Proteins combination on PHBV microsphere scaffold to regulate Hep3B cells activity and functionality: a model of liver tissue engineering system

The synergistic effects of extracellular matrix (ECM) protein combinations on Hep3B cell proliferation and functions are studied herein. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) microspheres were covalently conjugated with three types of proteins, collagen (type I), laminin, and fibronectin, using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide cross linkers. Successful conjugations of protein molecules were verified by the presence of nitrogen peaks in X-ray photoelectron spectroscopy. The densities of grafted proteins were quantified using Micro-BCA kit. A human hepatoma cell line, Hep3B, was then cultured in vitro on the ECM proteins-modified microspheres for 2 weeks. Cell proliferation was estimated using MTT method, and two hepatic functions, albumin secretion and P-450 activity, were evaluated using ELISA and EROD assays, respectively. The results indicated that combination of the three ECM proteins on microsphere surfaces has a significant effect on the proliferation of Hep3B cells, thus better mimicking the in vivo environment for liver tissue engineering.     

Apoptosis in dengue virus infected liver cell lines HepG2 and Hep3B

While both in vivo and in vitro evidence has suggested that liver cells undergo apoptosis in response to dengue virus infection, little is known about the mechanism of induction. Given that the p53 tumour suppressor gene is a key mediator of apoptosis, we sought to define the role of this gene in response to dengue virus infection. After infection, a p53 wild type liver cell line (HepG2) showed changes consistent with apoptosis including alterations of cell morphology, cellular detachment and DNA laddering. However, p53 was neither up-regulated, nor showed any evidence of complexing with dengue virus proteins as determined by immunoprecipitation. Infection of a p53 null liver cell line (Hep3B) also produced changes consistent with the induction of apoptosis. While the profile of the cells undergoing apoptosis in each cell line was similar as determined by flow cytometry, the absolute levels were markedly different with up to 90% of Hep3B cells undergoing apoptosis compared to only 20% of HepG2 cells at day 5 post infection. By day 7, all Hep3B infected cells were dead. In contrast, it proved possible to culture dengue virus infected HepG2 cells for 3 months. Viral progeny released from the p53 null cell line were nine-fold higher per attached cell than from the p53 wild type cell line. These results suggest that, while induction of apoptosis in liver cells is mediated by a non-p53 regulated pathway, p53 may play a role in restricting the level of viral progeny to below a critical level at which apoptosis is triggered.     

犬骨髓基质干细胞与聚羟基烷酸酯体外相容性的实验研究

目的：评价聚羟基烷酸酯（PHBV）作为组织工程支架与犬骨髓基质干细胞（BMSCs）的生物相容性。方法:原代培养犬BMSCs，传至3-4代后，接种至PHBV膜和泡沫样三维支架上，以接种至培养板上细胞为对照组，倒置显微镜下观察细胞形态；于培养1、2、3周分别采用4%多聚甲醛固定，常规组织切片，HE染色；在5、10、14 d用Hoechst33258荧光法定量测定细胞内DNA含量，BCA法测定蛋白质含量。结果:倒置显微镜观察PHBV 纤维较粗，且透光性差，在相差显微镜下不易观察。第3-4代BMSCs接种至PHBV膜上2 h后即大部黏附，3 d后伸展良好，呈纺锤形或梭形，在三维支架的孔隙内立体生长，1周开始细胞间连接，3周广泛连接，分泌大量基质；培养接种1周后，取二个膜状PHBV固定，HE染色后，见骨髓基质干细胞增殖。培养接种2周后，骨髓基质干细胞增殖明显，呈梭形密布于膜状PHBV上。培养接种3周后，骨髓基质干细胞增殖较第二周无明显变化。定量测定接种的细胞内DNA含量和蛋白质含量与对照组相比无显著差异。结论:PHBV作为BMSCs的组织工程支架材料，具有良好的生物相容性。     

Up-regulation of drug-metabolizing enzyme genes in layered co-culture of a human liver cell line and endothelial cells

Primary human hepatocytes are used extensively to study drug-metabolizing enzymes such as the cytochrome P450 (CYP) enzymes. However, the activities of these enzymes decrease rapidly during culture. In the present study, using a thermo-responsive culture dish, layered co-culture was achieved by placing a bovine pulmonary artery endothelial cell (BPAEC) sheet onto the human hepatoma cell line HepG2. In the BPAEC/HepG2 layered co-culture system, real-time polymerase chain reaction analysis showed that the expression levels of various CYP enzymes were more than 10 times greater 21 days after layering than with a HepG2 monolayer. The expression levels of CYP1B1, CYP2C9, CYP2E1, and CYP3A4 were up-regulated in a time-dependent manner, gradually increasing from day 10 after layering, and continuing to increase until at least day 21. The gene expression levels of the various CYP enzymes were almost identical to that of human liver. These results suggest that our layered co-culture system enhances the function of HepG2 cells and that our BPAEC/HepG2 layered co-culture system can serve as a useful model for the in vitro evaluation of CYP regulation.     

