Effects of Vitamin A and K3 on Immune Function and Intestinal Antioxidant Capacity of Aged Laying Hens

This study was conducted to investigate effects of vitamin A (VA) and vitamin K3 (VK3) on immune function and intestinal antioxidant capacity of aged laying hens. In a 3 × 3 factorial arrangement, the diets of 1080 Roman Pink laying hens (87 weeks old) was formulated with deficient, adequate and excess VA and VK3, including 0, 7000 and 14000 IU/kg VA and 0, 2.0 and 4.0 mg/kg VK3 for 8 weeks. Interactive effects between VA and VK3 were observed that VA and VK3 decreased the splenetic mRNA expression of inducible nitric oxide synthase (iNOS) and tumour necrosis factor α (TNF-α), but increased the plasma immunoglobulin G (IgG) content and jejunal mRNA expression of nuclear factor-like 2 (Nrf2). Hens fed adequate or excess VA had higher spleen index, mRNA expression of interleukin-10 (IL-10) in spleen, sIgA content, catalase (CAT), glutathione peroxidase and total dismutase (T-SOD) activity, and mRNA expression of polymeric immunoglobulin receptor (pIgR) in jejunum and lower mRNA expression of IL-1ß in jejunum and iNOS, TNF-α in spleen. Furthermore, adequate or excess VK3 significantly increased plasma IgG content, the CAT, T-SOD and total antioxidant capacity activities, up-regulated the mRNA expression of pIgR, Nrf2, SOD1 and CAT in jejunum and down-regulated the mRNA expression of iNOS and TNF-α in spleen.(AU)

Circulating non-coding RNAs as new biomarkers and novel therapeutic targets in colorectal cancer.

Colorectal cancer (CRC) is one of the most common malignant tumors, and a large number of patients are diagnosed and die every year. Due to the lack of appropriate diagnosis, prediction and treatment, early diagnosis rate of CRC is low and the prognosis is poor. Studies have found that abnormally expressed non-coding RNAs (ncRNAs) (including microRNAs (miRNAs), circular RNAs (circRNAs) and long non-coding RNAs (lncRNAs),etc.) play an important regulatory role in the occurrence and development of CRC. Some studies have shown that they are stable in the blood and can be detected repeatedly. They are expected to be non-invasive biomarkers for early diagnosis, prognosis evaluation, and prediction of drug sensitivity of CRC, as well as potential applications in the treatment of CRC.

HPV16 infection promotes an M2 macrophage phenotype to promote the invasion and metastasis of esophageal squamous cell carcinoma.

OBJECTIVES: High-risk human papillomavirus (HR-HPV) is an important risk factor for esophageal cancer. Macrophages constitute a crucial immune medium for regulating HPV-related tumors; however, the specific regulatory mechanisms remain unknown. Therefore, the purpose of our current study was to investigate the mechanism by which HPV16E6 regulates macrophages to promote the invasion and metastasis of esophageal cancer. METHODS: HPV16E6 infection was detected by polymerase chain reaction. Immunohistochemistry was used to verify the distribution of tumor-associated macrophages (TAMs) and MMP-9 expression in esophageal squamous cell carcinoma tissues (ESCCs), and cancer adjacent normal tissues (CANs) from Kazakh patients. ESCC cells were transfected with a plasmid over-expressing HPV16E6 and non-contact cocultured with macrophages. RESULTS: The infection rate of HPV16E6 in Kazakh ESCCs was clearly higher than that in CANs (P < 0.05). The density of CD163-positive TAMs was significantly positively correlated with HPV16E6 infection in ESCCs (P < 0.05). After coculturing macrophages and EC9706 cells transfected with the HPV16E6 plasmid, the phenotype of macrophages transformed into M2 macrophages. The migration and invasion ability of ESCC cells were higher in the HPV16E6-transfected and coculture group than in the HPV16E6 empty vector-transfected and non-cocultured HPV16E6-transfected groups (all P < 0.05). The density of M2-like TAMs in ESCCs was positively correlated with the level of MMP-9 expression. MMP-9 expression in the HPV16E6-ESCC coculture macrophages group was substantially higher than that in controls (all P < 0.05). CONCLUSIONS: HPV16 infection mediates tumor-associated macrophages to promote ESCC invasion and migration.

