Astrocytoma and Schwann cells in coculture

Glial fibrillary acidic protein (GFAP) is the principal intermediate filament protein found in mature astrocytes. Although the exact function of GFAP is poorly understood, it is presumed to stabilize the astrocyte’s cytoskeleton and help in maintaining cell shape. Previous studies from our laboratory have shown that when astrocytes were cocultured with primary Schwann cells (pSCs), astrocytes became hypertrophied and fibrous with intensely positive GFAP staining and segregated Schwann cells (SCs) into pockets. In order to understand the functional role of GFAP in this already established astrocyte-SC coculture model, we generated GFAP-negative cell lines from a GFAP-positive astrocytoma cell line and cocultured both the cell lines with pSCs. Our studies demonstrate that the GFAP-positive cell line put out processes toward the SCs, whereas the GFAP-negative cells did not form processes and the majority of the cells remained round. The most significant and interesting finding of this study, however, is the formation of elaborate processes by SCs when grown in coculture with the astrocytoma cells, unlike SCs cultured alone, which showed their typical bipolar spindle-shaped morphology. The extent of processes did not seem to be dependent on GFAP, since SCs cultured with both the cell lines formed similar processes. This coculture model may be useful in elucidating the factor(s) responsible for the formation of processes by SCs and can be further help in our understanding of the mechanism of morphological transformation of SCs.     

脂肪间充质干细胞向髓核样细胞定向分化研究

目的探讨SD大鼠来源的髓核(nucleus pulposus,NP)细胞促使脂肪间充质干细胞(adipose tissue-derived mesenchymal stem cells,AMSCs)向NP样细胞定向分化的分子机制。方法采用酶消化法取脂肪细胞,极限稀释法纯化细胞;采用组织块培养法培养NP细胞。利用流式细胞技术,免疫荧光及RT-PCR检测对AMSCs及NP细胞进行鉴定。结果 AMSCs中Sca-1和CD44的阳性率较高,而CD45和CD11b阴性,共培养组荧光强度明显亮于单纯AMSCs组,AMSCs在NP细胞的诱导下聚焦蛋白聚糖(Aggrecan)、Ⅱ型胶原蛋白(CollagenⅡ)、Sox-9等表达水平较对照组高。结论共培养环境中髓核细胞分泌的可溶性因子TGF-1能促使AMSCs向NP样细胞定向分化。     

生长板软骨细胞促进骨髓基质干细胞血管内皮生长因子的表达

目的观察骨髓基质干细胞(BMSCs)在生长板软骨细胞旁分泌作用下血管内皮生长因子(VEGF)的表达规律及其与成骨分化的相关性。方法大鼠BMSCs与生长板软骨细胞进行间接共培养,培养终末期做细胞化学染色,定量测定碱性磷酸酶(ALP)活性,用RT-PCR方法半定量检测VEGFmRNA的表达。结果生长板软骨细胞持续高表达VEGF。BMSCs随共培养时间的延长,ALP活性升高,BMSCs的VEGF的表达也逐渐增强。培养液加入两种分泌型VEGF中和抗体后,VEGF表达趋势不变,ALP活性仍为升高趋势,也不影响培养终末期钙化结节的形成。培养终末期BMSCs的CD31和CD34均阴性。结论BMSCs成骨分化过程中VEGF的表达符合成骨细胞分化基因的表达规律,与成骨细胞特征性基因的表达趋势一致,体外条件共培养条件下,中和VEGF后并不能阻碍BMSCs的成骨分化。     

The effects of human keratinocyte coculture on human adipose‐derived stem cells

The potential for adipose‐derived stem cells to differentiate into keratinocyte‐like cells has recently been receiving attention, stemming from the hypothesis that a bioengineered skin may be manufactured from these readily available mesenchymal stem cells. This study was conducted to evaluate the influence of human keratinocyte non‐contact coculture on hADSCs. Human epidermal keratinocytes and hADSCs obtained by lipoaspiration were cultured in keratinogenic growth media, which were divided into the following groups: human adipose‐derived stem cell (hADSC) monoculture, non‐contact coculture of hADSCs and human keratinocytes and keratinocyte monoculture. Cell proliferation was assessed, and keratogenicity was analysed through immunocytochemistry and polymerase chain reaction of early, intermediate and late keratogenic markers. hADSCs cocultured with keratinocytes displayed enhanced proliferation compared with the monoculture group. After a 7‐day coculture period, immunohistochemistry and polymerase chain reaction findings revealed the presence of specific keratinocyte markers in the coculture group. This study demonstrates that hADSCs cocultured with keratinocytes have the capacity to transdifferentiate into keratinocyte lineage cells, and suggests that adipose tissue may be a source of keratinocytes that may further be used in structuring the bioengineered skin.     