PHBV microspheres as neural tissue engineering scaffold support neuronal cell growth and axon-dendrite polarization

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) microspheres, with properties such as slower degradation and more efficient drug delivery properties, have important benefits for neural tissue engineering. Our previous studies have shown PHBV microspheres to improve cell growth and differentiation. This study aimed to investigate if PHBV microspheres would support neurons to extend these benefits to neural tissue engineering. PHBV microspheres' suitability as neural tissue engineering scaffolds was investigated using PC12 cells, cortical neurons (CNs), and neural progenitor cells (NPCs) to cover a variety of neuronal types for different applications. Microspheres were fabricated using an emulsion-solvent-evaporation technique. DNA quantification, cell viability assays, and immunofluorescent staining were carried out. PC12 cultures on PHBV microspheres showed growth trends comparable to two-dimensional controls. This was further verified by staining for cell spreading. Also, CNs expressed components of the signaling pathway on PHBV microspheres, and had greater axon-dendrite segregation (4.1 times for axon stains and 2.3 times for dendrite stains) than on coverslips. NPCs were also found to differentiate into neurons on the microspheres. Overall, the results indicate that PHBV microspheres, as scaffolds for neural tissue engineering, supported a variety of neuronal cell types and promoted greater axon-dendrite segregation.     

Effect of 5-azacytidine (5-aza) on UCP2 expression in human liver and colon cancer cells

The function of the uncoupling protein 2 (UCP2) is different for each cancer cell. However, the mechanism of expression is still unclear. DNA methylation affects protein expression and is one factor that transforms normal cells into cancer cells. In this study, the hepatocellular carcinoma Hep3B and HepG2 cells and colorectal cancer HT-29 cells were treated with 5-azacytidine (5-aza), a DNA demethylation agent, to observe the modification of UCP2 expression and the methylation degree in the UCP2 promoter region. Promoter basal activity and degree of UCP2 expression were measured in Hep3B, HepG2, and HT-29 cells. In addition, methylation-specific PCR (MSP) was performed to investigate the degree of methylation in the UCP2 promoter region. The methylation region in the UCP2 promoter was confirmed based on bisulfite sequencing. In Hep3B cells in which UCP2 mRNA was not transcribed, the promoter basal activity was significantly higher than in HT-29 or HepG2 cells in which UCP2 mRNA was transcribed. Treatment with 5-aza increased UCP2 expression in Hep3B and HT-29 cells; however, the expression in HepG2 cells was unchanged. The UCP2 promoter in Hep3B cells has numerous methylated regions compared with HT-29 and HepG2 cells. The results of the present study revealed that inhibition of UCP2 expression in Hep3B cells was due to methylation of the promoter region. Investigating the mechanism that induces UCP2 expression in cancer cells is important to understand the function of UCP2, which could aid in cancer treatment.     

In vitro characterization of hepatocyte growth factor release from PHBV/PLGA microsphere scaffold

Polymer scaffolds which can support cells to grow as well as deliver growth factors to the cells simultaneously have great potential for the successful regeneration of failed tissues. As popularly used vehicles to deliver anti-cancer drugs and growth factors, microspheres also show many advantages as substrates to guide the growth of cells. Therefore, we aimed to examine the feasibility of using microspheres as ideal scaffolds for liver tissue engineering. To determine the capabilities of previously used microsphere scaffold to deliver growth factors simultaneously, this work investigated a long-term (about three months) release of bovine serum albumin (BSA) from microsphere scaffolds fabricated by using two different polymers, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV, 8% PHV), poly(lactide-co-glycolide) acid (PLGA, 5050) and a blend of PLGA and PHBV. BSA served as a model for hepatocyte growth factor (HGF) since both proteins have similar molecular weights and hydrophilicity. Furthermore, HGF was encapsulated into the PLGA/PHBV composite microsphere with a core-shell structure, and sustained delivery of HGF with maintained bioactivity was achieved for at least 40 days. The moderate degradation rate (about 55% loss of the initial mass) and well-preserved structure after three months of incubation indicated that the PLGA/PHBV composite microspheres would therefore be more suitable than the pure PHBV or PLGA microspheres as a scaffold for engineering liver tissue.     

