Preparation and in vitro studies of microencapsulated cells releasing human tissue inhibitor of metalloproteinase-2

Objective: To prepare microencapsulated cells releasing human tissue inhibitor of metalloproteinase-2 (TIMP-2), and investigate their biological characteristics in vitro. Methods: Chinese hamster ovary (CHO) cells were stably transfected with a human TIMP-2 expression vector, encapsulated in barium alginate microcapsules and cultured in vitro. Morphological appearance of the microcapsules was observed under a light microscope. Cell viability was assessed using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Enzyme linked immunosorbent assay (ELISA) and reverse zymography were used to confirm the release of biologically active TIMP-2 from the microcapsules. Cryopreservation study of the microencapsulated cells was carried out using dimethyl sulfoxide (DMSO) as preservative agent. Results: The microcapsules appeared like a sphere with diameter of 300–600 μm. The surface of the capsule wall was clearly smooth. The microencapsulated cells survived well and kept proliferating over the 6 weeks observed. No significant difference in TIMP-2 secretion was found between encapsulated and unencapsulated cells. Reverse zymography confirmed the bioactivity of MMP (matrix metalloproteinase) inhibition of TIMP-2. The cryopreservation process did not damage the microcapsule morphology nor the viability of the cells inside. Conclusion: Microencapsulated engineered CHO cells survive at least 6 weeks after preparation in vitro, and secrete bioactive TIMP-2 freely from the microcapsules.     

Delivering MC3T3-E1 cells into injectable calcium phosphate cement through alginate-chitosan microcapsules for bone tissue engineering

研究目的：本研究应用海藻酸钠-壳聚糖微囊保护成骨细胞,接种到β-磷酸三钙/磷酸钙骨水泥（β-TCP/CPC）浆料中,使β-TCP/CPC骨修复材料具有一定的细胞活性,同时提高固化后材料的孔隙率和孔径,以最终实现提高β-TCP/CPC骨水泥的降解速度,加快成骨和骨修复。创新要点：本研究首次应用海藻酸钠-壳聚糖微胶囊包封成骨细胞与CPC浆料复合,复合后实现自动细胞释放,释放出的细胞具有良好的生物学活性。研究方法：（1）高压静电成囊法制备载小鼠成骨前体细胞（MC3T3-E1）的海藻酸钙和海藻酸钠-壳聚糖微胶囊;（2）微囊化MC3T3-E1细胞,进行体外培养,使用细胞计数试剂盒（CCK-8）检测细胞活性,并用钙黄绿素-AM（Calcein-AM）和碘化丙啶（PI）进行活死细胞双重染色;（3）微囊化MC3T3-E1细胞与β-TCP/CPC浆料复合培养后,激光共聚焦扫描显微镜和环境扫描电子显微镜观测细胞在材料上的释放、粘附,CCK-8法检测材料上细胞的活力,碱性磷酸酶（ALP）检测观察细胞的分化状况,茜素红染色观察释放细胞的矿化能力。重要结论：海藻酸钠-壳聚糖微胶囊可作为可注射磷酸钙骨水泥内部接种成骨细胞并实现细胞释放的良好载体,释放出的成骨细胞具有良好的生物学活性。     

Effects of moniliformin and selenium on human articular cartilage metabolism and their potential relationships to the pathogenesis of Kashin-Beck disease

Objective:To investigate the effects of mycotoxin moniliformin (MON) on the metabolism of aggrecan and type II collagen in human chondrocytes in vitro and the relationship between MON and Kashin-Beck disease (KBD).Methods:Human chondrocytes were isolated and cultured on bone matrix gelatin to form an artificial cartilage model in vitro with or without MON toxin.Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.The expression of aggrecan and type II col...     

Stability of β-carotene microcapsules with Maillard reaction products derived from whey protein isolate and galactose as coating materials

The stability of β-carotene microcapsules using Maillard reaction products (MRPs) derived from whey protein isolate (WPI) and galactose as coating materials, was studied under the varying environmental conditions of temperature, pH, air, incandescent light, and ultraviolet (UV) light. Scanning electron microscopy showed that microcapsules prepared by WPI-galactose MRPs displayed a smooth and less concave-convex surface and that the particle size (D₅₀) of the microcapsules made with WPI-galactose MRPs was smaller than those made with WPI-galactose mixture. The storage stability of β-carotene microencapsulated in WPI-galactose MRPs was remarkably better than that of β-carotene microencapsulated in the WPI-galactose mixture and that of β-carotene crystal, in respect of temperature, pH, air, incandescent light, and UV light measurements. When the storage temperature was increased from 5 to 105 °C, the retention rate of β-carotene microcapsules significantly decreased (P<0.05). When pH values were increased from 1 to 12, the β-carotene retention rate of the microcapsules significantly increased and afterward decreased. Compared with the retention rate of β-carotene microencapsulated in a WPI-galactose mixture, the retention rate of β-carotene microencapsulated in WPI-galactose MRPs was at a maximum between pH 8 and 9. Under the actions of air, incandescent light, and UV light, the retention rates of β-carotene microcapsules in WPI-galactose MRPs and WPI-galactose mixture, as well as in β-carotene crystal, decreased significantly as the storage time increased (P<0.05). Therefore, the use of WPI-galactose MRPs as coating materials can aid in improving the storage stability of β-carotene microcapsules.     

