Detection of Calreticulin as a Candidate Prognostic Biomarker in Invasive Breast Carcinoma from a Biological Scaffold-Based 3D Co-culture System

@#Introduction: Breast cancer is the most common cancer in women and the world’s second leading cause of death in women, after lung cancer. Calreticulin (CRT), an endoplasmic reticulum (ER) multipurpose protein, has been proposed as a potential biomarker for breast cancer. However, reports on the correlation between CRT expression and cell invasiveness in breast cancer micro-tissues are scarce. Thus, in the current study, we analyzed the potential correlation between CRT and invasiveness of breast cancer in a biological scaffold-based 3D co-culture system. Methods: MCF7, MDA-MB-231 and MCF-10A breast cell lines were co-cultured in a 3-dimensional (3D) system with MRC-5 lung fibroblast cell line in the cell density ratio of 3:1. Thereafter, calreticulin gene and protein expression levels were determined based on quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and immunohistochemistry, respectively. Moreover, via RT-qPCR analysis, the gene expression levels of calreticulin-related candidate metastasis genes in breast cancer micro-tissues were carried out. Results: The results showed occasional foci of lumen-like morphology in the non-cancerous breast micro-tissues and the formation of solid clusters for breast cancer micro-tissues. Moreover, immunohistochemistry results revealed protein expression of calreticulin in non-cancerous and cancerous breast micro-tissues with cytoplasmic and nucleic acid localizations. It was found that PCMT1 and ER-α genes were significantly downregulated (p < 0.01) in invasive breast cancer micro-tissues. Conclusion: This study suggests that CRT and CRT-related candidate metastasis genes may potentially serve as prognostic biomarkers in invasive breast carcinoma.

Using OMICS technologies to analyze the mechanisms of synthetic microbial co-culture systems: a review / 生物工程学报

In recent years, the interaction mechanisms underpinning the synthetic microbial co-culture systems have gained increasing attention due to their potentials in various biotechnological applications. Exploration of the inter-species mechanisms underpinning the synthetic microbial co-culture system could contribute to a better understanding of the theoretical basis to further optimize the existing co-culture systems, and design new synthetic co-culture system for large-scale application. OMICS technologies such as genomics, transcriptomics, proteomics, and metabolomics could analyze the biological processes in a high throughput manner. Multi-omics analysis could achieve a "global view" of various members in the microbial co-culture systems, which presents opportunities in understanding synthetic microbial consortia better. This article summarizes recent advances in understanding the mechanisms of synthetic microbial co-culture systems using omics technologies, from the aspects of metabolic network, energy metabolism, signal transduction, membrane transport, stress response, community stability and structural rationality. All these findings could provide important theoretical basis for future application of the microbial co-culture systems with the aids of emerging biotechnologies such as synthetic biology and genome editing.

Inhibitory effect of recombinant human semaphorin 3A on angiogenesis of gastric cancer and the associated mechanisms / 解剖学报

Objective To investigate the expression of semaphorin 3A (Sema3A) and its receptor neuropilin-1 (NRP-1) in gastric cancer and its correlation with microvessel density (MVD), and then to explore the effect of recombinant human Sema3A on angiogenesis of gastric cancer and the associated mechanisms. Methods Forty cases of gastric cancer tissues and its corresponding adjacent normal tissues were used to detecte the expression of Sema3A, NRP-1 and MVD in tissues by immunohistochemistry method . The expression level of Sema3A in serum of gastric cancer patient group and normal control group were measured by Enzyme-linked immuno-sorbent assay (ELISA). Western blotting was used to detect the expression of Sema3A and NRP-1 in five gastric cancer cell lines (MGC-803,HGC-27,MKN-28,SGC-7901,MKN-45) and human gastric mucosal epithelial cell (GES-1). Transwell chamber was used to construct non-contact in vitro co-culture system, in which the effects of different concentrations of recombinant human Sema3A on angiogenesis in gastric cancer were analyzed by tube formation assay preliminarily. The expression levels of vascular endothelial growth factor receptor 2 (VEGFR2) and NRP-1 in co-culture system were detected by Western blotting. Results The expression levels of Sema3A in gastric cancer tissues, cell lines and patient serum were significantly lower than that in the control group(P<0. 05), while the expression of NRP-1 in gastric cancer tissues and MKN-28 cells was significantly increased, and both of them were associated with TNM staging of gastric cancer (P < 0. 05) . In vitro co-culture system, The tube forming abilities of human umbilical vein endothelial cell (HUVEC) were decreased in recombinant human Sema3A treated group, and this phenomenon was concentration dependent. The expression of VEGFR2 protein was down-regulated by recombinant human Sema3A. Conclusion The expression of Sema3A was decreased in gastric cancer tissues, cell lines and patient serum, and negatively correlated with microvessel density. The recombinant human Sema3A could inhibit the angiogenesis of gastric cancer in vitro, which may be related to down-regulation of VEGFR2 protein expression.