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate)-based nanofibrous scaffolds to support functional esophageal epithelial cells towards engineering the esophagus

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and PHBV–gelatin were electrospun to obtain defect-free nanofibers by optimizing various process and solution parameters. Tensile strength, Young’s modulus, and wettability of PHBV–gelatin nanofibrous scaffold were determined and compared with PHBV nanofibrous scaffold. Our results demonstrate that PHBV–gelatin nanofibers exhibited higher tensile strength and Young’s modulus than the PHBV nanofibers. Human esophageal epithelial cells (HEEpiC) were cultured on PHBV and PHBV–gelatin nanofiber showed better cell proliferation in PHBV nanofibrous scaffold than the PHBV–gelatin scaffold after 7?days of culture. HEEpiC cultured on PHBV and PHBV–gelatin nanofibrous scaffold exhibited characteristic epithelial cobblestone morphology after 3 days of culture. Further, the HEEpiC extracellular matrix (ECM) proteins (collagen type IV and laminin) and phenotypic marker proteins (cytokeratin-4 and 14) expressions were significantly higher in PHBV–gelatin nanofibrous scaffold than the PHBV nanofiber scaffold. However, the long-term stability and functional state of the cells on the PHBV scaffold give it an edge over the blend scaffolds. Thus, PHBV-based nanofibrous scaffolds could be explored further as ECM substitutes for the regeneration of esophageal tissue.     

Protective effect of RIP and c-FLIP in preventing liver cancer cell apoptosis induced by TRAIL

TRAIL (TNF-related apoptosis-inducing ligand) is a member of the tumor necrosis factor superfamily that can induce tumor selective death by up-regulating death receptor 4 (DR4) and DR5 expression. The study aimed to explore the role of RIP and c-FLIP genes in TRAIL induced liver cancer cell HepG2 and Hep3B apoptosis and related mechanism. RIP and c-FLIP silenced HepG2 and Hep3B cell model were established through siRNA. Western blot was applied to test c-FLIP, RIP, DR4, DR5, FADD, Caspase-3/8/9, ERK1/2, and DFF45 protein expression. Caspase-8 kit was used to detect Caspase-8 expression. Flow cytometry was performed to measure cell apoptosis rate. Acid phosphatase method was applied to determine cell cycle. TRAIL had no significant effect on Caspase-3/8/9, DR4, DR5, ERK1/2, and DFF45 protein expression, but up-regulated c-FLIP and RIP protein expression and reduced FADD expression level. After treated by the chemotherapy drug mitomycin and adriamycin, c-FLIP and RIP expression decreased significantly, while FADD increased. After knockout c-FLIP and RIP gene, HepG2 and Hep3B cell apoptosis rate induced by TRAIL increased obviously. Meanwhile, cell subG1 percentage increased markedly and exhibited G1 phase growth retardation. In addition, after two kinds of gene knockout, Caspase-8 was activated and produce Caspase-3 P20 and P24, leading DFF45 appeared DNA fragment P17 and P25. c-FLIP and RIP can inhibit Caspase-8 activation and prompting HepG2 and Hep3B resistant to cell apoptosis induced by TRAIL.     

HLA-E在肝癌细胞系中的表达

目的: 研究人类白细胞抗原E(HLA-E)在肝癌细胞系中的表达情况。方法: 通过real-time PCR和Western blotting技术研究5种肝癌细胞系和胎肝细胞系中HLA-E mRNA 和蛋白的表达情况,进行相对定量分析。结果: Real-time PCR 检测显示,HepG2 细胞、Bel7402 细胞、 PLC 细胞、MHCC97细胞和Hep3B2.1-7 细胞这5种肝癌细胞系与L02胎肝细胞系的HLA-E mRNA 水平比较,除Hep3B2.1-7细胞表达几乎接近缺失(P<0.01)外,其余无显著差异(P>0.05)。HepG2细胞、Bel7402 细胞、 PLC 细胞、MHCC97 细胞和Hep3B2.1-7 细胞与L02 细胞的HLA-E 蛋白水平比较,差异显著(P<0.01),其中Hep3B2.1-7细胞表达缺失。结论: 肝癌细胞系的HLA-E mRNA和蛋白表达不同步,可能存在转录后调控。     

