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。     

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。     

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。     

人脐带间充质干细胞诱导为牙周韧带样细胞的实验研究

目的 建立人牙周韧带细胞(human periodontal ligament cell,hPDLC)与人脐带间充质干细胞(human umbilical cord mesenchymal stem cell,hUCMSC)体外非接触式共培养模型,研究hUCMSC定向分化为hPDLC的可能性,探索新的可用于牙周组织工程的种子细胞.方法 利用跨室培养装置(Transwell)培养板建屯hPDLC与hUCMSC体外非接触式共培养模型,免疫组织化学方法 检测其骨桥蛋白(osteopontin,OPN)、骨钙素(ostocalcin,OCN)及骨涎蛋白(bone sialopmtein,BSP)的表达情况,并采用蛋白质印迹法从蛋白水平定量分析诱导后hUCMSC在分子水平的改变.结果 hUCMSC在非接触式共培养体系中可以被hPDLC诱导为多角形或梭形,蛋白质印迹法检测结果 显示,共培养3、7、14及21 d后的hUCMSC在蛋白水平OCN和OPN表达上调[OCN共培养前(0.88±0.21),共培养21 d(1.42±0.17);OPN共培养前(0.93±0.13),共培养21 d(1.43±0.22)];BSP表达逐渐下调[共培养前(1.60±0.09),培养21 d(0.75±0.20)],与共培养前相比差异均有统计学意义(P＜0.05).结论 hUCMSC在一定条件下可向hPDLC定向分化,并有望成为牙周组织工程的种子细胞.     

Pressure-Loaded MSCs During Early Osteodifferentiation Promote Osteoclastogenesis by Increase of RANKL/OPG Ratio

Mechanical stress plays an important role in bone remodeling. However, it is still unclear whether mechanical stress regulates osteoclastogenesis mediated by mesenchymal stem cells (MSCs) during initial osteodifferentiation. We investigated the effects of static and dynamic pressures on osteoclast-inducing potential of MSCs during early osteodifferentiation. The osteoclastogenesis was examined using TRAP staining. The mRNA levels of receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) genes were analyzed using real-time RT-PCR. It was shown that MSCs exposed to either pressure during initial osteodifferentiation promoted osteoclastogenesis with the up-regulation of RANKL/OPG ratio. MSCs displayed diverse responses to pressures at different points of initial osteodifferentiation. The RANKL/OPG ratio was significantly increased after osteoinduction in the primary MSCs without pressures exposure, which contradicted the previous report. These results suggest novel mechanisms of the initial biological responses of bone remodeling upon mechanical stimuli.     

Clinical experience and perinatal outcome of blastocyst transfer after coculture of human embryos with human endometrial epithelial cells: a 5-year follow-up study

OBJECTIVE: To evaluate the reproductive and neonatal outcome of blastocyst transfer after coculture with human endometrial epithelial cells in IVF and oocyte donation. DESIGN: Retrospective study.Private assisted reproductive center. PATIENTS(S): Two hundred sixty women undergoing IVF and 469 oocyte recipients. INTERVENTION(S): IVF or intracytoplasmic sperm injection (ICSI) and transfer of at least one blastocyst after coculture with human endometrial epithelial cells. MAIN OUTCOME MEASURE(S): Blastocyst formation rate, implantation and pregnancy rates, neonatal outcome, and congenital birth defects. RESULT(S): Among patients who had transfer with their own oocytes, 1193 of 2349 cocultured embryos developed up to the blastocyst stage (50.8%), and pregnancy and implantation rates of 33.9% and 19.2%, respectively, were achieved. In the oocyte donation program, 1819 blastocysts were obtained from 3127 embryos (58.2%), with subsequent pregnancy and implantation rates of 57.0% and 31.0%, respectively. The blastocyst rate remained stable throughout the 5 years of the study, but the pregnancy and implantation rates increased dramatically. Of 139 deliveries, 57 (41.0%) were multiple pregnancies and 1 (0.7%) was a multifetal birth (four live born infants). Out of 200 children born, 59% were male, and congenital birth defects were observed in 2.5%. CONCLUSION(S): Coculture of human embryos with endometrial epithelial cells yields a blastocyst formation rate of 50.8% to 58.2% and encouraging implantation and pregnancy rates. This technique reduces the mean number of embryos transferred in each patient. The number of embryos implanted is more relevant to neonatal outcome than is the coculture system and blastocyst transfer used. The risk of congenital birth defects associated with this program is similar to that recorded in early ET in IVF or ICSI.