tissue sections and cultured cells at fourth passage.

Methods:

Human endometrial specimens were divided into two parts, one part for morphological studies and the other part for in vitro culture. Full thickness of human normal endometrial sections and cultured endometrial cells at fourth passage were analyzed via immunohistochemistry for CD146 and some stemness markers such as Oct4, Nanog, Sox2, and Klf4 and the expression of typical mesenchymal stem cell markers CD90, CD105.

Results:

11.88±1.29% of human endometrial cells within tissue sections expressed CD146 marker vs. 28±2.3% of cultured cells, CD90 and CD105 were expressed by functionalis stroma (85±2.4 and 89±3.2%) than basalis stroma (16±1.4 and 17±1.9%), respectively (P<0.05). Oct4 and Nanog-expressing cells comprise 1.43±0.08 and 0.54±0.01% of endometrial stromal cells in endometrial sections vs. 12±3.1% and 8±2.9% of cultured cells, respectively. They reside near the glands in the basal layer of endometrium. Sox2 and Klf4 were not commonly expressed in tissue samples and cultured cells. CD9 and EpCAM were expressed by epithelial cells of the endometrium, rather than by stroma or perivascular cells.

Conclusion:

The human endometrial stem cells and pluripotency markers may be localized more in basalis layer of endometrium. The immunostaining observations of endometrial cells at fourth passage were correlated with the immunohistochemistry data.Key Words: Endometrium, Immunohistochemistry, Mesenchymal stem cells     

Kinase Gene Expression and Subcellular Protein Expression Pattern of Protein Kinase C Isoforms in Curcumin-treated Human Hepatocellular Carcinoma Hep 3B Cells

Curcumin, a yellow component of turmeric or curry powder, has been demonstrated to exhibit anti-carcinogenic effects in vitro, in vivo, and in human clinical trials. One of its molecular targets is protein kinase C (PKC) which has been reported to play essential roles in apoptosis, cell proliferation, and carcinogenesis. In this study, PKC mRNA expression was significantly inhibited in curcumin-treated human hepatocellular carcinoma (HCC) Hep 3B cells identified using a kinase cDNA microarray. Furthermore, curcumin decreased total protein expression of all PKCs in a time-related manner by immunoblotting of whole cell lysates, nuclear, membrane, and cytosolic fractions. In cytosolic fraction, the expression of PKC-α was totally inhibited by curcumin. In contrast, the expression levels of PKC-ζ and -μ were dramatically increased. Increases in expression of PKC-δ and PKC-ζ in the membrane and nucleus, and PKC-ι in the membrane were detected. In summary, the changes in expression and distribution of subcellular PKC isoforms in curcumin-treated Hep 3B cells suggest possible PKC-associated anti-tumor mechanisms of curcumin and provide alternative therapies for human HCC.     

Evaluation of Neurogenic Potential of Human Umbilical Cord Mesenchymal Cells; a Time- and Concentration-Dependent Manner

Background:

Retinoic acid as one of the most important regulators for cell differentiation was examined in this study for differentiation of human umbilical mesenchymal cells (hUCM).

Methods:

After isolation, hUCM were evaluated for mesenchymal stem cell properties by flow cytometry and alkaline phosphatase assay. Also, doubling time of the cells and their differentiation potential into adipogenic and osteogenic cells were tested. hUCM were then cultured with different concentrations of retinoic acid, and on days 1, 7, and 12, the percentage of differentiated cells was determined by immunostaining for nestin, anti-microtubule associated protein 2 (MAP₂), glutamic acid decarboxylase (GAD), and gamma-aminobutyric acid (GABA) markers.

Results:

The isolated cells were negative for the hematopoietic markers and positive for the mesenchymal markers. They showed the population doubling time 60 ± 3 hours and differentiated into osteogenic and adipogenic cells. A descending trend in nestin and an ascending trend in MAP₂, GAD, and GABA expression were observed from the first day until the last day between different concentrations of retinoic acid.