Colorectal cancer cell growth inhibition by linoleic acid is related to fatty acid composition changes

Polyunsaturated fatty acids (PUFAs) possess anti-cancer action both in vitro and in vivo. In the present study, we detected cell viability with methyl thiazolyl tetrazolium (MTT) assay and cell membrane permeability with propidium iodide (PI) fluorescence dyeing, and calculated cell membrane fluidity change as fluorescence anisotropy. Fatty acid content in cells was measured by gas chromatography/mass spectroscopy (GC/MS), and the relationship between fatty acid composition and cell viability was studied. We observed that n-6 PUFA linoleic acid (LA) inhibited tumor cell growth at high concentrations (≥300 μmol/L), while low concentrations (100–200 μmol/L) seemed to promote cell proliferation. Analyses of cell membrane permeability, cell membrane fluidity, and cell fatty acid composition suggested that the anti-cancer action of LA could be related to changes in the ratio of n-6 to n-3 PUFAs. We observed that pre-incubation of cancer cells with 100 μmol/L LA for 24 h enhanced cell sensitivity to the cytotoxic action of LA, whereas undifferentiated cell line LoVo seemed to have a distinct path in LA-induced death. These results showed that one of the mechanisms by which supplementation of LA induces cancer cell death could be altering the ratio of n-6/n-3 PUFAs, and this may be related to cell differentiation status.     

Artesunate inhibits growth and induces apoptosis in human osteosarcoma HOS cell line in vitro and in vivo

This paper aims to investigate the effects of artesunate (ART) on growth and apoptosis in human osteosarcoma HOS cell line in vitro and in vivo and to explore the possible underlying mechanisms. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The induction of apoptosis was detected by light and transmission electron microscopy and flow cytometry. Western blot analysis was used to investigate the related mechanisms. Nude mice were further employed to investigate the antitumour activity of ART in vivo. MTT assay results demonstrated that ART selectively inhibits the growth of HOS cells in a dose- and time-dependent manner. Based on the findings of light and transmission electron microscopy, Hoechst 33258 staining, and fluorescein isothiocyanate (FITC)-annexin V staining, the cytotoxicity of ART in HOS cells occurs through apoptosis. With ART treatment, cytosolic cytochrome c was increased, Bax expression was gradually upregulated, Bcl-2 expression was downregulated, and caspase-9 and caspase-3 were activated. Thus, the intrinsic apoptotic pathway may be involved in ART-induced apoptosis. Cell cycle analysis by flow cytometry indicated that ART may induce cell cycle arrest at G₂/M phase. In nude mice bearing HOS xenograft tumours, ART inhibited tumour growth and regulated the expressions of cleaved caspase-3 and survivin, in agreement with in vitro observations. ART has a selective antitumour activity against human osteosarcoma HOS cells, which may be related to its effects on induction of apoptosis via the intrinsic pathway. The results suggest that ART is a promising candidate for the treatment of osteosarcoma.     

复发性自然流产患者血清RLX-2,TIMP-1,TIMP-2的表达及其临床诊断价值

目的检测复发性自然流产（RSA）患者血清RLX-2,TIMP1,TIMP2的表达水平,探讨RLX-2,TIMPs在RSA发生过程中的作用机制,及其对RSA的临床诊断价值。方法采用ELISA法对18例RSA患者与18例正常妊娠者不同妊娠期血清RLX-2,TIMP1,TIMP2的表达水平进行检测,采用受试者工作特性曲线对血清RLX-2,TIMP2及RLX-2/TIMP-2比值在RSA临床诊断的价值进行评价。结果血清RLX-2的水平在妊娠呈上升趋势,10～12周达到高峰;实验组血清TIMP-2水平在各妊娠期均高于正常组（P〈0.05）,各妊娠期RLX-2/TIMP-2比值的曲线下面积（AUC）均较RLX-2,TIMP-2高,其最佳拐点为≤0.1（P〈0.000 1）。结论RSA的发生可能与妊娠期RLX-2的分泌不足以及过高的血清TIMP-2浓度有关;妊娠10～12周时RLX-2/TIMP-2比值≤0.1对RSA的临床诊断价值最大。     