High-efficiency cell seeding method by relatively hydrophobic culture strategy

Cell adhesion efficiency is one of the key factors affecting the results of manufacturing tissue engineering constructs. High efficiency is required for seeding low proliferation cells onto scaffolds. In this study, we designed a strategy to improve the efficiency of cell adhesion using hydrophobic cell culture environment to enhance cells adhering to a scaffold. Cells have lower affinity to the surface of polydimethylsiloxane (PDMS) than tissue culture polystyrene (TCPS) plates. When cells were cultured with gelatin microspheres or chitosan films in a PDMS-coated plate instead of a normal TCPS plate, there was a significant increase in cell attachment efficiency. Cells cultured in the PDMS-coated system tended to selectively attach onto the gelatin microspheres or chitosan films, which are relatively more hydrophilic than the PDMS surface. However, minimal cell attachment on gelatin microspheres or chitosan films was observed when gelatin microspheres or chitosan films were placed in normal TCPS plate. Cell counting experiments with gelatin microspheres in the PDMS-coated system resulted in a cell attachment efficiency of 89.8% after 1 day of cultivation, whereas the cell attachment efficiency was less than 1% in normal TCPS plate. The results demonstrate that the method is easy to use and could be useful for fast cultivation of cell-scaffold constructs.     

Three dimensional culture upregulates extracellular matrix protein expression in human liver cell lines--a step towards mimicking the liver in vivo?

Extracellular matrix (ECM) in the liver affects the phenotype of both hepatocytes and non-parenchymal cells. To be able to mimic in vivo liver function for extracorporeal hepatic support using human cell lines, a necessary step is to upregulate the function normally seen in monolayer culture. 3-D spheroid colonies were formed by culturing single HepG2 cells encapsulated in alginate beads. ECM expression in these cultures was compared to monolayer Hep G2 cultures. The following ECM proteins were detected immunohistochemically:- collagens 1, III, V and VI, the glycoproteins fibronectin, tenascin and vitronectin, and the basement membrane protein laminin. In 3-D cultures, all proteins except tenascin were strongly expressed, as compared with weak or undetectable expression in monolayer cultures, even with 10-fold increases in the antibody concentration used. In conclusion, we have demonstrated that the 3-D environment created by alginate encapsulation of cell lines leads to cell behaviour mimicking that in vivo.     

SH2-B抗体对肝癌细胞脂肪沉积的影响

目的探讨SH2-B抗体对肝癌HepG2细胞内脂肪沉积的影响。方法实验分为对照组、高脂(HF)组、HF+SH2-B抗体组。将人肝癌细胞(HepG2)培养于DMEM培养基中,培养48h后用油红O染色法定性观察细胞内脂肪沉积,测定各组OD值,Western blot检测各组SH2-B蛋白表达,并用RT-PCR法检测各组JAK2mRNA表达。结果与对照组比较,HF组和HF+SH2-B抗体组肝癌细胞内脂肪含量增加(0.05),但HF组明显高于HF+SH2-B抗体组(0.05)。SH2-B蛋白和JAK2 mRNA表达在HF组明显高于HF+SH2-B抗体组和正常组(0.0 5)。结论 SH2-B蛋白及其下游分子JAK2在高脂状态下的肝细胞内表达上调,SH2-B抗体减轻肝细胞脂肪沉积可能与降低SH2-B和JAK2活性相关。     

Poly(beta-hydroxybutyrate-co-beta-hydroxyvalerate) supports in vitro osteogenesis

Studies have demonstrated that polymeric biomaterials have the potential to support osteoblast growth and development for bone tissue repair. Poly(beta-hydroxybutyrate-co-beta-hydroxyvalerate) (PHBV), a bioabsorbable, biocompatible polyhydroxy acid polymer, is an excellent candidate that, as yet, has not been extensively investigated for this purpose. As such, we examined the attachment characteristics, self-renewal capacity, and osteogenic potential of osteoblast-like cells (MC3T3-E1 S14) when cultured on PHBV films compared with tissue culture polystyrene (TCP). Cells were assayed over 2 weeks and examined for changes in morphology, attachment, number and proliferation status, alkaline phosphatase (ALP) activity, calcium accumulation, nodule formation, and the expression of osteogenic genes. We found that these spindle-shaped MC3T3-E1 S14 cells made cell-cell and cell-substrate contact. Time-dependent cell attachment was shown to be accelerated on PHBV compared with collagen and laminin, but delayed compared with TCP and fibronectin. Cell number and the expression of ALP, osteopontin, and pro-collagen alpha1(I) mRNA were comparable for cells grown on PHBV and TCP, with all these markers increasing over time. This demonstrates the ability of PHBV to support osteoblast cell function. However, a lag was observed for cells on PHBV in comparison with those on TCP for proliferation, ALP activity, and cbfa-1 mRNA expression. In addition, we observed a reduction in total calcium accumulation, nodule formation, and osteocalcin mRNA expression. It is possible that this cellular response is a consequence of the contrasting surface properties of PHBV and TCP. The PHBV substrate used was rougher and more hydrophobic than TCP. Although further substrate analysis is required, we conclude that this polymer is a suitable candidate for the continued development as a biomaterial for bone tissue engineering.     

