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2018 Vol. 48, No. 7 Published: 2018-07-25   469 Expression of EBV LMP1 C-terminal protein and preparation of its polyclonal antibody MAO Shanshan, WANG Lude, ZHOU Meng, LYU Kaiji, ZHU Jinshun, Kamara Saidu, YE Xiaoxian, ZHANG Lifang. Objective: To prepare EBV LMP1 C-terminal (187-386 aa) protein by prokaryotic expression system and polyclonal antibody against it and verify its biological character. Methods: A prokaryotic vector pET21a(+)/EBV LMP1 C-terminal (187-386 aa) was constructed and transformed into E.coli BL21 (DE3), and then the recombinant protein was induced by IPTG and purified by Ni-NTA affinity chromatography. SDS-PAGE and Western blot analysis demonstrated the purified LMP1 C-terminal protein was expressed correctly. To obtain the polyclonal antibody, the rabbit was immunized with the purified LMP1 C-terminal recombinant protein. An analysis of polyclonal antibody was made by ELISA, Western blot and immunofluorescence technique. Results: The recombinant protein of LMP1 C-terminal was successfully expressed and purified  by the prokaryotic expression system. The polyclonal antibody was obtained by immunizing the rabbits with the recombinant protein. Conclusion: The recombinant protein of LMP1 C-terminal has strong immunogenicity and the polyclonal antibody of LMP1 C-terminal has high titer and specificity. 2018 Vol. 48 (7): 469-473 [Abstract] ( 768 ) HTML (1 KB)  PDF (1311 KB)  ( 807 ) 474 The effects of human cytomegalovirus cancer suppressor UL138 gene expression on the immune-associated functions of gastric cancer cells YE Lele, ZHANG Jiali, CHEN Wenjing, SHEN Xian, HU Changyuan, DING Ning, XUE Xiangyang Objective: To investigate the effects of HCMV tumor suppressor gene UL138 gene expression on immune-related function in gastric cancer cell. Methods: The recombinant eukaryotic expression plasmid of UL138 gene was constructed and transfected into BGC-823 gastric cancer cells. The expression of UL138 and immunogenic death related protein were verified by Western blot and immunofluorescence. The effect of UL138 expression on gastric cancer cells was analyzed by gene chip, combined with GO, Pathway and other bioinformatics techniques. Fluorescence quantitative PCR was used to further verify the expression of immune related molecules in gastric cancer cells. Results: After being transfected with recombinant plasmid, the expression of UL138 in gastric cancer cells was confirmed by Western blot and immunofluorescence; in addition, Western blot analysis showed that the expression of UL138 up-regulated the expression of ERp57, an immunogenic death associated protein, but did not affect the expression of HSP70. Microarray analysis showed that the expression of UL138 up-regulated 370 genes and down regulated 206 genes. Bioinformatics analysis suggested that these differentially expressed genes were involved in cytoskeletal remodeling, cell adhesion and other pathways involved in gastric cancer and digestive system diseases. Quantitative PCR analysis showed that the expression of UL138 up-regulated the 16 inflammatory related cytokines expression of IL1A, IL6, IL11, IL8, IL1RL1, IL7R, IL13RA2, CCL3L1, CCL5, TNFAIP3, TNFRSF21, VEGFA, CD44, CD55, CD59, CD274 gene and down-regulated CXCL10, IFNAR1 gene. Conclusion: The expression of UL138 was involved in the process of apoptosis of gastric cancer cells, up-regulated expression of IL1A, IL6, IL8 gene of pro-inflammatory cytokines and immune related immunogenicity of death associated protein ERp57. 