Effects of intermittent negative pressure on osteogenesis in human bone marrow-derived stroma cells

Objective: We investigated the effects of intermittent negative pressure on osteogenesis in human bone marrow-derived stroma cells (BMSCs) in vitro. Methods: BMSCs were isolated from adult marrow donated by a hip osteoarthritis patient with prosthetic replacement and cultured in vitro. The third passage cells were divided into negative pressure treatment group and control group. The treatment group was induced by negative pressure intermittently (pressure: 50 kPa, 30 rain/times, and twice daily). The control was cultured in conventional condition. The osteogenesis of BMSCs was examined by phase-contrast mi-croscopy, the determination of alkaline phosphatase (ALP) activities, and the immunohistochemistry of collagen type I. The mRNA expressions of osteoprotegerin (OPG) and osteoprotegerin ligand (OPGL) in BMSCs were analyzed by real-time poly-merase chain reaction (PCR). Results: BMSCs showed a typical appearance of osteoblast after 2 weeks of induction by intermit-tent negative pressure, the activity of ALP increased significantly, and the expression of collagen type 1 was positive. In the treatment group, the mRNA expression of OPG increased significantly (P<0.05) and the mRNA expression of OPGL decreased significantly (P<0.05) after 2 weeks, compared with the control. Conclusion: Intermittent negative pressure could promote os-teogenesis in human BMSCs in vitro.     

Ds-echinoside A, a new triterpene glycoside derived from sea cucumber, exhibits antimetastatic activity via the inhibition of NF-κB-dependent MMP-9 and VEGF expressions

Ds-echinoside A (DSEA), a non-sulfated triterpene glycoside, was isolated from the sea cucumber Pearsonothuria graeffei. In vitro and in vivo investigations were conducted on the effects of DSEA on tumor cell adhesion, migration, invasion, and angiogenesis. In this study, we found that DSEA inhibited the proliferation of human hepatocellular liver carcinoma cells Hep G2, with a half-maximal inhibitory concentration (IC₅₀) of 2.65 μmol/L, and suppressed Hep G2 cell adhesion, migration, and invasion in a dose-dependent manner. DSEA also reduced tube formation of human endothelial cells ECV-304 on matrigel in vitro and attenuated neovascularization in the chick embryo chorioallantoic membrane (CAM) assay in vivo. Immunocytochemical analysis revealed that DSEA significantly decreased the expression of matrix metalloproteinase-9 (MMP-9), which plays an important role in the degradation of basement membrane in tumor metastasis and angiogenesis. DSEA also increased the protein expression level of tissue inhibitor of metalloproteinase-1 (TIMP-1), an important regulator of MMP-9 activation. From the results of Western blotting, the expressions of nuclear factor-kappa B (NF-κB) and vascular endothelial growth factor (VEGF) were found to be remarkably reduced by DSEA. These findings suggest that DSEA exhibits a significant antimetastatic activity through the specific inhibition of NF-κB-dependent MMP-9 and VEGF expressions.     

Evaluation of the major royal jelly proteins as an alternative to fetal bovine serum in culturing human cell lines

Royal jelly (RJ) is a well-known bioactive substance. It contains large amounts of major royal jelly proteins (MRJPs), which express growth-factor-like activity in several animal and human cell lines. However, the question on whether MRJPs possess growth-factor-like activity on all types of cell cultures remains. In order to determine whether MRJPs can be used as an alternative to fetal bovine serum (FBS) in different types of human cell culture, the proliferation of the complex serum with different ratios of MRJPs/FBS (M/F) was evaluated on five cell lines: 293T, HFL-I, 231, HCT116, and Changliver using MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay. The proliferation activity of the combination of the complex M/F serum with cytokines on the test cell lines was also measured. The results demonstrated that the complex serum with M/F 6/4 possessed the highest proliferation activity similar to or in excess of FBS. However, no activity of complex medium with M/F 6/4 was observed in 231 cells, indicating a selectivity of MRJPs on cell types. Compared with the complex medium with M/F 6/4, the complex medium with M/F 6/4 together with two cytokines, epidermal growth factor (EGF) and insulin-transferrin-selenium (ITS), promoted proliferations of Changliver, 293T, HCT116, and HFL-I by 18.73%?56.19% (P<0.01). Our findings demonstrate that MRJPs could partially replace FBS in culturing many human cell lines.     

