ABSTRACT
ObjectiveChronic pancreatitis (CP) is an irreversible pancreatic parenchymal disease with complicated etiology. Chymotrypsin C (CTRC) gene variation may be related to CP occurrence. The article analyzed the mutation of CTRC gene in Han population with CP in Sichuan, and discussed its clinical relevance.MethodsPeripheral blood samples were collected from 106 patients with CP and 148 healthy controls and DNA was extracted for whole exon sequencing of CTRC gene and analysis of clinical correlation.ResultsOne case of c.611G>A heterozygous missense mutation and three cases of single nucleotide polymorphism (SNP) site variation were found which included one new SNP c.40+133G>A and two known SNPs: rs6679763 and rs555015. The variation of c.40 +133G> A and rs6679763 showed a significant difference between case group and control group (P=0.029, P=0.011). Clinical baseline data showed significant differences between two groups on smoking (P=0.042), biliary disease (P=0.013), and blood glucose(P=0.017). When the confounding factors were eliminated, we found that smoking and rs6679763 variant site were significantly associated with CP risk (OR=2.817, 95%CI: 1.016-7.811, P=0.047;OR = 4.893, 95%CI: 1.152-20.781, P=0.031, respectively). There was no multiplicative interaction between gene mutation and environment or clinical data.ConclusionSmoking, biliary disease, blood glucose, c.611G>A mutation, rs6679763 and c.40+133G>A variation may be related to the occurrence of CP. Smoking and rs6679763 locus variation are independent risk factors for CP, and the CTRC gene SNP locus rs6679763 and c.40+133G>A and the point mutation site c.611G>A are predisposing loci in Chinese Han population of Sichuan province.
ABSTRACT
<p><b>BACKGROUND</b>The multidrug resistance (MDR) associated with the expression of the mdr1 gene and its product P-glycoprotein is a major factor in the prognosis of hepatocellular carcinoma cell (HCC) patients treated with chemotherapy. Our study was to establish a stable HCC MDR cell line where a de novo acquisition of multidrug resistance specifically related to overexpression of a transgenic mdr1.</p><p><b>METHODS</b>The 4.5-kb mdr1 cDNA obtained from the plasmid pHaMDR1-1 was cloned into the PCI-neo mammalian expression vector, later was transferred by liposome to human hepatocarcinoma cell line HepG2. Then the transfected HepG2 cells resisting G418 were clustered and cultured and the specific fragment of mdr1 cDNA, mRNA and the P-glycoprotein (Pgp) in these HepG2 cells were detected by PCR, RT-PCR and flow cytometry, respectively. The accumulation of the daunorubicin was determinated by flow cytometry simultaneously. The nude mice model of grafting tumour was established by injecting subcutaneously HepG2/mdr1 cells in the right axilla. When the tumour diameter reached 5 mm, adriamycin was injected into peritoneal cavity. The size and growth inhibition of tumour were evaluated.</p><p><b>RESULTS</b>The mdr1 expression vector was constructed successfully and the MDR HCC line HepG2/mdr1 developed. The PCR analysis showed that the specific fragment of mdr1 cDNA in HepG2/mdr1 cells, but not in the control group HepG2 cells. Furthermore, the content of the specific fragment of mdr1 mRNA and Pgp expression in HepG2/mdr1 cells were (59.7 +/- 7.9)% and (12.28 +/- 2.09)%, respectively, compared with (16.9 +/- 3.2)% and (3.07 +/- 1.06)% in HepG2 cells. In the nude mice HCC model, the tumour genes of both groups were identified. After ADM therapy, the mean size of HepG2 cell tumours was significantly smaller than HepG2/mdr1 cell tumours.</p><p><b>CONCLUSION</b>The approach using the transfer of mdr1 cDNA may be applicable to the development of MDR hepatocarcinoma cell line, whose MDR mechanism is known. This would provide the experimental basis of MDR research.</p>