Objective To induce skin cancer in BALB/c mice using DMBA as initiator and TPA as tumor promot-er. Through optimizing the doses and frequencies of DMBA administration to establish a stable skin cancer model with less time and causing less skin damage. Methods Shaving the back of mice to expose a piece of skin around 2 cm × 2 cm. The mice were divided into a blank control group and four treatment groups randomly. These four groups were given 1, 2, 4, 7 times 100μg/100μL DMBA/acetone, respectively, in the first week, and twice 4μg/100μL TPA/acetone per week in the next 11 weeks. The body weight changes, time of tumor formation and number of tumors formed were recorded during the experiment. The mice were sacrificed at 12th week and samples of tumor tissue and adjacent normal skin tissue were taken for pathological examination using HE staining. Results Tumors were observed at the 7th week in the group with once DMBA administration in the first week and at the 4th week in the group with twice DMBA administration in the first week. Skin cancers were formed also in the group with 4?time DMBA administration in the first week, however, with signif?icantly more severe skin damages. The mice receiving DMBA everyday in the first week died at the 3th week. Conclusions The best induction protocol for skin cancer in BALB/c mice should be twice DMBA in the first week followed by twice TPA

Objective To investigate the role of glucose-regulated protein 78 (GRP78) in sevoflurane preconditioning-induced inhibition of apoptosis in cardiomyocytes of rats.Methods The cultured cardiomyocytes of Sprague-Dawley rats were randomly divided into 5 groups (n =30 each) using a random number table:control group (group C),hypoxia-reoxgenation (H/R) group,sevoflurane preconditioning group (group S),siRNA-GRP78 group and siRNA control group.H/R was produced by 2 h exposure of cells to 95％ N2-5％ CO2 in an air-tight chamber at 37 ℃,followed by reoxygenation with 95％ O2-5％ CO2 in an air-tight chamber at 37 ℃ for 1 h.In group S,the cells were incubated with 2.5％ sevoflurane for 20 min,followed by 10-min washout before H/R.In siRNA-GRP78 group,the cells were transfected with siRNA-GRP78 100 nmol/L,and 24 h later preconditioning with sevoflurane was performed and H/R was produced.In siRNA group,cells were transfected with siRNA,and the other treatments were similar to those previously described in siRNA-GRP78 group.After treatment in each group,the expression of GRP78 in myocardial cells and cytochrome c in cytoplasm and mitochondria was detected by Western blot.Lactic dehydrogenase (LDH) and creatine kinase (CK) activities in the culture medium of each group were determined by ELISA.The apoptosis in myocardial cells was assessed by flow cytometry.Apoptotic rate was calculated.Intracellular free Ca2+ concentration ([Ca2+]i) was measured with the fluorescent probe Fura-2/ AM.The opening of mPTP was measured by fluorescence spectrophotometry.Results Compared to group C,the expression of GRP78 in myocardial cells and cytochrome c in cytoplasm was significantly up-regulated,LDH and CK activities in the culture medium,apoptotic rate and [Ca2+]i were increased,and the expression of cytochrome c in mitochondria was down-regulated in H/R group.Compared to group H/R,the expression of GRP78 in myocardial cells and cytochrome c in mitochondria was significantly up-regulated,LDH and CK activities in the culture medium,apoptotic rate,[Ca2+] i and opening of mPTP were decreased,and the expression of cytochrome c in cytoplasm was down-regulated in group S,and no significant change was found in the parameters mentioned above in siRNA group.Compared to group S,the expression of GRP78 in myocardial cells and cytochrome c in mitochondria was significantly down-regulated,LDH and CK activities in the culture medium,apoptotic rate,[Ca2+] i and opening of mPTP were increased,and the expression of cytochrome c in cytoplasm was up-regulated in group siRNA-GRP78.Conclusion GRP78 is involved in sevoflurane preconditioning-induced inhibition of apoptosis in cardiomyocytes of rats,and the mechanism is related to maintenance of intracellular Ca2+ stability and inhibition of opening of mPTP.