Imaging-anatomic Measurements of Carotid Artery Bifurcation

ABSTRACT Objective: To have anatomic measurements of carotid artery bifurcation (CAB) with 64-spiral computed tomography angiography (64-SCTA), and provide anatomic basis for related research. Methods: Imaging data of 92 subjects (45 males, 47 females, the age range 20-82 years and mean age 48.4 ± 6.1 years) without pathology of CAB, who underwent 64-SCTA in head and neck from June 1, 2008 to June 30, 2010, were selected from the Picture Archiving and Communication Systems in Zhongshan Hospital of Xiamen University, Fujian, China. On the 3D images, the angle and size of CAB were measured, and the statistical comparisons of measurements were made between the bilateral, sex and age groups. Results: The measurements of CAB were divided into young (≤ 40 years) and older (> 40 years) groups: bifurcation angle is 36.206° ± 10.210° and 49.343° ± 16.489°, respectively; the inner diameter of common carotid artery (CCA) is 6.820 ± 0.635 and 6.845 ± 0.838 mm, respectively; the proximal inner diameter of internal carotid artery (ICA) is 7.143 ± 0.992 and 7.476 ± 1.630 mm, of the enlargement is 7.568 ± 1.069 and 8.554 ± 1.733 mm, of the distal is 4.897 ± 0.508 and 5.123 ± 0.699 mm, respectively; the inner diameter of external carotid artery (ECA) is 4.324 ± 0.580 and 4.104 ± 0.638 mm, respectively. There were statistically significant differences in all the measurements between male and female groups, in the bifurcation angle, inner diameters of ICA and ECA between young and older groups, and in the bifurcation angle between the left and right (p < 0.05). Conclusion: A 64-SCTA with 3D image post-processing technique can clearly observe and show the CAB. All CAB measurements will provide the objective basis for applied anatomy, imaging diagnosis and surgery treatment.

Genetic Diversity and Phyletic Evolution of Eleven Chinese Indigenous and Three Commercial Chicken Breeds by mtDNA Sequences

Chinese indigenous chicken breeds are geographically widespread, and a total of 116 indigenous chicken breeds are listed as Chinese national genetic resources. However, these indigenous chicken breeds are facing serious challenges as declining population and germplasm degeneration because lots of commercial chicken breeds had been introduced. In this study, the genetic variations of eleven Chinese indigenous chicken breeds of Sichuan province and three commercial chicken breeds were investigated based on the partial mitochondrial DNA D-loop of 487bp in length. 147 individuals from 14 breeds were examined and 34 haplotypes were observed. Genetic diversity analysis showed that the highest haplotype diversity level was found in Dahen Chicken (DH) population, while the Arbor Acres Chicken (WF) and Roman layer (RM) showed lower genetic diversity levels. The long-term artificial selection may lead to reduced nucleotide diversity. Genetic population differentiation analysis indicated that most of the variation (80.80%) was attributed to variations among breeds. Phylogenetic analysis revealed that these individuals were divided into four distinct genetic clades, including cluster A, B, C and D. Eighteen haplotypes were classified as cluster A, eight haplotypes were classified as cluster B, five haplotypes were classified as cluster C and three haplotypes were classified as cluster D. There was no breed-specific clade. Our study firstly identified the populations genetic structure of Chinese indigenous chickens and the most important commercial breeds in Sichuan province, though the genetic diversity of indigenous breeds did not suffer obvious decrease, but could be helpful for efficient artificial breeding selection and genetic resources conservation.(AU)

Variations in Tissue-Specific Expression of Adipose Differentiation-Related Protein Gene in Two Native Yunnan Chicken Breeds

