Prevalence and clinical impact of sarcopenia in liver transplant recipients:A meta-analysis

BACKGROUND The prevalence of sarcopenia in patients undergoing liver transplantation(LT)remains to be determined partly because of different diagnostic criteria.Sarcopenia has recently been recognized as a new prognostic factor for predicting outcomes in LT candidates.AIM To estimate the prevalence of sarcopenia and evaluate its clinical effect on LT candidates.METHODS This systematic search was conducted in PubMed,Web of Science,Embase,and Cochrane Library for original English-language articles that investigated the prevalence and influence of sarcopenia in patients undergoing LT from database inception to November 30,2022.Cohort studies of the definition of sarcopenia that estimate sarcopenia prevalence and evaluate its effect on clinical outcomes and the risk of mortality were included.RESULTS Twenty-five studies involving 7760 patients undergoing LT were included.The pooled prevalence of sarcopenia in patients undergoing LT was 40.7%[95%confidence intervals(95%CI):32.1-49.6].The 1-,3-,and 5-year cumulative probab-ilities of post-LT survival in patients with preoperative sarcopenia were all lower than those without sarcopenia(P<0.05).Sarcopenia was associated with an increased risk of post-LT mortality in patients undergoing LT(adjusted hazard ratio:1.58;95%CI:1.21-2.07).Patients with preoperative sarcopenia had a longer intensive care unit stay,a high risk ratio of sepsis,and serious post-LT complications than those without sarcopenia.CONCLUSION Sarcopenia is prevalent in a substantial proportion of patients undergoing LT and is strongly and independently associated with higher a risk of mortality risk.

Abnormal splenic artery diameter/hepatic artery diameter ratio in cirrhosis-induced portal hypertension

AIM:To determine an optimal cutoff value for abnormal splenic artery diameter/proper hepatic artery diameter(S/P) ratio in cirrhosis-induced portal hypertension.METHODS:Patients with cirrhosis and portal hypertension(n = 770) and healthy volunteers(n = 31) underwent volumetric computed tomography threedimensional vascular reconstruction to measure the internal diameters of the splenic artery and proper hepatic artery to calculate the S/P ratio.The cutoff value for abnormal S/P ratio was determined using receiver operating characteristic curve analysis,and the prevalence of abnormal S/P ratio and associations between abnormal S/P ratio and major complications of portal hypertension were studied using logistic regression.RESULTS:The receiver operating characteristic analysis showed that the cutoff points for abnormal splenic artery internal diameter and S/P ratio were > 5.19 mm and > 1.40,respectively.The sensitivity,specificity,positive predictive value,and negative predictive value were 74.2%,45.2%,97.1%,and 6.6%,respectively.The prevalence of an abnormal S/P ratio in the patients with cirrhosis and portal hypertension was 83.4%.Patients with a higher S/P ratio had a lower risk of developing ascites [odds ratio(OR) = 0.708,95%CI:0.508-0.986,P = 0.041] and a higher risk of developing esophageal and gastric varices(OR = 1.483,95%CI:1.010-2.175,P = 0.044) and forming collateral circulation(OR = 1.518,95%CI:1.033-2.230,P = 0.034).After splenectomy,the portal venous pressure and maximum and mean portal venous flow velocities were reduced,while the flow rate and maximum and minimum flow velocities of the hepatic artery were increased(P < 0.05).CONCLUSION:The prevalence of an abnormal S/P ratio is high in patients with cirrhosis and portal hypertension,and it can be used as an important marker of splanchnic hemodynamic disturbances.

Anticoagulation therapy prevents portal-splenic vein thrombosis after splenectomy with gastroesophageal devascularization

