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Objective:To investigate the correlation between the expression of programmed death ligand-1 (PD-L1) and clinicopathological parameters and prognosis in hepatocellular carcinoma (HCC) tissues.Methods:From January 2008 to December 2016, 344 HCC patients underwent surgery in Nantong Third Hospital Affiliated to Nantong University were enrolled. The expression of PD-L1 in paraffin HCC tissues was detected by tissue microarray and immunohistochemistry. The correlation between the expression of PD-L1 in tumor cells, tumor-infiltrating immune cells and the clinicopathological parameters and prognosis of HCC patients were analyzed. And the related factors affecting the prognosis of patients were explored. Chi-square test, log-rank test and univariate and multivariate Cox regression analysis were used for statistical analysis.Results:Positive PD-L1 located in the membrane and/or cytoplasm of HCC tumor cells and tumor-infiltrating immune cells. The positive rate of PD-L1 expression in tumor cells was 21.8%(75/344). The expression of PD-L1 in tumor cells was related to histological grade and microvascular invasion, the positive rates of PD-L1 expression of patients with histological gradeⅠ, Ⅱ and Ⅲ were 7.7% (2/26), 16.5% (19/115) and 26.6% (54/203), respectively, the positive rates of PD-L1 expression of patients with or without microvascular invasion were 29.3% (34/116) and 18.0% (41/228), respectively, and the differences were statistically significant ( χ2=7.659 and 5.787, P=0.022 and 0.016). The positive expression rate of PD-L1 in tumor-infiltrating immune cells was 47.1% (162/344). The expression of PD-L1 in tumor-infiltrating immune cells was related with microvascular invasion, the positive rates of PD-L1 expression in patients with or without microvascular invasion were 56.9% (66/116) and 42.1% (96/228), respectively, and the differences were statistically significant ( χ2=6.751, P=0.009). The median survival time of patients with negative expression of PD-L1 in HCC tumor cells was 61 months (30 months, 92 months), while that of patients with positive PD-L1 expression was 16 months(6 months, 44 months), and the difference was statistically significant ( χ2=55.722, P<0.01). The expression of PD-L1 in HCC tumor cells was an independent risk factor affecting overall survival time (hazard ratio=3.090, P<0.01). Conclusions:The expression of PD-L1 in HCC tumor cells may be related to the malignancy and invasion of HCC, and may be a potential risk factor for prognosis.
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Objective@#To investigate the tumor necrosis factor receptor superfamily 1B gene (TNFRSF1B) polymorphism in relation to the outcomes of hepatitis C virus (HCV) infection.@*Methods@#One thousand six hundred and forty-five cases without HCV infection, 545 cases with HCV clearance, and 783 cases with chronic HCV infection were enrolled. TaqMan probe method was used to investigate genotype rs1061622 (T > G) and rs1061624 (G > A). Two single nucleotide polymorphisms (SNPs) sites were genotyped and haplotypes were constructed to evaluate their relation with the outcome of HCV infection.@*Results@#Logistic regression analysis showed that there was no relation to the two SNPs with HCV infection susceptibility and chronicity (P > 0.05). Haplotype analysis showed that carrier TA had an increased susceptibility to HCV infection [adjusted odds ratio (OR) = 1.15, 95% confidence interval (CI): 1.01 to 1.30, P = 0.038)]. Carrier TA and GG haplotypes were conducive to chronic HCV infection (adjusted OR = 1.28, 95% CI: 1.08 to 1.53, P = 0.006; OR = 1.31, 95% CI: 1.03 to 1.66, P = 0.026).@*Conclusion@#The combinational effects of rs1061622 and rs1061624 in TNFRSF1B gene may increase the risk of HCV chronicity and infection.