Adipose differentiation-related protein (ADFP) is a fatty acid-binding protein that can promote the absorption of long-chain fatty acids. However, few results have been published regarding its role in Yunnan Native chicken breeds. The aim of this study was to determine ADFP gene tissue-specific expression in Piao chickens (PC) and Wuliangshan black-bone chickens (WBC) by RT-qPCR. The ontogenetic expression levels of the ADFP gene were significantly different during growth and development phases in the subcutaneous fat, liver, and pectoralis muscle of PC, and in the subcutaneous fat, liver, and pectoralis muscle of WBC (p 0.05). Individual tissue-differential expression levelswere detectedon d 91 and 112 in PC, with highest levels determined in abdominal fat and subcutaneous fat, respectively. However, in WBC, the highest levels were determined on d 49, 91 and 112 d in the pectoralis muscle and liver. Correlation analysis revealed ADFP expression level in liver of WBC was significantly related with LW and HC (p 0.05), while no significant correlations with carcass fatness (CF) were found in PC (p>0.05). The results suggest ADFPdifferential expression in the liver and pectoral muscles of PC and WBC during the growth and development phases (p 0.05). The observed expression patterns indicate that the ADFP gene plays an important role in lipid metabolism of PC and WBC, and that these patterns are expressed differently in the tissues of different chicken genotypes.(AU)

Variations in Tissue-Specific Expression of Adipose Differentiation-Related Protein Gene in Two Native Yunnan Chicken Breeds

Adipose differentiation-related protein (ADFP) is a fatty acid-binding protein that can promote the absorption of long-chain fatty acids. However, few results have been published regarding its role in Yunnan Native chicken breeds. The aim of this study was to determine ADFP gene tissue-specific expression in Piao chickens (PC) and Wuliangshan black-bone chickens (WBC) by RT-qPCR. The ontogenetic expression levels of the ADFP gene were significantly different during growth and development phases in the subcutaneous fat, liver, and pectoralis muscle of PC, and in the subcutaneous fat, liver, and pectoralis muscle of WBC (p 0.05). Individual tissue-differential expression levelswere detectedon d 91 and 112 in PC, with highest levels determined in abdominal fat and subcutaneous fat, respectively. However, in WBC, the highest levels were determined on d 49, 91 and 112 d in the pectoralis muscle and liver. Correlation analysis revealed ADFP expression level in liver of WBC was significantly related with LW and HC (p 0.05), while no significant correlations with carcass fatness (CF) were found in PC (p>0.05). The results suggest ADFPdifferential expression in the liver and pectoral muscles of PC and WBC during the growth and development phases (p 0.05). The observed expression patterns indicate that the ADFP gene plays an important role in lipid metabolism of PC and WBC, and that these patterns are expressed differently in the tissues of different chicken genotypes.

Genetic Diversity and Phyletic Evolution of Eleven Chinese Indigenous and Three Commercial Chicken Breeds by mtDNA Sequences

Chinese indigenous chicken breeds are geographically widespread, and a total of 116 indigenous chicken breeds are listed as Chinese national genetic resources. However, these indigenous chicken breeds are facing serious challenges as declining population and germplasm degeneration because lots of commercial chicken breeds had been introduced. In this study, the genetic variations of eleven Chinese indigenous chicken breeds of Sichuan province and three commercial chicken breeds were investigated based on the partial mitochondrial DNA D-loop of 487bp in length. 147 individuals from 14 breeds were examined and 34 haplotypes were observed. Genetic diversity analysis showed that the highest haplotype diversity level was found in Dahen Chicken (DH) population, while the Arbor Acres Chicken (WF) and Roman layer (RM) showed lower genetic diversity levels. The long-term artificial selection may lead to reduced nucleotide diversity. Genetic population differentiation analysis indicated that most of the variation (80.80%) was attributed to variations among breeds. Phylogenetic analysis revealed that these individuals were divided into four distinct genetic clades, including cluster A, B, C and D. Eighteen haplotypes were classified as cluster A, eight haplotypes were classified as cluster B, five haplotypes were classified as cluster C and three haplotypes were classified as cluster D. There was no breed-specific clade. Our study firstly identified the populations genetic structure of Chinese indigenous chickens and the most important commercial breeds in Sichuan province, though the genetic diversity of indigenous breeds did not suffer obvious decrease, but could be helpful for efficient artificial breeding selection and genetic resources conservation.

