| BackgroundHepatocellular carcinoma (HCC) is the most common malignancy worldwide, which hepatitis B virus (HBV) is the major underlying etiology of HCC. In recent studies conducted in Asia and Northern America, it was estimated that the lifetime risk of developing HCC is increased by 25-37 times in HBsAg carriers compared to non-infected populations. But didn't know the exact mechanism of a high incidence of hepatitis b patients with liver cancerHowever, the exact mechanism of a high incidence of hepatitis B virus patients with liver cancer remains unclear. In recent years, new research confirms microenvironment play an important role in the development of cancer.Above all, the cell-mediated immunity in microenvironment plays an even more crucial role in the occurrence of tumor. Prendergast GC put forward an important concept:Cancer is a microenvironment and immune disease. According to the concept, the scholars also discussed a lot. At present, most of the studies were focused on the imbalance of Thl and Th2 cell, regulatory T cells, Th17 cell, macrophage and NK cell.etc. Nevertheless, the research about integral functional status of cellular immunity is rare. T cell receptor (TCR) complementarity determining region 3 (CDR3) spectratyping is a good new technique to reflect of immune function. This study makes use of the technology to analysis the difference between integral and partial cellular immune function and to probe the role of partial cellular immune function play in pathogenesis of HBV-RHCC.ObjectivesWe investigated the patterns of Complementarity Determining Region3 (CDR3) length distribution for all 24 T cell receptor beta variable (TCRBV) gene families in the peripheral blood lymphocytes (PBLs), tumor-free liver (TFL) and tumor-infiltrating T lymphocytes (TILs) of ten patients with HBV-RHCC by immunoscope spectratyping technique. To probe the difference between integral and partial cellular immune function, it may helpful to reveal the role of partial cellular immune function in pathogenesis of HBV-RHCC. Furthermore, it might be an ideal objective evaluation methodology for individualized immunotherapy of HBV-RHCCMethods(1)Ten patients with HBV-RHCC were enrolled in this study.Samples of heparinized peripheral blood (10-20ml) who underwent radical resection from March to Jury in 2015 from the Xinxiang Central Hospital were collected during the preoperative period. Paired fresh liver tumor and tumor-free liver (TFL) tissue at the maximum distance from the tumor were collected. (2) RNA extraction, synthesis of the first cDNA and PCR amplification:Total RNA was extracted from PBMCs, liver tumor and tumor-free liver (TFL) tissue using total RNA isolation reagent and immediately reverse-transcribed to cDNA using cDNA synthesis kit. The specific primers for 24 TCRBV gene families were designed and synthesized, and ? chain constant region gene (BC) design a common fluorescent-labeled primer depend on previously literature described. By reverse transcription polymerase chain reaction amplification TCR CDR3 region of each gene family BV, the amplified products were conducted 1.5% agarose gel electrophoresis; (3) Analysis of TCRBV families by immunoscope technology Six microliters of fluorescent PCR products loaded onto the 6% acrylamide sequencing gel and then run for 2h on Applied Biosystems model 373A DNA Sequencer. The data were analyzed by Peak Scanner Software v1.0. The Twist Rate, the Miss Rate, the Normal Distribution Rate, the Single Rate and the Complexity Score were used to comprehensively analyze the integrality and diversity of CDR3 repertoires; (4) The PCR products of the preponderant usage BV family were PCR amplification again by the same sense primers and anti-sense primers (without FAM-labeled) at the same PCR condition, sequenced on an ABI 377DNA Sequencer. (5) Statistical analysis:The SPSS 21.0 software was used to calculate statistical significance for the differences of particular measurements between groups. Two-tailed unpaired t test were used; P values less than 0.05 were considered to be statistically significant.Results(1) The patterns of CDR3 length distribution for all TCR BV family (Products of PCR) in the peripheral blood lymphocytes (PBLs),tumor-free liver (TFL) and tumor-infiltrating T lymphocytes (TILs) of ten patients with HBV-RHCC were analyzed on a 1.5% agarose gel by gelred staining, we found majority of of PCR products of TCR BV families have a single band, but a minority of the products of TCRBV family did not or a faint band be seen in the gel; (2) When it come s to analysis the TCR-CDR3 BV families profile of th peripheral blood, tumor-free tissue and tumor tissues of ten patients with HBV-RHCC, a majority of BV families present twist distribution, such as monoclonal peak, oligo-clonal peak and skewed peaks; A part of BV families show the normal distribution distribution; a minority of BV families expressed very low frequency or no peak in graph.(3) Given that CDR3 profile in different BV family and different patients were varied in a certain degree, the Twist Rate, the Miss Rate, the Normal Distribution Rate, the Single Rate and the Complexity Score were used to further comprehensively analyze the integrality and diversity of CDR3 repertoires. Although there were no significant difference among the Twist Rate, the Miss Rate, the Normal distribution Rate of CDR3 profile between TFL and TILs (P>0.05), a great discrepancy between the two compartments were found in the attribution of the skewing peak, Normal distribution and absent peak. Additionally, there were a higher number of BV families with Gaussian distribution in PBLs compartment compared to the TFLs'(P=0.041), and there was no significant difference in the other indexes. Moreover, when TILs were compared with the corresponding liver tumors, we observed a more diversity profile in the PBL (P=0.042);(4)CDR3 profile relative to family history of HCC, HBV DNA and AFP level When it comes to the interplay between skewed TCRBV gene families and family history of HCC was analyzed, the complexity score in family history group of PBLs was obviously higher than that in the group without family history (p=0.016).In contrast, with respect to HBV DNA and AFP level, there was no difference in last two groups (p>0.05) (date not shown).(5) Sequencing of the CDR3 gene.We found that the monoclonal populations expressing TCR-BV1,-BV3,-BV7, BV-14 and -BV21 molecule were more prevalent compared with other TCRBV families. To further analyze the preponderant usage, some of the CDR3 segments of the monoclonal T cells of ten patients were sequenced. The CDR3 Nucleic sequences of preponderant usage BV families in PBL TFL and TIL of FIBV-RHCC were obtained. We discovered a series of laws via comparing the amino acid sequences between different samples and different patients, for instance:? All the shared motifs were found in the same BV family;?The shared motifs were discovered in the same tissue.?The identical motifs were explored in different sample and different tissue.ConclusionOur study investigated the patterns of Complementarity Determining Region3 (CDR3) length distribution for all 24 T cell receptor beta variable (TCRBV) gene families in the peripheral blood lymphocytes (PBLs), tumor-free liver (TFL) and tumor-infiltrating T lymphocytes (TILs) of ten patients with FIBV-RHCC by immunoscope spectratyping technique. In addition, the consensus sequences were found distributed between PBLs and TILs compartments in different patients. We found a remarkable different function in cellular immunology in PBLs, TFL and TILs, and confirmed the apparent clonal proliferation in TFL and TILs compartments. The discovery of consensus sequences provide fundamental basis and a potential target spot for targeted therapy in HBV-RHCC patients. |
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