OBJECTIVE:To evaluate oncogenicity of HRAS (V112A) and its G12C, G12C,Q61R,G12C/G12/Q61R mutants in NIH mice. METHODS:HRAS (V112A) containing the V112A mutation was amplified from the DiFi human colon cancer cells. G12C[HRAS(V112A/G12C)],G12C[HRAS(V112A/G12C)],Q61R[HRAS(V112A/Q61R)] and 3 sites joint[HRAS(V112A/G12C/G12C/Q61R)] mutants were generated with site directed mutation PCR. After HRAS (V112A) and the above 4 mutants were inserted into pCDNA3.1(+) and the in vitro expression was verified with Western blot,each plasmind was injected into 8 6-8 weeks aged female NIH mice intracutaneously with a dosage of 100 μg per mouse to observe the expression of tumorgenicity. Pathological tissues was assayed with PCR and Western blot was used to detect the presence of the HRAS gene and protein. RESULTS:HRAS(V112A) and the 4 designed mutants were amplified successfully. After transfection of L929,HRAS expression was detected. Four months after the injection,4 mice from the HRAS (V112A) injected group developed hyperplastic lesions and 1 mouse from the HRAS(V112A/Q61R) group developed tumor. No lesions were observed in the HRAS (V112A/G12C),HRAS(V112A/G12C) and HRAS(V112A/G12C/G12C/Q61R) groups. PCR and Western blot results show the existence and expression of the corresponding HRAS genes. CONCLUSION:HRAS containing the V112A mutation could induce hyperplastic lesions in mice,while HRAS containing V112A and Q61R mutations could induce tumors in NIH mice. The HRAS(V112A) containing G12C,G12C and G12C/G12C/Q61R did not induce observable lesions.

OBJECTIVE:To establish and to validate a droplet microfluidic single-cell transcriptome detection platform for cancer research. METHODS:Using a droplet microfluidic system,a single cell and a single barcoded microparticle were wrapped together in an oil-water emulsion droplet. The mixed human acute promyelocytic leukemia cell line HL-60 and the mouse melanoma cell line B16-F10 were used as simulative sample to evaluate the technical indexes of the system. RESULTS:When the oil and the water phase flow rates were 200 and 30 μL/min,respectively,and the cell and microparticle concentrations were at 250 and 400/μL,the optimum cell labeling rate was reached at (11.87±3.75)%. With a simulative sample which contained 75 thousand cells and the theoretical cell labeling rate of 11%,the transcriptome data of 7 535 cells was obtained,among which there were 70 mixed human and mouse transcriptome cells accounting for only 0.9% of all cells. Transcriptome differences were used to distinguish the two types of cells and to screen out the specific expression markers among them. CONLUSION:In this study,a droplet microfluidic single-cell transcriptome detection platform was initiated and validated. The platform meets the requirements of oncology laboratory and shows extensive potential for tumor research.

OBJECTIVE:To investigate expression of key signaling molecules MEK and ERK in the MAPK/ERK signaling pathway and effect of PD98059 on MAPK/ERK pathway's biological function in the gastric cancer SGC-7901 cells. METHODS:Gastric cancer cell line SGC-7901 was cultured in vitro. Cell proliferation rates were detected by the CCK-8 method after treatment with different concentrations of PD98059 at 0,25,50,100,200,300 and 400 μmol/L for 24 h. Expression of MEK and ERK mRNA was detected by real-time quantitative PCR (qPCR) after treatment with 0,25,50 and 100 μmol/L PD98059 for 24 h. Expression of MEK and ERK proteins was detected by Western blot. Cell cycle and apoptosis were detected by flow cytometry. The normal gastric mucosal epithelial GES-1 cells were used as controls. RESULTS:Compared with the normal cells,expression of MEK and ERK mRNA and p-MEK and p-ERK proteins in the gastric cancer cells was significantly increased (P < 0.05). After treatment with PD98059,the cell proliferation rates were decreased with increasing concentration and in a dose-dependent manner. When the concentration of PD98059 was between 200 and 400 μmol/L,the inhibition became stabilized. Expression of the MEK and ERK mRNA was lower than that of the control group after treatment with 0-100 μmol/L PD98059 (P < 0.05). With increasing concentrations of PD98059,expression of ERK mRNA was gradually reduced (P < 0.05). Western blot analyses show that expression of p-MEK1/2 and p-ERK1/2 protein decreased after treatment with 50 and 100 μmol/L PD98059 (P < 0.05). Moreover,PD98059 caused G0/G1 phase arrest and induced apoptosis. CONCLUSION:MAPK/ERK signaling pathway was active in the gastric cancer cells. PD98059 inhibited activities in the MAPK/ERK signaling pathway and affected the biological function of the gastric cancer cells.