Conclusion:

hUCM cells may have the potential to differentiate into neural cells in the presence of different incubation period and concentration of retinoic acid.Key Words: Cell differentiation, Neural stem cells, Retinoic acid     

Rate of Cooling Alters Chip Color,Sugar Contents,and Gene Expression Profiles in Stored Potato Tubers

When stored at temperatures below 10 °C, potatoes accumulate sucrose and the reducing sugars glucose and fructose. This process, cold-induced sweetening, has been studied extensively because potatoes with elevated reducing sugar contents produce undesirable, dark-colored products and acrylamide, a suspected carcinogen, during high-temperature cooking. Potatoes in commercial storages are cooled slowly, but many research studies have used potatoes cooled rapidly. In this study, effects of cooling rate and variety on chip color, sugars, and gene expression were examined. Sucrose and reducing sugar contents were substantially lower in slowly cooled than in rapidly cooled tubers of ‘Snowden’ and “MegaChip’ for the first 11 weeks after cooling to 3 °C began. Differences in gene expression for VInv, β-amylase, SPS, AGPase and GBSS were observed between cooling treatments and varieties. Overall, the data showed that cooling rate, time in storage, and variety influenced multiple aspects of cold-induced sweetening.     

Sol-Gel Synthesis,in vitro Behavior,and Human Bone Marrow-Derived Mesenchymal Stem Cell Differentiation and Proliferation of Bioactive Glass 58S

Background:Bioactive glasses 58S, are silicate-based materials containing calcium and phosphate, which dissolved in body fluid and bond to the bone tissue. This type of bioactive glass is highly biocompatible and has a wide range of clinical applications. Methods:The 58S glass powders were synthesized via sol-gel methods, using tetraethyl orthosilicate, triethyl phosphate, and calcium nitrate, as precursors. Upon the analyses of phase and chemical structures of bioactive glass in different gelation times (12, 48, and 100 h), the appropriate heat treatment (at 525, 575, and 625 °C) was performed to eliminate nitrate compounds and stabilize the glass powder samples. The in vitro assay in SBF solution revealed the bioactivity of the synthesized 58S glass through the morphological (SEM), chemical structure (FTIR), release of calcium, phosphorous and silicon elements, pH variations, and weight loss measurements. The behavior of MSCs in the presence of bioactive glass powders was studied by MTT cytotoxicity, cell staining, ALP activity and biomineralization tests, as well as by the evaluation of ALP, osteocalcin, osteonectin, collagen I, and RUNX2 gene expression. Results: The results confirmed a gelation time of 100 h and a calcination temperature of 575 °C at optimal conditions for the synthesis of nitrate-free bioactive glass powders. Conclusion:The glass spherical nanoparticles in the range of 20-30 nm possess the improved bioactivity and osteogenic properties as demanded for bone tissue engineering. Key Words: Bioactive glass 58S, Gene expression, Mesenchymal stem cells     

in vitro Anti-Proliferative Effect of Adiponectin on Human Endometriotic Stromal Cells through AdipoR1 and AdipoR2 Gene Receptor Expression

Background:

Endometriosis is a complex disorder in reproductive age women which consist of stromal and epithelial cells implantation outside the uterine cavity. Adiponectin is a member of cytokine family with various metabolic roles and proliferation inhibition of many cancer cells. The aim of the present research was to determine adiponectin effect on human endometriotic stromal cells (ESCs) proliferation and their expression of adiponectin receptors.

Methods:

In this experimental study, endometrial biopsies (n=7) were taken. ESCs isolation was done by enzymatic digestion and cell filtrations. ESCs of each biopsy were divided into four groups: 0 (control), 10, 100, and 200 ng/ml adiponectin concentrations in three different times (24, 48, or 72 h). The effect of adiponectin on ESC viability and expression of mRNA Adipo receptor1 (R1) and Adipo receptor2 (R2) was determined by Trypan blue staining and semi-quantitative RT-PCR, respectively. Data were analyzed by one-way ANOVA and unpaired student’s t-test, and P<0.05 was considered statistically significant.

Results:

Adiponectin inhibited human endometriotic stromal cell proliferation in time- and dose-dependent manners significantly (P=0.001). Expression of AdipoR1 and AdipoR2 gene receptors was increased in human ESCs significantly (P<0.05).