Deep sequencing reveals the molecular pathology characteristics between primary uterine leiomyoma and pulmonary benign metastasizing leiomyoma.

PURPOSE: Pulmonary benign metastasizing leiomyoma (PBML), a rare condition of smooth muscle tumor, originates from women with a history of uterine leiomyoma (LM). Numerous genetic studies of uterine LM have been reported; however, there are few cytogenetic and molecular descriptions of PBML. Therefore, molecular subtyping is necessary to understand the pathogenesis of metastasizing sites. METHODS: Driver gene exon-capture sequencing was performed on one patient's peripheral blood, paraffin samples from primary uterine LM, and lung metastasizing leiomyoma 8 years later. RESULTS: The results showed that the same missense mutations of BLMH, LRP2, MED12, SMAD2, and UGT1A8 were concurrently mutated in the primary uterine LM and the PBML. Moreover, a splice mutation of PTEN (c.492+1G>A) was uniquely identified in the lung metastasis of the patient. CONCLUSION: This study indicates that the metastatic lung lesions were derived from the same malignant cell clone of uterine LMs and later acquired the novel driver mutations in the evolution of the tumor. In addition, driver gene sequencing can discriminate somatic driver mutations as biological indicators of potential malignant leiomyoma and can identify pathogenic variation driver mutations, which could be used for individualized therapy.

Analysis of serum microRNA expression in male workers with occupational noise-induced hearing loss.

Occupational noise-induced hearing loss (ONIHL) is a prevalent occupational disorder that impairs auditory function in workers exposed to prolonged noise. However, serum microRNA expression in ONIHL subjects has not yet been studied. We aimed to compare the serum microRNA expression profiles in male workers of ONIHL subjects and controls. MicroRNA microarray analysis revealed that four serum microRNAs were differentially expressed between controls (n=3) and ONIHL subjects (n=3). Among these microRNAs, three were upregulated (hsa-miR-3162-5p, hsa-miR-4484, hsa-miR-1229-5p) and one was downregulated (hsa-miR-4652-3p) in the ONIHL group (fold change >1.5 and Pbon value <0.05). Real time quantitative PCR was conducted for validation of the microRNA expression. Significantly increased serum levels of miR-1229-5p were found in ONIHL subjects compared to controls (n=10 for each group; P<0.05). A total of 659 (27.0%) genes were predicted as the target genes of miR-1229-5p. These genes were involved in various pathways, such as mitogen-activated protein kinase (MAPK) signaling pathway. Overexpression of miR-1229-5p dramatically inhibited the luciferase activity of 3' UTR segment of MAPK1 (P<0.01). Compared to the negative control, HEK293T cells expressing miR-1229-5p mimics showed a significant decline in mRNA levels of MAPK1 (P<0.05). This preliminary study indicated that serum miR-1229-5p was significantly elevated in ONIHL subjects. Increased miR-1229-5p may participate in the pathogenesis of ONIHL through repressing MAPK1 signaling.

The effect of aspirin on circulating tumor cells in metastatic colorectal and breast cancer patients: a phase II trial study.

PURPOSE: Aspirin could reduce the risk of cancer metastasis. Circulating tumor cells (CTCs) are a key factor of cancer metastasis, but no evidence has revealed how aspirin affects CTCs and its epithelial-mesenchymal transition (EMT). Here, we conducted a clinical trial to investigate how aspirin affects CTCs in metastatic colorectal cancer (MCC) and breast cancer patients (MBC). METHODS: The trial is retrospective registered at clinicaltrials.gov (NCT02602938). The eligible patients are given 100 mg aspirin q.d. for 8 weeks, and CTCs are evaluated at baseline, 4 and 8 weeks for absolute number, phenotype (epithelial type, E+, mesenchymal type, M+, and biophenotypic type, B+), and vimentin expression. RESULTS: Data on 21 MCC and 19 MBC patients are analyzed, and it revealed that the CTC numbers decreased with aspirin treatment in MCC (p < 0.001) but not MBC (p = 0.0532); besides, ratio of E+ CTCs increased (p = 0.037) and M+ CTCs decreased at 2 months in MCC (p = 0.013), but neither the ratio of E+ or M+ CTCs changes significantly in MBC; vimentin expression of M+ CTCs is higher than E+ and B+ CTCs either in MBC or MCC patients at baseline (p < 0.01); and aspirin suppresses the vimentin expression in M+ (p = 0.002)and B+ (p = 0.006) CTCs of MCC and M+ CTCs of MBC (p = 0.004); besides it find vimentin expression in B+ (p = 0.004) or M+ (p < 0.001), CTCs are markedly decreased in patients with total CTC numbers declined. CONCLUSION: Aspirin could decrease CTCs numbers and block EMT transition in MCC patients and part of MBC patients.

