Purpose:To construct replication-defective adenovirus which was recombinated with Egr-1 promoter and to Smad7, and to study whether it can express exogenous Smad7 protein in cytoplasm.Methods:Based on Adeno-X~(TM) expression system, the CMV promoter of the pShuttle vector was replaced by Egr-1 promoter, and the Smad7 cDNA was subcloned into the MCS(multiple cloning site) of pShuttle. The recombinant pShuttle was then subcloned into the Adeno-X~(TM) genome, which was transformed into E.coli to get recombinant Adeno-X~(TM) plasmid DNA. The recombinant adenovirus was made and amplified in the transfected HEK 293 cells before it was purified and tested for viral titer. Then the Smad7’s location in cells was determined by immunocytochemistry.Results:Identified by restriction endonuclease analysis and PCR, recombinant adenovirus with Egr-1 promoter and Smad7 cDNA was constructed successfully, with a viral titer of 1.0?10~(11) TCID50/ml. Both endogenous and exogenous Smad7 protein was found to be located in cytoplasm of fibroblast cells.Conclusions:With Adeno-X~(TM) expression system, utilizing the techniques of molecular cloning, recombinant adenoviral vector could be quickly and efficiently constructed which could be packaged into replication-defective Adenovirus and amplified in HEK293 cells. The recombinant adenovirus could express exogenous Smad7 protein in fibroblast cells.[

Objective To explore the role of serotonin(5-HT) in obsessive compulsive disorder(OCD) and the difference in platelet 5-HT content between OCD and healthy controls, the obsession and the compulsion subgroup. Methods The concentration of serotonin (5-HT) in twenty-seven patients with OCD and twenty-seven patients without OCD were detected in the study. Results Platelet serotonin level in patients with OCD ( ( 139 ±172 ) μg/L) was lower than that in patients without OCD ( ( 248 ± 215 ) μg/L), and the differences were significant (P＜0.05). Conclusion The present results support the hypothesis that 5-HT hypofunctionality contribute to OCD. And the differences between the obsession and the compulsion subgroup in the role of 5-HT are significant.

Objective To study whether the expression Smad 7 protein by the recombinant adenovirus with Egr-1 promoter and Smad 7 cDNA in fibroblast cell can block the signal transduction pathway of transforming growth factor-beta1 (TGF-?1) under irradiation thereby inhibiting collagen synthesis in vitro. Methods The location of endogenous Smad 7 and exogenous Smad 7 protein in recombinant adenovirus infected fibroblast cells(3T6) were determined by immunocytochemical method. The infected 3T6 cells were irradiated and then cultured with TGF-?1 4 hours after irradiation. The activity of preventing radiation-induced fibrosis by expression Smad 7 protein was evaluated by the amount of collagen synthesis and proliferation of 3T6 cells. The amount of collagen synthesis was shown by the coruscant per minute (cmp) through the 3?H-Proline incorporation technique. Results The endogenous Smad 7 and exogenous Smad 7 protein both were located in the cytoplasm. When cultured with TGF-?1 4 hours after irradiation, the amount of collagen synthesis in the 3T6 cells infected with the recombinant adenovirus was significantly less than that in the cells without infecting adenovirus after irradiation(P=0.001), But, there was no difference in the proliferation of 3T6 cells between those with and without adenovirus infection (P= 0.312 ). Conclusions The Egr-1 promoter in the recombinant adenovirus can regulate the expression of downstream Smad 7 cDNA in 3T6 cells. The expression Smad 7 protein could block the TGF-?1 signal transduction pathway thereby inhibiting the collagen synthesis. The mechanism of inhibiting the collagen synthesis may be accomplished at the transcription level.

