Protective Mechanism of Paeoniflorin on Mice with Ulcerative Colitis Based on AMPK/mTOR Autophagy Pathway / 中国实验方剂学杂志

ObjectiveTo explore the protective mechanism of paeoniflorin on mice with ulcerative colitis （UC） through the adenosine monophosphate-activated protein kinase （AMPK）/mammalian target of rapamycin （mTOR） autophagy pathway. MethodUC mouse model was established by allowing mice freely drink 4% DSS， and 56 BALB/c male mice were randomly divided into model group， AMPK inhibitor group （20 mg·kg-1）， paeoniflorin （50 mg·kg-1） + inhibitor （20 mg·kg-1） group， and high dose （50 mg·kg-1）， medium dose （25 mg·kg-1）， and low dose （12.5 mg·kg-1） paeoniflorin groups. After seven days of drug intervention， the protective effect of paeoniflorin on mice with UC was determined by comparing the body weight， disease activity index （DAI） changes， and Hematoxylin-eosin （HE） staining results. Enzyme linked immunosorbent assay （ELISA） was used to detect the levels of tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6） in the serum of mice in each group， and immunofluorescence was utilized to detect microtubule-associated protein 1 light chain 3 （LC3） content in the colon， AMPK， mTOR proteins， and their phosphorylated proteins including p-AMPK and p-mTOR in the colon tissue were detected by Western blot， and the mRNA expression levels of AMPK， mTOR， Beclin1， LC3， and p62 were detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. ResultCompared with the blank group， the model group showed a decrease in body mass， an increase in DAI score， and severe pathological damage to the colon. The levels of inflammatory factors including TNF-α and IL-6 increased in serum （P<0.01）， while the protein levels of LC3 and p-AMPK/AMPK were down-regulated in colon tissue， and those of p-mTOR/mTOR were up-regulated （P<0.01）. The mRNA expression levels of AMPK and LC3 were down-regulated， while the mRNA expression levels of mTOR and p62 were up-regulated （P<0.01）. Compared with the model group and the paeoniflorin + inhibitor group， the mice treated with paeoniflorin showed an increase in body mass， a decrease in DAI score， a reduction in pathological damage to colon tissue， and a reduction in the levels of inflammatory factors of TNF-α and IL-6 in serum （P<0.05）. The protein levels of LC3 and p-AMPK/AMPK in colon tissue were up-regulated， while the protein levels of p-mTOR/mTOR were down-regulated （P<0.01）. The mRNA expression levels of AMPK， Beclin1， and LC3 were up-regulated， while the mRNA expression of mTOR and p62 were down-regulated （P<0.01）. The colon tissue of the inhibitor group was severely damaged， and the trend of various indicators was completely opposite to that of the high dose paeoniflorin group. ConclusionPaeoniflorin can enhance autophagy and reduce inflammatory damage in mice with UC by activating the AMPK/mTOR signaling pathway and thus play a protective role.

Application of 18F-FDG PET in the diagnosis and evaluation of SARS-CoV-2 infection and related events / 中华核医学与分子影像杂志

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly ravaged the world and infected hundreds of millions of people since its outbreak. Early diagnosis of COVID-19 is conducive to the control of virus transmission and the timely treatment of patients. Among the imaging techniques, chest CT is an important basis for the diagnosis and evaluation of COVID-19. 18F-FDG PET is not generally recommended as a routine diagnostic tool for COVID-19, but it plays an important role in the assessment of SARS-CoV-2-related events based on the characteristic of whole-body multi-systemic scan and functional imaging diagnosis. In this paper, the application of 18F-FDG PET in COVID-19 diagnosis, prognostic evaluation and long-term sequelae evaluation, and clinical performance of 18F-FDG PET after COVID-19 vaccine are summarized on the basis of literature research and clinical reports analysis. Furthermore, the application and development direction of other new molecular probes for nuclear medicine in COVID-19 are prospected.