Analysis of serum microRNA expression in male workers with occupational noise-induced hearing loss

Occupational noise-induced hearing loss (ONIHL) is a prevalent occupational disorder that impairs auditory function in workers exposed to prolonged noise. However, serum microRNA expression in ONIHL subjects has not yet been studied. We aimed to compare the serum microRNA expression profiles in male workers of ONIHL subjects and controls. MicroRNA microarray analysis revealed that four serum microRNAs were differentially expressed between controls (n=3) and ONIHL subjects (n=3). Among these microRNAs, three were upregulated (hsa-miR-3162-5p, hsa-miR-4484, hsa-miR-1229-5p) and one was downregulated (hsa-miR-4652-3p) in the ONIHL group (fold change >1.5 and Pbon value <0.05). Real time quantitative PCR was conducted for validation of the microRNA expression. Significantly increased serum levels of miR-1229-5p were found in ONIHL subjects compared to controls (n=10 for each group; P<0.05). A total of 659 (27.0%) genes were predicted as the target genes of miR-1229-5p. These genes were involved in various pathways, such as mitogen-activated protein kinase (MAPK) signaling pathway. Overexpression of miR-1229-5p dramatically inhibited the luciferase activity of 3′ UTR segment of MAPK1 (P<0.01). Compared to the negative control, HEK293T cells expressing miR-1229-5p mimics showed a significant decline in mRNA levels of MAPK1 (P<0.05). This preliminary study indicated that serum miR-1229-5p was significantly elevated in ONIHL subjects. Increased miR-1229-5p may participate in the pathogenesis of ONIHL through repressing MAPK1 signaling.

Long non-coding RNA LINC00261 sensitizes human colon cancer cells to cisplatin therapy

Colon cancer is one of the most common digestive tumors. The present study aimed to explore the functional role, as well as the underlying mechanism of long non-coding RNA LINC00261 in colon cancer. Expression of LINC00261 was analyzed in colon cancer cell lines and human normal cell lines. Acquired resistance cell lines were then built and the acquired resistance efficiency was detected by evaluating cell viability. Thereafter, the effects of LINC00261 overexpression on cisplatin-resistant colon cancer cells were measured, as well as cell apoptosis, viability, migration, and invasion. Subsequently, we investigated the interaction of LINC00261 and β-catenin. The results showed that the LINC00261 gene was down-regulated in colon cancer cell lines and tissues, and in cisplatin-resistant cells. LINC00261 overexpression might relieve cisplatin resistance of colon cancer cells via promoting cell apoptosis, and inhibiting cell viability, migration, and invasion. Moreover, LINC00261 might down-regulate nuclear β-catenin through restraining β-catenin from cytoplasm into nuclei or it could also promote β-catenin degradation and inhibit activation of Wnt pathway. Finally, LINC00261 reduced cisplatin resistance of colon cancer in vivo and enhanced the anti-colon cancer effect of cisplatin through reducing tumor volume and weight.

Long non-coding RNA LINC00261 sensitizes human colon cancer cells to cisplatin therapy.