BACKGROUND:As current studies on isolation, culture andcryopreservation of human amniotic epithelial cells (hAECs) are relatively scattered, it is difficult to form a comprehensive and effective solution to meet the clinical needs of stem cells for transplantation in future.OBJECTIVE:To establish the technology of isolation, culture and cryopreservation of hAECs, and to study the biological characteristics of hAECs.METHODS:Orthogonal method was used to study the effects of different factors on the separation, culture and cryopreservation, and range method was adopted to analyze the data to optimize the separation, culture and cryopreservation. We performed cell primary and passage cultures, morphology observed by microscope, drawn cell growth curve and flow cytometry assay, immunofluorescence staining, hepatocyte like cell differentiation to study the biological characteristics of hAECs.RESULTS AND CONCLUSION:(1) The optimal hAECs separation conditions were as follows:trypsin digestions were conducted at a concentration of 0.25%, four times, once for 20 minutes digestion; optimal conditions of culture were 4×108/L cell seeding density, 10 μg/L epidermal growth factor, 5% serum; optimal conditions of cryopreservation were 1×1010/L cell cryopreservation density, 10% dimethyl sulfoxide, 80% serum. (2) The primary cells were adhered to the wall in 2-3 days, exhibiting irregular polygon, paving stone-like growth. Cell adherence and growth rate were accelerated after subculture, and the growth and proliferation ability of passage 2 cells were not significantly decreased after cryopreservation and resuscitation. (3) Immunofluorescence staining showed that the primary cells strongly expressed SSEA-4 and CK19, but did not express Vimentin, CD45 and HLA-DR. The immunophenotype statistics of the primary and passage 4 cells showed the epithelial mesenchymal transition of hAECs in culture process. (4) Immunofluorescence staining showed that the liver cell marker expression of ALB, CK18 was significantly increased after hAECs were induced to differentiate into hepatocyte-like cells. Glycogen staining revealed glycogen synthesis in hAECs after 3 weeks of induction. To conclude, hAECs are easy to obtain and have strong proliferation ability in vitro, and express surface markers for undifferentiated embryonic stem cells.

Objective To study the effect of different dose fractionation on overall survival in patients with limited-stage small cell lung cancer (LS-SCLC). Methods LS-SCLC patients treated with radical combined chemotherapy and radiotherapy (RT) between January 2001 and Dec 2007 were analyzed retrospectively. According to the dose fractionation schemes, patients were divided into three groups:conventional fractionated RT (1. 8 -2.0 Gy,once daily), hyperfractionated RT (1.4 Gy, twice daily) and hypofractionated RT (2. 5 Gy,once daily). Overall survival, disease free survival and pattern of failures of the three groups were compared. A total of 177 patients were enrolled, including 63 patients in conventional fractionated RT group, 79 in hyperfractionated RT group and 35 in hypofractionated RT group. Results The overall follow-up rate was 96. 6%. The patient numbers with follow-up of more than 2 and 5 years were 153 and 92, respectively. The median survival time of the entire group was 22. 4 months, and the 2-and 5-year survival rates were 43.4% and 23. 5%, respectively. The 2-year survival rates for three groups were 31%, 46% and 59% (x2 =7.94,P=0.019), respectively. The 2-year disease free survival for three groups were 20%, 31% and 40% ( x2 = 4. 86, P = 0. 088 ), respectively. In the pairwise comparisons,patients in hypofractionated RT group have better survival than those in conventional fractionated RT group ( x2 = 7. 81, P = 0. 005 ), the effect of hyperfractionated RT group lies between the hypo-and the conventional fractionated RT groups, but no significant differences were detected ( x2 = 2. 31, P = 0. 128; x2 = 2. 95, P =0. 086). The mildest side effect was found in the hypofractionated RT group. No statistically significant differences were found in the patterns of first failure. Conclusion The hypofractionated RT scheme showed potential survival benefits for patients with LS-SCLC and should be considered in the setting of randomized clinical trials.

OBJECTIVE:To investigate clinical efficacy and safety of meglumine adenosine cyclophosphate in the treatment of acute myocardium infarction. METHODS:A total of 80 patients with acute myocardium infarction in our hospital during May 2015-Jan. 2016 were selected and divided into control group and observation group according to random number table,40 cases in each group. Control group was given conventional treatment. Observation group was additionally given Meglumine adenosine cyclo-phosphate injection 120 mg added into 5% Glucose injection 250 mL,ivgtt,qd,on the basis of control group. Both groups re-ceived treatment for 7 d. Clinical efficacies as well as the levels of serum hs-CRP and NT-proBNP before and after treatment were observed in 2 groups,and the occurrence of ADR was compared between 2 groups. RESULTS:Total response rate of observation group(92.50%)was significantly higher than that of control group(75.00%),with statistical significance(P<0.05). Before treat-ment,there was no statistical significance in the serum levels of hs-CRP or NT-proBNP between 2 groups(P>0.05). After treat-ment,the serum levels of hs-CRP and NT-proBNP in 2 groups were decreased significantly,and the observation group was signifi-cantly lower than the control group,with statistical significance(P<0.05). There was no statistical significance in the incidence of ADR between 2 groups(P>0.05). CONCLUSIONS:Meglumine adenosine cyclophosphate shows significant therapeutic efficacy for acute myocardium infarction,reduces serum levels of hs-CRP and NT-proBNP significantly with good safety.