2018 Vol. 48 (7): 474-478 [Abstract] ( 731 ) HTML (1 KB)  PDF (1452 KB)  ( 821 ) 479 The mitochondrial function of mitochondrial tRNAMet4435A>G mutation combined with YARS2 c.5750A>C mutation associated with essential hypertension REN Xiaoyan, CHEN Yaru, SHI Wenwen, ZHENG Binjiao, XUE Ling, GUAN MinXin Objective: To explore the combined effects of m.4435A>G mutation and YARS2 c.5750A>C mutation on the mitochondrial function in patients with essential hypertension. Methods: Based on clinical manifestations, complete mitochondrial DNA sequences and YARS2 exon sequencing results, we searched and identified the proband and screened the extended family members with m.4435A>G gene mutation. Peripheral blood samples of the confirmed pedigree were collected and blood lymphocytes were translated into immortalized lymphoblastoid cell lines. After a period of culture, a series of experiments reflecting cell function were performed, including mitochondrial membrane potential (MMP) detection, intracellular reactive oxygen test (ROS) and Northern blot test. Results: Mitochondrial DNA sequence amplification was used to identify the proband and family members with m.4435A>G gene mutation. Further phylogenetic analysis revealed that the conservatism index of the 4435 loci reached 100%. With the successful establishment of immortalized cell lines, tRNA results showed that the steady-state level of the samples carrying m.4435A>G mutation decreased significantly, and tRNATyr steady-state level of the samples carrying YARS2 c.5750A>C mutation also significantly reduced. The extent to which tRNAMet decreased in the double mutant samples was greater than single mutation samples. Compared with the control group, the ROS levels containing m.4435A>G and YARS2 c.5750A>C gene mutant cell lines increased significantly, and the increased level of double mutants was higher than single mutations, with statistical difference (P<0.05). The decreased level of the mitochondrial membrane potential in cell lines of m.4435A>G and YARS2 c.5750A>C gene mutation was significantly higher than in the control group. Conclusion: The mutation of m.4435A>G and YARS2 c.5750A>C caused abnormal metabolism of tRNAMet and tRNATyr respectively, which finally led to cell dysfunction resulting from mitochondrial translation defects, increased ROS level and decreased MMP plus the increased mitochondrial mutation due to nuclear gene mutation. The results suggest that m.4435A>G and YARS2 c.5750A>C mutation plays an important role in the occurrence of essential hypertension in the family with essential hypertension. 2018 Vol. 48 (7): 479-484 [Abstract] ( 690 ) HTML (1 KB)  PDF (1569 KB)  ( 749 ) 485 The different injection site determine the result of bone marrow mesenchymal stem cells transplantation for myocardial infarction JIN Peifeng, Wang Yehuan, JIANG Sheng, ZHANG Hao, SUN Chengchao. Objective: To determine whether the injection sites of myocardial infarcted area had a various effect on the bone marrow mesenchymal stem cells (BMSCs) survival and efficacy of cell transplantation. Methods: Myocardial infarction (MI) was induced by left anterior descending artery ligation in female rats. 3 weeks after MI, 3×106 bromodeoxyuridine (Brdu) labeled male BMSCs were directly intramyocardial injected into the borderline (BZC group) or central zone (CZC group) of the MI area. In the control groups, the same volume of phosphate buffered saline (PBS) was injected into the borderline (BZP) or the central zone of the myocardial infarction (CZP), respectively. Cell survival at the 24 hours and 4 weeks after transplantation was assayed by quantitative real-time PCR to identify Y chromosome gene. Heart function was evaluated by echocardiography before and after cell transplantation. Ventricular remodeling was analysis by using HE staining with measurement of the expansion index and scar thickness. Cell transplantation induced angiogenesis was tested by VIII factor staining. Results: The detectable transplanted cell survival rate in the BZC group were significantly higher than that of CZC group both at the time of 24 h (15.74%±4.13% vs. 8.2%±2.63%, P<0.01) and 4 weeks (5.57%±1.13% vs. 1.72%±0.41%, P<0.01) after cell transplantation. Compared with the CZC group, improvement of left ventricular ejection fraction (LVEF) and fractional shortening (P<0.05, respectively) were observed in BZC group. Additionally, BMSCs transplanted in the borderline zone also induced augmentation of capillary density than that in the CZC group (P<0.01). Left ventricular remodeling (scar thickness expansion index) in BZC group was significantly improved (P<0.01), and no improved expansion index was identified in CZC group. Conclusion: Borderline area was more suitable than central zone of MI for the survival of the transplanted cells and thereby promoted the angiogenesis and restored the damaged heart function. 