Preparation of Al 18F-PSMA-11 and its preliminary PET/CT imaging / 中华核医学与分子影像杂志

Objective:To optimize the preparation conditions and methods of Al 18F-prostate specific membrane antigen (PSMA)-11 and evaluate the feasibility of clinical transformation. Methods:PSMA- N, N′-bis(2-hydroxy-5-(carboxyethy)benzyl)ethylenediamine- N, N′-diacetic acid (HBED-CC) dissolved in CH 3COONH 4 buffer (pH=4.8) was reacted with AlCl 3·3H 2O dissolved in pure water at a molar ratio of 1∶1 (60 ℃, 10 min), and then purified by tC18 column and freeze-dried to obtain [Al]-PSMA-11. [Al]-PSMA-11 was labeled by 18F - and the effects of reaction temperature and pH value on the labeling rate were investigated. The labeled products were purified by tC18 column and filtered through sterile filter to obtain Al 18F-PSMA-11. The comparison of biodistribution between Al 18F-PSMA-11 and 68Ga-PSMA-11 was analyzed on 5 healthy volunteers (age (56±8) years). The differences of SUV max between two groups were analyzed by independent-sample t test. Besides, the early and delayed imaging of Al 18F-PSMA-11 PET/CT were performed on a patient (70 years old) with recurrent prostate cancer for assessment of its potential for prostate cancer recurrence monitoring. Results:The labeling rate was (42.3±3.2)% reacting in aqueous phase (60 ℃, pH=4.8) for 15 min. After being purified with tC18 cartridge, the radiochemical purity of the product was still more than 95% after placement at room temperature for 3 h. Preliminary application demonstrated that there was no significant difference in the biodistribution of Al 18F-PSMA-11 and 68Ga-PSMA-11 among lacrimal gland, parotid gland, submandibular gland, liver, spleen, kidney, bladder and part of intestine and SUV max of targeted organs were also not different ( t values: 0.19-1.95, all P>0.05) between two groups. Multiple bone metastases were observed by Al 18F-PSMA-11 PET/CT delayed imaging (3 h) in a patient with recurrent prostate cancer. Conclusion:Al 18F-PSMA-11 produced with pre-conjugated [Al]-PSMA-11 meets the requirement of the PET imaging application, and it has good potential of localization and imaging for prostate cancer metastatic lesions.

Serum LAPTM4B-35 protein as a novel diagnostic marker for hepatocellular carcinoma / 北京大学学报(医学版)

OBJECTIVE@#LAPTM4B-35 protein is one of the isoforms that are encoded by a cancer driver gene, LAPTM4B. This gene was primarily found and identified in our lab of Peking University School of Basic Medical Sciences. The LAPTM4B-35 protein and its encoded mRNA are significantly over-expressed in a variety of cancers, such as hepatocellular carcinoma (HCC), lung cancers (including non small-cell lung cancer and small-cell lung cancer), stomach cancer, colorectal carcinoma, pancreatic cancer, gallbladder cancer, cholangiocarcinoma, breast cancer, prostate cancer, ovarian cancer, cervical cancer, endometrial cancer, and so on. It has firmly demonstrated through lab experiments either in vivo or in vitro, as well as clinical studies that the over-expression of LAPTM4B-35 can promote cancer growth, metastasis, and multidrug resistance. Specially, the expressive level of LAPTM4B-35 is associa-ted with recurrence of HCC. The aim of this study is to identify the release of LAPTM4B-35 protein from hepatocellular carcinoma into blood of HCC patients and into the medium of cultured HCC cells, and to identify its possible form of LAPTM4B-35 protein existed in blood and cell culture medium, as well as to explore the possibility of LAPTM4B-35 protein as a novel HCC biomarker for diagnosis of HCC and prognosis of HCC patients.@*METHODS@#Immunobloting (Western blot) and enzyme-linked immunosorbent assay (ELISA) were used for identification of LAPTM4B-35 protein in the blood of HCC patients and normal individuals. Ultrafiltration and ultracentrifugation were used to isolate and purify exosomes from the culture medium of HCC cells.@*RESULTS@#LAPTM4B-35 protein existed in the blood from HCC patients and normal donors that were demonstrated through Western blot and ELISA. LAPTM4B-35 was also released into the culture medium of HCC cells in the form of exosomes. Preliminary experiments showed that the average and the median of LAPTM4B-35 protein level in the blood of HCC patients (n=43) were both significantly higher than that in the blood of normal donors (n=33) through sandwich ELISA.@*CONCLUSION@#It is promising that the LAPTM4B-35 protein which is released from HCC cells in the form of exosomes into their extraenvironment may be exploited as a novel cancer biomarker for HCC serological diagnosis.

