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Objective:To establish the hepatic organoid of hepatitis B virus (HBV) infection on the basis of induced pluripotent stem cells (iPSC) and an inverted colloidal crystal polyethylene glycol scaffold (ICC), and to evaluate the antiviral effect of nucleoside drugs.Methods:iPSC was differentiated into hepatocyte-like cells (HLC), and inoculated into ICC to construct a hepatic organoid. The relative mRNA expressions of Nanog homeobox (NANOG), sex determining region Y-box (SOX) 2, SOX17, forkhead box protein A2 (FOXA2), alpha fetoprotein (AFP) and albumin (ALB) were detected by real time quantitative polymerase chain reaction (RT-qPCR). Confocal laser microscopy was used to photograph the three-dimension (3D) structure of organs. The expression of sodium taurocholate cotransporting polypeptide (NTCP) in HLC was analyzed by Western blot and immunofluorescence. HepG2.2.15 cells were used to extract HBV virus particles to infect hepatic organoid. The relative expression of HBV pregenome RNA (pgRNA) in cells was detected by RT-qPCR. The expressions of hepatitis B core antigen (HBcAg) and hepatitis B surface antigen (HBsAg) in cytoplasm were observed under confocal laser microscopy. A total of 0.5 μmol/L entecavir and 0.5 μmol/L lamivudine were used to treat the infected cells respectively. The relative expression of HBV pgRNA in infected and uninfected cells was detected by RT-qPCR. Independent sample t test and one-way analysis of variance were used for statistical analysis. Results:Within 21 days of iPSC differentiation, the mRNA expressions of NANOG and SOX2 in stem cells markers decreased ( F=158.90 and 8.31, respectivley; P<0.001 and P=0.002, respectively), while the mRNA expressions of SOX17 and FOXA2 in the endoderm increased first and then decreased ( F=37.23 and 82.57, respectively, both P<0.001). In the later stage of differentiation, the mRNA expressions of AFP and ALB in liver cells increased ( F=4.65 and 34.64, respectively, P=0.012 and P<0.001, respectively), and all differences were statistically significant. NTCP was highly expressed in differentiated cells detected by Western blot and fluorescence microscopy, the protein expression level was 0.803±0.099. Confocal laser microscopy confirmed that the differentiated cells expressed ALB and presented spherical structure in ICC. The expression of HBV pgRNA and the immunostaining of HBsAg and HBcAg confirmed that HBV successfully infected hepatic organoid. Three days after the application of entecavir and lamivudine, the HBV pgRNA level decreased significantly both in entecavir group (0.665±0.220) and lamivudine group (0.503±0.117) compared to the uninfected cells (3.347±0.454), and the differences were both statistically significant ( t=10.53 and 12.72, respectively, both P<0.001). Conclusions:HLC display hepatic specific genes ALB and NTCP. Hepatic organoids constructed with iPSC and ICC have human liver function and can be infected by HBV. Entecavir and lamivudine could effectively inhibit the replication of HBV in hepatic organoids.
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Objective:To explore the effect and mechanism of dendritic cell derived exosomes (Dexs) loading ubiquitinated (Ub) hepatitis D antigen (HDAg) on activating specific cytotoxic T lymphocytes (CTL).Methods:Ub-S-HDAg-Dexs were co-cultured with dendritic cells (DC) which were from the femora of C57BL/6 mice for 48 h, then flow cytometry was used to detect the maturity of DC (CD86, CD80 and major histocompatibility complex (MHC) Ⅱ). The spleen-derived T lymphocytes from C57BL/6 mice were added in vitro to activate DC and co-cultivated for 72 h. The T cells were divided into Ub-S-HDAg-Dexs group (add 50 μg/mL Ub-S-HDAg-Dexs), Blank-Dexs group (add 50 μg/mL DC derived exosomes without plasmid transfection), Con-Dexs group (add 50 μg/mL DC derived exosomes transfected by cantrol plasmid), PBS group (add 50 μL/mL phosphate buffered saline), and Ub-S-HDAg-Dexs+ AG490 group (add 50 μg/mL Ub-S-HDAg-Dexs, DC and T lymphocytes stimulated by exosomes, and 50 μmol/L AG490 was also added to the cell mix). Flow cytometry was used to detect CD8 + T cells secreting interferon-gamma, non-radioactive lactate dehydrogenase release test to detect the killing activity of specific CTL. Real-time quantitative polymerase chain reaction (PCR) and Western blotting were used to detect the mRNA and protein expressions of JAK kinase (JAK) 2, GATA-binding protein 3 (GATA3), T-bet, signal transduction and activator of transcription (STAT) 1 and STAT4. Independent sample t test were used for statistical analysis. Results:The positive rates of the surface molecules CD80, CD86, MHCⅡof DC stimulated by Ub-S-HDAg-Dexs were 83.850%±0.219%、68.910%±0.134%、84.320%±0.445%, respectively.In the Ub-S-HDAg-Dexs group, the rate of CD8 + T cells secreting interferon-gamma