Effect of Serum from Kidney Yang Deficiency Rats on Expression of β-catenin/OPG/RANKL in OB-OC Co-culture System and Regulatory of Icariin / 中国实验方剂学杂志

Objective:To observe the effect of serum of kidney Yang deficiency rats on the expression of β-catenin，osteoprotegerin(OPG) and nuclear transcription factor-κB receptor activator ligand (RANKL) in the co-culture system and regulatory of icariin on it， and to explore the possible mechanism of inducing osteoporosis.Method:The 16 male SD rats were randomly divided into blank group and model group， 8 rats in each group. 10 mL·kg-1 adenine was administrated to stomach to establish kidney yang deficiency model. Serum was separated and extracted after the model was established successfully. Isolation and culture of osteoblast（OB） and osteoclast（OC） in vitro， OB was observed and identified by alkaline phosphatase（ALP），alizarin red and Giemsa staining， OC was identified by tartrate resistant acid phosphatase（TRAP） staining， OB-OC co-culture system was established in transwell cell， icariin group（100 μmol·L-1）， blank group， icariin（100 μmol·L-1） + serum group， serum group and Dickkopf1（DKK-1） drug（100 μg·L-1） were set up in group ， 2 days after intervention of co-culture system， OC was counted， ALP and TRAP in supernatant were detected by microplate enzyme labeling， and the expression of OPG，β-catenin and RANKL in each group was detected by Western blot.Result:Compared with blank group， the ALP activity，β-catenin and OPG protein expression in serum group were significant reduction （P<0.05）， while the OC quantity， TRAP activity and RANKL protein expression were marked increase （P<0.05）. Compared with serum group， ALP activity of icariin group decreased significantly （P<0.01）， Compared with icariin group， ALP activity and OPG protein expression decreased （P<0.05）， trap activity and RANKL expression increased （P <0.05） in icariin + serum group.Conclusion:The serum of kidney Yang deficiency rats can induce the occurrence of osteoporosis， and the mechanism of action may be through inhibition of ALP activity， down regulating the expression of β-catenin and OPG protein， increasing the activity of TRAP and the expression of RANKL protein.

Effects and potential mechanism of bone marrow-derived stem cells on hepatic stellate cells in two contact ;coculture system / 中华传染病杂志

Objective To investigate the effects of bone marrow-derived stem cells (BMMSC)on the expressions of inflammatory cytokines and Toll-like receptor (TLR)4 pathway in primary hepatic stellate cells (HSC)with direct and indirect contact coculture system.Methods Purified HSC were separately treated with LPS in the concentrations of 0,50,100 and 150 g/L for 48 h.Proliferation ratio was tested with cell counting kit-8 to determine the optimal concentration.HSC in LPS were divided into three groups,including HSC alone group,cocultured with BMMSC at 1∶1 group and cocultured with transwell group at the optimal concentration.The supernatants were collected to detect the concentrations of interleukin-6 (IL-6)and tumor necrosis factor (TNF)-α.Cells were further divided into seven groups, including BMMSC without LPS group,HSC without LPS group,BMMSC with LPS group,HSC with LPS group,BMMSC in transwell system group,HSC in transwell system group,all cells in transwell system group and direct contact system group.The mRNA expressions ofα-smooth muscle actin (α-SMA) and collagen I were detected by quantitative real-time PCR,and protein expressions of TLR4,myeloid differentiation factor 88 (MyD88)and nuclear factor-κB (NF-κB)were analyzed by Western blot.Data were analyzed with one-way ANOVA analysis.Results The proliferation rate of HSC in 50,100 and 150μg/L LPS were (129.77±11 .26)%,(162.90±13.15 )% and (55 .12 ±11 .6)%,respectively compared with HSC without LPS group.The differences were statistically significant (t =5 .91 ,10.70 and 8.65 , respectively,all P <0.05).The concentrations of IL-6 and TNF-αin HSC alone group,directly/indirectly cocultured group were (252.02 ±30.94)ng/L and (148.00 ± 10.27 )ng/L,(88.52 ±6.61 )g/L and (72.63±5 .54)ng/L,(103.74±7.14)ng/L and (81 .79 ±6.92 )ng/L,respectively.The differences between HSC alone group and directly/indirectly cocultured group were significant (t=12.66 and 15 .82, 11 .81 and 12.34,respectively,all P < 0.05 ).The directly and indirectly cocultured groups were significantly different (t=3.83 and 2.53,respectively,both P <0.05 ).The mRNA expressions of α-SMA and collagen I in HSC with LPS were remarkably increased compared with HSC without LPS (t =14.16 and 11 .84,respectively,both P <0.05 )and reversed by cocultured with BMMSC (t =11 .98 and 4.47,respectively,P <0.05).All cells in transwell group expressed moreα-SMA and collagen I than the direct contact group (t=3.70 and 3.19,respectively,P <0.05 ).The TLR pathway associated protein expressions,TLR4,MyD88,and NF-κB in HSC in transwell group were significantly down-regulated compared with HSC with LPS group.And all cells in transwell system had higher level of protein expressions compared with direct contact system (P < 0.05 ).Conclusions BMMSC are effective in inhibiting HSC activation and inflammatory cytokines excretion,which may be modulated through TLR4 pathway and cell to cell contact.

