Plant bioreactor is a new production platform for expression of recombinant protein, which is one of the cores of molecular farming. In this study, the anti DYKDDDDK (FLAG) antibody was recombinantly expressed in tobacco (Nicotiana benthamiana) and purified. FLAG antibody with high affinity was obtained after immunizing mice for several times and its sequence was determined. Based on this, virus vectors expressing heavy chain (HC) and light chain (LC) inoculated into Nicotiana benthamiana leaves by using Agrobacterium-mediated delivery. Accumulation of the HC and LC was analyzed by SDS/PAGE followed by Western blotting probed with specific antibodies from 2 to 9 days postinfiltration (dpi). Accumulation of the FLAG antibody displayed at 3 dpi, and reached a maximum at 5 dpi. It was estimated that 66 mg of antibody per kilogram of fresh leaves could be obtained. After separation and purification, the antibody was concentrated to 1 mg/mL. The 1:10 000 diluted antibody can probe with 1 ng/mL FLAG fused antigen well, indicating the high affinity of the FLAG antibody produced in plants. In conclusion, the plant bioreactor is able to produce high affinity FLAG antibodies, with the characteristics of simplicity, low cost and highly added value, which contains enormous potential for the rapid and abundant biosynthesis of antibodies.
Animals ; Mice ; Antibodies ; Nicotiana/genetics* ; Agrobacterium/genetics* ; Bioreactors ; Blotting, Western

In recent years, series of driver genes, such as EGFR, KRAS/NRAS, BRAF, PIK3CA, ALK and ROS1 and so on, have been found in non-small cell lung cancer (NSCLC) one after another with the development of molecular detecting technology. Targeted drugs bring benefits for these NSCLC patients with driver gene variations. However, some NSCLC did not have any known driver gene variations; we called it pan-negative lung cancer. In this paper, we summarize the concept, clinical pathological characteristics, the epidemiological characteristics, treatment and prognosis of pan-negative NSCLC.
Carcinoma, Non-Small-Cell Lung ; diagnosis ; drug therapy ; genetics ; pathology ; Humans ; Lung Neoplasms ; diagnosis ; drug therapy ; genetics ; pathology ; Mutation ; Prognosis

Objective To investigate the expression of apolipoprotein (Apo) F in hepatocellular carcinoma (HCC) and its application value in the prognosis of patients with HCC. Methods 50 HCC samples were procured from patients undergoing surgical resection in the Third Affiliated Hospital of Sun Yat-sen University between September 2015 and September 2016, and all the samples were confirmed by postoperative pathological examination. The informed consents of all patients were obtained and the local ethical committee approval was received. There were 37 males and 13 females, aged from 31-67 with a median age of 53 years old. The expression of ApoF mRNA in HCC tissues was detected by RT-PCR. The expression profile was analyzed by using data from the Gene Expression Omnibus (GEO). The expression of ApoF between two groups were compared by t test. Correlation analysis of clinical related parameter was conducted by Chi-square test, and survival prognosis was analyzed by Kaplan-Meier test and Log rank test. Results The average relative expression of ApoF mRNA in HCC tissues was 0.15±0.07, significantly lower than 0.55±0.09 in the adjacent tissues (t=-6.26, P<0.05). GEO online analysis showed that expression of ApoF was significantly correlated with the status of liver cirrhosis, and most HCC patients with liver cirrhosis presented low expression of ApoF (χ2=4.626, P<0.05). The 5-year disease-free survival was respectively 55.9% and 32.0% in ApoF high expression group and low expression group, where significant difference was observed (χ2=3.939, P<0.05). Conclusions Low expression of ApoF exists in HCC tissues, and it is related to the liver cirrhosis status of patients. Patients with low ApoF expression present poorer prognosis. ApoF plays a role in inhibiting the cancer.