was 6.420%±0.028%, which was higher than those of other groups, including PBS group, Blank-Dexs group, Con-Dexs group and Ub-S-HDAg-Dexs+ AG490 group ( t=90.78, 30.32, 63.06 and 85.42, respectively, all P<0.001). The cytotoxicity of T cells in the Ub-S-HDAg-Dexs group was 82.4%±3.9%, which was higher than those of other groups ( t=17.28, 9.74, 3.95 and 15.89, respectively, all P<0.050). The relative mRNA expressions of JAK2, T-bet, STAT1, STAT4 in Ub-S-HDAg-Dexs group were higher than those in other groups, including Con-Dexs group ( t=10.74, 32.34, 13.00 and 16.28, respectively, all P<0.001), Blank-Dexs group ( t=15.05, 21.51, 6.46 and 13.12, respectively, all P<0.050), PBS group ( t=21.83, 41.42, 7.30 and 17.50, respectively, all P<0.050), Ub-S-HDAg-Dexs+ AG490 group ( t=35.75, 20.69, 17.02 and 17.07, respectively, all P<0.001), and the differences were all statistically significant. The protein expressions of T-bet, STAT1, STAT4 in Ub-S-HDAg-Dexs group increased compared with those in PBS group ( t=346.70, 57.54 and 55.81, respectively, all P<0.001), with statistical significance. In the presence of AG490, the protein expressions of T-bet, STAT1 and STAT4 decreased compared with those in Ub-S-HDAg-Dexs group, and the differences were statistically significant ( t=355.40, 52.79 and 126.10, respectively, all P<0.001). Conclusions:Ubiquitinated HDAg transported by exosomes could effectively promote DC maturation, induce T lymphocyte differentiation, and generate specific CTL responses, which provides a new idea for the treatment of hepatitis D.
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Objective To evaluate the degree of psychological distress and analyze the influential factors of cancer patients receiving radiotherapy,aiming to provide theoretical basis for individualized psychological interventions.Methods A cross-sectional survey of 157 cancer patients treated with radiotherapy in Sun Yat-sen Memorial Hospital of Sun Yat-sen University in the previous year was conducted in terms of data collection and mean score of NCCN distress thermometer by using the convenience sampling method.Tumor types included nasopharyngeal cancer,breast cancer and cervical cancer,etc.The influential factors were analyzed by using logistic regression method.Results The mean score of NCCN distress thermometer in 157 cancer patients was 4.07± 0.19.Among them,87 cases (55.41％) experienced significant psychological distress (NCCN distress thermometer score ≥ 4).The main influential factors of psychological distress consisted of depression,loneliness,fear,appearance changes,urination changes,nausea,poor appetite,eating trouble,diarrhoea,constipation,pain and radiodermatitis.Logistic regression analysis demonstrated that nausea and urination changes were the independent influential factors of psychological distress.Conclusions Over half of cancer patients undergoing radiotherapy present with evident psychological distress.Somatic problems and emotional situations are the main factors of psychological pain.Consequently,it is highly necessary to emphasize the psychological health and actively resolve the somatic and emotional symptoms of cancer patients receiving radiotherapy.
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Objective@#To evaluate the degree of psychological distress and analyze the influential factors of cancer patients receiving radiotherapy, aiming to provide theoretical basis for individualized psychological interventions.@*Methods@#A cross-sectional survey of 157 cancer patients treated with radiotherapy in Sun Yat-sen Memorial Hospital of Sun Yat-sen University in the previous year was conducted in terms of data collection and mean score of NCCN distress thermometer by using the convenience sampling method. Tumor types included nasopharyngeal cancer, breast cancer and cervical cancer, etc. The influential factors were analyzed by using logistic regression method.@*Results@#The mean score of NCCN distress thermometer in 157 cancer patients was 4.07±0.19. Among them, 87 cases (55.41%) experienced significant psychological distress (NCCN distress thermometer score≥4). The main influential factors of psychological distress consisted of depression, loneliness, fear, appearance changes, urination changes, nausea, poor appetite, eating trouble, diarrhoea, constipation, pain and radiodermatitis. Logistic regression analysis demonstrated that nausea and urination changes were the independent influential factors of psychological distress.@*Conclusions@#Over half of cancer patients undergoing radiotherapy present with evident psychological distress. Somatic problems and emotional situations are the main factors of psychological pain. Consequently, it is highly necessary to emphasize the psychological health and actively resolve the somatic and emotional symptoms of cancer patients receiving radiotherapy.