2018 Vol. 48 (7): 485-489 [Abstract] ( 781 ) HTML (1 KB)  PDF (1397 KB)  ( 771 ) 490 The correlation between abdominal fat distribution and colorectal polyps YE Huajun, XU Hanyan, JIANG Xuepei, HE Xixi, HUANG Zhiming. Objective: To investigate the correlation between distribution of adipose tissue and the risk of colorectal polyps and to provide clinical evidence for the etiology of colorectal polyps and building efficient prophylactic measures. Methods: 330 patients with colorectal polyps (Polyps group) and 546 controls without colorectal polyps (non-polyp group) were retrospectively analyzed. All subjects underwent computerized tomography (CT). With the MATLAB2013 analysis software and obesity was evaluated by visceral adipose tissue (VAT), subcutaneous adipose tissue (SAT), total adipose tissue (TAT), VAT/TAT, body mass index (BMI). The obesity between different gender, different pathological type, different number of polyp, different size of polyp was compared. Results: There were significant differences in the VAT, VAT/TAT and BMI between polyps group and non-polyp group. There were no significant differences in the levels of VAT, SAT, VAT/TAT between colorectal polyps with different pathological type, size and number (P>0.05). Multivariate analysis showed a higher VAT (OR=2.544, 95%CI: 1.551-4.173) and VAT/TAT (OR=2.103, 95%CI: 1.262-3.516) were independently associated with the risk of colorectal polyps in male (P<0.05), while no significant difference in female (P>0.05). Conclusion: VAT and SAT are not correlated with the pathological type, size and number of colorectal polyps. VAT and SAT are not correlated with colorectal polyps in female. Visceral adiposity measured by VAT, VAT/TAT is associated with an increased risk of colorectal polyps in male, while the SAT is not associated with the risk of colorectal polyps in male. 2018 Vol. 48 (7): 490-494 [Abstract] ( 709 ) HTML (1 KB)  PDF (1196 KB)  ( 741 ) 495 The regulatory effect of survivin in amyloid precursor protein metabolism JIANG Weiqing, YUAN Xin, LIU Chen, REN Xiao, WANG Yonggang Objective: To explore the regulatory effect of survivin in amyloid precursor protein (APP) metabolism. Methods: Immunofluorescence technique was conducted to observe whether there was colocalization of survivin and APP through confocal imaging. Cell transfection with APP or APP-survivin plasmids were conducted to detect: whehter survivin affected the expression of APP through western blot analysis, and whether survivin inhibited the cell apoptosis induced by overexpression of APP though flow cytometry. Results: Survivin colocalized with APP in cultured rat primary hippocampal neurons. Survivin inhibited the expression of APP in PC12 cells. Moreover, survivin inhibited the cell apoptosis induced by overexpression of APP. Conclusion: Survivin could inhibit the cellular protein expression of APP and could inhibit the cell apoptosis induced by overexpression of APP. 2018 Vol. 48 (7): 495-498 [Abstract] ( 778 ) HTML (1 KB)  PDF (1435 KB)  ( 692 ) 499 Diagnostic role of Wnt pathway gene promoter methylation in non-small-cell lung cancer CHENG Guangjia, LIU Shunlin, ZHU Guoliang, WANG Jinzhi. Objective: The gene in the Wnt signaling pathway was analyzed to improve its role as a biomarker for the diagnosis of non-small-cell lung cancer (NSCLC). Methods: The methylation levels of SFRP1, SFRP2, WIF1 and PRKCB in 111 NSCLC patients were evaluated by quantitative methylation-specific PCR (qMSP), and combined with clinical data and TCGA database analysis. Results: Promoter methylation levels of four candidate genes were significantly higher in tumor tissues compared with the adjacent tissues. SFRP1, SFRP2 expression level in male patients was higher than female; the sensitivity and specificity were 83.3% and 96.0% combined with the methylation level of four in the diagnosis of NSCLC. Conclusion: A panel of Wnt signal pathway genes (SFRP1, SFRP2, WIF1 and PRKCB) had the potential as methylation biomarkers in the diagnosis of NSCLC. 