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Objective@#To explore the relationship between the tumor necrosis factor receptor superfamily members 11A (TNFRSF11A) and 11B (TNFRSF11B) gene polymorphisms and the outcome of hepatitis C virus (HCV) infection.@*Methods@#In this case-control study, 749 cases of persistent HCV infection, 494 cases of spontaneous clearance and 1 486 control subjects were included from 2008 to 2016. TaqMan-MGB probe method was used to detect the genotype of TNFRSF11A rs1805034 and TNFRSF11B rs2073617. The genotypes distribution of the two single nucleotide polymorphisms (SNP) were analyzed in different populations.@*Results@#Co-dominant model showed that individuals carrying the rs2073617 CC genotype were prone to have chronic HCV infection, compared with individuals carrying the rs2073617 TT genotype (OR=1.517, 95%CI: 1.055-2.181, P=0.024). Recessive model results showed that individuals carrying rs2073617 CC genotype were more likely to develop chronic HCV infection compared with individuals carrying rs2073617 TT or TC genotype (OR=1.435, 95%CI: 1.033-1.996, P=0.032). Additive model showed that the risk for chronic HCV infection increased with the increase of the number of rs2073617 C alleles (OR=1.204, 95%CI: 1.013-1.431, P=0.035).@*Conclusion@#The genetic polymorphism of TNFRSF11B rs2073617 might be related with the chronicity of HCV infection.
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Drug-induced liver injury ( DILI) and autoimmune hepatitis ( AIH) have many similar clinical and histological manifestations , which brings difficulties to clinicians in differential diagnosis .This article elaborates on the association between DILI and AIH and their simi-larities and differences in clinical and histological manifestations , in order to help clinicians with the differential diagnosis of DILI and AIH . This article reviews the selection of therapeutic regimens for DILI and AIH and introduces how to select therapeutic strategies based on patient conditions and make a definite diagnosis when there are difficulties in differential diagnosis .
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Objective To explore the association between nuclear factor kappa-light-chainenhancer of activated genetic polymorphisms in B cells (NF-κB) and the HCV susceptibility,among the Chinese population.Methods A total of 1 679 participants were enrolled;including 503 drug users and 1 176 other participants at risk under the exposure for blood.By using the logistic regression analysis,related risk factors for HCV infection among subjects were analyzed.Two NF-κB pathway variants,NF-κB1 rs72696119 and REL rs13031237 were then genotyped by TaqMan assay method.Logistic regression analysis was performed to analyze the association between gene polymorphisms and the susceptibility on HCV.Results Among the drug users,women (OR=0.408,95％CI:0.308-0.767) appeared to be associated with the decreased risk for HCV infection,while factors as drug injection (OR=8.817,95％CI:5.577-13.937) and the duration of drug-intake ＞5.5 years (OR=2.891,95％CI:1.824-4.583) were associated with the increased risk for HCV infection.Among the participants who had been exposed to blood,women (OR=3.431,95％ CI:2.360-4.988) were associated with the increased risk for HCV infection,while the levels of education beyond elementary school (OR =0.613,95％ CI:0.429-0.876) were associated with the decreased risk for HCV infection.Compared to the reference NF-κB1 rs72696119 CC genotype,the carriage of GG genotype was associated with an increased risk of susceptibility on HCV (OR=1.412,95％ CI:1.035-1.927) among the total study population.Results from the interaction analysis showed that there was no interactive effects appeared between rs72696119 and route of infection,or between rs72696119 and gender among the total population under study (all P＞0.05).Conclusion NF-κB1 polymorphism rs72696119 and related factors seemed associated with the susceptibility to HCV infection among high-risk Chinese populations.