AIM:To compare the incidence of early portal or splenic vein thrombosis(PSVT) in patients treated with irregular and regular anticoagulantion after splenectomy with gastroesophageal devascularization.METHODS:We retrospectively analyzed 301 patients who underwent splenectomy with gastroesophageal devascularization for portal hypertension due to cirrhosis between April 2004 and July 2010.Patients were categorized into group A with irregular anticoagulation and group B with regular anticoagulation,respectively.Group A(153 patients) received anticoagulant monotherapy for an undesignated time period or with aspirin or warfarin without low-molecular-weight heparin(LMWH) irregularly.Group B(148 patients) received subcutaneous injection of LMWH routinely within the first 5 d after surgery,followed by oral warfarin and aspirin for one month regularly.The target prothrombin time/international normalized ratio(PT/INR) was 1.25-1.50.Platelet and PT/INR were monitored.Color Doppler imaging was performed to monitor PSVT as well as the effectiveness of thrombolytic therapy.RESULTS:The patients' data were collected and analyzed retrospectively.Among the patients,94 developed early postoperative mural PSVT,including 63 patients in group A(63/153,41.17%) and 31 patients in group B(31/148,20.94%).There were 50(32.67%) patients in group A and 27(18.24%) in group B with mural PSVT in the main trunk of portal vein.After the administration of thrombolytic,anticoagulant and antiaggregation therapy,complete or partial thrombus dissolution achieved in 50(79.37%) in group A and 26(83.87%) in group B.CONCLUSION:Regular anticoagulation therapy can reduce the incidence of PSVT in patients who undergo splenectomy with gastroesophageal devascularization,and regular anticoagulant therapy is safer and more effective than irregular anticoagulant therapy.Early and timely thrombolytic therapy is imperative and feasible for the prevention of PSVT.

Chinese guidelines on management of hepatic encephalopathy in cirrhosis

The Chinese Society of Hepatology developed the current guidelines on the management of hepatic encephalopathy in cirrhosis based on the published evidence and the panelists’ consensus. The guidelines provided recommendations for the diagnosis and management of hepatic encephalopathy (HE) including minimal hepatic encephalopathy (MHE) and overt hepatic encephalopathy, emphasizing the importance on screening MHE in patients with end-stage liver diseases. The guidelines emphasized that early identification and timely treatment are the key to improve the prognosis of HE. The principles of treatment include prompt removal of the cause, recovery of acute neuropsychiatric abnormalities to baseline status, primary prevention, and secondary prevention as soon as possible.

Chinese guidelines on the management of liver cirrhosis(abbreviated version)

Based on reviews of the literature and experts’consensus,the Chinese Society of Hepatology developed guidelines for the diagnosis and treatment of liver cirrhosis,in order to improve clinical practice.In addition to what has been covered in previously published guidelines on the management of cirrhosis complications,these guidelines add new sections and provide updates.The guidelines emphasize the early diagnosis of the cause and assessment of complications.Comprehensive treatments including etiological treatment and complication management should be initiated immediately.In addition,regular monitoring,especially surveillance of hepatocellular carcinoma,is crucial for managing patients.

Current understanding of the metabolism of micronutrients in chronic alcoholic liver disease

Alcoholic liver disease(ALD)remains an important health problem worldwide.Perturbation of micronutrients has been broadly reported to be a common characteristic in patients with ALD,given the fact that micronutrients often act as composition or coenzymes of many biochemical enzymes responsible for the inflammatory response,oxidative stress,and cell proliferation.Mapping the metabolic pattern and the function of these micronutrients is a prerequisite before targeted intervention can be delivered in clinical practice.Recent years have registered a significant improvement in our understanding of the role of micronutrients on the pathogenesis and progression of ALD.However,how and to what extent these micronutrients are involved in the pathophysiology of ALD remains largely unknown.In the current study,we provide a review of recent studies that investigated the imbalance of micronutrients in patients with ALD with a focus on zinc,iron,copper,magnesium,selenium,vitamin D and vitamin E,and determine how disturbances in micronutrients relates to the pathophysiology of ALD.Overall,zinc,selenium,vitamin D,and vitamin E uniformly exhibited a deficiency,and iron demonstrated an elevated trend.While for copper,both an elevation and deficiency were observed from existing literature.More importantly,we also highlight several challenges in terms of low sample size,study design discrepancies,sample heterogeneity across studies,and the use of machine learning approaches.

Histopathological characteristics of needle core biopsy and surgical specimens from patients with solitary hepatocellular carcinoma or intrahepatic cholangiocarcinoma