CCR5稳定表达的CHO细胞的构建

在人体内,CCR5与许多免疫疾病有关,CCR5有望成为众多药物的作用靶点。将ccr5基因与真核表达载体pBBS242构建成重组质粒pBBS242-ccr5,转染CHO细胞,并经潮霉素B筛选。流式细胞仪检测结果表明CCR5在CHO细胞得到了稳定表达。     

银杏叶总黄酮对人肝癌HepG2细胞增殖与凋亡的影响

目的探讨银杏叶总黄酮对体外培养的人肝癌细胞HepG2增殖与凋亡的影响。方法将银杏叶总黄酮作用于体外培养的人肝癌细胞HepG2,MTT法检测其对HepG2细胞增殖的影响,缺口末端核苷标记（TUNNEL）法检测其对HepG2细胞凋亡的影响。结果银杏叶总黄酮对体外培养的人肝癌细胞HepG2的增殖效率下降,使凋亡细胞数增加（P〈0．01）,且呈剂量依赖效应。结论银杏叶总黄酮对体外培养的人HepG2细胞增殖有抑制作用,并能诱导细胞凋亡。     

Antitumor activities of D-glucosamine and its derivatives

The growth inhibitory effects of D-glucosamine hydrochloride （GlcNH2-HCl）, D-glucosamine （GlcNH2） and N-acetyl glucosamine （NAG） on human hepatoma SMMC-7721 cells in vitro were investigated. The results showed that GlcNH2.HCl and GlcNH2 resulted in a concentration-dependent reduction in hepatoma cell growth as measured by MTT （3-（4,5-dimethylthiazol- 2-yl）-2,5-diphenyltetrazolium bromide） assay. This effect was accompanied by a marked increase in the proportion of S cells as analyzed by flow cytometry. In addition, human hepatoma SMMC-7721 cells treated with GlcNH2-HCl resulted in the induction of apoptosis as assayed qualitatively by agarose gel electrophoresis. NAG could not inhibit the proliferation of SMMC-7721 cells. GlcNH2-HCl exhibited antitumor activity against Sarcoma 180 in Kunming mice at dosage of 125-500 mg/kg, dose of 250 mg/kg being the best. GlcNH2-HCl at dose of 250 mg/kg could enhance significantly the thymus index, and spleen index and could promote T lymphocyte proliferation induced by ConA. The antitumor effect of GlcNH2-HCl is probably host-mediated and cytocidal.     

银杏叶槲皮素对人肝癌HepG2细胞增殖与凋亡的影响

目的探讨银杏叶黄酮单体成分槲皮素体外对人肝癌细胞HepG2增殖与凋亡的影响。方法将银杏叶黄酮单体成分槲皮素作用于体外培养的人肝癌细胞HepG2,MTT法检测其对HepG2细胞增殖的影响,缺口末端核苷标记（TUNNEL）法检测其对HepG2细胞凋亡的影响。结果银杏叶黄酮单体成分槲皮素使体外培养的人肝癌细胞HepG2的增殖效率下降,使凋亡细胞数增加（P〈0.01）,两者均呈剂量依赖效应。结论银杏叶黄酮单体成分槲皮素具有与银杏叶总黄酮相似的作用,对体外培养的人HepG2细胞增殖有抑制作用,并能诱导细胞凋亡。     

NDV感染体外诱导胃癌细胞BGC-823的凋亡

目的 :研究新城疫病毒在体外抗胃癌细胞活性及其与细胞凋亡的关系。方法 :应用倒置显微镜观察细胞形态、MTT法测NDV在体外对BGC - 82 3的抑制和杀伤作用 ,同时用流式细胞术检测胃癌细胞凋亡情况及细胞分裂周期各时象的变化。结果 :NDV在体外可使BGC - 82 3胃癌细胞形成明显的细胞病变效应、细胞生长抑制及细胞凋亡 ,且细胞凋亡率与感染时间呈正相关。G2、S期细胞减少 ,增殖指数 (PI)降低 ,与阴性对照组比较有显著性差异 (P<0 .0 1)。结论 :NDV具有显著的抗BGC - 82 3胃癌细胞活性。     

增强型绿色荧光蛋白基因转染CHO细胞研究

目的：探讨增强型绿色荧光蛋白（enhanced green fluorescent protein, EGFP）在中华仓鼠卵巢细胞（CHO-DHFR）细胞中的作用。方法：将增强型绿色荧光蛋白基因的真核表达载体pcDNA3．1（＋）-EGFP，转染至培养的中华仓鼠卵巢细胞（Chinese Hamster Ovary, CHO-DHFR^- ）中。结果：成功表达并产生绿色荧光。结论：证明EGFP是一种良好的报告基因和筛选标志．为进一步研究应用最广泛的哺乳动物细胞表达系统一CHO细胞表达系统奠定了基础。