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Objective To explore the association between nuclear factor kappa-light-chainenhancer of activated genetic polymorphisms in B cells (NF-κB) and the HCV susceptibility,among the Chinese population.Methods A total of 1 679 participants were enrolled;including 503 drug users and 1 176 other participants at risk under the exposure for blood.By using the logistic regression analysis,related risk factors for HCV infection among subjects were analyzed.Two NF-κB pathway variants,NF-κB1 rs72696119 and REL rs13031237 were then genotyped by TaqMan assay method.Logistic regression analysis was performed to analyze the association between gene polymorphisms and the susceptibility on HCV.Results Among the drug users,women (OR=0.408,95％CI:0.308-0.767) appeared to be associated with the decreased risk for HCV infection,while factors as drug injection (OR=8.817,95％CI:5.577-13.937) and the duration of drug-intake ＞5.5 years (OR=2.891,95％CI:1.824-4.583) were associated with the increased risk for HCV infection.Among the participants who had been exposed to blood,women (OR=3.431,95％ CI:2.360-4.988) were associated with the increased risk for HCV infection,while the levels of education beyond elementary school (OR =0.613,95％ CI:0.429-0.876) were associated with the decreased risk for HCV infection.Compared to the reference NF-κB1 rs72696119 CC genotype,the carriage of GG genotype was associated with an increased risk of susceptibility on HCV (OR=1.412,95％ CI:1.035-1.927) among the total study population.Results from the interaction analysis showed that there was no interactive effects appeared between rs72696119 and route of infection,or between rs72696119 and gender among the total population under study (all P＞0.05).Conclusion NF-κB1 polymorphism rs72696119 and related factors seemed associated with the susceptibility to HCV infection among high-risk Chinese populations.
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Objective To explore a time series based meta-analysis of randomized clinical trials (RCTs) and observational studies which examined differences in mortality in acute-on-chronic liver failure (ACLF) patients treated with artificial liver support system(ALSS) or not.Methods Medline,Embase,Ovid,and Cochrane library database was systemically searched up to December 2014.The outcome measure was mortality at different follow-up endpoints.Relative risks (RRs) were pooled for analysis.Results Ten studies,involving a total of 1682 ACLF patients,among whom 842 were treated with ALSS.ALSS was found to reduce the risk of 1-month and 3-month mortality for patients with ACLF by nearly 16.4％ and 13.2％,respectively.Randomized trials and observational studies provided good internal and external validity,respectively.Conclusions ALSS therapy could reduce short-term mortality in patients with ACLF.Clinical utility of this system for survival benefit may be implied.
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<p><b>OBJECTIVE</b>To investigate the role of Src family kinases (Fgr, Hck, Lyn) and the major protein kinase C substrate SSeCKS in non-alcoholic steatohepatitis (NASH) and determine the possible mechanism regulating differential expression.</p><p><b>METHODS</b>Kupffer cells were stimulated with CCL4 and effect on SSeCKS, Hck, Fgr, and Lyn expression was detected by real-time reverse transcription-PCR. Male Sprague-Dawley rats were used to create a NASH model by feeing a high fat diet. The modeled rats were divided into a model group and a normal group. After sacrifice, the extent of hepatic steatosis and inflammation was assessed, and the expression levels of SSeCKS and Hck, Fgr, Lyn were detected by immunohistochemical staining.</p><p><b>RESULTS</b>Expression of Lyn and Hck was decreased in the CCL4-stimulated Kupffer cells and the change in expression level was positively associated with levels of inflammatory stimuli (P < 0.01). The change in expression of SSeCKS in the CCL4-stimulated Kupffer cells was negatively correlated with inflammatory stimuli (P < 0.01). Fgr expression was very low in the unstimulated Kupffer cells and was not affected by the exposure to inflammatory stimuli. The number of inflammatory cells in the liver tissues of rars were negatively correlated with expression of Lyn, Hck and SSeCKS (P < 0.01), with low negative correlation for Lyn (r =-0.398, P < 0.01) and moderate negative correlation for Hck (r=-0.508, P < 0.01); the Lyn and Hck expression levels were highly positively correlated (r =0.942, P < 0.01).</p><p><b>CONCLUSION</b>Src family kinases (Lyn, Hck and Fgr) and SSeCKS are involved in development and progression of NASH, and their differential expression patterns are associated to a certain extent. The factors may represent potential targets of therapy for NASH-related inflammation.</p>