Objective To explore the impact of telomerase regulation factor PinX1 to the proliferation and invasion ability of hepatoma cells. Methods Hepatoma cells PinX1-7721 (experimental group) with stable expression of PinX1 as well as control cell VECTOR-7721 (control group) were constructed. The expression of PinX1 mRNA was detected by RT-PCR. The proliferation ability and clonality of hepatoma cells were detected by CCK-8 method and plate clonality assay, and the invasion ability of hepatoma cells by Transwell assay. Comparison of the experiment data was conducted by t test. Results Expression level of PinX1 mRNA in experiment group was (13.9±2.0)×10-3, which was significantly higher than (1.1±0.2)×10-3in control group (t=10.98, P<0.05). A450of the cells on 1-7 d in experiment group was respectively 0.260±0.004, 0.340±0.008, 0.450±0.040, 0.500±0.020, 0.730±0.030, 1.350±0.040 and 1.640±0.050, which were significantly lower than 0.280±0.009, 0.410±0.007, 0.680±0.044, 0.730±0.029, 0.850±0.070, 1.700±0.020 and 2.080±0.280 in control group (t=-5.82, -12.99, -6.36, -5.96, -28.42,-18.98, -5.08; P<0.05). The plate clonality assay results showed that the clone formation quantity of cells in experiment group was 143±32, which was significantly lower than 305±25 in control group (t=-6.91, P<0.05).Transwell assay results showed that the quantity of trans-membrane cell in experiment group was 230±16, which was significantly lower than 650±30 in control group (t=-21.40, P<0.05). Conclusion PinX1 could inhibit the proliferation and invasion ability of hepatoma cells.

Objective To investigate expression of plasmalemmal vesicle-associated protein (PLVAP) in hepatocellular carcinoma (HCC) tissues and its relationship with clinicopathological features.Methods Tissue specimens were collected from 108 patients with HCC in the Third Affiliated Hospital of Sun Yat-sen University from January 2013 to December 2015.92 patients were male and 16 female,aged (48±5) years on average.The informed consents of all patients were obtained and the local ethical committee approval was received.The expression level of PLVAP was analyzed based on the data of HCC in public databases.The expression level of PLVAP mRNA in HCC and paracarcinoma tissues was detected by RT-PCR,and the relationship between the expression of PLVAP and clinicopathological characteristics of HCC was analyzed.The relationship between PLVAP and prognosis of HCC patients was investigated with the data from cancer genome atlas (TCGA) database.The expression levels of PLVAP mRNA between HCC tissues and para-carcinoma tissues were compared by Kruskal-Wallis rank-sum test.Correlation analysis was performed by Chi-square test.Survival analysis was conducted by Kaplan-Meier survival curve and Log-rank test.Results According to Human Protein Atlas and Oncomine databases,the expression level of PLVAP in HCC tissues was significantly higher than that in normal liver tissues.RT-PCR showed that the median expression level of PLVAP mRNA in HCC tissues was 0.172(0.004-0.607),significantly higher compared with 0.091(0.002-0.513) in para-carcinoma tissues (Z=6.839,P＜0.05).The expression level of PLVAP in HCC patients was significantly correlated with TB,tumor size and microvascular invasion (x2=4.183,3.924,6.075;P＜0.05).In PLVAP high expression group,the overall survival and tumor-free survival were 58.8(0.5-107.0) and 42.2(0.1-67.2) months,where no significant difference from 55.7(0.2-120.7) and 20.9(0.1-109.4) months in PLVAP low expression group (x2=0.054,0.065;P＞0.05).Conclusions The expression level of PLVAP is significantly correlated with the development and progression of HCC,whereas it is probably not associated with the prognosis of HCC patients.