Objective:To investigate the effect of gemcitabine on myeloid derived suppressor cells (MDSC) in the spleen of B lymphoma cell-bearing mice, and the therapeutic effect of gemcitabine combined with intratumoral injection of dendritic cells (DCs) in treatment of large B lymphoma. Methods: BALB/c mice were inoculated subcutaneously with B lymphoma A20 cells; large tumors were formed 30 d after inoculation. Gr-1~+ CD11b~+ MDSC proportion in the spleen was analyzed by flow cytometry before and after gemcitabine treatment. Splenic MDSC sorted by immunomagnetic beads was further treated with gemcitabine, and then the apoptosis of MDSC was examined by Annexin-V/PI staining. Tumor growth and survival time of A20 tumor-bearing mice were observed after treatment with gemcitabine and intratumoral injection of DCs. Results: Splenic Gr-1~+ CD11b~+ MDSC ratio in A20 cell-bearing mice was 10 times higher than that in the normal mice. Gemcitabine induced apoptosis and necrosis of purified MDSC in vitro in a time-dependent manner. The percentage of MDSC in the spleen of A20 tumor-bearing mice was decreased after injection of a single dose of gemcitabine. Gemcit-abine or intratumoral injection of DCs alone inhibited growth of tumor to a certain degree, with the mean survival periods of mice in the gemcitabine, DCs, and untreated groups being (48.8±3.6) d, (47.2±7.4) d, and (38.8±2.2) d, respectively. Gemcitabine chemotherapy combined with intratumoral DC injection resulted in continuous shrink of the tumors, and 60% of the mice survived for more than 90 d. Conclusion: Gemcitabine can effectively eliminate splenic MDSC in tumor-bearing mice. Gemcitabine chemotherapy and DCs immunotherapy can work synergistically in the treat-ment of huge lymphoma. These results provide an experimental basis for the comprehensive chemotherapy and immunotber-apy of relapsed or refractory lymphoma.

Objective To design a visual terrain classification algorithm to facilitate the robot to make appropriate movement strategy by perceiving the surrounding environment.Methods Bag of words (BOW) and support vector machine (SVM) were used to develop a simple and effective terrain classification algorithm.The BOW model involved in feature extraction,codebook generation and feature coding.The mid-level feature developed by BOW model was then fed into SVM classifier to obtain the terrain classification result.Results The quadruped robot platform was applied to performing visual terrain classification experiment in the natural environment.The test environment included floor,asphalt,sand and grass.Good experimental results were achieved,and the classification accuracy was above 90％ (the beat was 97.54％ for grass).Conclusion The algorithm can effectively and accurately distinguish all kinds of terrains,with high accuracy and good stability.The key frame selection method needs researching in the future.

Objective To discuss the influence of eldepryl on the expressions of glial fibrillary acidic protein (GFAP) and cd11b in substantia nigra and striatum in the rats with Parkinson’s disease (PD),and to clarify the regulatory role of eldepryl in the gliacytes.Methods 72 SD rats were randomly divided into control group,PD model group and eldepryl group,and each group was divided randomly into 4 d and 8 d subgroups (n=12)after the success of model preparation.The PD rat models were established by injecting rotenone in subcutaneous.The number of GFAP and cd11b positive cells and the expressions of GFAP and cd11b were detected by immunohistochemistry and Western blotting method.Results The GFAP and cd11b positive cells were all in a resting state in control group, the GFAP-positive cell body was slender and irregular and had elongated protrusions;the cd1 1 b-positive cell body was small and branch-like,and it had more slender protrusions.The GFAP and cd11b positive cells were all in a active state in model group, the GFAP-positive cell body was hypertrophy, the proj ections increased thickening;the cd1 1 b-positive cell body was more bigger, the proj ections were shorter and thicker, and the number was increased.Compared with model group, the GFAP-positive cell body and protrusions were more slender, the CD11b-positive cell body was more smaller,the projections were more slender,and the number was decreased in eldepryl group.There were a small amount of expression of GFAP and cd11b positive cells in substantia nigra and striatum in the rats in control group,and there was no significant difference between 8 d group and 4 d group(P>0.05). The number of GFAP and cd11b positive cells and the protein expression levels were significantly increased in model group compared with control group(P<0.01);there was more expression in 8 d group compared with 4 d group,but there was no significant difference(P>0.05).The number of GFAP and cd11b positive cells and the protein expression levels in eldepryl group were significantly reduced compared with model group(P<0.01);there were less expression in 8 d group compared with 4 d group, and there was significant difference (P<0.05 ). Conclusion There are activation and proliferation of the gliacytes in substantia nigra and striatum in the rats with PD,and eldepryl can inhibit the activation and proliferation of gliacytes.