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Animals , Male , Rats , A Kinase Anchor Proteins , Cell Cycle Proteins , Fatty Liver, Alcoholic , Inflammation , Rats, Sprague-Dawley , src-Family KinasesABSTRACT
Background and purpose: Arginase-1 (Arg-1) is an enzyme involved in the urea cycle. Research has shown that changed expression of Arg-1 plays an important role in the cellular metabolism and growth. The purpose of this research was to investigate the expression of Arg-1 in hepatocellular carcinoma (HCC) and to analyze its correlation with clinicopathological features. Methods: The expression of Arg-1 protein and mRNA in 31 samples of HCC, paracancerous liver tissues and 12 samples of normal liver was detected by Western blot and reverse transcription-polymerase chain reaction (RT-PCR). The expression profiles of Arg-1 at protein level in 158 samples of HCC and paracancerous liver tissues were detected with high-throughput tissue microarray technique and immunohistochemistry. The relationships between Arg-1 expression and clinicopathological features were also analyzed. Results:The expression of Arg-1 mRNA and protein was signiifcantly decreased in HCC compared with the paracancerous liver tissues and normal liver tissues (F=57.83, 160.89; all P<0.01). The expression of Arg-1 in HCC was related to differentiation degree (F=10.41, 30.03; all P<0.01). And with tumor differentiation decreased, the expression level down-regulated. Immunohistochemistry revealed that Arg-1 protein was mainly located in the cytoplasm and nuclear. The expression rates of Arg-1 were 88%, 98.7%and 100%in HCC, paracancerous tissues and normal liver tissues, respectively. Statistical analysis revealed that Arg-1 protein staining rate was signiifcantly lower in tumor tissues than that in paracancerous tissues (χ2=14.7416, P<0.01) and normal liver tissues (χ2=4.1415, P<0.05). The Arg-1 expression was correlated with differentiation degree of HCC, vascular invasion and recurrence after operation (χ2=22.8459, 10.2639, 10.6368 respectively;all P<0.05), but not correlated with age, gender, the hepatitis B virus, the level of serum AFP, liver cirrhosis, diameter of tumor and tumor number. Conclusion:The expression level of Arg-1 is much lower in HCC than that in the paracancerous liver and normal liver. Moreover, Arg-1 expression level is closely related to tumor differentiation degree, metastasis and relapse. These data demonstrate that Arg-1 may play a negative role in the development of HCC.
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Objective To explore the value of arginase-1(Arg-1) and glypican-3 (GPC-3) combined examination in the differential diagnosis of hepatocellular carcinoma (HCC),metastatic carcinoma (MC) of liver and intrahepatic cholangiocarcinoma (ICC).Methods From January 2005 to December 2011,a total of 54 patients with HCC were selected,including 10 cases with high differentiation,25 cases with moderate differentiation and 19 cases with poor differentiation.At the same time,25 patients with MC of liver and 20 patients with ICC were selected.A total of 31 normal liver specimens were set as control.The expressions of Arg 1 and GPC-3 in the above tissues were detected by immunohistochemistry method.The sensitivity and specificity of the examination in the diagnosis of HCC were analyzed.Chi-square test was performed for count data analysis.Results The positive expression rate of Arg-1 in HCC,MC of liver,ICC and normal liver tissues was 87.0％ (47/54),4.0％ (1/25),5.0％ (1/20) and 100.0％ (31/31),respectively.The Arg 1 positive expression rate in HCC tissues was higher than that in other tumor tissues of non-HCC,and the difference was statistically significant (x2 =66.98,P＜0.05).The positive expression rate of GPC-3 in HCC,MC of liver,ICC and normal liver tissues was 70.4％ (38/54),12.0％ (3/25),5.0％ (1/20) and 0 (0/31),respectively.The GPC-3 positive expression rate in HCC tissues was higher than that in other tumor tissues of non-HCC and the difference was statistically significant (x2=37.98,P＜0.05).The sensitivity and specificity of Arg-1 or GPC-3 positive in HCC diagnosis was 92.6％ (50/54) and 86.7％ (39/45).The sensitivity and specificity of both Arg 1 and GPC-3 positive in HCCdiagnosis was 64.8％ (35/54) and 100.0％ (45/45).Conclusion Arginase-1 and glypican-3 combined examination has an important value in HCC diagnosis and differential diagnosis.