Objective To observe the changes of microRNA‐214 (miR‐214) expression in rat pulmonary artery smooth mus‐cle cells (PASMCs) induced by different hypoxia time ,and lay the foundation to explore the effect and mechanism of regulation of miR‐214 on PASMCs proliferation .Methods The primary cultured PASMCs were cultured under hypoxic 0 h ,6 h ,12 h ,24 h ,48 h ,respectively .The real time quantitative PCR was used to detect miR‐214 expression in each group PASMCs .Results The ex‐pression of miR‐214 in hypoxia group PASMCs was sustained as time increased ,apart from hypoxic hypoxia 6h group and 0h group ,the expression of miR‐214 was no significant difference (P>0 .05);the expression of miR‐214 among other groups PASMCs was significantly different (P<0 .05) .Conclusion The expression of miR‐214 in PASMCs increased after induction of hypoxia .We speculated that miR‐214 may be involved in the regulation of hypoxia induced PASMCs proliferation .

Objective:To summarize the clinical features and genetic characteristics of Zellweger spectrum disorder caused by PEX6 gene variation.Methods:This was a case series research. Clinical date and genetic results of 2 neonatal cases of Zellweger syndrome caused by PEX6 gene variation in Wuhan Children′s Hospital, Tongji Medical College, Huazhong University of Science & Technology and Affiliated Hospital of Guangdong Medical University from July 2021 to July 2022 were retrospectively collected and analyzed. Literature up to August 2023 was searched from electronic databases of China National Knowledge Infrastructure (CNKI), Wanfang Data and PubMed with the combined keywords of "Zellweger syndrome" "Zellweger spectrum disorder", and "PEX6 gene" both in Chinese and English. The main clinical features and genetic characteristics of Zellweger spectrum disorder caused by PEX6 gene variation were summarized.Results:The 2 male neonates both developed clinical manifestations as dyspnea, hypotonia, feeding difficulties, enlarged fontanelle, and high palatine arch after birth. Biochemical parameters indicated elevated bile acids, and the cranial ultrasound showed the enlarged bilateral ventricles and subependymal cyst in both 2 neonates. Zellweger syndrome was confirmed by whole exome sequencing, and the results revealed PEX6 gene variation in the 2 neonates, including compound heterozygous variants c.315G>A and c.2095-3T>G, and homozygous variant c.506_507del. Case 1 was hospitalized for 5 days, and case 2 for 32 days; they both died shortly after being discharged (the specific time is unknown). Literature review found 26 patients, including 2 neonates in this study, with Zellweger spectrum disorder caused by PEX6 gene defect reported in 1 Chinese article and 11 English articles. Clinical features included hearing loss (19 cases), developmental delay (19 cases), vision impairment (19 cases), elevated very long chain fatty acids (17 cases), brain malformations (15 cases), hypotonia (12 cases), hepatic insufficiency (12 cases), distinctive facies (10 cases), and dental impairment (9 cases). Compound heterozygous variations dominated the variation types (15 cases), and the frameshift variations (16 cases) were the main pathogenic variations.Conclusions:Zellweger spectrum disorder should be considered when neonates show hypotonia, feeding difficulty, distinctive facial appearance, brain malformations and failure of hearing screening, or when older children show retinitis pigmentosa, sensorineural hearing loss, amelogenesis imperfecta and developmental delays. Detection of genetic variation in the PEX gene is crucial for definitive diagnosis.