OBJECTIVE:To study protective effect of Trollius altaicus extract powder (TAEP) on PM2.5-induced acute lung injury (ALI) in rats. METHODS:Sixty SD rats were randomly divided into normal control (normal saline),model (normal saline),TAEP 125 mg/kg,250 mg/kg,500 mg/kg,and dexamethasone positive control (2 mg/kg) groups. Each dose group was orally administered with the test substance for 30 days. Except the normal control group,the other groups were instilled with PM2.5 suspension on the 30th day,causing acute lung injury in rats. Blood samples were collected from the abdominal aorta. Leukocyte count was performed and serum lactate dehydrogenase(LDH),superoxide dismutase(SOD),glutathione(GSH) and malondialdehyde(MDA) were analyzed. Nitric oxide(NO),interleukin-6(IL-6),interleukin-1β(IL-1β),TNF-alpha(TNF-α) were measured. The right upper lobe was taken for histopathological examination,the lower lobe was weighed to calculate the wet/dry weight ratio (W/D),the left lung was taken for alveolar lavage,and the alveolar lavage fluid (BALF) was collected to detect the contents of IL-6,IL-1beta and TNF-alpha. RESULTS:Compared with the normal control group,the white blood cell count,IL-6,IL-1β and TNF-α levels in serum and BALF were increased (P < 0.01),and serum LDH,MDA and NO levels were increased (P < 0.01),the W/D of lung tissue increased (P < 0.01),and the content of SOD and GSH decreased (P < 0.01). Compared with the model group,the white blood cell count of each dose of TAEP,IL-6 and IL in serum and BALF -1β,TNF-α decreased (P < 0.05 or P < 0.01),serum LDH,MDA,NO content decreased (P < 0.05 or P < 0.01),and lung tissue W/D decreased (P average < 0.05 or P < 0.01),SOD and GSH levels increased (P < 0.05 or P < 0.01); TAEP 500 mg/kg group decreased white blood cell count,serum,BALF IL-1β,IL-6,TNF -α content decreased,serum LDH,MDA,NO content decreased,SOD,GSH content increased,lung W/D decreased compared with dexamethasone group (P < 0.05). CONCLUSION:The results indicate that the Trollius altaicus extract powder has a protective effect on PM2.5-induced lung injury in rats.

OBJECTIVE:To investigate the liver cancer cell HepG2-dervied immunogenic substances as tumor antigens for dendritic cell-(DC) based immunotherapy of liver cancer. METHODS:Changes in morphology and protein expression of HepG2 cells were determined by transmission electron microscopy and western blot,respectively; and in surface molecules of DCs by flow cytometry using exosomes with a final concentration of 8 μg/mL. In addition,exosome-pulsed DCs were co-cultured with T lymphocytes for 5 days. Proliferation of lymphocytes was detected by CSFE staining,and their killing of tumors was detected by Annexin-V/PI double staining. RESULTS:The hepatoma cell HepG2 exosomes expressed the marker proteins CD63 and CD81,carried a large number of tumor antigens AFP and heat shock proteins HSP70 and HSP90,but did not express the cellular protein calnexin. Compared with the DC-MOCK group,exosome-pulsed DCs up-regulated the expression of surface molecules such as CD83,CD80,and CD86; promoted T lymphocyte proliferation (P < 0.01),and increased T cell-mediated tumor-specific and non-specific killing effects (P < 0.01). CONCLUSION:Hepatoma cell HepG2 exosomes carried a large number of tumor antigens and stimulated the maturation of DCs,which may serve as a potential antigenic spectrum of DCs-based immunotherapy for liver and other cancers.