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Objective To construct small hairpin RNA(shRNA) lentiviral vectors targeting human a proliferation-inducing ligand(APRIL) gene and detect the titer of virus and suitable multiplicity of infection (MOI) after 293T cells were infected by the lentival vectors. Methods Three RNA interference targeting sequences of APRIL gene were screened including shAPRIL1210, shAPRIL1754 and shAPRIL1604. Both sense and antisense Oligo DNA of the targeting sequences were synthesized and cloned into the pGCL-GFP vector, respectively. The resulting lentiviral vectors containing shAPRIL were named LV-shAPRIL1210, LV-shAPRIL1754, LV-shAPRILI604. Then they were confirmed by PCR and DNA sequencing. 293T cells were co-transfected with LV-shAPRIL, pHelper 1.0 and pHelper 2. 0 to product lentivirus, respectively. The titer of virus and suitable MOI were tested according to the expression level of GFP in the 293T cells. Results PCR analysis and DNA sequencing confirmed that three shAPRIL DNA were successfully inserted into the lentiviral vectors. The titers of concentrated virus were 5 × 107, 6 × 107 and 4 × 107(transduction units )TU/ml, respectively, and the suitable MOI was 5. Conclusions Three shRNA lentiviral vectors targeting human APRIL gene have been successfully constructed, which lays a foundation for future studying APRIL's gene silencing in related target cells.
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Objective To construct of shRNA lentiviral expression vector targeting APRIL (aproliferation-inducing ligand) gene in CFPAC-1 cell of human pancreatic cancer. Methods We used gene engineering to screen RNA interference targeting sequence of APRIL gene. The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was designed, synthesized and cloned into the pGCL-GFP vector. The resulting lentiviral vector containing shAPRIL were named LV-shAPRIL. Then it was conformed by PCR and DNA sequencing identification. 293T cells were eotransfected with LV-shAPRIL,pHelper 1.0 and pHelper 2.0 to product ientivirus. The titer of virus was tested according to the expression level of GFP in the 293T cells. After recombinant lentivirus infection into CFPAC-1 cells, we used real-time RT-PCR and Western blotting to examine APRIL mRNA and protein expression at different cell culture period.Results PCR analysis and DNA sequencing conformed that shAPRIL DNA was successfully inserted into the lentiviral vector. The titer of concentrated virus were 5 × 107 TU/ml. APRIL expression in CFPAC-1 cells were inhibited significantly at both mRNA and protein level. APRIL mRNA expression were decreased 73%, 70%and 71% , respectively, after the infection of 4 days, 4 weeks and 8 weeks by LV-shAPRIL. APRIL protein expression were decreased 66%, 63% and 62%, respectively , after the infection of 4 days , 4 weeks and 8weeks by LV-shAPRIL. Conclusions ShRNA lentiviral expression vector targeting APRIL gene has been successully constructed, and it can effectively inhibit the expression of APRIL gene in CFPAC-1 ceils. This study lays a foundatin for in vivo research APRIL gene scilence in pancreatic cancer cell using the model of nude mice.
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Objective To investigate the change and effect of SSeCKS(src suppressed c kinase substrates)in the activation of hepatic stellate cells(HSCs).Methods HSCs were isolated from normal rats,the change of SSeCKS mRNA expression on HSCs culture in vitro was determined using real.time PCR.protein level was determined by Western blot and immunofluorescence methods.A rat model of liver fibrosis was established.The expression and location of SSeCKS and α-SMA(α-smooth muscle actin)in liver tissues were detected by immunofluorescence methods.Results SSeCKS mRNA expression WaS loW in freshly isolated HSCs cell and the expression increased in activated HSCs in vitro.In liver fibrosis tissue,the number of SSeCKS-positive cells was increased and these cells were distributed along the sinusoids which also contained α-SMA positive cells.Conclusion The expression of SSeCKS was increased in activated HSCs in vitro.Therefore.SSeCKS may be involved in the liver inflammation and fibrosis.