FGF-2 combined with TGF-β1 induces bone marrow mesenchyma stem cells differentiating into cardio-myocyte-like cells in vitro / 实用医学杂志

Objective To expore the effects of FGF-2 and TGF-β1 on the differentiation of rat bone mar-row mesenchymal stem cells(BMSCs)towards cardiocytes. Methods BMSCs were isolated from bones of SD rats and cultured and identified by flow cytometry.The cells were divided into four groups:group A(FGF-2),group B (TGF-β1),group C(FGF-2+TGF-β1)and group D(blank control).The morphological changes were observed by the microscope. The expressions of Desmin,α-sarcomeric actin and cTnI were detected by immunocytochemical stainning.The expressions of GATA-4 and Nkx2.5was detected by RT-qPCR.Result The positive expression rate of CD90,CD29 and CD45 was 84.7%,86.5%,0.3% respectively. After induction,group C presence of myotube structures and the shape is fusiform.The positive rate of combined induction group was higher than the others (P < 0.05). The expression of GATA-4 and Nkx2.5 genes was higher than that in the other groups(P < 0.05). Conclution FGF-2 and TGF-β1 can be used to induce BMSCs into cardiocytes,and the combined group has the better effect.

Expression of ASPP2 and P16INK4a and its relationship with apoptosis in esophageal carcinoma / 实用医学杂志

Objective To study the expression of ASPP2 and P16INK4a in esophageal carcinoma and their relationship to the apoptosis and related clinicopathological characteristics. Methods Immunohistochemistry S-P method was used to examine the expression of ASPP2 and P16INK4a in the pathological specimens of 112 esophageal carcinoma cases and 31 cases of normal esophageal mucosa. TUNEL was also employed to detect the rate of apopto-sis in 37 esophageal carcinoma cases and 12 cases of normal esophageal mucosa. Results The difference between the expression of ASPP2 and P16INK4a in esophageal carcinoma and normal esophageal mucosa was statistically signif-icant(P < 0.05). Their abnormal expressions were all related to lymph node metastasis and differentiation degree (P < 0.05). The difference between the positive rate of apoptosis in esophageal carcinoma and normal esophageal mucosa was statistically significant(P < 0.05). The abnormal expression of ASPP2 and P16INK4a was all related to apoptosis in esophageal carcinoma. Conclusions The different expression of ASPP2 and P16INK4a may cooperatively play a role in differentiation degree ,lymph nodes metastasis and apoptosis in esophageal carcinoma. Co-examina-tion of them may be useful for the diagnosis and guiding the clinical treatment in esophageal carcinoma.

Expression of ASPP2 and P16INK4a and its relationship with apoptosis in esophageal carcinoma / 实用医学杂志

Objective To study the expression of ASPP2 and P16INK4a in esophageal carcinoma and their relationship to the apoptosis and related clinicopathological characteristics. Methods Immunohistochemistry S-P method was used to examine the expression of ASPP2 and P16INK4a in the pathological specimens of 112 esophageal carcinoma cases and 31 cases of normal esophageal mucosa. TUNEL was also employed to detect the rate of apopto-sis in 37 esophageal carcinoma cases and 12 cases of normal esophageal mucosa. Results The difference between the expression of ASPP2 and P16INK4a in esophageal carcinoma and normal esophageal mucosa was statistically signif-icant(P < 0.05). Their abnormal expressions were all related to lymph node metastasis and differentiation degree (P < 0.05). The difference between the positive rate of apoptosis in esophageal carcinoma and normal esophageal mucosa was statistically significant(P < 0.05). The abnormal expression of ASPP2 and P16INK4a was all related to apoptosis in esophageal carcinoma. Conclusions The different expression of ASPP2 and P16INK4a may cooperatively play a role in differentiation degree ,lymph nodes metastasis and apoptosis in esophageal carcinoma. Co-examina-tion of them may be useful for the diagnosis and guiding the clinical treatment in esophageal carcinoma.

cAMP responsive element binding protein-1 is a transcription factor of lysosomal-associated protein transmembrane-4 Beta in human breast cancer cells.