Genetics of Naphthalene Catabolism in Pseudomonads

At present, the modern two-step fermentation process is one of the major approaches for the industrial production of vitamin C. The key step in this process is the conversion of L-sorbose to 2-keto-L-gulonic acid (2-KLG), the vitamin C precursor, which is accomplished by an artificial microbial ecosystem consisting of Ketogulonicigenium vulgare and Bacillus megaterium. This review describes current progress in understanding this ecosystem, not only the individual physiological characteristics of the two strains, but also the interactions between them. Special emphasis is placed on recent systems biology studies of the ecosystem. We also discuss the regulation and improvement of this ecosystem, including analysis of the fermentation medium components and genetic engineering and optimum fermentative strategies. Finally, perspectives on the knowledge and engineering of this important artificial microbial ecosystem are discussed.     

Characterization of cathepsin X in colorectal cancer development and progression

The lysosomal cysteine carboxypeptidase cathepsin X (CTSX), localized predominantly in immune cells, has been associated with the development and progression of cancer. To determine its specific role in colorectal carcinoma (CRC), we analyzed CTSX expression in non-malignant mucosa and carcinoma of 177 patients as well as in 111 adenomas and related it with clinicopathological parameters. Further, the role of CTSX in the adhesion and invasion of the colon carcinoma cell lines HT-29 and HCT116 was investigated in an in vitro culture cell system with fibroblasts and monocytes, reflecting the situation at the tumor invasion front.Epithelial CTSX expression significantly increased from normal mucosa to adenoma and carcinoma, with highest expression levels in high grade intraepithelial neoplasia and in early tumor stages. Loss of CTSX occurred with tumor progression, and correlated with advanced local invasion, lymph node and distal metastasis, lymphatic vessel and vein invasion, tumor cell budding and poorer overall survival of patients with CRC. The subcellular distribution of CTSX changed from vesicular paranuclear expression in the tumor center to submembranous expression in cells of the invasion front. Peritumoral macrophages showed highest expression of CTSX. In vitro assays identified CTSX as relevant factor for cell–cell adhesion and tumor cell anchorage to fibroblasts and basal membrane components, whereas inhibition of CTSX caused increased invasiveness of colon carcinoma cells in mono- and co-culture. In conclusion, CTSX is involved in early tumorigenesis and in the stabilization of tumor cell formation in CRC. The results suggest that loss of CTSX may be needed for tumor cell detachment, local invasion and tumor progression. In addition, CTSX in tumor-associated macrophages indicates a role for CTSX in the anti-tumor immune response.     

肌源性干细胞分离培养及诱导分化为平滑肌细胞的研究

目的探讨兔肌源性干细胞分离、鉴定及诱导分化为平滑肌细胞的方法。方法取1．5个月龄新西兰兔大腿肌肉，采用Preplate技术分离培养肌源性细胞，并进行流式细胞仪（FCM）、免疫细胞化学、Western blot等确定细胞表型。采用添加血管内皮生长因子（VEGF）和与兔阴茎海绵体平滑肌细胞（CCSMC）共培养的方法诱导分离而得的细胞分化为平滑肌细胞并运用免疫细胞化学和Western blot检测特异性α-平滑肌肌动蛋白（α—SMA）的表达。结果经过连续6d的差速贴壁分离得小圆形或短梭形的小体积细胞，FCM结果显示这些细胞〉80％为desmin＋，〉70％为bcl-2^＋，〉95％为CD45^-。免疫细胞化学定性结果显示desmin＋。高汇合度（〉50％）或低血清培养时容易融合形成肌管或肌细胞核链。诱导处理后分离的细胞表达α—SMA。结论采用Preplate技术能很好地分离出肌源性干细胞，并能在体外诱导分化为平滑肌细胞，为组织工程提供种子细胞。     