Effect of Catalpol on the Activity and Apoptosis of Osteoclast in the Osteoblasts-osteoclasts Co-culture Sys-tem and Its Mechanism Study / 中国药房

OBJECTIVE:To study the effect of catalpol on the activity and apoptosis of osteoclasts (OC) in the osteoblasts (OB)-OC co-culture system and its mechanism. METHODS:OB and OC were isolated respectively from the SD rats of 1-3 days and 5-7 days old to establish OB-OC co-culture system. After treated with 0(blank control),0.05,0.5,5,50 and 100 mg/L catal-pol for 48,72 and 96 h,the number of bone absorption lacuna for OC was observed by inverted microscope to reflect OC activity. After treated with 0(blank control)and 0.05 mg/L catalpol for 48,72 and 96 h,the activity of tartrate resistant acid phosphatase (TRACP)in OC was detected,and the apoptosis rate of OC was calculated. After treated with 0(blank control)and 0.05 mg/L ca-talpol,mRNA expression of osteoprotegerin(OPG)in OB was detected. RESULTS:In OB-OC co-culture system,the number of bone absorption lacuna in 0.05-50 mg/L catalpol groups was significantly lower than blank control group(P<0.01),indicating ca-talpol could inhibit OC activity,especially 0.05 mg/L catapol. Compared with blank control,0.05 mg/L catapol lowered the activity of TRACP but increased the apoptosis rate of OC(P<0.05);mRNA expression of OPG was up-regulated in OB(P<0.01). CON-CLUSIONS:In OB-OC co-culture system,catalpol can inhibit the activity of OC and induce the apoptosis of OC,and its mecha-nism may be associated with the mRNA expression up-regulation of OPG in OB.

Research progress of cell co-culture method / 中华危重病急救医学

Cell culture technology is the most commonly used method in the in vitro experiments at present. However, monolayer cell culture technology has been unable to meet the demand of the researchers. This is because that monolayer cell culture cannot mimic the cellular environment in which multiple cells interact with each other in the body. We cannot discuss the relationship of many cells, because we do not know the relationship between cells through a single kind of cell. So cell co-culture medicine arises at the historic moment for the demand. With the development of research method in recent years, cell co-culture method also has been improved in practice: from direct contact co-cultures to indirect contact co-cultures, from two-dimensional co-cultures to three-dimensional co-cultures. Cell co-culture method is closer to the human body. It is also more advantageous to study the interaction among cells. Nowadays, there are more researchers tend to select this method to study the physiological and pathological in vitro model, tissue engineering, and cell differentiation research. At the same time, it has become the focus of drug research and development, drug analysis, mechanism of drug action, and drug targets. This article will review the studies of cell co-culture method, summarize advantages and disadvantages of various methods, so as to promote improvement of cell culture methods, to build cells co-culture system that more close to human body, and build the in vitro model that simulate internal circulation of human body further.

Relaxation of Danhong Injection in vascular smooth muscle cells in co-culture system and study on its mechanism / 中草药