Colon cancer is one of the most common digestive tumors. The present study aimed to explore the functional role, as well as the underlying mechanism of long non-coding RNA LINC00261 in colon cancer. Expression of LINC00261 was analyzed in colon cancer cell lines and human normal cell lines. Acquired resistance cell lines were then built and the acquired resistance efficiency was detected by evaluating cell viability. Thereafter, the effects of LINC00261 overexpression on cisplatin-resistant colon cancer cells were measured, as well as cell apoptosis, viability, migration, and invasion. Subsequently, we investigated the interaction of LINC00261 and ß-catenin. The results showed that the LINC00261 gene was down-regulated in colon cancer cell lines and tissues, and in cisplatin-resistant cells. LINC00261 overexpression might relieve cisplatin resistance of colon cancer cells via promoting cell apoptosis, and inhibiting cell viability, migration, and invasion. Moreover, LINC00261 might down-regulate nuclear ß-catenin through restraining ß-catenin from cytoplasm into nuclei or it could also promote ß-catenin degradation and inhibit activation of Wnt pathway. Finally, LINC00261 reduced cisplatin resistance of colon cancer in vivo and enhanced the anti-colon cancer effect of cisplatin through reducing tumor volume and weight.

Clinical significance of fascin-1 and laminin-5 in non-small cell lung cancer.

Lung cancer is the leading cause of cancer death in men and the second leading cause of cancer death in women worldwide. Fascin-1 and laminin-5 were associated with the invasiveness and prognoses of several cancers. The expression and the serum levels of fascin-1 and laminin-5 in patients with non-small cell lung cancer (NSCLC) were analyzed in this study. The expression of fascin-1 and laminin-5 were examined in 378 patients and their serum level was measured in 154 patients. The health of all patients was followed post-surgery. The expression of fascin-1 (P = 0.000) and lanminin-5 (P = 0.001) and the serum levels of fascin-1 (P = 0.015) and laminin-5 (P = 0.046) were related to the relapse of patients with NSCLC. Both serum levels and expression of fascin-1 and laminin-5 can be used to effectively evaluate the prognoses of patients with NSCLC.

Investigation on ERCC5 genetic polymorphisms and the development of gastric cancer in a Chinese population.

Our study aimed to investigate the role of 2 ERCC5 promoter SNPs (rs2094258 and rs751402) in the development of gastric cancer in the Chinese population. The present hospital-based case-control study consisted of 155 patients with gastric cancer and 246 healthy controls recruited between March 2012 and December 2014. Genotyping for the rs2094258 and rs751402 polymorphic sites was carried out using polymerase chain reaction-restriction fragment length polymorphism. Statistical analyses were conducted using the SPASS version 16.0 software (SPSS, Inc., Chicago, IL, USA). As determined by the chi-square test, there was a significant difference in the genotype distributions of rs751402 between patients and controls (X2 = 6.74, P = 0.03). By unconditional logistic regression analysis, we observed that the TT genotype in rs751402 was significantly associated with increased risk to gastric cancer as compared with the CC genotype, and the adjusted OR (95%CI) was 2.17 (1.15-4.09). Moreover, subjects carrying the T allele in rs751402 had elevated risk of developing gastric cancer when compared with those carrying the C allele, with an adjusted OR value (95%CI) of 1.47 (1.09-1.99). In conclusion, we suggest that the ERCC5 rs751402 gene polymorphism may influence the susceptibility to gastric cancer in the Chinese population.

An improved DNA marker technique for genetic characterization using RAMP-PCR with high-GC primers.

Random amplified polymorphic DNA (RAPD) is a widely used molecular marker technique. As traditional RAPD has poor reproducibility and productivity, we previously developed an improved RAPD method (termed RAMP-PCR), which increased the reproducibility, number of bands, and efficiency of studies on polymorphism. To further develop the efficiency of this method, we used high-GC content primers for improved RAMP-PCR with DNA samples from Lonicera japonica. Comparison of amplification profiles obtained by standard RAPD primers with those obtained by regular PCR and RAMP-PCR, and high-GC primers with regular PCR and RAMP-PCR showed that the average number of bands and polymorphisms per primer gradually and significantly increased (from 6.4 to 15.0 and from 4.6 to 10.2, respectively). Cluster dendrograms showed similar results, indicating that this new method is consistent and reproducible. A total of 22 samples from different species, including plants, animals, and humans, were used for RAMP-PCR with high-GC primers. Multiple bands were successfully amplified from all samples, demonstrating that this method is a reliable technique with consistent results and may be of general interest in studies on different genera and species. We developed highly effective DNA markers, which can provide a more effective and potentially valuable approach than traditional RAPD for the genetic identification of various organisms, particularly of medicinal plants.