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Objective@#To evaluate the effect of tumor shape and location on pulmonary dose-volume parameters by intensity-modulated radiation therapy (IMRT) in patients with non-small cell lung cancer (NSCLC), aiming to provide a reference basis for establishing limits of the pulmonary dose-volume parameters during IMRT.@*Methods@#Clinical data of 208 NSCLC patients undergoing radical IMRT from June 2009 to June 2016 were retrospectively analyzed. According to the tumor shape and location, 208 cases were divided into the vertical bar group (n=127) and the horizontal bar group (n=81), the superior lung group (n=103) and the inferior lung group (n=105). Regression model curve was used to evaluate the effect of tumor shape and location upon the common pulmonary dose-volume parameters(V5, V20, MLD, AVS5 and AVS20).@*Results@#In all groups, the fitting curves of V5, V20 and MLD were manifested in the quadratic equation pattern, and AVS5 and AVS20 in the logarithmic equation manner. In the vertical bar group, the V5(P=0.015), V20(P=0.047) and MLD (P=0.012) were significantly higher, whereas the AVS5(P=0.044) was significantly lower compared with those in the horizontal bar group. No statistical significance was observed in AVS20 between two groups (P=0.490). The tumor location exerted significant effect upon V5 alone (P=0.009).@*Conclusions@#When the tumors presents in the vertical bar shape, the limits of the common lung dose-volume parameters are likely to exceed those of tumors in the vertical bar shape. Lung tumors located in the inferior lobe exerts a more significant effect upon the low-dose region volume compared with the tumors in the superior lobe.
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Objective:To explore the effectof tumor volume on pulmonary dose-volume parameters by intensity-modulated radiation therapy (IMRT) in non-small cell lung cancer (NSCLC),and to provide a basis for pulmonary dose parameters in IMRT treatment.Methods:A total of 204 patients with NSCLC received IMRT were retrospectively analyzed from June,2009 to October,2013.The prescribed dose of planning target volume (PTV) for primary tumor was 60-66Gy (2.00-2.25 Gy,27-33 times in all).The fractional volume percent of the lung received a dose ＞5 or 20 Gy (V5,V20),and absolute volume of lung received a dose ＜5 Gy (AVS5).The mean lung dose (MLD) in normal tissues were analyzed.Regression model curve was used to analyze them along with the change of primary tumor volume.Results:With the increase in lung tumor volume,the V5,V20 and MLD presented quadratic equation curve,and AVS5 presented logarithmic equation.When the tumor volume,less than a certain value (294.6,283.2,304.9 cm3,respectively),the V5,V20 and MLD increased with tumor size and presented an increased quadratic curve;when the tumor volume was higher than a certain value (294.6,283.2,304.9 cm3 respectively),the V5,V20 and MLD was declined.The AVS5 was declined in a logarithmic curve along with the increase of tumor volume.Conclusion:With the increase in lung tumor volume,the change in rule ofV5,V20,MLD and AVS5 is not completely equivalent.When the tumor volume exceeds a certain boundary value (about 300 cubic centimeter),the corresponding tumor diameter is about 7-8 cm.In addition to the focus on pulmonary V5,V20 and MLD,we should also pay more attention to AVS5 restrictions in establishment ofIMRT in NSCLC.
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Objective The aim of this study was to explore the inhibitory effect of alcohol extracts of Narcissus bulb on melanogenesis in zebrafish embryos.Methods Zebrafish embryos were exposed to different concentrations of alcohol extracts of Narcissus (0, 50, 100, 200, and 500 μg/mL), and then the formation of melanin was observed.Furthermore, we measured the enzyme activity of tyrosinase (TYR), which plays a pivotal role in melanogenesis.Meanwhile, the spatial and temporal pattern of melanin-specific marker genes were detected.Arbutin (Ar) was used as a positive control in all these experiments.Results The treatment of alcohol extracts of Narcissus bulb inhibited melanogenesis in the zebrafish embryos in a dose-dependent manner.Furthermore, the mRNA expression levels of melanin-related genes such as tyrosinase (TYR), silver (SILV) and Mitfa were significantly reduced after treatment with different concentrations of Narcissus extracts by in-situ and semi-quantitative PCR.Finally, the enzyme activity of tyrosinase was also gradually decreased with increasing concentrations of the alcohol extracts.Conclusions Narcissus alcohol extracts can effectively inhibit the production of melanin in zebrafish embryos.This study provides potential evidence and approaches for the screening of natural whitening compounds using zebrafish models.