Objective:This study was performed using preclinical transplanted animal experiments to analyce the effects and mechanisms of third-generation EGFR-TKIs combined with anti-angiogenic therapy, thereby providing theoretical basis for further clinical trials. Methods:Researchers constructed the transplant BALB/C nude mice models with H1975 lung adenocarcinoma cell line (EGFR T790M) and divided the mice into four groups and treated them with osimertinib (2.5 or 5 mg/kg/day, gavage) alone or plus bevacizumab (5 mg/kg/twice weekly, i.p.) when the tumors reached approximately 0.4-0.6 cm3 in volume. The tumor growth curve of tumor volume was drawn according to the time in every group. After 2 weeks of treatment, the mice were killed and the tumors were processed for immunohistochemical staining and Western blot analysis. Immunostaining was performed to detect:HIF-1α, VEGF, and microvessel density (MVD) by using SP method on paraffin sections. Western blot analysis was used to analyze the protein expression levels of EGFR, AKT, and ERK signal transduction pathways. Results:After 2 weeks of treatment in high-and low-dose osimertinib alone, tumor volume in the high-dose group was significantly less than in low-dose osimertinib-alone group (P<0.05). VEGF, HIF-1αexpression, and MVD were significantly low in the high-dose osimertinib-alone group (P<0.05). Increasing doses of osimertinib induced dose-dependent weakening of the p-EGFR, p-AKT, and p-ERK expression levels (P<0.05). In the low-dose osimertinib-plus-bevacizumab group and low-dose osimertinib-alone group, no significant difference in tumor volume and the above factors was observed. In the low-dose osimertinib-plus-bevacizumab group and high-dose osimertinib-alone group, tumor volumes did not exhibit significant difference (P=0.178). Moreover, VEGF, HIF-1αexpression, and MVD exhibited no significant difference. No significant difference in the p-EGFR, p-AKT, and p-ERK expression levels was found between high-dose osimertinib-alone group and low-dose osimertinib-plus-bevacizumab group (P>0.05). In the high-dose osimertinib-plus-bevacizumab group, tumor growth was not significantly greater than that in the high-dose osimertinib-alone group (P=0.642). No significant difference was observed in the above factors.In the high-and low-dose osimertinib-plus-bevacizumab groups, tumor volume and the above factors did not exhibit significant differences (P>0.05). Conclusion:Osimertinib has obvious antitumor effects in EGFR-mutant lung adenocarcinoma with T790M mutation cell xenografts. Bevacizumab has a synergetic inhibitory effect with osimertinib against EGFR-mutant lung adenocarcinoma with T790M mutation cell xenografts. Bevacizumab enhanced the antitumor effects of osimertinib by reducing VEGF expression and the microvascular density of the tumor, thereby improving the tumor microenvironment. Bevacizumab can enhance the effect of osimertinib by suppressing EGFR, ERK, and AKT phosphorylation, thereby synergistically inhibiting EGFR activation and downstream signaling.

Objective To evaluate effects of daily average temperature on the occurrence of urticaria in Lanzhou city,and to analyze differences in the effects between different populations.Methods Time-series data on daily outpatient visits for urticaria between January 1,2007 and December 31,2013 were collected from the First Hospital of Lanzhou University and Lanzhou University Second Hospital.Daily meteorological data during this peroid were obtained from the Gansu Meteorological Bureau.Distributed lag non-linear models were used to analyze the association between daily average temperature and occurrence of urticaria,and the analysis was stratified by age and gender.Results The association between daily average temperature and daily number of outpatient visits for urticaria was nonlinear.Low temperature had significant lag effects on the daily number of outpatient visits for urticaria,with the maximum relative risk (RR) value (1.014 [95％ CI 1.000-1.023]) observed at 6 ℃ on lag day 18.Stratification analysis demonstrated that the effects of high temperature on the number of outpatient visits for urticaria were apparent on the day of exposure in age groups of 0-18 and 19-64 years,but decreased on the day of exposure in the age group ≥ 65 years.The effects of low temperature,which showed similar trends along with the increment of lag days in all groups,were relatively delayed and occurred 2 to 4 days after exposure.Conclusions Air temperature affects the occurrence of urticaria in Lanzhou city.Low temperature has evident lag effects on the occurrence of urticaria,while high temperature does not have.

Objective To investigate the effect and mechanism of long noncoding RNA (lncRNA) PTEN pseudogene 1 (PTENP1) on the proliferation and migration of hepatocellular carcinoma (HCC) cells. Methods Lentiviral vectors expressing PTENP1 were constructed. HCC cells BEL-7404 were infected with LV003-GFP-PTENP1 and control vectors LV003-GFP. BEL-7404 cells stably expressing PTENP1 were constructed and the experimental and control groups were established. The proliferation and clone formation abilities of HCC cells in two groups were detected by CCK-8 assay and clonogenic assay. The migration ability of HCC cells was detected by wound healing assay. The expression of p44/42 mitogen-activated protein kinase (MAPK) and p38 MAPK proteins were detected by Western blot. Results The absorbance values A450 of the cells at 48 and 72 h in the experimental group were 1.4±0.3 and 2.3±1.1, signiifcantly lower compared with 3.2±1.7 and 3.4±1.1 in the control group (t=-5.78,-4.23;P<0.05). The number of cell clone formation in the experimental group was 55±12, signiifcantly less than 154±45 in the control group (t=-3.98, P<0.05). The percentage of cell migration in the experimental group was (21.7±2.6)%, signiifcantly lower than (57.7±4.9)%in the control group (t=-8.34, P<0.05). Western blot revealed that the expression of p44/42 MAPK and p38 MAPK proteins in the experimental group was significantly down-regulated compared with those in the control group. Conclusion lncRNA PTENP1 can inhibit the proliferation and migration of HCC cells probably through regulating MAPK signaling pathway.