2018 Vol. 48 (7): 499-503 [Abstract] ( 671 ) HTML (1 KB)  PDF (1372 KB)  ( 721 ) 504 Diagnositic value of KDIGO acute kidney disease definition on hospitalized patients with acute kidney failure ZONG Xue, LIN Fujun, JIANG Gengru Objective: To investigate the incidence and clinical characteristics of acute renal impairment in hospitalized patients using the 2012 Kidney Disease Improving Global Outcomes (KDIGO) definition-acute kidney disease (AKD) and assessed the risk factors associated with the adverse outcome of AKD. Methods: A retrospective analysis of 1984 patients receiving treatment in the ward of Renal Division of Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine from January 1st 2016 to December 31st 2016, using the 2012 KDIGO AKD/AKI definitions to identify patients with AKD (AKI and non-AKI AKD). The incidence, clinical characteristics and prognosis of AKI and AKD non-AKI patients were compared. Logistic regression was used to identify risk factors associated with adverse outcomes of AKD. Results: A total of 105 patients (5.29%) were diagnosed with AKD, of which 70 met the AKI criteria and 35 were classified as non-AKI AKD. Pre-renal, intrinsic and post-renal AKD accounted for 43.8%, 40.0% and 3.8% respectively. Compared with AKI patients, patients diagnosed with non-AKI AKD had higher proportion of co-morbidity and higher 24-hour proteinuria, lower serum albumin and slower serum creatinine (Scr) rising velocity. Logistic regression revealed that systolic blood pressure (OR=1.03), CKD history (OR=4.95), Scr rising velocity (OR=0.98) and hemoglobin (OR=0.96) were independent risk factors for adverse renal outcomes of AKD. Conclusion: The 2012 KDIGO AKD criteria identified more patients with acute renal impairment. Higher systolic blood pressure, CKD history, lower Scr rising velocity and lower Hemoglobin were independent risk factors for adverse outcome of AKD. AKD patients diagnosed as non-AKI had the same renal prognosis compared with the patients who met the AKI criteria. The results of this study underline the value of AKD diagnostic criteria and support its application in the clinical practice. 2018 Vol. 48 (7): 504-507,516 [Abstract] ( 761 ) HTML (1 KB)  PDF (1225 KB)  ( 648 ) 508 Study on isolation and culture of rat bone marrow-derived dendritic cells: regulatory effect of HMGB1/TLR4 signaling pathway WANG Hanlei, XUE Jiyang, GE Hanwei, XIA Jie, LIN Wei, ZHAO Qifeng. Objective: To investigate the regulatory effect of HMGB1/TLR4 signaling pathway on the expression of MyD88, NF-κB P65, cytokine and costimulatory molecules in rat bone marrow-derived dendritic cells (mDCs). Methods: Dendritic cells were isolated, cultured, purified and identified in vitro from bone marrow stem cells of rats, then randomly divided into 5 groups: mDCs cultured with HMGB1 only (H group), HMGB1 specific neutralizing antibody (H-Ab group), TLR4 antagonist (TLR4-A group) and control IgG (IgG group) 30 min after HMGB1, separately, or with RPMI l640 serum-containing medium as control group (C gruop). Each group was observed at different time points of 24 h, 48 h and 72 h by detecting the expression of MyD88, NF-κB P65 protein and mRNA, costimulatory molecules (CD80, CD86) and the levels of cytokine (IL-6, IL-8, IL-12, TNF-α) in the supernatant. Results: MyD88 and NF-κB P65 protein and mRNA, costimulatory molecules and cytokine levels in mDCs stimulated with HMGB1 were significantly higher than control group at each time point (P<0.01). The levels were significantly decreased after being treated with HMGB1 specific neutralizing antibody or TLR4 antagonist (P<0.01). Conclusion: Data analysis suggests that HMGB1 plays a regulatory role in mDCs by affecting the downstream MyD88, NF-κB P65 protein and mRNA expression through TLR4, stimulating the secretion of cytokine and promoting the expression of costimulatory molecules (CD80, CD86). 