In order to decrease the potential side-effects of human basic fibroblast growth factor (hbFGF) caused by its broadspectrum mitogenic activity, a single residue of hbFGF, the residue serine 108, was replaced with neutral alanine residue to construct a mutant of hbFGF (mhbFGF) with reduced mitogenic activity. The mutant was overexpressed in Escherichia coli BL21(DE3) by IPTG induction. The expression level of mhbFGF was about 30% of the total cellular protein. The expressed mhbFGF was purified by ionic exchange and heparin affinity chromatography from the supernatant of bacteria lysate. Measured by MTT method, the effect of mhbFGF on Balb/c 3T3 cell proliferation was much lower than that of wild-type hbFGF. The purified recombinant mhbFGF was prepared and sufficient for the following pharmacological and safety studies.

AIM:To study the mechanism of the unique export of one of human basic fibroblast growth factor (hbFGF) forms lacking the N-terminal nuclear localization signal (NLS),we high expressed and purified this hbFGF form in E.coli strain BL21(DE3).METHODS:The cDNA fragment of the hbFGF amplified by polymerase chain reaction (PCR) was cloned into the expression vector pET3c and expressed in BL21(DE3) by IPTG induction.The expressed hbFGF was purified by ionic exchange and heparin affinity chromatography from the supernatant of bacteria lysate.The mitogenic activity was measured by MTT.RESULTS:The expression level of hbFGF in E.coli was about 20% of the total cellular protein.The appreciable mitogenic activity of the purified hbFGF was comparable to that of hbFGF standard.CONCLUSION:The BL21(DE3)/ pET3c expression system could be used to high express hbFGF lacking NLS.The purified recombinant hbFGF was prepared and sufficient for further study.

AIM:To synthesize the human platelet factor-4(hPF4) gene with a convenient and effective approach, and high express the hPF4 gene in E. coli BL21 (DE3). METHODS: According to the primary structure of hPF4, the nucleotide sequence was synthesized using touch-down PCR method. The resultant gene fragment containing EcoR Ⅰ and Xho Ⅰ overhangs at 5' and 3' ends was cloned into the expression vector pGEX-4T-1 to construct the recombinant plasmid pGEX-4T-1-hPF4,which was then transformed into the E. coli strain BL21 (DE3). RESULTS: hPF4 gene was successfully synthesized by touchdown PCR method. A fusion protein composed of glutathione S-transferase (GST) and the recombinant hPF4 was expressed in BL21(DE3) by IPTG induction. The expression level of the fusion protein in E. coli was about 30% of the total cellular protein. CONCLUSION: Touch-down PCR may provide a convenient and effective approach to obtain other target genes. The expressed fusion protein forms the inclusion bodies, providing sufficient material for further purification and biological activities process.

Objective To investigate the clinical efficacy of paclitaxel combined with three-dimensional conformal ra-diotherapy(3DCRT) for advanced non-small cell lung cancer(NSCLC) in the elderly. Methods From June 2011 to June 2013, 51 elderly patients with NSCLC were randomly divided into chemoradiotherapy group (n=26) and chemotherapy group(n=25). All patients were treated with weekly 60 mg/m2 of paclitaxel intravenously for 3 weeks, and patients in the chemoradiotherapy group were also treated with three-dimensional conformal radiation (1.8~2.0 Gy per fraction, 5 times per week, total dose of 60~66 Gy). The clinical efficacy and adverse reactions were compared between two groups. Re-sults In chemoradiotherapy group, the response rate was 84.6% and the overall efficiency rate was 96.2%, which were higher than 60.0% and 84.0% in chemotherapy group (P<0.05). In chemoradiotherapy group, the one year survival rate was 79.2%, which was higher than 61.5% in chemotherapy group (P<0.05), so did one year progression free survival rates (67.4% vs 40.0%)(P<0.05). The main side effects were gastrointestinal toxicity, radiation esophagitis and bone-marrow suppression. Conclusion Paclitaxel combined with three-dimensional conformal radiotherapy for advanced NSCLC in the elderly are safe and effective, the toxicity is tolerable.

Objective To explore the clinical application value of second-generation dual-source CT combined with intelligent modulation and iterative reconstruction in emergency aortic dissection imaging.Methods A total of 40 emergency patients with clinical suspected aortic dissection were included in this study.Conventional scanning was performed in the control group,and large-pitch intelligent modulation and iterative reconstruction were performed in the test group.The mean CT value,mean noise,signal noise ratio(SNR),contrast noise ratio(CNR),effective dose,image quality and aortic root image quality were evaluated and analyzed.Results Totally 40 patients successfully completed CT aortic dissection imaging.There was no difference in image quality between the two groups (P＞ 0.05).The quality of aortic root images in the test group was better than that in the control group,and the difference was statistically significant(x2=22.556,P＜0.05).The mean CT value and mean noise of aorta in the control group were slightly higher than those in the test group.However,SNR and CNR in the test group were higher than those in the control group,and the difference was statistically significant (t =-21.042,-15.924,8.530,11.495,P＜0.05).The effective dose of the control group [(10.59±3.89)mSv] was significantly higher than that [(6.39±0.81) mSv] of the test group,the difference was statistically significant (t =-12.327,P＜0.05).Conclusions The combined intelligent modulation technique and iterative reconstruction technique with dual-source CT large pitch scanning can meet the requirements of image quality and reduce the effective dose,and can be used as a conventional imaging method for emergency CT of aortic dissection.

