Biliary tract cancer is characterized by high invasiveness, occult early clinical manifestations and rapid progression. Surgical resection typically fails to achieve satisfactory outcomes. Biliary tract cancer exhibits low sensitivity to radiotherapy and chemotherapy. The prognosis of patients is extremely poor. Genomics research based on next-generation sequencing technology has made some advances. The gene mutation spectrum of biliary tract cancer has been preliminarily revealed, which lays a foundation for the study of molecular typing. This review summarizes the research progress and clinical application of gene mutation spectrum of biliary tract cancer in recent years, aiming to provide reference for the clinical diagnosis, treatment and basic research.

BACKGROUND:There are a lot of studies on isolation and culture methods of human placental chorionic-derived mesenchymal stem cells (hpcMSCs), but how to simply and efficiently harvest a large amount of primary MSCs has not been resolved. OBJECTIVE:To optimize the tissue explants method of isolating and culturing hpcMSCsin vitro. METHODS:Human placental chorionic villi were collected from full-term deliveries under aseptic condition and isolated by electric homogenizer. hpcMSCs were prepared by tissue explants method. The fluid and tissue of the primary culture flask and douching normal saline of the initial culture were centrifuged and prepared for secondary culture. RESULTS AND CONCLUSION: It saved time and effort to treat human placental chorionic villi with electric homogenizer, with good effects on tissue dispersion and removal of red blood cells. The average time of cell acquisition in initial culture and secondary culture was (17.73±1.14) and (10.03±1.30) days, respectively. The yields of primary cultured cells in initial culture and secondary culture were (6.97±0.98)×105 and (13.82±1.44)×105per Φ100 mm culture dish, respectively. The adherent cells showed fibroblast-cell-like shape, which were in parallel or circinate arrangement. Highly expressed CD73, CD105 and CD90 could be detected in the third generation of hpcMSCs, but CD34, CD45, CD14, CD19 and HLA-DR were negative. Following induction, alizarin red staining and oil red O staining produced a strong reaction in cells. In a word, the optimized method is a simple and efficient method for obtaining a large amount of primary hpcMSCs.

Objective To establish a cultivating method for obtaining a large number of P0 generation human placental chorionic-derived mesenchymal stem cells (hpcMSCs).Methods The hpcMSCs were isolated from human placental chorion.After primary culturing and culturing for seven days,the culture medium,the non-adherent tissue and the douching normal saline of the primary culture were centrifuged and re-cultured twice.Cell morphology was observed by an inverted microscope.CCK-8 was used to measure the cell growth curve.Flow cytometry was used to detect cell surface markers.Adipogenic and osteogenic differentiation kits were used to assess the cell differentiation potential.Results The obtained hpcMSCs were fibroblast-like adherent cells and (25.54±3.38)×106 cells were obtained per placenta.The total yield of the primary culture,secondary culture and tertiary culture were (11.73±2.09)×106,(11.12±1.42)×106 and (2.69±0.71)×106,respectively,and the incubation time were (12.00±0.64) d,(8.87±0.63) d and (12.33±0.80) d.There was significant differences in incubation time between the secondary culture and the primary culture as well as the tertiary culture (all P＜0.05),and there was no significant difference between the primary culture and the tertiary culture.However,the incubation time of the tertiary culture had an increasing trend (P＞0.05).The yield per culture flask of the primary culture,secondary culture and tertiary culture were (1.12±0.15) × 106,(2.10±0.16)×106 and (1.04±0.16)×106,respectively.There was significant differences in the yield per culture flask between the secondary culture and the primary culture as well as the tertiary culture (all P＜0.05),and there was no significant difference between the primary culture and the tertiary culture.However,the yield per culture flask of the tertiary culture had a decreasing trend (P＞0.05).There was no difference among the three cultures in the growth curve and the expression of surface markers,and the osteogenic and adipogenic differentiation were all positive.Conclusions The P0 generation hpcMSCs isolated from a choriocarcinoma sample can be doubled by the three cultures compared with the primary culture,which can provide plenty stem cell source for the regenerative medicine.

OBJECTIVETo determine the immune repertoires of peripheral CD4+T cell receptor (TCR) Vb CDR3 in primary biliary cirrhosis (PBC) and analyze TCR diversity and preferred usage at sequence-level resolution.

METHODSARM-PCR and high-throughput sequencing were used to obtain millions of TCR Vb CDR3 sequences from peripheral CD4+T cells isolated from 7 patients with PBC and healthy volunteers. All sequencing data were analyzed, together with corresponding clinical information, by bioinformatic software. The Mann-Whitney U test was used for statistical analysis.

RESULTSThe PBC patients showed a lower level of diversity among the peripheral CD4+TCR Vb CDR3 than the healthy volunteers, and patients with higher level progression of the disease showed a greater lack of diversity. In addition, 4 specific preferred-usage amino acid sequences were discovered for the PBC patients: ASSFTGGPVEQY, ASSLISSGNNEQF, ATSRDTLAGGPGDTQY, and SASLEGNTEAF; these sequences were also found in higher frequencies in patients with later stages of PBC.

CONCLUSIONSDecreased TCR Vb CDR3 diversities and specific preferred usage of TCR CDR3 sequences in peripheral CD4+T lymphocytes in PBC suggest that clonal expansion of a large number of CD4+T cells may be an important factor for PBC progression. These data provide a better understanding about the general characteristics of CD4+T cells in PBC patients and related to pathogenesis of the disease, and may provide useful insights into potential targets for immunotherapy.

Amino Acid Sequence ; CD4-Positive T-Lymphocytes ; High-Throughput Nucleotide Sequencing ; Humans ; Liver Cirrhosis, Biliary ; Polymerase Chain Reaction ; Receptors, Antigen, T-Cell

The human genome is not a linear structure, but a three-dimensional structure through complex folding and assembly. Chromosome structure capture technology can detect the three-dimensional construction of chromatin. Hi-C sequencing data of various tumors indicate that the chromatin topology associated domains changed during tumor progression and is related to copy number variation. In addition, transformation of the genomic compartment is related to gene expression. However, current researches on three-dimensional structures of tumoral chromatin are still in the stage of exploration, and some conclusions are too superficial to be applied to the clinic immediately, which requires further study.

With the development of precision medicine and individualized treatment, tissue biopsy in cancer patients diagnosis and therapy has been broadly used. However, because it’s hard to collect enough samples for biliary tract tumors, liquid biopsy was broadly applied for the diagnosis. In liquid biopsy, circulating tumor cells, circulating tumor DNA, and tumor-derived exosomes carrying tumor-specific information are released from tumor tissue into blood, bile, and other body fluids, which makes tumor biopsy samples easily to be obtained in a non-invasive way. At the same time, through a series of morphological and molecular measurements as well as genetic characterization, liquid biopsy can be used to look for the new early diagnostic markers, and therapeutic targets, monitoring progression and prognosis of diseases. This article outlined the current technology used to detect circulating tumor cells, circulating tumor DNA, and tumor-derived exosomes, and summarizes the latest advances in the clinical application of liquid biopsy in biliary tract cancers.