Lysosomal-associated protein transmembrane-4 beta (LAPTM4B) is a potential proto-oncogene, whose overexpression is involved in cancer occurrence and progression. Its transcript is up-regulated in various types of solid tumors including breast cancer. However, its transcriptional regulation mechanism is still unclear. To investigate the mechanism of transcriptional regulation of LAPTM4B in human breast cancer cells, a series of luciferase reporter constructs and construct with mutated binding site for cAMP responsive element binding protein-1 (CREB1) were generated by PCR amplification and transiently transfected into breast cancer cells to determine the transcriptional activities of different promoter regions. The +10+292 promoter region was possessed the highest transcriptional activity. The ability of CREB1 to bind the LAPMT4B promoter was confirmed by electrophoretic mobility shift assay, super-shift and RNA interference experiments. Our study identified the core promoter region responsible for constitutive expression of LAPTM4B and clarified that CREB1 played an important role in LAPTM4B transcriptional regulation in human breast cancer cells.

Upregulation of LAPTM4B-35 promotes malignant transformation and tumorigenesis in L02 human liver cell line.

Hepatocellular carcinoma (HCC) is one of the most frequent malignant neoplasms worldwide and is the second leading cause of cancer death in China. We have previously demonstrated that LAPTM4B-35, encoded by lysosomal protein transmembrane 4 beta gene, is overexpressed in over 80% of HCCs and is a novel-independent prognostic factor for metastasis, recurrence, and postoperative survival in HCC. In this study, we investigated the role of LAPTM4B-35 in malignant transformation and tumorigenesis using L02 cells, a cell line originated from human normal liver cells. Our data show that replication-deficient adenovirus vector-mediated upregulation of LAPTM4B-35 promotes anchorage-independent proliferation and resistance to adriamycin-induced apoptosis. Study of the underlying mechanisms demonstrated alterations of molecular events involved in these processes, which included the activation of phosphoinositide 3-kinases (PI3K)/serine/threonine protein kinase B (PKB/AKT)/bcl-xL/bcl-2-associated death promoter homolog (Bad) signaling pathway, inhibition of caspase-3 activation, upregulation of Bcl-2, and downregulation of Bax. In addition, upregulation of LAPTM4B-35 in L02 cells resulted in tumorigenesis in 100% (6/6) of inoculated nude mice and accelerated the death of mice with xenografts in vivo. In conclusion, LAPTM4B-35 promotes malignant transformation and tumorigenesis in human liver L02 cell line through promotion of deregulated proliferation and inhibition of apoptosis. These findings suggest that overexpression of LAPTM4B-35 may play a critical role in hepatocarcinogenesis and therefore, may be a therapeutic target for HCC.

Overexpression of LAPTM4B-35 attenuates epirubucin-induced apoptosis of gallbladder carcinoma GBC-SD cells.

BACKGROUND: It was shown previously that LAPTM4B promoted growth of gallbladder carcinoma (GBC) cells and predicted poor prognosis in GBC; however, its roles and relative mechanisms in apoptosis of GBC cells remain unknown. METHODS: The plasmids, pcDNA3-AE, containing the complete open reading frame of LAPTM4B and Mock (pcDNA3), were transfected transiently into GBC-SD cells, followed by induction of apoptosis by epirubicin. Cell apoptosis was determined by Hoechst 33258 staining, propidium iodide (PI) staining, and Annexin V/PI double staining flow cytometry. Protein expression was detected by immunoblotting. RESULTS: Overexpression of LAPTM4B-35 was observed in cells transfected with pcDNA3-AE. These cells possessed significantly less apoptosis ratios compared with cells transfected with the Mock plasmid, although the values were still greater than those in parent cells. Of the apoptosis-related molecules, expression of Bcl-2 and Bcl-xL was up-regulated in cells transfected with pcDNA3-AE, whereas expressions of Bax, Bid, and cleaved caspase-9 and -3 were down-regulated compared with their expression in other kinds of cells. CONCLUSION: Our data show that LAPTM4B-35 attenuated epirubicin-induced apoptosis of GBC-SD cells in vitro through a mitochondria-dependent pathway. Therefore, the protein LAPTM4B-35 might be associated with the chemoresistance of GBC.