Objective To visualize and analyze the studies on spleen-strengthening and dampness-expelling method for treating skin diseases in the past 20 years by using CiteSpace 6.3.R1 software,and to understand current status and hot spots of research on spleen-strengthening and dampness-expelling method for treating skin diseases,so as to provide reference for the treatment and research of skin diseases. Methods Literatures on the spleen-strengthening and dampness-expelling method for skin disease treatment,which were published from January 1,2004 to June 1,2024,were retrieved from China National Knowledge Network Infrustruction. Then,CiteSpace 6.3.R1 software was adopted to visualize the authors,distribution of research institutes,and keywords. Results A total of 1326 valid papers were included,and there was an overall increase in literature publication. The author with the largest number of publications was Liu Hongxia (31 papers) and Lu Chuanjian (11 papers),the institution with the largest number of publications was Beijing University of Traditional Chinese Medicine(66 papers),followed by Guangzhou University of Chinese Medicine (62 papers). In the keyword analysis,the current hot keywords in this field are Wuling San,external treatment method,treatment from the spleen,spleen deficiency and dampness accumulation,etc. The research hot spots in the literature mainly include the experience of famous doctors,the law of medication and the research on the mechanism of action,and the frontier trends mainly include the mechanism of action,inflammatory factors,and the same treatment of heterogeneous diseases. Conclusion The method of spleen-strengthening and dampness-expelling is an important rule of traditional Chinese medicine in treating dermatological diseases. The main research content in recent 20 years is clinical observation,experience of famous doctors and mechanism of action. The mechanism of action,inflammatory factors,and heterogeneity of the same treatment are the hot spots of the research. The multi-disciplinary integration may be future research direction. However,there are currently few core authors and institutions involved in the research,and the cooperation between regions needs to be strengthened.

Objective:To construct preeclampsia (PE) prediction models using information from the hospital electronic medical information and clinical laboratory data through logistic regression (LR) and machine learning algorithms, and to compare their predictive performance.Methods:The study was conducted based on the information from Rouji Pregnancy Test Database and the perinatal data of women who visited the Third Affiliated Hospital of Guangzhou Medical University from January 1, 2012, to December 31, 2019. Drawing upon clinical treatment guidelines and related literature, 28 clinical indicators from 2 736 pregnant women at 24 to 28 weeks of gestation were selected after a thorough integration and used for the construction of the PE prediction model dataset. Patients diagnosed with PE comprised the PE group ( n=245), while another 255 cases from the rest who did not have PE were selected, with undersampling method, as the control group. The Random Forest algorithm (RF), eXtreme Gradient Boosting (XGB) algorithm, and LR model were each employed to develop predictive models for PE. Following the construction of the models, external validation of PE prediction accuracy was carried out using data acquired from an independent prospective cohort study on PE that was conducted from June 2019 to December 2022, in which 38 PE cases and 80 controls were chosen. The performance of predictive models were evaluated using metrics such as accuracy, sensitivity, specificity, and the area under the curve (AUC) of receiver operating characteristic. Results:Indicators included in the construction of the three predictive models suggested that uric acid, creatinine, maternal age, early pregnancy body mass index, urea, triglycerides, red blood cell count, eosinophil count, total cholesterol, neutrophil count, urine protein, alanine aminotransferase, and urine occult blood were influential in PE prediction models. The AUCs for RF, XGB, and LR models in the training and test sets were 0.851 (95% CI:0.730-0.891), 0.955 (95% CI:0.865-0.987), 0.884 (95% CI:0.767-0.923) vs. 0.845 (95% CI:0.723-0.868), 0.907 (95% CI:0.791-0.919), 0.851 (95% CI:0.755-0.893), respectively. In the test set, the accuracy, sensitivity, and specificity for RF, XGB, and LR models were 0.803, 0.607, 0.958, 0.864, 0.790, 0.927, and 0.832, 0.661, 0.971, respectively. In the external validation of the RF, XGB and LR predictive models, the accuracy were 0.822, 0.814, and 0.763; the sensitivity were 0.737, 0.789, and 0.605, and the specificity were 0.863, 0.825, and 0.838, respectively. Among them, XGB model showed the highest Youden's index (0.614). Conclusion:Compared to traditional methods of model construction, machine learning algorithms can establish more effective PE prediction models using real clinical data.