Objective: To explore the feasibility, safety and long-term efficacy of laparoscopic total gastrectomy combined with distal pancreaticosplenectomy for the treatment of T4b gastric cancer. Methods: A retrospective cohort study was performed. Clinical data of consecutive patients with T4b gastric cancer invading pancreatic tail undergoing laparoscopic or open total gastrectomy combined with distal pancreaticosplenectomy from January 2010 to December 2014 were analyzed retrospectively. Enrollment criteria: (1) primary gastric cancer confirmed by pathology as T4b adenocarcinoma; (2) chest+abdominal+pelvic enhanced CT indicated cancer invading pancreatic tail without distant metastasis, and R0 resection was evaluated as feasible before operation; (3) physical status was ECOG score 0 to 2, and was tolerant to operation. Patients with peritoneal implant metastasis and tumor invasion of other organs during operation, or changes in surgical methods for other reasons were excluded. All the operations were performed by the same surgical team, which had the experiences of more than 100 cases of laparoscopic and 100 cases of open radical gastrectomy with D2 lymph node dissection. The choice of surgical procedure was discussed by the surgeon and the patient, and decided according to the patient′s intension. Patients were divided into the laparoscopic group and open group according to the surgical method. Intraoperative and perioperative findings were compared between the two groups. The 3-year disease-free survival rate were analyzed with Kaplan-Meier survival curve and compared by using log-rank test. Results: A total of 37 consecutive patients were enrolled, including 21 in the laparoscopic group and 16 in the open group, and no one receiving laparoscopic procedure was converted to open surgery. The baseline data of two groups were comparable (all P>0.05). Compared with the open group, the laparoscopic group had significantly longer operation time [(264.0±35.1) minutes vs. (226.6±49.9) minutes, t=2.685, P=0.011], significantly less intraoperative blood loss [(65.7±37.4) ml vs. (182.2±94.6) ml, t=-4.658, P<0.001], significantly shorter time to postoperative flatus [(2.8±0.7) days vs. (4.1±0.7) days, t=-5.776, P<0.001] and significantly shorter postoperative hospital stay [(13.3±2.8) days vs. (16.6±4.3) days, t=-2.822, P=0.008]. Morbidity of postoperative complications, including anastomotic leakage, pancreatic fistula, abdominal abscess, intraperitoneal hemorrhage and duodenal stump leakage, in two groups was similar [19.0% (4/21) vs. 4/16, P=0.705]. There were no cases of anastomotic bleeding or stenosis. The 30-day postoperative mortality was 0 in the laparoscopic group and 1/16 in the open group, respectively (P=0.432). The 3-year disease-free survival rates were 38.1% and 37.5% in the laparoscopic and open group, respectively (P=0.751). Conclusion Laparoscopic total gastrectomy combined with distal pancreaticosplenectomy performed by experienced surgeons for T4b gastric cancer is safe and effective.

OBJECTIVETo evaluate the clinical application of carbon nanoparticles labeled lymph node staining in curative laparoscopic resection for colorectal carcinoma.