Conclusions:

Adiponectin can suppress endometriosis by inhibiting ESC proliferation and increased AdipoR1 and AdipoR2 expression.Key Words: Adiponectin, Adiponectin receptors, Endometriosis, Stromal cells     

6-BA浓度及基因型对大豆胚尖诱导丛生芽的影响

以吉林35、吉林47等5个大豆品种为材料,胚尖为外植体,研究不同浓度6-BA对丛生芽诱芽率和平均芽数的影响,同时测定吉林35在胚尖诱导丛生芽的过程中对潮霉素的耐受性.结果表明:3.0 mg·L<,-1>6-BA浓度下吉林35的诱芽率最高,综合考虑丛生芽诱芽率和平均芽数2个性状,吉林35更加适合于大豆胚尖再生系统;在遗传...     

大豆Usp1基因的克隆和表达分析

在对大豆根系盐胁迫抑制差减杂交文库EST序列分析的基础上,利用RT-PCR技术克隆得到了一个大豆Us-pA基因,命名为GmUsp1,其编码一个包含164个氨基酸残基的多肽链。多序列比对和编码蛋白结构分析表明,GmUsp1属于泛应激蛋白(Universal Stress Protein)家族成员之一。其氨基酸序列与蚕豆Vf_enod18序列相似度最高,属于MJ-0577类中含有ATP结合位点的UspA亚族,与MJ-0577有相似的蛋白二级结构。分别采用250 mmol.L-1NaCl、100μmol.L-1ABA和30%PEG 6000进行胁迫处理,耐盐品种文丰7号和盐敏感品种Union的GmUsp1均被诱导表达,二者对胁迫诱导响应时间和表达量存在差异,说明GmUsp1可能参与大豆对非生物逆境胁迫的应答调控。     

卡那霉素和草铵膦对不同基因型大豆胚尖不定芽诱导的影响

以合丰35、黑农44和吉林35的胚尖为外植体,分别考察了不同浓度的卡那霉素和草铵膦对不同基因型大豆胚尖不定芽诱导的影响。结果表明,不定芽诱导率合丰35和吉林35在卡那霉素100 mg.L-1时与对照差异不显著,黑农44在100 mg.L-1处理时显著低于对照;芽数吉林35在卡那霉素100 mg.L-1时显著低于对照并高于其他浓度,而合丰35与黑农44在100 mg.L-1时芽数与对照差异不显著;3种基因型大豆胚尖不定芽伸长均在卡那霉素浓度为100 mg.L-1时受到显著抑制。草铵膦浓度在0.2～1.2 mg.L-1时对合丰35和吉林35的芽数没有影响,但黑农44在1.0 mg.L-1时芽数开始显著低于对照;芽长合丰35、黑农44和吉林35分别在0.6、0.2和0.2 mg.L-1时显著低于对照。因此,合丰35、黑农44和吉林35的适宜卡那霉素的筛选浓度为100 mg.L-1,草铵膦浓度分别为0.6、0.2和0.2mg.L-1。     

大豆GmPM13基因的克隆及原核表达

通过对大豆(Glycine max)油体钙蛋白(caleosin)基因GmPM13的克隆及原核表达,为今后利用基因工程方法检测转GmPM13基因提供抗体。运用RT-PCR技术克隆得到大豆油体钙蛋白GmPM13基因,构建原核表达载体,命名为p ET-28a-pm13。并转化到Ecdi和Rosetta中,经异丙基硫代半乳糖苷(IPTG)诱导后,SDS-PAGE分析GmPM13蛋白的表达情况。大豆GmPM13基因全长c DNA序列为738 bp,包括一个720 bp的开放阅读框,编码239个氨基酸,油体钙蛋白的分子量为27.1 k Da,诱导表达产物的大小与预计蛋白大小相符。在28℃,1.5 mol·L~(-1)IPTG浓度下,诱导12 h蛋白的表达量最高,占总蛋白的39.25%。     

The Effect of Melanocyte Stimulating Hormone and Hydroxyapatite on Osteogenesis in Pulp Stem Cells of Human Teeth Transferred into Polyester Scaffolds