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Objective:To explore the regular variation pattern of tumor volumes of the patients with non-small cell lung cancer (NSCLC) before and after targeting treatment of epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI),and to clarify its clinical value.Methods:The materials of 39 NSCLC patients with EGFR-TKI targeting treatment were retrospectively analyzed. The tumor volumes were detected by volume measurement software of TPS and Image J image processing software,then the absolute and relative tumor volume changes of the NSCLC patients before and after targeting treatment were analyzed by paired sample comparison symbol Wilcoxon rank test. Results:The absolute tumor volumes (mm3 )of the patients with NSCLC before and 1 month after targeting treatment were 14 822.11 (7 524.73,54 999.41)and 7 954.42 (3 499.73,29 396.83),respectively, and there was statistically significant difference (Z=-3.257,P=0.001);the absolute tumor volumes of the patients with NSCLC 1 and 2 months after targeting treatment were 8 358.47 (4 394.36,24 430.05)and 7 028.76 (3 634.98,21 056.71),respectively,and there also was statisticaliy significant difference (Z=-2.213,P=0.027).When the original tumor volume before targeting treatment was regarded as 1,the relative tumor volume of 1 month after targeting theatment was 0.612 6 (0.313 8,0.853 7),and there was significant difference (Z=-3.855,P0.05);the changes of tumor relative volume presented platform stage after 3 months.The tumor relative volumes of 7-9 months after EGFR-TKI treatment reached the bottom.Conclusion:The average primary tumor volume of the NSCLC patients is obviously reduced 1 and 2 months after TKI targeting treatment. It may be optimal to carry out radiotherapy in 3-9 months after EGFR-TKI targeting treatment.
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Objective In order to investigate the impact of interactive online teaching in Laboratory Diagnosis, a randomized experimental study was conducted among the medical students in Grade 2009.Methods A total of 65 subjects were randomized into the experimental group (N=31) and the control group (N=34).In addition to the standard in-class presentation/discussion and school-wide refined online courses offered in the control group, interactive online teaching was added to experimental group.After completion of the course, the impact of interactive online teaching in the experimental group was compared to that in the control group through a final exam and a question-naire concentrating on motivation of study, problem solving and team working skills.Statistical signifi-cance of the comparison was evaluated using t-test.Results The results from the final exam showed that the average score in the experimental group was higher than the control group (87.20 ± 5.25 vs.82.69 ± 5.76), It is statistically significant (P=0.002).The average scores of short answer and case study questions in the experimental group was higher than the control group (26.16 ± 1.57 vs.25.31 ± 1.73, 18.94 ± 1.05 vs.15.53 ± 1.15), It is statistically significant (P=0.043,P=0.000).The satisfaction evaluation of interest in learning, timely answers to questions, learning, analytical skills, teamwork culture reflected by the questionnaire indicated statistically higher (P=0.041,0.000,0.000,0.000,0.000) average scores in the experimental group as compared to its counterparts in the control group (4.30 ± 0.65 vs.3.97 ± 0.62, 4.63 ± 0.49 vs.3.25 ± 0.88, 4.43 ± 0.57 vs.3.49 ± 0.65, 4.46 ± 0.57 vs.3.37 ± 0.73, 4.33 ± 0.66 vs.3.68 ± 0.58).Conclusion Interactive online teaching helps to improve the study performance of medical students in Laboratory Diagnosis.Incorporating interactive online teaching into the current course regarding Laboratory Diagnosis merits recommendation.
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OBJECTIVE:To study the adhibition of misoprostol combined with palace cavity attraction tube in the treatment of painless abortion after cesarean section. METHODS:214 patients who received painless abortion after cesarean section were ran-domly divided into group A(71 case),group B(71 case)and group C(72 case). After all patients were given conventional intrave-nous anesthesia,Group A was given misoprostol 0.6 mg for oral administration in preoperative 2 h. Group B was given Misoprostol tablet 0.2 mg abrasive powder put in posterior fornix in preoperative 3 h. Group C did not give any drugs. All the groups used dis-posable suction curettage tube. The efficacies of sedation and cervical dilatation,operation time,intraoperative blood loss,recovery time,dosage of propofol and incidences of complications and adverse reactions in the 3 groups were observed. RESULTS:The op-eration time,intraoperative blood loss in group A were lower than group B and lower than group C,the differences were statistical-ly significant(P0.05). The total effective rates of sedation and cervical dilatation in group A and B were significantly higher than group C,and the difference was statistical-ly significant(P0.05). The incidence of compli-cations in group A was lower than group B and lower than group C,and the difference was statistically significant (P0.05). CONCLUSIONS:The efficien-cy and safety of misoprostol for oral administration or medicated vaginal suppository combined with palace cavity attraction tube in the treatment of painless abortion after cesarean section is similar,but oral administration can reduce the dosage of anesthetics,op-eration time and smaller intraoperative blood loss volume.