LAPTM4B-35 overexpression is an independent prognostic marker in endometrial carcinoma.

BACKGROUND: Lysosomal protein transmembrane 4 ß-35 (LAPTM4B-35), a novel oncoprotein that belongs to the mammalian 4-tetratransmembrane spanning protein superfamily, has been implicated in oncogenesis and cancer progression in several solid malignances. However, the expression of LAPTM4B-35 and its role in endometrial cancer progression remain unknown. MATERIALS AND METHODS: We investigated the expression of the LAPTM4B-35 protein by immunohistochemistry in 30 normal endometrium specimens and 165 endometrial carcinomas and analyzed its correlation with various clinicopathologic features, including patient outcome. RESULTS: LAPTM4B-35 immunoreactivity was overexpressed in endometrial carcinoma cases compared with normal endometrium (P < 0.001). High LAPTM4B-35 expression was found in 117 (70.91%) of these 165 carcinomas and was positively correlated with the International Federation of Gynecology and Obstetrics stage, histological grade, depth of myometrial invasion, lymph node metastasis, lymph vascular space involvement, and recurrence, but not with age and histological type. Patients with high LAPTM4B-35 expression had significantly poorer overall survival and disease-free survival compared with patients with low expression of LAPTM4B-35 (P = 0.001 and P = 0.002, respectively). Multivariate analysis showed that high LAPTM4B-35 expression was an independent prognostic factor for both overall survival and disease-free survival of patients with endometrial carcinoma (both P = 0.005). CONCLUSIONS: These results showed that high LAPTM4B-35 expression was associated with progression and prognosis of endometrial carcinoma.

Expression of LAPTM4B in gallbladder carcinoma cells: the role in invasive potential.

BACKGROUND/AIMS: It was previously established that LAPTM4B-35 highly expressed in gallbladder carcinoma and being of clinicopathological and prognostic significances. However, expression of LAPTM4B gene in gallbladder carcinoma (GBC-SD), a gallbladder carcinoma cell line, and its role in invasive potential remain unclear. METHODOLOGY: Expression of LAPTM4B in GBC-SD cells was first detected. Plasmids, pcDNA3-AE (containing complete open reading frame of LAPTM4B) and Mock (pcDNA3), were transiently transfected into GBC-SD cells. Invasive phenotypes (migration and invasion) and relative molecules were then shown by transwell assay, crossing river test and Western blot analysis. RESULTS: Immunocytochemical staining revealed that LAPTM4B-35 positively expressed in cytoplasm of GBC-SD cells. But LAPTM4B-35 expression was obviously weaker in GBC-SD cells than that in BEL-7402 cells (positive control). Besides, cells transfected with pcDNA3-AE presented shorter crossing river time, less migrated and invaded cell numbers, compared with cells transfected with the Mock plasmid and parent cells. Finally, increased expressions of active uPA, MMP-9, pro MMP-2 and active MMP-2 were also observed in cells transfected with pcDNA3-AE. CONCLUSIONS: Our data suggested that LAPTM4B expressed in GBC-SD cells at a relatively low level. Forced overexpression of LAPTM4B increased invasive potential of GBC-SD cells, through modulating molecules associated with degradation of extracellular matrix.

Overexpression of LAPTM4B promotes growth of gallbladder carcinoma cells in vitro.