Presently, tissue engineering is employed in the restoration and repair of tissue defects. Degradable scaffolds, stem cells and stimulating factors are employed in this method. In this study, the effect of melanocyte-stimulating hormone (MSH) and/or hydroxyapatite (HA) on proliferation, osteoblast differentiation, and mineralization of human dental pulp stem cells (hDPSCs) seeded on PLLA-PCL nanofibrous scaffolds was evaluated. For this aim, PLLA-PCL-HA nanofibrous scaffolds were fabricated using electrospinning method. FE-SEM images exhibited that all nanofibers had bead-free morphologies with average diameters ranging from 150–205 nm. Human DPSCs seeded into PLLA-PCL nanofibers were treated with MSH. Cell viability, proliferation, morphology, osteogenic potential, and the expression of tissue-specific genes were assessed by means of MTT assay, FE-SEM, alizarin red S staining, and RT-PCR analysis. hDPSCs exhibited improved adhesion and proliferation capacity on the PLLA-PCL-HA nanofibers treated with MSH compared to other groups (p<0.05). Additionally, PLLA-PCL-HA nanofibers treated with MSH exhibited significantly higher mineralization and alkaline phosphatase activity than other groups. RT-PCR results confirmed that PLLA-PCL-HA nanofibers enriched with MSH could significantly unregulated the gene expression of BMP2, osteocalcin, RUNX2 and DSPP that correlated to osteogenic differentiation (p<0.05). Based on results, incorporation of HA nanoparticles in PLLA-PCL nanofibers and addition of MSH in media exhibited synergistic effects on the adhesion, proliferation, and osteogenesis differentiation of hDPSCs, and therefore assumed to be a favorable scaffold for bone tissue engineering applications.     

拟南芥AtLACS9基因的克隆及其植物表达载体构建

将大豆种子凝集素基因的启动子(lec)从pBl-lec载体上酶切下来,连接到植物表达载体pCAMBIA3301(p3301)的多克隆位点上,然后运用RT-PCR方法扩增了拟南芥AtLACS9基因的编码区cDNA,并将其克隆到改造后的p3301载体的lec启动子之后,为进一步转化大豆、获得油份含量提高的转基因大豆做准备....     

Sugars Profiles of Different Chestnut (Castanea sativa Mill.) and Almond (Prunus dulcis) Cultivars by HPLC-RI

Sugar profiles of different almond and chestnut cultivars were obtained by high-performance liquid chromatography (HPLC), by means of a refractive index (RI) detector. A solid-liquid extraction procedure was used in defatted and dried samples. The chromatographic separation was achieved using a Eurospher 100-5 NH₂ column using an isocratic elution with acetonitrile/water (70:30, v/v) at a flow rate of 1.0 ml/min. All the compounds were separated in 16 min. The method was optimized and proved to be reproducible and accurate. Generally, more than 95% of sugars were identified for both matrixes. Sugars profiles were quite homogeneous for almond cultivars; sucrose was the main sugar (11.46 ± 0.14 in Marcona to 22.23 ± 0.59 in Ferragnes g/100 g of dried weight), followed by raffinose (0.71 ± 0.05 in Ferraduel to 2.11 ± 0.29 in Duro Italiano), glucose (0.42 ± 0.12 in Pegarinhos two seeded to 1.47 ± 0.19 in Ferragnes) and fructose (0.11 ± 0.02 in Pegarinhos two seeded to 0.59 ± 0.05 in Gloriette). Commercial cultivars proved to have higher sucrose contents, except in the case of Marcona. Nevertheless, chestnut cultivars revealed a high heterogeneity. Sucrose was the main sugar in Aveleira (22.05 ± 1.48), Judia (23.30 ± 0.83) and Longal (9.56 ± 0.91), while glucose was slightly prevalent in Boa Ventura (6.63 ± 0.49). The observed variance could serve for inter-cultivar discrimination.     

巴西橡胶树葡萄糖/核糖醇脱氢酶基因的克隆和表达分析

割胶显著刺激新开割橡胶树胶乳产量,前期的比较蛋白质组学的研究中,已通过双向电泳的方法分离到一个随割胶下调的葡萄糖/核糖醇脱氢酶(GRDH,glucose/ribitol dehydrogenase)。通过搜索本实验室的橡胶树EST数据库和PCR扩增得到该蛋白点对应的全长c DNA序列并命名为Hb GRDH1。序列分析显示,c DNA的编码序列(CDS)为882 bp,推测编码294个aa,分子量大小为32.2 ku,等电点为5.18,基因组序列包含4个外显子和3个内含子。通过Realtime PCR和Solexa高通量转录组测序分析发现,Hb GRDH1主要在胶乳和雌花中表达,在叶片发育稳定期呈现明显的下调表达趋势;在新开割树胶乳中,前4刀该基因表达变化不明显,而第5刀开始呈现上调表达趋势;在胶乳中,乙烯利处理对该基因表达前12 h无明显影响,在24 h时呈明显的下调表达。本研究结果有助于进一步阐明橡胶树中Hb GRDH1基因的功能,特别是其参与橡胶树胶乳再生调控的分子机理。     