2018 Vol. 48 (7): 508-516 [Abstract] ( 759 ) HTML (1 KB)  PDF (1985 KB)  ( 792 ) 517 Protective effect of tanshinone IIA on cognitive impairment in chronic renal failure rats and its influence on endoplasmic reticulum stress-related apoptosis and oxidative stress ZHU Ming, WANG Xiaotong, MIN Jingjing, CHEN Qi, Wang Xiaoyi Objective: To investigate the neuroprotective effect and the potential mechanism of Tan IIA on the cognitive impairment in chronic renal failure (CRF) rats. Methods: Sixty male SD rats were randomly divided into 5 groups with 12 rats in each group. They were grouped as follows: the control group, the model group, the TM group, the TM+Tan IIA group, and the Tan IIA group. The 5/6 renal excision method were adopted to simulate the CRF model. From the 4th weeks to 12th weeks, the TM group and the TM+Tan IIA group were injected TM intra-peritoneal twice per week with the dosage of 4.5 mg/kg, meanwhile the TM+Tan IIA group and the Tan IIA group were injected Tan IIA intraperitoneally once daily with the dosage of 15 mg/kg. The control group was injected intra-peritoneat with an equal volume of saline. Morris water maze test was applied to test the learning and memory abilities of the rats after modeling. HE staining was used to observe the morphology of hippocampal neurons. Tunel staining was used to detect the apoptosis index of hippocampal neurons. MDA content and SOD activity in Serum and hippocampal tissues were detected by the kit. Western blot was used to detect the expression of GRP78, CHOP and Caspase-12 proteins. Results: Compared with the control group, the learning and memory abilities of the model rats in the Morris water maze were significantly decreased, and the decline was more obvious after the TM intervention. Meanwhile, the level of GRP78, CHOP, Caspase-12 protein expressions and the percentage of apoptotic cells increased, accompanied with the increased MDA content reduced SOD levels and disrupted cell structure. In the ERS agonist TM intervention group, increased ERS-associated apoptosis, increased oxidative stress levels, and more pronounced cell structure destruction were observed compared with the model group. The Tan IIA intervention significantly decreased the expression of ERS-associated apoptosis, increased the ability of anti-oxidative stress, and significantly ameliorated the cell structure. Moreover the learning and memory ability of the rats in the Morris water maze was significantly improved. Conclusion: The ERS related excessive apoptosis may be involved in CRF-induced cognitive impairment in rats. Tan IIA probably plays a role in neuroprotection and improves the cognitive function by inhibiting the ERS-related apoptosis and antioxidative stress. 2018 Vol. 48 (7): 517-523，528 [Abstract] ( 724 ) HTML (1 KB)  PDF (1782 KB)  ( 756 ) 524 The diagnostic value of serum pepsinogen in gastric ulcer  CHEN Lei, GU Yunfeng, ZHAN Aixia, QIAN Dingliang, ZHENG Jingwei, ZHENG Meiqin Objective: To evaluate the diagnostic value of serum pepsinogen in gastric ulcer and find out the optimal PG cutoff value for determining gastric ulcer. Methods: A total number of 388 patients with stomach discomfort treated in the Third Affiliated Hospital of Wenzhou Medical University from January 2016 to February 2017 were enrolled in this study. Before gastroscopy, fasting serum pepsinogen was analyzed by ELISA. The efficacy of pepsinogen was evaluated according to endosopic and pathological results. Results: According to the results of the pathological diagnosis, 388 cases were divided as superficial gastritis group (control group, 132 cases), atrophic gastritis group (168 cases), gastric ulcer group (48 cases) and gastric cancer group (40 cases). Serum PG I and PG II levels were significantly increased, PGR (PG I/PG II) significantly decreased (P<0.05) in gastric ulcer group compared with the control group. Serum PG I and PG II levels were significantly increased (P<0.05) in gastric cancer group compared with atrophic gastritis group. No statistical difference (P<0.05) in serum PG I, PG II and PGR levels were found between gastric ulcer group and gastric cancer group. Serum PG II levels were higher and PGR levels lower in gastric cancer group compared with the control group (P<0.05). PG I, PG II, PGR and AUC for gastric ulcer was 0.688, 0.704, 0.627 and 0.709 respectively. The cut-off value of PG I, PG II, PGR was 164.1 μg/L, 15.05 μg/L and 11.76. The test combined with PG I, PG II and PGR diagnosed for gastric ulcer was defined as PG I>165.7 μg/L, PG II>19.1 μg/L and PGR<8.68. The sensitivity and specificity were 68.8% and 65.9%. Conclusion: High PG I and PG II but low PGR are indicators of gastric ulcer. Combined diagnosis of PG I, PG II and PGR shows the best diagnostic value for gastric ulcer. Gastric ulcer can be screened in large scale by the combination of PG I and PG II and PGR in to improve the early diagnosis of gastric ulcer and precancerous lesion. 