BACKGROUND Pathological manifestations of hepatic tumours are often associated with prognosis. Although surgical specimens(SS) can provide more information,currently, pre-treatment needle core biopsy(NCB) is increasingly showing important value in understanding the nature of liver tumors and even in diagnosis and treatment decisions. However, the concordance of the clinicopathological characteristics and immunohistochemical(IHC) staining between NCB and SS from patients with hepatic tumours were less concerned.AIM To introduce a more accurate method for interpreting the IHC staining results in order to improve the diagnostic value of hepatic malignancy in NCB samples.METHOD A total of 208 patients who underwent both preoperative NCB and surgical resection for hepatocellular carcinoma(HCC) or intrahepatic cholangiocarcinomaRESULTS Morphologically, the presence of compact tumour nests or a cord-like structure in NCB was considered the primary cause of misdiagnosis of HCC from ICC. The kappa statistic showed a moderate agreement in histomorphology(k = 0.504) and histological grade(k = 0.488) between NCB and SS of the tumours. A 4-tier(+++,++, +, and-) scoring scheme that emphasized the focal neoplastic cell immunoreactivity of tumour cells revealed perfect concordance of CK19, GPC3 and HepPar1 between NCB and SS(k = 0.717; k = 0.768; k = 0.633). Furthermore,with the aid of a binary classification derived from the 4-tier score, a high concordance was achieved in interpreting the IHC staining of the three markers between NCB and final SS(k = 0.931; k = 0.907; k = 0.803), increasing the accuracy of NCB diagnosis C(k = 0.987; area under the curve = 0.997, 95%CI: 0.990-1.000; P< 0.001).CONCLUSION These findings imply that reasonable interpretation of IHC results in NCB is vital for improving the accuracy of tumour diagnosis. The simplified binary classification provides an easy and applicable approach.

Retrospective observation of therapeutic effects of adult auxiliary partial living donor liver transplantation on postpartum acute liver failure: A case report

We present a female patient with preterm labor, severe viral hepatitis B of acute phase, hepatic encephalopathy stage Ⅲ and coma.After delivery, the illness was exacerbated and the patient presented with clinical signs of vital organ dysfunctions such as acute respiratory distress syndrome, cerebral edema and hypoxemia that needed mechanical ventilation.Emergency liver transplantation was recommended after multidisciplinary panel consultations.The donor, her mother, consented to donate her right liver.Auxiliary partial orthotopic living donor liver transplantion(APOLDLT) was performed.After operation, the patient was on triple medication of tacrolimus plus mofetil mycophenolate and prednisone for immunosuppression.The combination of antihepatitis B virus(HBV) immunoglobulin and entecavir was initiated for anti-HBV therapy.Both the patient and the donor recovered well without any complications.The patient was followed up regularly.Her liver function, clinical signs and symptoms improved significantly.Until now, the recipient has been living for more than 78 mo free of any complications.The APOLDLT is a life-saving modality for rescuing patients with high-risk acute liver failure following HBV infection without available donor and hence is recommended under standardized antiviral therapy coverage as stated above.

Characterization of metabolic landscape in hepatocellular carcinoma

Hepatocellular carcinoma(HCC)is one of the most prevalent cancers worldwide,accounting for approximately 75%-85%of primary liver cancers.Metabolic alterations have been labeled as an emerging hallmark of tumors.Specially,the last decades have registered a significant improvement in our understanding of the role of metabolism in driving the carcinogenesis and progression of HCC.In this paper,we provide a review of recent studies that investigated the metabolic traits of HCC with a specific focus on three common metabolic alterations involving glycolysis,lipid metabolism,and glutamine addiction which have been gaining much attention in the field of HCC.Next,we describe some representative diagnostic markers or tools,and promising treatment agents that are proposed on the basis of the aforementioned metabolic alterations for HCC.Finally,we present some challenges and directions that may promisingly speed up the process of developing objective diagnostic markers and therapeutic options underlying HCC.Specifically,we recommend future investigations to carefully take into account the influence of heterogeneity,control for study-specific confounds,and invite the validation of existing biomarkers.

Detection of Antibody to Hepatitis Delta Virus in Human Serum by Double Antigen Sandwich ELISA

A simple rapid detection of antibody to hepatitis delta virus (anti-HDV) in human serum was developed by using double antigen sandwich ELISA. HDV gene fragment encoding HDAg was isolated from a Chinese patient infected with HDV by RT-PCR, and a high-efficient expression HD-PQE31 strain was constructed with the fragment. We obtained high titer and good quality hepatitis delta virus protein purified by Ni-NTA metal-affinity chromatography, which was identified by Western blot and ELISA, then we set up the double antigen sandwich ELISA for detection of anti-HDV in human serum, and the performance of the sandwich ELISA was evaluated in terms of specificity and sensitivity. Results were: 1) The purified HDAg protein’s purity was 90%, and its ELISA titer was 1/100 000. 2) 42 anti-HDV positive sera were detected and showed that the sensitivity of sandwich ELISA was higher than that of competitive ELISA (t=2.44, p<0.01). 3) The inhibitory rates for 2 anti-HDV positive sera by the specific HDAg were 74% and 93% respectively. 4) For the assay of specificity, all 60 samples infected by other hepatitis viruses and 30 normal samples were negative for anti-HDV. These results suggested that the double antigen sandwich ELISA with purified recombinant HDAg showed higher specificity and sensitivity, It can be used in routine laboratories to diagnose the HDV infection.