METHODSSixty-five patients undergoing curative laparoscopic resection for colorectal carcinoma in the Sun Yat-sen Memorial Hospital between September 2011 and June 2013 were prospectively enrolled and randomly divided into label group (with carbon nanoparticles, n=34) and control group (without carbon nanoparticles, n=31). Association between labeled lymph nodes and metastasis was analyzed. The total number of retrieved lymph nodes and lymph nodes metastatic ratio were compared between the two groups.

RESULTSMean number of retrieved lymph node of the label group was higher as compared to the control group (22.3±4.2 vs. 15.4±3.5, P<0.05). The total number of retrieved lymph node was 725 in the label group and 478 in the control group. Among them, lymph node < 5 mm accounted for 4.6% (33/725) in the label group, which was higher than 2.0% (10/478) (P=0.025) in the control group. The number of black stain label lymph node was 412, with black stain ratio 56.8% (412/725) in the label group. Metastatic ratio of black stain nodes was significantly higher than that of non-stain nodes [28.6% (118/412) vs. 19.5% (61/313), P=0.005].

CONCLUSIONSThe technique of carbon nanoparticles labeled lymph node staining in curative laparoscopic resection for colorectal carcinoma is easy and effective, which can increase the retrieved number of lymph nodes, especially for nodes < 5 mm. The black stain lymph nodes indicate higher risk of metastasis.

Objective To evaluate the role of peroxisome proliferator-activated receptor gamma (PPARγ) in exogenous protectin D1 ( PD1)-induced reduction of endotoxin-induced acute lung injury (ALI) in mice. Methods Thirty-two clean-grade healthy male BABL∕C mice, weighing 20-25 g, aged 6-8 weeks, were divided into 4 groups (n＝8 each) using a random number table method: sham operation group (group S), ALI group, PD1 group and PPARγ antagonist GW9662 group. Mice underwent oral tra-cheal intubation, normal saline was instilled, and 1 h later normal saline was injected via the tail vein in group S. Mice underwent oral tracheal intubation, lipopolysaccharide (LPS) 3 mg∕kg was instilled, and 1 h later normal saline was injected via the tail vein in group ALI. Mice underwent oral tracheal intubation, LPS 3 mg∕kg was instilled, and 1 h later PD1 200 ng was injected via the tail vein in group PD1. Mice un-derwent oral tracheal intubation, LPS 3 mg∕kg was instilled, and 1 h later GW9662 1 mg∕kg and PD1 200 ng were injected via the tail vein in group GW9662. Mice were sacrificed at 24 h after intratracheal instilla-tion of LPS, the left lung was lavaged with phosphate buffer solution, and the broncho-alveolar lavage fluid (BALF) was collected for determination of neutrophil count and concentrations of interleukin-1beta ( IL-1β), tumor necrosis factor-alpha ( TNF-α) and IL-6 ( by enzyme-linked immunosorbent assay). Right lung tissues were obtained and cut into sections which were stained with haematoxylin and eosin and exam-ined with a light microscope for microscopic examination of the pathological changes which were scored (lung injury score) and for determination of the expression of PPARγ in lung tissues. Results Compared with group S, the neutrophil counts in BALF, concentrations of IL-1β, TNF-α and IL-6 and lung injury score were significantly increased, and the expression of PPARγ was down-regulated in group ALI ( P＜0. 01). Compared with group ALI, the neutrophil counts in BALF, concentrations of IL-1β, TNF-α and IL-6 and lung injury score were significantly decreased, and the expression of PPARγ was up-regulated in group PD1 (P＜0. 01). Compared with group PD1, the neutrophil counts in BALF, concentrations of IL-1β, TNF-α and IL-6 and lung injury score were significantly increased, and the expression of PPARγ was down-regulated in group GW9662 ( P＜0. 01). Conclusion PPARγ activation is involved in exogenous protectin D1-induced reduction of LPS-induced ALI in mice.