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Objective To investigate the inhibitive effect of shRNA (short hairpin RNA) targeting APRIL gene on the pancreatic cancer cells in vitro and in vivo, in order to explore the feasibility of gene therapy for pancreatic cancer. Methods The LV-shAPRIL targeting APRIL gene had been constructed before, and was used to infect the CFPAC-1 cells. Cell proliferation and apoptosis were examined by MTT and flow cytometry. Then CFPAC-1 cells were used to construct the model of transplantation tumor into the nude mice, the tumor growth was assessed after LV-shAPRIL treatment. Results 96 hours after the LV-shAPRIL infection into CFPAC-1 cells, the cell proliferation was significantly inhibited when compared with control group and lentivirus infection group (P＜0.05 ). Flow cytometry showed the apoptosis ratio of the CFPAC-1 cells was (17.35±0.96)% in LV-shAPRIL group, which was higher than that in control group and lentivirus infection group (P＜0.05 ). After LV-shAPRIL injection into the model of nude mice, the tumor growth was slower than that in the two control groups. The tumor's volume of the LV-shAPRIL group was(821.8±123.3) mm3 and the mass was (2.16±0.18)g at 27 day, and were obviously depressed, when compared with two control groups (P±0.05). Conclusions LV-shAPRIL targeting APRIL gene can inhibit the growth of the CFPAC-1 cells in vitro and vivo. This may provide a new gene therapy approach for pancreatic cancer.
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Aim To elucidate the polymorphism of hepatic lipasegene gene and the relation to coronary heart disease. Methods CHD group had one hundred and fifty-six patients, and each subgroup was: myocardium infarction CHD subgroup included eighty-four patients; non-myocardium infarction subgroup included seventy-two patients; pure CHD subgroup comprised sixty-five patients and hypertension and CHD subgroup comprised ninety-one patients. Phenol-Chloroform method was used to extract DNA from human peripheral blood, and a combination of polymerasechain reaction and restriction fragment length polymorphism were used to analyze the distribution of genotypes and alleles of the polymorphism site of hepatic lipase. Results The genotype and allele distribution of HL-514C/T polymorphism were significantly different between the whole CHD group and control group(P
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Objective To clone human pancreatic cancer gene PTCH,construct the recombinant(expression) plasmid PET22b/PTCH and express the fusion protein.Methods The PTCH gene was(amplified) by RT-PCR from the total RNA extracted from human pancreatic cancer strain SW1990. The amplified product was inserted into the vector PET22b to construct the recombinant expression plasmid PET22b/PTCH,which was transformed into E.coliBL21-CodonPlus~(TM)-RP and then identified by(sequence) analysis.The expression of fusion protein was induced with IPTG and verified by Western blot method.Results A human pancreatic cancer gene with a reading frame of 789 bp was successfully cloned from human pancreatic cancer strain SW1990,which had the same sequence as that of PTCH gene in Genbank.The expression of PET22b/PTCH was proved by Western blot.Conclusion Human(pancreatic) cancer gene PTCH was successfully cloned and constructed with PET22b plasmid.The(prepared) fusion protein lays the basis for further study.
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Objective:To analyze 916 cases of endoscopically diagnozed reflux esophagitis(RE) and to explore the endoscopic and clinicopathologic characteristics of RE.Methods:Totally 916 RE patients from Lixiahe district in Jiangsu province(1997 2002) were graded with criterion made in Chinese Yantai in 1999.Some cases were subjected to pathology,age,sex,symptoms,endoscopic manifestations and pathology variety examination.Results:The incidence of RE was for 7.4% in endoscopic checked patients(916/12 376).The male to female ratio was 2.2∶1,mean age was (54.42?15.05),patients above 60 years old accounted for 46.5%.Only 32.6% of RE patients had typical reflux symptoms(426/916,299/916).Endoscopic diagnosis focused on Chinese Yantai standardⅠ Ⅱ (85.3%,781/916),pathology diagnosis focused on light to moderate degree,secondary RE was common in severe degree.Some patients were accompanied by bile reflux,esophagus gap hernia and peptic ulcer.Conclusion:The incidence of RE in Lixiahe district in Jiangsu province is rather high,it is common in middle aged and senior male patients(Ⅰ Ⅱ).Pathology variety is chiefly scalelike epithelial proliferation filled with chronic inflammatory cells.It was partly accompanied by atypical proliferation,erosion and ulcer,appearance of early cancer was occasionally found.The sensitivity of clinic symptoms diagnosis is lower than endoscopic diagnosis which is simple and of great value.