BACKGROUND: The overexpression of LAPTM4B-35 in gallbladder carcinoma (GBC) and its clinicopathologic and prognostic significance have been previously shown. Thus, this gene may play a role in the growth of GBC cells. METHODS: The pcDNA3-AE containing the complete open reading frame of LAPTM4B (lysosome-associated protein transmembrane-4beta) and mock (pcDNA3) plasmids were transiently transfected into GBC-SD cells. Cell proliferation, cell cycle distribution, and protein expression were evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium assay, flow cytometry, and Western blot, respectively. RESULTS: Cells transfected with pcDNA3-AE revealed accelerated proliferation, less serum dependence, and significant cell cycle progression compared with cells transfected with mock plasmid and parent cells. These phenotypes were accompanied by upregulated expression of C-myc, c-Fos, c-Jun, cyclin D1, and cyclin E and downregulated expression of P16 and P-27. CONCLUSIONS: LAPTM4B overexpression promotes the growth of GBC cells in vitro by regulating the expression levels of some proliferation-associated proteins. Therefore, the LAPTM4B gene might be used as a novel therapeutic target of GBC.

LAPTM4B-35 overexpression is a risk factor for tumor recurrence and poor prognosis in hepatocellular carcinoma.

PURPOSE: Lysosomal protein transmembrane 4 beta-35 (LAPTM4B-35) is a tetra-transmembrane glycoprotein that is abundantly localized on membrane-bound organelles including endosomes and lysosomes, and promotes cell proliferation and tumorigenesis through regulation of cell cycle and signaling pathways. The aim of the present study is to determine the potential clinical implications of LAPTM4B-35 expression in hepatocellular carcinoma (HCC). METHODS: Immunohistochemistry assay was used to determine the expression of LAPTM4B-35 protein in normal and HCC tissues from 71 patients. The correlations of LAPTM4B-35 expression with clinicopathological parameters, including gender, age, background liver, viral status, tumor size, portal vein invasion, histopathological differentiation, serum AFP level, TNM staging and recurrence of HCC were assessed by Chi-squared test. Patient survival and their differences were determined by Kaplan-Meier method and log-rank test. Cox regression (Proportional hazard model) was adopted for multivariate analysis of prognostic factors. RESULTS: LAPTM4B-35 immunoreactivity was negative or low in normal liver tissues, but high in HCC tissues (51/71, 71.8%). The overexpression of LAPTM4B-35 was significantly associated with recurrence, TNM staging and portal vein invasion of HCC. Patients with high LAPTM4B-35 expression had significantly poorer overall survival (OS) and disease-free survival (DFS) (both P < 0.001) when compared with patients with the low expression of LAPTM4B-35. On multivariate analysis, LAPTM4B-35 expression was found to be an independent prognostic factor for OS and DFS (P = 0.018 and P = 0.001, respectively). CONCLUSION: LAPTM4B-35 expression showed a strong association with the potencies of recurrence and metastasis and progression of HCC, and that may be applied as a novel marker for the prediction of recurrence and metastasis potency of HCC, and helpful for improving the diagnosis, prognosis and treatment of HCC.

Reduced expression of ASS is closely related to clinicopathological features and post-resectional survival of hepatocellular carcinoma.

Argininosuccinate synthetase (ASS) has previously been proven to be reductively expressed in hepatocellular carcinoma (HCC) and various types of HCC cell lines. Arginine, the product of ASS, has been used as a target in HCC by recombinant human arginase or arginine deiminase, which is now in the phase II clinical trial stage. This study aimed to present the levels of ASS expression in HCCs and its correlation with clinicopathological features and prognosis of HCC patients. Immunohistochemical detection of ASS was performed on samples from 71 patients with HCC. Positive staining was found in 21 HCCs, with a score of 2, as well as in normal liver tissues. Reduced ASS staining was found in 70.4% (50/71) of HCC tissues, including 21 with a score of 0 and 29 with a score of 1. The staining score in cancer tissues was significantly associated with gender, background liver, histopathological differentiation, recurrence, TNM staging and portal vein invasion (P<0.05), but not with age, viral status, tumor size and serum α-fetoprotein level. Patients with a high ASS expression had significantly poorer overall and disease-free survival (P<0.001 and P<0.001, respectively). These data showed that ASS was reductively or negatively expressed in a large portion of HCC, and that ASS levels in HCCs correlated inversely with prognosis. In conclusion, a high expression of ASS may be a novel marker of poor prognosis of patients presenting with HCC.