Objective:To investigate the effect of conventional treatment plus long-term transcranial direct current stimulation (tDCS) on consciousness recovery in patients with minimally conscious state (MCS) after traumatic brain injury (TBI).Methods:A retrospective cohort study was conducted to analyze the clinical data of 60 patients with MCS after TBI admitted to Zhejiang Armed Police Corps Hospital from January 2019 to December 2020, including 38 males and 22 females, aged 19-60 years [(45.7±11.4)years]. Course of disease was 3-6 months [(4.6±0.9)months]. Of all, 30 patients received internal medicine, hyperbaric oxygen, rehabilitation and other conventional treatment (conventional treatment group), and 30 patients received tDCS stimulation on the basis of conventional treatment (tDCS treatment group). The tDCS stimulation contained 4 cycles for 28 days with each cycle lasting for 7 days (stimulation for 5 days, rest for 2 days). Coma recovery scale-revised (CRS-R) total score, brainstem auditory evoked potential (BAEP) score and clinical effictive rate (significantly effective+effective) were compared between the two groups before treatment, during 4 cycles of treatment and at 6 months and 12 months after treatment. Complications induced by tDCS were also evaluated.Results:There was no significant difference in CRS-R total score and BAEP score between the two groups before treatment (all P>0.05). CRS-R total score and BAEP score in tDCS treatment group were significantly higher than those in conventional treatment group during 4 cycles of treatment and at 6 months and 12 months after treatment (all P<0.05). CRS-R total score and BAEP score in both groups gradually increased during 4 cycles of treatment and at 6 months and 12 months after treatment (all P<0.05). The clinical effective rate in tDCS treatment group was 73% (22/30) when compared to 57% (17/30) in conventional treatment group ( P<0.05). In tDCS treatment group, 10 patients had local reversible slight redness at the cathodal position, while no other serious adverse effects, such as local burns, ulceration, exudation or epilepsy. Conclusion:Compared with conventional treatment, conventional treatment plus long-term tDCS can be more effective in improving the state of consciousness without serious adverse effects for MCS patients after TBI.

Objective:To study the expression level of programmed death ligand 1 (PD-L1) in combined hepatocellular-cholangiocarcinoma (cHCC-CCA) and its correlation with the clinical characteristics and prognosis.Methods:The clinical data of 75 patients with cHCC-CCA undergoing surgery in Tianjin Medical University Cancer Institute and Hospital from January 2011 to December 2019, including 61 males and 14 females, with a median age of 55 years (36 to 77). Immunohistochemistry was conducted to determine the PD-L1 expression in tumor. The status of PD-L1 expression, clinicopathological data and prognosis of patients were analyzed.Results:In low-differentiated cHCC-CCA tissues, the proportion of PD-L1 expression (21.1%, 8/38) was higher than that in moderately to well-differentiated cHCC-CCA tissues (2.70%, 1/37, χ2=4.366, P=0.037). The median disease-free survival (DFS) and overall survival (OS)of PD-L1 positive patients were 12.3 and 15.1 months, respectively, lower than those of PD-L1 negative patients (14.4 and 23.3 months). The difference of DFS was statistically significant ( χ2=4.052, P=0.044). In multivariate analysis, major vascular invasion (DFS: HR=1.965, 95% CI: 1.119-3.450, P=0.019; OS: HR=1.781, 95% CI: 1.022-3.105, P=0.042) and lymph node metastasis (DFS: HR=2.451, 95% CI: 1.1033-5.814, P=0.042; OS: HR=2.652, 95% CI: 1.120-6.279, P=0.027) were identified as independent prognostic factors affecting DFS and OS. Conclusions:The proportion of PD-L1 positive is higher inthe low-differentiated cHCC-CCA tissue compared to that in moderately to well-differentiated cHCC-CCA. The major vascular invasion and lymph node metastasis are independent factors affecting the prognosis of patients with cHCC-CCA.