TGF-β1联合内脏内胚层END-2细胞诱导胚胎干细胞分化为心肌细胞及与凋亡的关系

目的:探讨体外诱导胚胎干细胞(ESCs)向心肌细胞分化过程中分化与凋亡的关系,为优化组织工程的种子细胞提供依据。方法:先将ESCs悬浮培养形成2-3 d拟胚体(embryoid bodies,EBs),再向培养液中添加转化生长因子-β1(TGF-β1),以及与内脏内胚层样END-2细胞条件培养液共培养联合诱导ESCs向心肌细胞分化。以自然分化为对照组。激光共聚焦显微技术检测心肌细胞特异性肌钙蛋白T(TnT)的表达,透射电镜观察分化心肌细胞的超微结构。用AnnexinⅤ/PI双染后,在流式细胞仪上检测ESCs分化过程中的凋亡情况。结果:EBs经TGF-β1诱导后,有(43.00±2.08)%的拟胚体出现自发节律性收缩,表达心肌细胞特异性蛋白TnT,以及观察到心肌样超微结构,尤其是联合TGF-β1和END-2细胞条件培养液诱导,自发性收缩的拟胚体高达(88.00±1.42)%(P<0.01),收缩区域较大且拥有较成熟的心肌样结构。然而自然分化组发生自发节律性收缩的拟胚体只有(12.00±1.53)%。经2种诱导条件诱导后,检测两者的凋亡率分别为(5.58±0.65)%,(9.60±0.75)%(P<0.05)。结论:联合TGF-β1和内脏内胚层样END-2细胞条件培养液对心肌细胞分化有较高的诱导率,两者发挥协同诱导作用,可能机制是直接诱导ESCs分化或促进不向心肌细胞分化的ESCs凋亡。     

Neurite Outgrowth is Directed by Schwann Cell Alignment in the Absence of Other Guidance Cues

Schwann cells enhance axonal regeneration following nerve injury in vivo and provide a favorable substrate for neurite outgrowth in vitro. However, much remains unknown about the nature of interactions that occur between Schwann cells and growing neurites. In this paper, we describe direct evidence of the ability of Schwann cell alignment alone to direct neurite outgrowth. Previously, we reported that laminin micropatterns can be used to align Schwann cells and thus create oriented Schwann cell monolayers. In the current study, dissociated rat spinal neurons were seeded onto oriented Schwann cell monolayers, whose alignment provided the only directional cue for growing neurites, and neurite alignment with the underlying Schwann cells was analyzed. The orientation of neurite outgrowth mimicked that of the Schwann cells. Associations observed between neurites and Schwann cells suggest that Schwann cells may guide neurite outgrowth through both topographical and molecular mechanisms. This work demonstrates that Schwann cell alignment can direct neurite outgrowth in the absence of other directional cues, and provides a new method for examining neuronal–Schwann cell interactions in vitro.     

Paracrine crosstalk between human hair follicle dermal papilla cells and microvascular endothelial cells

Human follicle dermal papilla cells (FDPC) are a specialized population of mesenchymal cells located in the skin. They regulate hair follicle (HF) development and growth, and represent a reservoir of multipotent stem cells. Growing evidence supports the hypothesis that HF cycling is associated with vascular remodeling. Follicular keratinocytes release vascular endothelial growth factor (VEGF) that sustains perifollicular angiogenesis leading to an increase of follicle and hair size. Furthermore, several human diseases characterized by hair loss, including Androgenetic Alopecia, exhibit alterations of skin vasculature. However, the molecular mechanisms underlying HF vascularization remain largely unknown. In vitro coculture approaches can be successfully employed to greatly improve our knowledge and shed more light on this issue. Here we used Transwell‐based co‐cultures to show that FDPC promote survival, proliferation and tubulogenesis of human microvascular endothelial cells (HMVEC) more efficiently than fibroblasts. Accordingly, FDPC enhance the endothelial release of VEGF and IGF‐1, two well‐known proangiogenic growth factors. Collectively, our data suggest a key role of papilla cells in vascular remodeling of the hair follicle.