OBJECTIVE:To explore effects of formaldehyde (FA) on intra-hepatocellular free cholesterol content in HepG2 cells. METHODS:HepG2 cells were treated with formaldehyde at 0.004,0.02 and 0.1 mmol/L concentrations for 24 and 48 hours,respectively. The content of intra-hepatocellular free cholesterol was detected by using free the cholesterol GPO-POD assay kit. Western blot was used to detect protein expression of the sterol regulatory element-binding protein (SREBP) -2,hydroxy mothylglutaryl coenzyme A reductase (HMGCR),acyl coenzyme A-cholesterol acyltransferase (ACAT) and low-density lipoprotein receptor (LDLR). RESULTS:Compared with negative control,the contents of intra-hepatocellular FC were increased significantly after treatments with FA for 24 h and 0.02 and 0.1 mmol/L for 48 h (P < 0.05). HMGCR expression levels were significantly increased after exposure to formaldehyde for 24 and 48 h (P < 0.05). After treatment with FA for 24 h,expression levels of ACAT and LDLR were significantly down-regulated (P < 0.05). After 48 h,expression levels of LDLR were decreased significantly,whereas the levels of ACAT were decreased significantly after treatment with 0.02 and 0.1 mmol/L FA (P < 0.05). CONCLUSION:FA exposure increased intra-hepatocellular free cholesterol contents in HepG2 cells. The increase could be caused by elevated de novo synthesis of cholesterol and decreased cholesterol esterification.

OBJECTIVE:To investigate changes of serum oxidative stress index in rats after their exposure to fire drill prop smoke. METHODS:40 rats were divided randomly into five groups:1 unexposed and 4 exposure groups with each group investigated at 1,6,24 and 168 h after the exposure. Exposures were carried out in a smoke producing device according to the GB/T 20285-2006 Classification of Smoke Toxicity Risk of Material. Blood samples were collected from femoral arteries to detect changes of GSH,MDA and SOD. Trachea and lung tissues were collected and stained by HE staining for histopathological examinations. RESULTS:After exposure,rats showed obvious injury symptoms in the nervous and respiratory systems. In addition,their weights were reduced,to a significant level for the 168 h group. Histopathological examination revealed significant inflammatory damage to the tracheas and lungs. Serum GSH concentrations of rats in the 6,24 and 168 h groups at 1.177,1.109 and 1.076 ng/mL,respectively,were significantly lower than those in the blank control group (P < 0.05 or P < 0.01). The serum MDA concentration in the 168 h group was 1.008 nmol/mL,which was significantly higher than that of the blank control group (P < 0.01). The serum SOD concentrations in the 24 and 168 h groups were 1.294 and 1.260 ng/mL,respectively,which were significantly lower than those in the blank control group (P < 0.05). CONCLUSION:Acute exposure to fire drill prop smoke can cause significant damage to the body. In addition,the oxidative stress index in serum changed obviously. Thus,oxidative stress reactions may be an important injury mechanism for excessive fire drill prop smoke exposure.

OBJECTIVE:To investigate protective effects of lycium barbarum polysaccharides (LBP) on radiation-injured spinal cord neurons in vitro. METHODS:SCN cells from spinal cords were cultured in vitro and were injured by exposure to different doses of X-ray irradiation (0,2,6,10 Gy). MTT assay was used to detect cell viability and to determine the best radiation dose. A radiation injury model was established. Irradiated cells were exposed to different concentrations of LBP (15,25,40 mg/L) to detect its protective effects. MTT assay was used to detect the cell survival rate and to determine the optimal dose of LBP intervention. The number of autophagy lysosomes was observed by electron microscope,and expression of the LC3 Ⅱ/I of autophagy-related genes was detected by Western blot and immunohisto-chemistry. RESULTS:Radiation exposure significantly reduced the survival rate of spinal cord nerve cells as shown by data from the MTT assay (P < 0.05). At the concentration of 40 mg/L,Western blot and immunohistochemistry results show that expression of gene LC3 Ⅱ/I was increased in the LBP + radiation group,with statistical significance difference (P < 0.05). CONCLUSION:LBP protected spinal cord neurons from radiation injury in vitro which may be related to its promotion of autophagy-related protein LC3Ⅱ/I expression.