Expression of LAPTM4B-35: a novel marker of progression, invasiveness and poor prognosis of extrahepatic cholangiocarcinoma.

LAPTM4B was proven to overexpress in hepatocellular carcinoma and relate to differentiation. We immunohistochemically investigated the expression and potential clinicopathological and prognostic significance of LAPTM4B encoded protein, LAPTM4B-35, in extrahepatic cholangiocarcinoma (EHCC) for specimens from consecutive 81 patients. LAPTM4B-35 staining was positive in cancer tissues from 59 patients (72.8%), including 12 with score 1, 22 with score 2 and 25 with score 3. No positive staining was found in non-cancer epithelia. The staining score in cancer tissues was not only significantly associated with TNM staging, histological grade, perineural and lymph node invasion (P<0.05), but also of comprehensive prognostic implications, including integrated estimation with CA19-9. These data established that LAPTM4B-35 positively expressed in a great portion of EHCC and might be a novel molecular maker of progression, invasiveness and poor prognosis.

[Relationship between LAPTM4B gene polymorphism and susceptibility of lung cancer].

OBJECTIVE: To investigate the possible association between the allelic variation of LAPTM4B and the genetic susceptibility of lung cancer. METHODS: The genotype of LAPTM4B was analyzed in 134 unrelated healthy adult individuals and 166 patients with lung cancer by utilizing polymerase chain reaction based on special primers. The genotypical distribution of LAPTM4B was analyzed by chi2 test. RESULTS: The allelic frequencies of the *2 were 40.1% and 28.0% in the lung cancer group and the healthy control group respectively, which was significantly different between the two groups (P=0.002). There was a significant difference in the overall genotypical distribution between the patients and the controls (P=0.005). The risk of suffering from lung cancer was increased 1.91 times in the individuals of the *1/2 genotype (95%CI: 1.178-3.110) and 3.26 times in the individuals of the *2/2 genotype of LAPTM4B (95%CI: 1.338-7.929) compared with the *1/2 genotype. No association was observed between the genotypical distribution of LAPTM4B and the clinical information on patients of lung cancer such as gender, age, pathological type, differentiation classification of TNM and infection of HBV. CONCLUSION: This study suggests that the allele *2 of LAPTM4B might be the risk factor of lung cancer, which could be associated with genetic susceptibility of lung cancer.

Expression of lysosome-associated protein transmembrane 4B-35 in cancer and its correlation with the differentiation status of hepatocellular carcinoma.

AIM: To produce high-quality polyclonal antibody to lysosome-associated protein transmembrane 4B-35 and to identify LAPTM4B-35 expression in cancer tissues and its correlation with differentiation status of hepatocellular carcinoma (HCC). METHODS: The 297 bp 5' end of LAPTM4B cDNA was obtained by PCR and inserted into prokaryotic expression vector pGEX-KG. Then the recombinant pGEX-KG-N(1-99) was transformed into E.coli JM109 to express GST-fusion protein. The fusion protein was purified by glutathione sepharose(TM) 4B agarose. The purified GST-LAPTM4B-N(1-99) was characterized by SDS-PAGE, and used to immunize rabbits. The titer and specificity of antisera were detected by ELISA and Western blot, respectively. The correlation between the expression levels of LAPTM4B-35 and the differentiation status of HCC was analyzed via Western blot. The expression of LAPTM4B-35 in HCC and other six cancer tissues was investigated via tissue chip and immunohistochemical analysis. RESULTS: About 6.2 mg of pure GST-LAPTM4B-N(1-99) was isolated from 1 L of bacteria. The GST-LAPTM4B-N(1-99) produced high titer antisera in rabbits and showed good immunity. Western blot showed specific reactions for the antibody to the LAPTM4B-35 in the total proteins from HCC tissues and BEL-7402 cells, also to the fusion protein purified or in the transformed bacteria. LAPTM4B-35 was remarkably expressed in several cancers, such as HCC, breast cancer, gastric carcinoma, lung cancer, and colon carcinoma, but not commonly expressed in esophageal cancer and rectum carcinoma. Notably, the expression levels of LAPTM4B-35 were significantly and inversely correlated to the differentiation of HCCs in a 20 case analysis. CONCLUSION: Specific polyclonal antibody (LAPTM4B-N(1-99)-pAb) to LAPTM4B-35 was produced. It identified the expression of LAPTM4B-35 in some cancer tissues originated from single layer cuboidal and columnar epithelial cells and firmly demonstrated that the expression of LAPTM4B-35 in HCC was inversely correlated with the differentiation of HCC.