Frequency and Absolute Number of FoxP3^+ Regulatory T Cells Correlate with Disease Progression of Chronic HIV-1 Infection

CD4+CD25+ Regulatory T cells (Treg) have been found to down-regulate immune activation in HIV-1 infection. However,whether the depletion of Treg benefits to the disease status of HIV infection remains undefined. To address this issue,we enumerated the Treg absolute counts and frequency in 75 antiviral-nave HIV-1-infected individuals in this study. It was found that HIV-infected patients displayed a significant decline in Treg absolute counts but a significant increase in Treg frequency. In addition,with disease progression indicated by CD4 T-cell absolute counts,circulating Treg frequency gradually increased; while Treg absolute counts were gradually decreased,suggesting that the alteration of Treg number closely correlated with disease progression in HIV infection. Functional analysis further showed that Treg efficiently inhibit both CD4 and CD8 T cell proliferation in vitro. Thus,our findings indicates that Treg actively participate in pathogenesis of chronic HIV infection,influencing the disease progression.

The Comparative Study on Two Models of Syndrome Differentiation of the Hand, Foot and Mouth Disease: An Investigation Analysis of the Signs and Symptoms on 2325 Cases

Objective To realize the characteristics of "zheng" differentiation-treatment for hand, foot and mouth disease(HFMD), a new methodology of syndrome differentiation for different stages of HFMD has been explored. Methods Total of 2 325 cases with HFMD were recorded by distributing them into exterior syndrome stage, interior syndrome stage, severe syndrome stage and recovered syndrome stage, respectively, and the main symptoms and subsidiary symptoms of different stages of HFMD have been observed. The major and minor pathogenesis of HFMD in different stages were obtained, and compared with the "2010 Guideline for the Diagnosis and Treatment of HFMD". Results It was found that the major pathogenesis of exterior stage was defined as "the invation of the wenevil to the defender of the body with the collaterals got involved ", and the minor as "qi deficiency"; in interior stage, "the fury of Gan-Yang" was the main pathogenesis, and "qi in chaos and qi deficiency" was the minor; in severe syndrome stage, "the damage of heart, liver and lung" was the main pathogenesis, and "qi in chaos" was the minor; and the pathogenesis of recovered stage was "qi-yin deficiency". Compared with the "2010 Guideline for the Diagnosis and Treatment of HFMD", it showed that "the obstruction of the fei-pi qi by the mixture of shi-re evil" and "the mixture of shi-re" in vivo was quite difficult to be explained in completely different context in the general situation; in the severe stage, the TCM clinical characteristics of syndrome differentiation might lose; in the early acute severe cases, the phenomenon that xin-yang and fei-qi almost ran out was difficult to be observed, then, the line between the severe and the acute severe became vague.Conclusions The theory of syndrome differentiation by stages of HFMD was reasonable in the actual situation of clinical description on HFMD which was expected to be further tested and widely applied in the "zheng" differentiation-treatment of HFMD in the future.

Characterization of Side Cell Populations Obtained from Human Amnion Mesenchymal Cells

Human amnion mesenchymal cells (AMCs) contain multipotent cells. To enrich such multipotent stem cells, we applied to AMCs the new method for the isolation of side population (SP) cells used for the enrichment of multipotent stem cells from many tissues. We succeeded in obtaining SP cells from AMCs (AMC-SP cells). AMC-SP cells were found in 0.2% of AMCs, irrespective of the length of pregnant period, ranging from 37 to 40 weeks. Cell cycle analyses suggested that AMC-SP cells belonged to a cell population that proliferated very slowly and/or was in a quiescent state in the amniotic membrane. Upon culturing, they proliferated with 40 to 80 cell doublings. However, they did not form colonies in a soft agarose culture, whereas HepG2 cells, representative human hepatoma cells formed many large colonies. These results suggest that AMC-SP cells that have considerable value for the use of regenerative medicine can be managed safely in vitro.