Relationship between genetic polymorphisms of methylenetetrahydrofolate reductase and breast cancer chemotherapy response.

Activity of methylenetetrahydrofolate reductase (MTHFR), an enzyme involved in folate metabolism, is influenced by mutations in the corresponding gene, contributing to a decrease in 5,10-MTHF. Due to such polymorphisms, individuals differ in MTHFR enzyme activity and plasma folate levels. We investigated the relationship between two common MTHFR polymorphisms (C677T and A1298C) and breast cancer (BC) chemotherapy response. From February 2013 to January 2016, 148 advanced BC patients at the Center Hospital of Cangzhou were enrolled and treated with six different chemotherapy regimens. Subjects were genotyped using polymerase chain reaction-restriction fragment length polymorphism. Forty-one (27.7%), 70 (47.3%), and 37 (25.0%) patients carried the C/C, C/T, and T/T C677T genotypes, respectively; 101 (68.2%), 42 (28.4%), and 5 (3.4%) had the A/A, A/C, and C/C genotypes of A1298C, respectively. Total chemotherapy efficacy was 66.9% (99/148), with 7 (4.7%), 92 (62.2%), 36 (24.3%), and 13 (8.8%) cases showing complete response, partial response, no change, and progressive disease, respectively. Chemotherapy regimens did not differ in effectiveness (P > 0.05). Efficacy rates associated with C677T C/C, C/T, and T/T genotypes were 58.5, 58.6, and 91.9%, respectively, with T/T carriers exhibiting significantly better responses than the C/C (P < 0.05) and C/T groups (P < 0.05). Effectiveness among A1298C A/A, A/C, and C/C carriers was 70.6, 64.3, and 0.0%, respectively, but no difference was established between these genotypes in this regard (P > 0.05). The MTHFR C677T genotype may be associated with BC chemotherapy response, and could be of great value in guiding individualized treatment for this disease.

In vitro expansion and differentiation of rat pancreatic duct-derived stem cells into insulin secreting cells using a dynamicthree-dimensional cell culture system.

In this study, a dynamic three-dimensional cell culture technology was used to expand and differentiate rat pancreatic duct-derived stem cells (PDSCs) into islet-like cell clusters that can secrete insulin. PDSCs were isolated from rat pancreatic tissues by in situ collagenase digestion and density gradient centrifugation. Using a dynamic three-dimensional culture technique, the cells were expanded and differentiated into functional islet-like cell clusters, which were characterized by morphological and phenotype analyses. After maintaining 1 x 108 isolated rat PDSCs in a dynamic three-dimensional cell culture for 7 days, 1.5 x 109 cells could be harvested. Passaged PDSCs expressed markers of pancreatic endocrine progenitors, including CD29 (86.17%), CD73 (90.73%), CD90 (84.13%), CD105 (78.28%), and Pdx-1. Following 14 additional days of culture in serum-free medium with nicotinamide, keratinocyte growth factor (KGF), and b fibroblast growth factor (FGF), the cells were differentiated into islet-like cell clusters (ICCs). The ICC morphology reflected that of fused cell clusters. During the late stage of differentiation, representative clusters were non-adherent and expressed insulin indicated by dithizone (DTZ)-positive staining. Insulin was detected in the extracellular fluid and cytoplasm of ICCs after 14 days of differentiation. Additionally, insulin levels were significantly higher at this time compared with the levels exhibited by PDSCs before differentiation (P < 0.01). By using a dynamic three-dimensional cell culture system, PDSCs can be expanded in vitro and can differentiate into functional islet-like cell clusters.