Liver injury after intermittent or continuous hepatic pedicle clamping and its protection by reduced glutathione.

BACKGROUND: The debate is still going on about selection of several clamping patterns during hepatectomy. The aim of this study was to assess the safety and preference of normothermic intermittent or continuous hepatic pedicle clamping and confirm the protective effect of reduced glutathione (GSH). METHODS: Thirty-two adult male healthy Sprague-Dawley (SD) rats were divided into groups of intermittent clamping and GSH absent (IA), continuous clamping and GSH absent (CA), intermittent clamping and GSH present (IP) and continuous clamping and GSH present (CP). The clamping manners were successively 40 minutes in continuous clamping groups and two cycles of 20 minutes with an interval of 5 minutes in intermittent clamping groups, and reperfusion periods were 60 minutes. Experimental parameters included levels of malonaldehyde (MDA) and Cu/Zn superoxide dismutase (SOD), pathological and ultrastructural changes in liver tissues, activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in sera. RESULTS: In the same group, the activities of ALT and AST were significantly higher in post-clamping rats than in pre-clamping rats (P<0.05), but no significant differences were noted in levels of MDA and Cu/Zn SOD (P>0.05). The differences of all values between post-reperfusion rats and pre-clamping rats were significant (P<0.05). Pathological and ultrastructural changes could be observed, but no irreversible injury was present. The comparison of the groups showed that the values at relevant time points between the intermittent and continuous groups were not significantly different (P>0.05). The values were significantly different between the GSH absent and present groups after reperfusion (P<0.05). The morphological damages were also obviously alleviated in the GSH present group. CONCLUSIONS: Normothermic intermittent or continuous hepatic pedicle clamping could cause reversible liver ischemia/reperfusion injury when the clamping time lasts 40 minutes. The injury extent seems to be similar. Continuous clamping should be regarded as a proper method in liver surgery. GSH has been confirmed as an effective agent in preventing post-clamping liver injury.

Structure analysis and expressions of a novel tetratransmembrane protein, lysosoma-associated protein transmembrane 4 beta associated with hepatocellular carcinoma.

AIM: To analyze the structure and expressions of the protein encoded by an HCC-associated novel gene, lysosome-associated protein transmembrane 4 beta (LAPTM4B). METHODS: Primary structure and fundamental characteristics of LAPTM4B protein were analysed with bioinformatics. Expressions of LAPTM4B in HCC tissues and various cell lines were detected using polyclonal antibodies and Western blot. RESULTS: LAPTM4B encoded two isoforms of proteins with molecular masses 35-ku and 24-ku, respectively. The expression level of LAPTM4B-35 protein in HCC tissues was dramatically upregulated and related to the differentiation status of HCC tissues, and it was also high in some cancer cell lines. Computer analysis showed LAPTM4B was an integral membrane protein with four transmembrane domains. LAPTM4B showed relatively high homology to LAPTM4A and LAPTM5 in various species. CONCLUSION: LAPTM4B gene encoded two isoforms of tetratransmembrane proteins, LAPTM4B-35 and LAPTM4B-24. The expression of LAPTM4B-35 protein is upregulated and associated with poor differentiation in human HCC tissues, and also at high levels in some cancer cell lines. LAPTM4B is an original and conserved protein.