LONP1 ameliorates liver injury and improves gluconeogenesis dysfunction in acute-on-chronic liver failure

Background:Acute-on-chronic liver failure(ACLF)is a severe liver disease with complex pathogenesis.Clinical hypoglycemia is common in patients with ACLF and often predicts a worse prognosis.Accumulating evidence suggests that glucose metabolic disturbance,especially gluconeogenesis dysfunction,plays a critical role in the disease progression of ACLF.Lon protease-1(LONP1)is a novel mediator of energy and glucose metabolism.However,whether gluconeogenesis is a potential mechanism through which LONP1 modulates ACLF remains unknown.Methods:In this study,we collected liver tissues from ACLF patients,established an ACLF mouse model with carbon tetrachloride(CCl 4),lipopolysaccharide(LPS),and D-galactose(D-gal),and constructed an in vitro hypoxia and hyperammonemia-triggered hepatocyte injury model.LONP1 overexpression and knockdown adenovirus were used to assess the protective effect of LONP1 on liver injury and gluconeogenesis regulation.Liver histopathology,biochemical index,mitochondrial morphology,cell viability and apoptosis,and the expression and activity of key gluconeogenic enzymes were detected to explore the underlying protective mechanisms of LONP1 in ACLF.Results:We found that LONP1 and the expressions of gluconeogenic enzymes were downregulated in clinical ACLF liver tissues.Furthermore,LONP1 overexpression remarkably attenuated liver injury,which was characterized by improved liver histopathological lesions and decreased serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in ACLF mice.Moreover,mitochondrial morphology was improved upon overexpression of LONP1.Meanwhile,the expression and activity of the key gluconeogenic enzymes were restored by LONP1 overexpression.Similarly,the hepatoprotective effect was also observed in the hepatocyte injury model,as evidenced by improved cell viability,reduced cell apoptosis,and improved gluconeogenesis level and activity,while LONP1 knockdown worsened liver injury and gluconeogenesis disorders.Conclusion:We demonstrated that gluconeogenesis dysfunction exists in ACLF,and LONP1 could ameliorate liver injury and improve gluconeogenic dysfunction,which would provide a promising therapeutic target for patients with ACLF.

Application of Global Leadership Initiative on Malnutrition criteria in patients with liver cirrhosis

Background:The Global Leadership Initiative on Malnutrition(GLIM)criteria were published to build a global consensus on nutritional diagnosis.Reduced muscle mass is a phenotypic criterion with strong evidence to support its inclusion in the GLIM consensus criteria.However,there is no consensus regarding how to accurately measure and define reduced muscle mass in clinical settings.This study aimed to investigate the optimal reference values of skeletal muscle mass index for diagnosing sarcopenia and GLIM-defined malnutrition,as well as the prevalence of GLIM-defined malnutrition in hospitalized cirrhotic patients.Methods:This retrospective study was conducted on 1002 adult patients with liver cirrhosis between January 1,2018,and February 28,2022,at Beijing You-An Hospital,Capital Medical University.Adult patients with a clinical diagnosis of liver cirrhosis and who underwent an abdominal computed tomography(CT)examination during hospitalization were included in the study.These patients were randomly divided into a modeling group(cohort 1,667 patients)and a validation group(cohort 2,335 patients).In cohort 1,optimal cut-off values of skeletal muscle index at the third lumbar skeletal muscle index(L3-SMI)were determined using receiver operating characteristic analyses against in-hospital mortality in different gender groups.Next,patients in cohort 2 were screened for nutritional risk using the Nutritional Risk Screening 2002(NRS-2002),and malnutrition was diagnosed by GLIM criteria.Additionally,the reference values of reduced muscle mass in GLIM criteria were derived from the L3-SMI values from cohort 1.Multivariate logistic regression analysis was used to analyze the association between GLIM-defined malnutrition and clinical outcomes.Results:The optimal cut-off values of L3-SMI were 39.50 cm 2/m 2 for male patients and 33.06 cm 2/m 2 for female patients.Based on the cut-off values,31.63%(68/215)of the male patients and 23.3%(28/120)of the female patients had CT-determined sarcopenia in cohort 2.The prevalence of GLIM-defined malnutrition in cirrhotic patients was 34.3%(115/335)and GLIM-defined malnutrition was an independent risk factor for in-hospital mortality in patients with liver cirrhosis(Wald=6.347,P=0.012).Conclusions:This study provided reference values for skeletal muscle mass index and the prevalence of GLIM-defined malnutrition in hospitalized patients with liver cirrhosis.These reference values will contribute to applying the GLIM criteria in cirrhotic patients.