OBJECTIVE:To study the mutagenicity of cigarette smoke condensates(CSC) in the TK gene mutation assay and to compare the mutagenicity of TK gene in cigarette samples with different tar release. METHODS:3 commercial cigarettes of different tar releases,5 mg(1^#),8 mg(2^#) and 11 mg(3^#),were tested. The mouse lymphoma cell line L5178Y tk^+/-3.7.2C was treated with 20 μg/mL,40 μg/mL,60 μg/mL,80 μg/mL,100 μg/mL and 150 μg/mL CSC from the 3 cigarette samples. 15 μL/mL DMSO treatment was used as the solvent control,and 3 μg/mL cyclophosphamide treatment was used as the positive control. The test protocol includeds the 4-h treatment,2-day expression and 12-day cloning phases. Then the mutation frequency of trifluorothymidine resistance (TMF) of each dose group was calculated. Finally,the mutagenic potency of different cigarette samples was compared. RESULTS:At the dose of 150 μg/mL of 1^#,100 μg/mL of 2^# and 3^#,the TMF were more than 3 times higher than that in the solvent control group. In addition,the TMF increased with increase of the CSC doses. The mutagenic potency of the CSCs for 3 cigarette samples were 0.37,0.36 and 0.38,respectively. CONCLUSION:All CSCs induced dose-dependent increases of mutagenicity in the mouse lymphoma assay. Comparing the mutagenicity of the 3 cigarette samples,there was no significant correlation between TK gene mutagenic potency and tar release in cigarette smoke.

OBJECTIVE:To investigate involvement of the ROS/Caspase-3 signaling pathway in INH-induced apoptosis in L-02 cells,and the protective effect of quercetin. METHODS:L-02 cells were randomly divided into control and treatment groups. The latter groups were treated with INH (10 mmol/L INH),25 μmol/L quercetin (10 mmol/L INH and 25 μmol/L quercetin) and 50 μmol/L quercetin group (10 mmol/L INH and 50 μmol/L quercetin). Vitality of L-02 cells was detected by the MTT method. LDH activity in supernatant fluid was measured by colorimetric method. Apoptosis of L-02 was determined by Hoechst 33258 fluorescent staining. Mitochondria of L-02 cells,reactive oxygen species (ROS) level and mitochondrial membrane potential (△Ψm) were analyzed with fluorescent probe DCFH-DA and Rho-123. Protein expression of Caspase-3 was analyzed with western blot method. RESULTS:Compared with the controls and with L-02 cells treated with INH and quercetin,cell vitality of the INH group was significantly declined (P < 0.01). Compared with the INH group,cell vitality of the 50 μmol/L quercetin group was markedly increased (P < 0.01). Activities of LDH,cell apoptosis rate and level of mitochondrial ROS in cells of the INH group were significantly increased over the control group (P < 0.01). Activities of LDH,cell apoptosis rates and levels of mitochondrial ROS in the 25 μmol/L and 50 μmol/L quercetin groups were significantly decreased compared with that of the INH group (P < 0.05 or P < 0.01,respectively),and the effects in the 50 μmol/L quercetin group were more obvious. The mitochondrial membrane potential of the INH group was significantly lower than that of the control group (P < 0.01). The mitochondrial membrane potential of the 25 μmol/L and 50 μmol/L quercetin groups were markedly higher than that of the INH group (P < 0.05 or P < 0.01,respectively),and the 50 μmol/L quercetin group was more effective compared with the control group. Protein expression of Caspase-3 in the INH group was markedly increased (P < 0.01),compared with the INH group. Protein expressions of Caspase-3 of the 25 μmol/L and 50 μmol/L quercetin groups were significantly reduced (P < 0.05 or P < 0.01,respectively),and the 50 μmol/L quercetin group had more obvious effect. CONCLUSION:INH induced cell apoptosis in the L-02 cells and ROS/Caspase-3 signaling pathway was involved in this process.Quercetin had a protective effect against the INH-induction of cell apoptosis,the mechanism may be related to reducing ROS release,and inhibiting the ROS/Caspase-3 signaling pathway.