ObjectiveTo investigate the clinical characteristics of primary liver cancer (PLC) complicated with non-Hodgkin’s lymphoma.MethodsClinical data of 2 patients with PLC complicated with non-Hodgkin's lymphoma admitted and treated in the Third Afifliated Hospital of Sun Yat-sen University between January 2006 and July 2015 as well as 18 patients reported by the literature were retrospectively analyzed. The informed consents of all patients were obtained and the local ethical committee approval was received. The incidence, diagnosis and treatment process, therapeutic regimen, curative effect and prognosis were observed. Relevant literature in PubMed database from January 1994 to December 2015 was searched for literature review.ResultsAmong the patients, 17 were males and 3 were females with the onset age ranging from 35 to 80 years old and the median of 64 years old. Nineteen cases were with hepatocellular carcinoma and 1 with mixed type liver cancer. Hepatitis virus infection was found in 90%(18/20) of the patients with 8 cases of hepatitis B virus (HBV) infection and 10 of hepatitis C virus (HCV) infection. All patients were complicated with B-cell non-Hodgkin's lymphoma. The main type was diffuse large B-cell lymphoma, accounting for 60%(12/20). And the other types were follicular lymphoma (n=5), mucosa-associated lymphoid tissue (MALT) lymphoma (n=1), marginal zone lymphoma (n=1) and unspeciifed lymphoma (n=1). The main lesions of non-Hodgkin's lymphoma respectively located in the liver (n=9), spleen (n=3), lymph node (n=3), stomach (n=3), vertebral body (n=1), other non-tissue and visceral organ (n=1). The therapeutic regimens were operation + chemotherapy (n=9), radiofrequency ablation or transcatheter arterial chemoembolization (TACE) + chemotherapy (n=6), liver transplantation (n=1) and palliative treatment (n=4). The median postoperative survival time of the patients was 48(5-105) months, while the survival time of the patients receiving palliative treatment was less than 1 month.ConclusionsMost PLC patients complicated with non-Hodgkin's lymphoma are male, and the pathological type of all the non-Hodgkin's lymphoma is B-cell type. The morbidity is closely associated with HBV infection. There are no speciifc clinical manifestations, and the conifrmed diagnosis depends on the pathological examination. Radical resection is the ifrst choice for treatment.

Objective To investigate the effect and mechanism of liver cancer derived mesenchymal stem cell (LCMSC) on the invasion of liver cancer cells. Methods The expressions of interleukin (IL)-6, IL-8 and chemotactic factors CXCL1, CXCL5 and CXCL12 mRNA in the bone marrow derived mesenchymal stem cell (BMSC) and LCMSC were detected by reverse transcription-polymerase chain reaction (RT-PCR). The expression of protein CXCL12 in the supernate of LCMSC was detected by enzyme-linked immunosorbent assy (ELISA) and Western blot. Different cells were co-cultured and divided into the HepG2+BMSC, HepG2+LCMSC and HepG2+LCMSC+siRNA-CXCL12 groups. The effect of CXCL12 on the invasion of liver cancer HepG2 cells were detected by Transwell migration assay. The experiment data were compared using one way analysis of variance and LSD-t test or t test. Results The expression of CXCL12 mRNA in LCMSC was 60.3±2.4, significantly higher than 13.8±1.8 in BMSC (t=15.68, P<0.05). The expression of protein CXCL12 in the supernate of LCMSC was (31.5±1.7) ng/L, significantly higher than (14.3±1.5) ng/L in BMSC (t=7.60, P<0.05). And the expression of protein CXCL12 was up-regulated. Transwell migration assay indicated that the quantity of membrane-invasion cells in the HepG2+LCMSC group was 110±12, significantly higher than 65±9 in the HepG2+BMSC group and 76±7 in the HepG2+LCMSC+siRNA-CXCL12 group (LSD-t=5.25, 4.86; P<0.05). Conclusion CXCL12 is highly expressed in LCMSC. LCMSC may enhance the invasion of HepG2 cells through up-regulating the expression of CXCL12. The invasion of liver cancer cells can be effectively weakend by silencing the CXCL12 gene with siRNA.