bFGF缓释微球的制备及其促雪旺细胞分裂增殖的初步研究

研究碱性成纤维细胞生长因子(bFGF)缓释微球的制备方法及其对雪旺细胞的促分裂增殖作用。以聚乳酸-羟基乙酸共聚物(PLGA)为载体材料,采用复乳包囊法制备bFGF-PLGA缓释微球,并对微球的形态学、粒径分布、载药量和包封率、及体外释药进行研究。将bFGF、bFGF-PLGA微球分别加入不同组的雪旺细胞培养液中,分别测定雪旺细胞的数量、活力和细胞周期。结果显示,复乳包囊法制备的bFGF-PLGA缓释微球表面光滑圆整,球体均匀度好;微球平均粒径为1.552±0.015μm,平均径距为1.310±0.010;载药量和包封率分别为(27.18×10-3)%±(0.51×10-3)%、66.43%±1.24%;微球的体外释药过程较为稳定,11d释药率为72.47%。体外细胞试验中,培养1、2d时,bFGF组的细胞计数、吸光度明显高于bFGF缓释微球组;培养3、4d时,bFGF组和bFGF缓释微球组的细胞计数、吸光度无统计学差异;培养6、8d时,bFGF缓释微球组的细胞计数、吸光度明显高于bFGF组。流式细胞仪检测结果显示,培养2d后,bFGF组的G2/M S期百分数高于bFGF缓释微球组;培养4、8d后,bFGF缓释微球组的G2/M S期百分数高于bFGF组,差异具有统计学意义。说明采用复乳包囊法制备bFGF-PLGA缓释微球的工艺可行,微球中bFGF的生物活性保存良好,能缓慢持续释放活性bFGF,促进雪旺细胞的分裂增殖。     

Cryopreservable and tumorigenic three-dimensional tumor culture in porous poly(lactic-co-glycolic acid) microsphere

In vitro tumor models that mimic in vivo conditions may be ideal for screening anticancer drugs and their formulations and developing tumors in animal models. Three-dimensional (3-D) culture of cancer cells on polymeric scaffolds can be an option for such models. In the present study, porous poly(lactic acid-co-glycolic acid) (PLGA) microsphere was used both as a cancer cell culture substrate to expand cells and as a cancer cell transplantation vehicle for tumor construction in mice. MCF-7 cells cultured on porous PLGA microspheres in stirred suspension bioreactors expanded by 2.8-fold over seven days and maintained viability. At three months after inoculation with 2 × 10⁶ cells/site, the tumor formation by MCF-7 cells cultured on microspheres was much more effective (4 tumors/5 mice) than its counterpart cultured on plates (1/5). More importantly, cell viability and metabolic activity were not significantly changed even after one freeze–thaw cycle of the 3-D culture. MCF-7 cells cultured on the microspheres and the cells in 3-D after cryopreservation were more resistant to doxorubicin than MCF-7 cells cultured on plates.     

Rotary cell culture system (RCCS): a new method for cultivating hepatocytes on microcarriers

The Rotary Cell Culture System (RCCS) is a new technology for growing anchorage dependent or suspension cells in the laboratory. The RCCS is a horizontally rotated, bubble free disposable culture vessel with diffusion gas exchange. The system provides a reproducible, complex 3D in vitro culture system with large cell masses. During cell growing the rotation speed can be adjusted to compensate for increased sedimentation rates. The unique environment of low shear forces, high mass transfer, and microgravity, provides very good cultivating conditions for many cell types, cell aggregates or tissue particles in a standard tissue culture laboratory. The system enables to culture HepG2 cells on Cytodex 3 microcarriers (mcs) to high densities. We inoculated 2 x 10(5)/ml HepG2 cells and 200 mg Cytodex 3 mcs in 50 ml Williams E medium (incl. 10% FCS) allowing them to attach to the mcs in the rotating vessel (rotation rate 14-20 rpm). HepG2 cells readily attached to the mcs while the vessel was rotating. Attachment of HepG2 to the mcs was about 50% after 24 hrs and 100 % within 48 hrs. After 72 hrs of rotary culturing small aggregates of Hep G2 on mcs were built. HepG2 cells and the aggregates rotated with the vessel and did not settle within the vessel or collide with the wall of the vessel. We conclude that this new RCCS is an excellent technology for culturing HepG2 cells on Cytodex 3 mcs. The system is easy to handle and enables to culture anchorage dependent cells to high densities in a short period.