Objective: In this study, we examined whether Danhong Injection (DHI) exerted a relaxation effect in vascular smooth muscle cells (A7r5) in co-culture system by human umbilical vein endothelial cells (Ea.Hy926) and studied its mechanisms. Methods: First of all, Western blotting assay was carried out to quantify the protein expression of MLC and P-MLC in A7r5 cells. Secondly, calcium assay kit was used to detect the changes in intracellular calcium while the changes of CaM mRNA expression were subsequently detected by RT-PCR. MTT assay was used to detect the effect of DHI in proliferation of A7r5 cell, which is induced by PDGF-BB. Finally, the co-culture system of EA.Hy926 and A7r5 cells was constructed by a Transwell. After the treatment of Hy926 cells with DHI for co-culture system, the protein expression of MLC and P-MLC in A7r5 cells in co-culture system was also quantified by Western blotting. Cell-based ELISA was used to observe the cAMP generation in A7r5 cells. Results: DHI (10 μL/mL) contributed to the decrease of the ratio of P-MLC/MLC (P < 0.05) and CaM mRNA expression (P < 0.01) in A7r5 cells, which are cultured individually, but had no significant effect in intracellular calcium concentration and the expression of MLC proteins. In co-culture system, DHI (10 μL/mL) could still decrease the ratio of P-MLC/MLC (P < 0.05) and increase the secretion of cAMP (P < 0.01) in A7r5 cells. In addition, DHI contributed to suppress the proliferation of A7r5 cells which were induced by the PDGF-BB (P < 0.01). Conclusion: DHI can directly relax vascular smooth muscle cells by reducing the ratio of P-MLC/MLC and the expression of CaM mRNA, meanwhile indirectly relaxing the vascular smooth muscle cells in co-culture system by promoting the secretion of cAMP which is increased by vasodilatory factors in endothelial cells.

Application of cell co-culture models in absorption of oral drug / 药学实践杂志

Cell co-culture system can better simulate the inner environment of human body ,predict drug transport and metabolism in intestinal environment and increase the relation between in vitro cell model and integral animal test .In recent years ,co-culture cell model plays an increasingly important role in evaluating the absorption of oral drugs ,which becomes the highlight in the evaluation of drug oral absorption during new drug discovery .This essay summarized co-culture cell model which simulates intestinal environment and their application ,and looked into the future of their application in evaluating oral drug intestinal absorption during oral drug discovery .

Effect of hypoxia and hyperglycemia on retinal pigment epithelial cells co-cultured with human mesenchymal stem cells / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To evaluate cell proliferation, apoptosis and migration of human retinal pigment epithelial cells ( RPE) when co - cultured with human marrow mesenchymal stem cells ( hMSCs ) in condition of hypoxia and hyperglycemia so as to explore possible mechanisms of diabetes aggravating choroidal neovascularization ( CNV) preliminarily. METHODS:Both hMSCs and RPE cells were co-cultured in a transwell system. The experiment was divided into four groups: 21% O2 with 5. 56mmol/L glucose ( control group, A ), 21% O2 with 30mmol/L glucose ( hyperglycemia and normoxia group, B ) , 5% O2 with 5.56mmol/L glucose ( normoglycemia and hypoxia group, C ) and 5% O2 with 30mmol/L glucose ( hyperglycemia and hypoxia group, D) . Cell Counting Kit-8 (CCK-8) was used to detect the proliferation of RPE cells in each group at 12, 24 and 48h respectively. Flow cytometry was performed to observe apoptosis of RPE cells at 24h. Additionally, we assessed migration capabilities of RPE via transwell assay under the condition of hyperglycemia and hypoxia by co-culturing of hMSCs.RESULTS:In this co-culturing system, at 12, 24 and 48h, group B (1. 61±0. 41, 1. 80±0. 34;1. 91±0. 35), C (1.34±0. 46, 1. 94±0. 40, 2. 14±0. 41) and D (1. 98±0. 47, 2.26±0.42, 2. 55±0. 40) showed significantly higher proliferation rate than group A (0. 92±0. 45, 1. 27±0. 32, 1.59±0. 41, P0. 05). CONCLUSION:By coexistence with hMSCs, the synergy of hyperglycemia and hypoxia can improve migration and proliferation of RPE cells, and have no effect on apoptosis of RPE cells within short period.

BMP2 expression impacts the growth and osteogenic differentiation of BMSCs in the co-culture system / 中华显微外科杂志

Objective To investigate the effects of BMP2 in the co-culture system of hBMSCs and hUVECs.Methods Based on the co-culture system of cells,a simple culture group (hBMSCs group),a combined culture group (BMSCs + hUVECs group) and a BMP2 silenced group (BMSCs + BMP2 silenced hUVECs) were established.On days 4,6,8,10,cells were counted for drawing cell growth curves.In addition,ALP and OC expression in BMSCs was detected in each group.Results At each time point,cell number in the BMSCs + hUVECs group were significantly greater than in the BMSCs group,while the BMP2 silenced group were in the median.ALP and OC expression in the BMSCs + hUVECs group were significantly greater than in the BMSCs group,while the BMP2 silenced group were in the median.Conclusion BMP2 plays an important role in the co-culture system,which can promote osteogenic differentiation and the growth of BMSCs.