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Objective To study the setup errors in three-dimensional conformal radiotherapy (3DCRT) for thoracic esophageal carcinoma using electronic portal imaging device(EPID) and calculate the margins from CTV to PTV. Methods Forty-one patients with thoracic esophageal carcinoma who received 3DCRT were continuously enrolled into this study. The anterior and lateral electronic portal images (EPI) were aquired by EPID once a week. The setup errors were obtained through comparing the difference between EPI and digitally reconstructed radiographs(DRR). Then the setup margins from CTV to PTV were calculat-ed. By using self paired design,22 patients received definitive radiotherapy with different margins. Group A: the margins were 10 mm in all the three axes;Group B: the margins were aquired in this study. The differ-ence were compared by Paired t-test or Wilcoxon signed-rank test. Results The margins from CTV to PTV in x,y and z axes were 8.72 mm, 10.50 mm and 5.62 mm, respectively. Between the group A and group B, the difference of the maximum dose of the spinal cord was significant(4638.7 cGy±1449.6 cGy vs. 4310.2 cGy±1528.7 cGy; t=5.48, P=0.000), and the difference of NTCP for the spinal cord was also significant (4.82%±5.99% vs. 3.64%±4.70%;Z=-2.70,P=0.007). Conclusions For patients with tho-racic esophageal carcinoma who receive 3DCRT in author's department,the margins from CTV to PTV in x, y and z axes were 8.72 mm, 10.50 mm and 5.62 mm, respectively. The spinal cord could be better protected by using these setup margins than using 10 mm in each axis.
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ACKGROUND:Autism is a kind of neurobiological disease.The increase of plasma concentration of 5 hydroxytryptamine(5 HT) is considered to be one of study results,which is the most definite and has the best repetition,for the biological investigation of autism. Whether the season of birth and maternal mood during pregnancy are correlated with the occurrence of autism? OBJECTIVE:To compare the differences of the plasma concentration of 5 HT,season of birth and the maternal mood during pregnancy between autistic and normal children,and probe into the cause of increased 5 HT in autism. DESIGN:An observational comparative study taking autistic and normal children as the subjects. SETTINGS:A clinical psychological department of a university hospital;Mental Health Institute of the Xiangya Second Hospital;A rehabilitation center for dumb children of city grade. PARTICIPANTS:Children in the autistic group(n=54),who were in accordance with the diagnostic criteria of autism in Diagnostic and Statistical Manual of Mental Disorders Fourth Edition(DSM IV) and had the total score of social adaptive ability scale(SAAS) over 40, were selected from the Outpatient Clinic for Children of the Mental Health Institute,Xiangya Second Hospital of Central South University and the training class of autism of Shenzhen Special Education School;Children in the normal control group,who had no obvious somatic and neurological diseases,and developing delay detected by physical,neurological and mental status examinations, rough detection of developing level,were selected from the primary school and kindergarten in Changsha and Shenzhen cities. INTERVENTIONS:The plasma level of 5 HT was examined, season of birth and maternal mood during pregnancy were investigated in autistic and normal children.The children with autism were accessed with SAAS. MAIN OUTCOME MEASURES:The plasma level of 5 HT,the season of birth and the maternal mood during pregnancy in both groups. RESULTS:The score of SAAS in children with autism was 67.65± 1.67.The plasma level of 5 HT was(0.78± 0.32) μ mol/L in the autistic group,which was significantly higher than that in the normal control group [(0.62± 0.16) μ mol/L](t=3.17,P=0.002). There were 17 children with autism had increased 5 HT.Compare with normal children,most children with autism were born in winter and spring,and their mother had obvious mood problems during pregnancy. CONCLUSION:The 5 HT level is higher in children with autism than in normal children.Most children with autism were born in winter and spring,and their mother had obvious emotional problems during pregnancy.There are phase of high functioning of 5 HT in mother in key period of fetus cerebra development that may be one of the causes of hyperserotonemia in autism,and that is possibly one of the etiologies of autism.