茶树ACC氧化酶基因全长cDNA的克隆与表达分析

ACC(1-氨基环丙烷-1-羧酸)氧化酶是植物乙烯合成过程中的关键酶之一,对乙烯的合成具有重要的调控作用。在茶树新梢cDNA文库测序所获得ESTs基础上,利用RT-PCR技术,克隆得到编码ACC氧化酶基因的全长序列,GenBank登录号为DQ904328。该基因长1232bp,编码320个氨基酸残基,预测分子量为36.2kD,等电点5.41。多序列联配表明茶树ACC氧化酶具有高度保守区域,基于邻接法的进化树显示与柿树ACC氧化酶的亲缘关系最近。对经高、低温后的不同品种进行RT-PCR分析,结果发现ACC氧化酶基因的表达量与品种的抗逆性有一定的相关性。     

Mixed Lineage Kinase Domain-Like Pseudokinase (MLKL) Gene Expression in Human Atherosclerosis with and without Type 2 Diabetes Mellitus

Background:MLKL, one of the main downstream components of the necroptosis or programmed necrosis has recently been demonstrated in advanced atherosclerotic lesions. However, its precise role in the atherosclerosis pathogenesis still requires more elucidation. Our study was set to delineate both the changes in peripheral MLKL gene expression and its influence on disease severity in atherosclerotic patients with and without type 2 DM. Methods:The study involved 50 patients (20 non-diabetics and 30 diabetics) undergoing coronary artery bypass graft and 20 apparently healthy controls. Taqman RT-PCR was used to quantify MLKL mRNA expression levels, while ELISA was employed to estimate serum insulin and hsCRP levels. Results:Compared with the control group, MLKL gene was up regulated significantly in CVD (p ≤ 0.001). Higher MLKL expression was demonstrated in diabetic CVD group than non-diabetic group (p < 0.05). Correlation studies reported positive associations between MLKL and markers of dyslipidemia, inflammation, and insulin resistance. Multiple regression analysis revealed that FBG levels, hsCRP levels, and total WBCs count were significant predictors for MLKL levels. ROC showed a significant diagnostic value of MLKL for CVD. Moreover, regression analysis demonstrated that MLKL and hsCRP were independent predicting factors for the severity of CVD. Conclusion:MLKL is linked to hallmarks of atherosclerosis and could explain increased cardiovascular risk in diabetic patients. Thus, it can be a potential drug target for treatment of atherosclerotic patients. Key Words: Atherosclerosis, Diabetes mellitus, Inflammation, Insulin resistance, Necroptosis     

Effects of Peptone Supplementation in Different Culture Media on Growth,Metabolic Pathway and Productivity of CHO DG44 Cells; a New Insight into Amino Acid Profiles

Background:

The optimization of bioprocess conditions towards improved growth profile and productivity yield is considered of great importance in biopharmaceutical manufacturing. Peptones as efficient sources of nutrients have been studied for their effect on media development; however, their role on metabolic pathway is not well understood.

Methods:

In the present study, the effect of different concentration of peptones on a recombinant Chinese hamster ovary (CHO) cell line grown in three serum-free suspension cultures was determined. Six peptones of different origins and available amino acid profiles were investigated regarding their impact on cell growth, productivity, and metabolic pathways changes.

Results:

In optimized feeding strategies, increases of 136% and 159% in volumetric productivity (for a low-nutrient culture media) and 55% (for a high-nutrient culture media) were achieved. Furthermore, particular sources of peptones with specific amino acid profile developed preferential results for each different culture medium. Two peptones, SoyA2SC and SoyE-110, were the only hydrolysates that showed production improvement in all three media. Casein Peptone plus Tryptone N1 and SoyA3SC showed different improved results based on their implemented concentration for each individual basal medium.

Conclusion:

The amino acid profile of peptones may provide clues to identify the most effective feeding strategies for recombinant CHO cells.Key Words: CHO cells, Culture media, Peptones, Recombinant proteins