2018 Vol. 48 (7): 524-528 [Abstract] ( 770 ) HTML (1 KB)  PDF (1210 KB)  ( 976 ) 529 Prediction and analysis of progestin primed ovarian stimulation in patients who failed in implantation of GnRH agonist long protocol undergoing IVF/ICSI ZHANG Yijia, CHEN Qiuju, KUANG Yanping. Objective: To analyze the clinical outcomes of progestin primed ovarian stimulation in patients with no less than two implantation failures in long protocol. Methods: A retrospective study including 278 patients who had ≥2 implantation failures in long protocol and received PPOS subsequently was performed. The ovarian stimulation and pregnancy outcomes were compared between PPOS and previous long protocol. Results: For patients with ≥2 implantation failures of long protocol, the number of retrieved oocytes, viable embryos and the viable embryo rate per oocyte in PPOS were less than long protocol (P<0.05). In FET cycles of PPOS, these patients transferred less embryos per cycle than long protocol (P<0.01), with the clinical pregnancy rate, implantation rate and live birth rate of 49.2%, 31.4% and 41.7%. Among these specific women, patients who only received FET in previous long protocol gained the clinical pregnancy rate, implantation rate and live birth rate of 45.2%, 30.2% and 41.1% in FET cycles of PPOS. Conclusion: Patients with no less than two implantation failures in long protocol can obtain acceptable pregnancy outcomes in PPOS, and the possible reason may be the improvement in quanlity of oocytes and embryos. PPOS can be an efficient alternative protocol for these patients. 2018 Vol. 48 (7): 529-534 [Abstract] ( 702 ) HTML (1 KB)  PDF (1207 KB)  ( 928 ) 535 MRI analysis of benzene toxic encephalopathy XIE Pinnan, CHEN Qi, XU Jingxuan, WANG Yingying, TAO Yuanping, XIE Yibing, WU Aiqin, XU Chongyong.  Objective: To study the dynamic MRI characteristics of benzene toxic encephalopathy. Methods: Complete imaging and clinical data of 35 cases with benzene toxic encephalopathy were collected from 4 hospitals of Wenzhou between March 2009 and November 2016. The imaging characteristics of benzene toxic encephalopathy were retrospectively analyzed. Results: All patients had a definite history of benzene exposure, acute or chronic onset. The clinical manifestations included the damage of central nerve system, the decline of routine blood leucocytes, the abnormality of EEG and the rise of cerebrospinal fluid pressure in lumbar puncture, et al. The arc zone of subcortical white matter fiber was invaded in 35 cases of benzene toxic encephalopathy on MRI scan, demonstrating a sign of “kwai petals” or “flame” shape. “Butterfly” change on both dentate nuclei of cerebellum was seen in 33 cases. The number of external capsule and lentiform nucleus was involved in 28 cases and 16 cases. All lesions demonstrated low signal on T1WI, high signal on T2WI, FLAIR and DWI. High or iso-signal on ADC images appeared in 26 cases and low signal in 9 cases. In 3 cases of MRS examination, the peak of Cho and Cr slightly elevated in 1 case, and no abnormality in 2 cases. No obvious changes appeared on MRI contrast enhancement, SWI and MRA. Conclusion: MRI manifestations of benzene toxic encephalopathy have some characteristics. Combined with the history, MRI can reduce the misdiagnosis of the disease and provide reference for clinical diagnosis and treatment. 2018 Vol. 48 (7): 535-538 [Abstract] ( 824 ) HTML (1 KB)  PDF (1287 KB)  ( 827 ) 544   Guide::    2018 Vol. 48 (7): 544-547 [Abstract] ( 705 ) HTML (1 KB)  PDF (1114 KB)  ( 803 )

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