Guidelines for the Prevention and Treatment of Chronic Hepatitis B(version 2022)

To facilitate the achieving of the goal of“eliminating viral hepatitis as a major public health threat by 2030”set by the World Health Organization,the Chinese Society of Hepatology together with the Chinese Society of Infectious Diseases(both are branches of the Chinese Medical Association)organized a panel of experts and updated the guidelines for prevention and treatment of chronic hepatitis B in China(version 2022).With the support of available evidence,this revision of the guidelines focuses on active prevention,large scale testing,and expansion of therapeutic indication of chronic hepatitis B with the aim of reducing the hepatitis B related disease burden.

Guidelines on the Diagnosis and Management of Primary Biliary Cholangitis (2021)

In 2015, the Chinese Society of Hepatology and the Chinese Society of Gastroenterology published a consensus on pri-mary biliary cholangitis (PBC). In the past years, numerous clinical studies have been published in the field of PBC. To guide the clinical diagnosis and management of PBC patients, the Chinese Society of Hepatology invited a panel of experts to assess the new clinical evidence and formulate the current guidelines.

Guidelines for the Prevention and Treatment of Chronic Hepatitis B(Version 2022)

To facilitate the achieving of the goal of“eliminating viral hepatitis as a major public health threat by 2030”set by theWorld Health Organization,the Chinese Society of Hepatology together with the Chinese Society of Infectious Diseases(both are branches of the ChineseMedical Association)organized a panel of experts and updated the guidelines for prevention and treatment of chronic hepatitis B in China(version 2022).With the support of available evidence,this revision of the guidelines focuses on active prevention,large-scale testing,and expansion of therapeutic indication of chronic hepatitis B with the aim of reducing the hepatitis B–related disease burden.

Natural herbal medicine Lianhuaqingwen capsule anti-influenza A （H1N1） trial： a randomized, double blind, positive controlled clinical trial

Background The 2009 influenza A （H1N1） virus infection is associated with the high risk of severe complications and is spreading more rapidly throughout the world than other reported seasonal influenzas. This study aimed to evaluate the efficacy and safety of the nature herbal medicine Lianhuaqingwen capsule （LHC） in patients infected with influenza A （H1N1） virus. Methods A total of 244 patients aged 16-65 years confirmed with influenza A （H1N1） virus infection by the real time RT-PCR were randomized to one of two treatment groups of 122 patients each. Each group assigned to receive either LHC or Oseltamivir for five days and observation for seven days. The patients were enrolled within 36 hours of illness onset if they had an axillary temperature of ≥37.4℃ and with at least one of the following symptoms： nasal obstruction, runny nose, cough, sore throat, fatigue, headache, myalgia, chills and sweating. The primary end point was the duration of illness. Results Of 244 patients, 240 （98.36%） patients with a median age 21 years completed the study between October 24, 2009 and November 23, 2009. There were no significant overall differences between LHC treated and Oseltamivir treated patients in the median duration of illness （LHC 69 hours vs. Oseltamivir 85 hours P 〉0.05） or the median duration of viral shedding （LHC 103 hours vs. Oseltamivir 96 hours, P 〉0.05）. However, it was worthwhile to note that LHC significantly reduced the severity of illness and the duration of symptoms including fever, cough, sore throat, and fatigue （P〈0.05）. Both study medications were well tolerated. No drug related serious adverse events occurred during the study. Conclusions Compared with Oseltamivir, LHC achieved a similar therapeutic effectiveness reduction of the duration of illness and duration of viral shedding. Therefore, LHC might be an alternative therapeutic measure for influenza A （H1N1） virus infections.

Establishment of hepatocellular carcinoma multidrug resistant monoclone cell line HepG2/mdr1

Background 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. Methods The 4.5-kb mdrl cDNA obtained from the plasmid pHaMDR1-1 was cloned into the PCl-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. Results The mdr1 expression vector was constructed successfully and the MDR HCC line HepG2/mdr1 developed. The PCR analysis showed that the specific fragment of mdrl 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. Conclusion 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.