Objective: To develop the Adolescent Mental Health Literacy Assessment Questionnaire (AMHLAQ), and to evaluate its reliability and validity among undergraduates. Methods: On the basis of the definition of mental health literacy (MHL) and the Knowledge, Attitudes and Practices (KAP) theory, this study constructed a total of 36 items consisting of four dimensions, and scores were measured according to a five point Likert type scale. Using a cluster sampling method, a questionnaire survey was conducted among 3 826 freshmen and sophomore students from two medical schools in Anhui Province. The items were screened by performing t tests, Pearson s correlation coefficient analysis and factor analysis. The reliability and validity of the questionnaire were evaluated using indicators including homogeneity reliability, the split half reliability coefficient, and construct validity. Results: Factor analysis revealed that the AMHLAQ consisted of 22 questions grouped into four domains. The variance cumulative contribution rate was 62.213%. The reliability result showed that the Cronbach s alpha coefficient of the total questionnaire was 0.897, the split half reliability was 0.800, the Cronbach s coefficient of each dimension was 0.796 to 0.885, the split half reliability of each dimension was 0.725 to 0.846, and the indicators had a high level of reliability. Confirmatory factor analysis showed that the model fit was good ( χ 2/df =19.319, P <0.01; RMSEA=0.069). Conclusion AMHLAQ is consistent with the evaluation standard of psychometrics, has good reliability and validity, and can be used to estimate the level of MHL among undergraduates.

Objective To summarize the clinical,electrophysiological and imaging features of frontal opercular epilepsy.Methods A retrospective analysis was conducted on 5 cases with frontal opercular epilepsy,who were treated at the Department of Functional Neurology,Shenzhen Second People's Hospital from December 2020 to December 2022.Among these cases,4 cases were underwent stereotactic-EEG(SEEG)guided radiofrequency thermocoagulation.The clinical,electrophysiological,and imaging characteristics of these 5 cases were summarized.Results The 5 cases had an onset age ranging from 2 to 17 years and a disease duration ranging from 1 to 20 years.All of them experienced daily seizures,especially at night.The seizure duration was less than 30 seconds,and consciousness recovered rapidly.Among the cases,3 exhibited hypermotor seizures of typeⅠorⅡ,characterized by body turning along the horizontal body axis.Two of them experienced laughter during the seizures,while 1 showed a fearful expression.The remaining 2 cases presented with symmetric tonic seizures,involving the facial muscles.One case reported indescribable aura,and 2 cases had autonomic symptoms.During the interictal period,all 5 cases showed epileptic discharges predominantly in the frontal region on EEG,with lateralization value present in only 2 cases.During the ictal period,4 cases demonstrated general low volatility and fast activity(LVFA),while 1 case showed low-frequency rhythmic sharp and slow waves originating from the lesioned side.Four cases underwent SEEG,which revealed seizure starting from the frontal operculum and adjacent electrodes with LVFA,rapidly spreading to the insula,insular opercular,and medial frontal lobe.Positive changes were observed in the MRI of 4 cases,including 2 cases with possible cortical dysplasia,1 case with tuberous sclerosis,and 1 case with encephalomalacia foci.All 4 cases underwent SEEG guided radiofrequency thermocoagulation,resulting in seizure frequency reduction.Conclusions Frontal opercular epilepsy is mainly characterized by hypermotor seizure with body turning along the horizontal body axis or symmetric tonic seizure.These seizure may be accompanied by emotional symptom or facial muscle tonic,but aura and autonomic symptom are less common.The lateralization value of EEG is limited in frontal opercular epilepsy.SEEG indicates early involvement of the insula,insular opercular,and medial frontal lobe.

Objective:To investigate the genetic safety of allogeneic bone marrow mesenchymal stem cells (BMSCs) transplantation in traumatic brain injury (TBI).Methods:(1) In vivo experiment: BMSCs from male SD rats were isolated and cultured. Moderate TBI models were prepared by implanting and fixing micro-drug injection cannula into the left ventricle of 12 female SD rats, and 3 d after that, striking the right cerebral cortex of the rats with pneumatic precision percussion device was performed. Four h, and 3, 6, 9, and 12 d after modeling, TBI rats were given a single/multiple BMSCs infusion (2.5×10 5/time, total volume 10 μL) by cannula; 48 and 72 h, and 10 and 14 d after modeling, brain tissues of TBI rats (3 at each time point) were prepared into paraffin specimens. Immunofluorescent staining was used to detect the microglia activation, and RNAscope ? technology was used to detect the co-localization of astrocytes, neurons, microglia and transplanted BMSCs to observe whether the allogeneic BMSCs were integrated with the host brain cells after transplantation into TBI host. (2) In vitro experiment: the frozen and revived microglial cell line BV2 was transfected with green fluorescent protein (GFP)-positive lentiviral particles, and then, BMSCs prelabeled with pHrodo RED probe and BV2 cells pretreated with lipopolysaccharide were co-cultured in a certain ratio (BV2:BMSCs=1:1, 1:2, 2:1); after 36 and 72 h of co-culture, the phagocytosis between the 2 kinds of cells was observed under confocal fluorescence inverted microscope to observe the specific action forms of microglia on BMSCs. Results:(1) In vivo experiment: 48 and 72 h, and 10 and 14 d after modeling, no colocalization of transplanted BMSCs with astrocytes or neurons was found in paraffin sections of brain tissue in TBI rats; however, 10 and 14 d after modeling, microglia in TBI rats were obviously activated and migrated to the left lateral ventricle and choroid plexus, and co-localization of microglia with transplanted BMSCs was observed. (2) In vitro experiment: phagocytosis occurred after co-culture of BV2 cells at different proportions with BMSCs for 36 and 72 h. Conclusion:After transplantation, allogeneic BMSCs do not integrate with astrocytes or neurons of the TBI host, but they could be phagocytosed by microglia, indicating that allogeneic BMSCs transplantation for TBI is genetically safe.