OBJECTIVE:The aim of this study was to investigate changes of MnSOD expression and its activity in liver regeneration after partial hepatectomy. METHODS:Using the classic mouse partial hepatectomy model,38 male BALB/c mice were randomly divided into 30% partial hepatectomy group (30%PH) and 70% partial hepatectomy group (70%PH). There were 18 mice in each experimental group,and 2 as sham operation control group. 3 mice was randomly selected from the two hepatectomy groups at 6 h,1 d,2 d,3 d,5 d and 7 d after the operation,and the control group was executed immediately after the sham operation. Liver tissues in the PH groups were harvested at 6 h,1 d,2 d,3 d,5 d and 7 d respectively. Frozen sections from collected tissues were used to detect the level of reactive oxygen species by the DHE dyeing method and using laser confocal microscopy. MnSOD mRNA was measured by Real Time Q-PCR. Expression of MnSOD protein was determined by Western blot,and activity of MnSOD was assessed using the MnSOD kit. RESULTS:Compared with the control group,the ROS levels in liver tissues from PH mice increased at 6 h and 1 d after operation,expression level of MnSOD mRNA increased (P < 0.05),but protein content of MnSOD did not change significantly in the 30%PH group (P > 0.05). Activities of MnSOD was higher on 1 d and 2 d,lower on 3 d and 5 d,and then recovered on 7 d after 30%PH. In the 70%PH group,ROS level in liver tissues increased from 1 d to 5 d after operation; MnSOD mRNA levels decreased first and then gradually recovered; protein content of MnSOD decreased; and activities of MnSOD increased at 6 h and 1 d,and then decreased during 2 d to 7 d (all P < 0.05). CONCLUSION:Liver regeneration started rapidly after hepatectomy in mice,especially after 70%PH. The hepatocytes rapidly proliferated,and then gradually returned to a resting state after a period of time. The observed changes might be related to the down-regulation of MnSOD content and activity,which led to the increase of ROS.

OBJECTIVE:To study the accumulation and migration of strontium by Epipremnum aureum,Chlorophytum comosum (Thunb.) Baker. and Tradescantia zebrine Bosse.,and the effect of strontium exposure on absorption of other six main nutrients. METHODS:Three kinds of plants were maintained by hydroponic culture. Each kind was treated as control and 3 treatment groups with strontium concentrations of 1,2 and 3 mmol/L,respectively. Each treatment was experimentally cultured with 4 plants of Epipremnum aureum and Tradescantia zebrine,and 3 plants of Chlorophytum comosum. The entire experiment was carried out in the light incubator,and the experimental conditions of each treatment were consistent. The lasted 28 d. The contents of calcium,iron,potassium,magnesium,phosphorus,sulfur and strontium in plant tissues were determined by ICP-AES. RESULTS:The content of strontium in three kinds of plant tissues treated with 3 mmol/L strontium was significantly different from that of control (P < 0.05). Strontium concentrations among the plant tissues were Tradescantia zebrine leaves > Epipremnum aureum leaves > Chlorophytum comosum leaves,Tradescantia zebrine stems > Epipremnum aureum stems,Tradescantia zebrine roots > Chlorophytum comosum roots. With the increase of strontium,the contents of calcium and iron in plant tissues were decreased significantly (P < 0.05). In this experiment,the transportation factor (TF) of Tradescantia zebrine leaves,stems and Chlorophytum comosum leaves were between 0.36 and 2.54. The TF of Tradescantia zebrine leaves and stems were the highest under 1 mmol/L strontium treatment,reaching 2.54 and 2.33,respectively. With the increase of strontium concentration in hydroponic solution,the TF showed a downward trend. CONCLUSION:Compared with the 3 experimental plants,Tradescantia zebrine can be used as an alternative plant for strontium phytoremediation in contaminated water. The increase of strontium content can reduce the absorption and accumulation of calcium and iron in plants.

OBJECTIVE:To explore the effects from exposure to nano-TiO₂ on the immune system of offspring of rats. METHODS:SD rats were divided into nano-TiO₂ and control groups with 30 females and 15 males in each group. Exposed groups were given nano-TiO₂ by gastric filling method at the doses of 250 mg/kg,and the control group was given equal volume of NaCl. The exposure was continued for 2 weeks before mating,and throughout pregnancy and lactation. Offspring were infused nano-TiO₂ or NaCl into stomach from weaning to execution. Immune function of offspring was observed on PND56. Observation indexes included organ coefficients of liver,spleen and thymus,and splenic and paneth lymphocyte typing and antibody-producing cell assay. RUSULTS:Organ coefficients of spleen in the nano-TiO₂ treated female rats group were significantly higher than that of the control group (P < 0.05). Organ coefficients of liver in nano-TiO₂ treated male rats group were significantly lower than that of the control group (P < 0.05). However,no abnormalities were found in histopathological examinations. No differences were observed on lymphocyte typing and antibody-producing cell assays between the nano-TiO₂ and the control groups (P > 0.05). CONCLUSION:The result suggests that,under the present experimental conditions,nano-TiO₂ has no effect on the immune system of offspring of rats.