OBJECTIVE To prepare the Graves disease (GD) mouse model through porcine thyroid globulin (PTG) injection and investigate the morbidity and stability of the model. METHODS C57BL6/N mice in model group received multi-point subcutaneous injection of PTG 25μg each week,six times in all. After the end of immunization,their heart rate and oxygen consumption were measured and serum triiodothyronine(T3)level was determined every two weeks. A model was considered successful if serum T3 level was higher than x+3s of the control group. Observation of the model lasted 12 weeks. At the 12th week,spleen and thymus gland indices,serum thyroid globulin antibodies and thyroid peroxidase antibodies were measured,and the thyroid glands were taken for pathological observation. RESULTS After six times of immunization,mice in model group showed increased heart rate(P<0.01),oxygen consumption(P<0.01)and T3 level(P<0.01)compared with control group. The morbidity was 77.7%for male mice and 88.8%for females. In addition,T3 level in model group remained higher than that in control group within 12 weeks after immunization. The T3 level tended to decrease in male mice,but remained at a relatively stable higher level in females. CONCLUSION This method is suitable for GD modeling due to its short model-making time,high morbidity and long durability.

Objective miRNA-122 levels may correlate with liver cancer prognosis,and therefore understanding its expression is crucial for future treatment.This study investigates the effect of DNA methylation on the expression of liver specific miRNA-122 and its effects on proliferation and apoptosis of hepatocellular carcinoma cells.Methods Methylation sequencing detected the methylation of the miRNA-122 promoter region,and the level of miRNA-122 expression was measured by using real-time quantitative PCR.The proliferation and apoptosis of hepatocellular cell lines were detected by flow cytometry and CCK8.Results Compared with human primary hepatocytes [(21.9 ± 11.4)％],the level of miRNA-122 promoter methylation in Huh7,HepG2,and QSG-7701 cell lines were (87.6±9.3) ％,(89.0 ± 14.3)％,and (69.5 ±11.5)％,respectively.This represents a significant increase (P=0.000),especially in Huh7 and HepG2 cell lines.Compared with human primary hepatocytes (2.83× 104 ±3746),the levels of miR-122 expression in the above three cell lines were significantly decreased,especially in Huh7 and HepG2 cell lines (P=0.007).After treatment with 5-Aza-dc,the degree of methylation in Huh7 and HepG2 cell lines were significantly lower than that of the blank group (P=0.038,P=0.025),and the levels of miRNA-122 expression were significantly elevated (P=0.008,P=0.003).Also,compared with the control groups,the apoptosis of Huh7 cells and HepG2 cells were significantly increased (P=0.001,0.027).Conclusion The expression of miRNA-122 is regulated by DNA methylation and correlated with the apoptosis of liver cancer cells.Therfore,the methylation regulation of miRNA-122 expression might be involved in the occurrence and development of hepatocellular carcinoma.

BACKGROUND: A skull defect is inevitable after decompression treatment for traumatic brain injury. Titanium mesh as the most recognized skull repair material has good biocompatibility and has been widely used in clinical practice. However, the timing for skull repair after brain injury is still in dispute.OBJECTIVE: To compare the changes of brain perfusion and the recovery of neurological function in patients with skull defects before and after early and late-stage titanium mesh repair based on CT perfusion technique.METHODS: This was a single-center, prospective, observational clinical trial that was completed at the Taihe Hospital,Hubei University of Medicine in Hubei Province, China. Eighty-six patients with craniocerebral injury who had undergone decompression with removal of bone flap from January 2013 to January 2016 were recruited and subjected to skull repair using titanium mesh. All the patients were randomized into two groups: test group (n=40) with early skull repair within 1-3 months after decompression and control group (n=46) with late-stage skull repair within 6-12 months after decompression. CT perfusion technology was used to observe changes of brain perfusion at 3 days operatively and at 10 days postoperatively. The Barthel index was evaluated at 30 days postoperatively. The trial was registered with ClinicalTrial.gov (identifier: NCT03222297) on July 12th, 2017. The study protocol was approved by the Ethics Committee of Taihe Hospital with the approval No. 2012 (08), and performed in accordance with the Declaration of Helsinki,formulated by the World Health Organization and the hospital's ethical requirements for human research. All the patients and their families were voluntary to participate in the trial, were fully informed of the trial process, and then signed the informed consent prior to the initialization of the trial.RESULTS AND CONCLUSION: The postoperative cerebral blood volume and cerebral blood flow at the parietal cortex on the side of skull defect and at the cortex in the defect region were significantly higher in the two group than the baseline (P < 0.05), while the time to peak was lower than the baseline (P < 0.05). Compared with the control group,significantly higher cerebral blood volume and cerebral blood flow as well as shorter time to peak were observed in the test group (P < 0.05). The Barthel index of the test group was also significantly higher than that of the control group at 30 days postoepratively (P < 0.05). Overall, early skull repair with titanium mesh is helpful to improve the cerebral blood perfusion at the affected side and the recovery of neurological function. In addition, CT perfusion technology is a safe and effective method to monitor hemodynamic changes in the brain.

Objective To evaluate the etiology, pathogenesis, clinical manifestation, imaging features, treatments and factors related to prognosis of acute spinal spontaneous hematoma.Methods The clinical data of 38 patients with acute spinal hematoma treated in our hospital from 2011 till now were analyzed retrospectively.Duration of follow-up was 6 months.The factors influencing the prognosis were analyzed.Results Acute epidural hematomas (n=29) were much more common than subdural (n=5), subarachnoid (n=1) and intramedullary (n=3).Most hematomas were located in the cervical and thoracic vertebra regions.The etiology of acute spinal spontaneous hematoma was unknown in most patients.Twenty-nine patients were dealt with surgical intervention and 9 patients were treated conservatively.After 6-month follow up, recovery rate measured by JOA score in patients of spinal injury ASIA level A and B was (51.26 ±38.97), and level C, D and E was (80.33 ±25.83), P<0.05.Recovery rate in patients with hematoma discovered in less then 24 hours treated with surgical decompression was (64.79 ±36.10), and that in those with hematoma present over 24 hours was (34.54 ±30.17), P<0.05.Conclusions Acute spinal hematoma always caused by unknown etiology, and usually manifests itself in a sudden onset of pain and neurological deficits.The early diagnosis mainly depends on MRI.Patients presenting with severe neurologic dysfunction or showing signs of progressive deficit should have immediate surgical intervention. The status of neurological deficits before surgery and the length of interval between onset and surgical intervention are associated with recovery.

In this study, the effect of heparin-derived oligosaccharide (HDO) on platelet-derived growth factor (PDGF) induced vascular smooth muscle cells (VSMCs) proliferation and the related signal transduction mechanisms were investigated. MTT assays were used to measure VSMCs proliferation. Cell cycle distribution was analyzed by flow cytometry. The level of key regulatory proteins in PKC, MAPK and Akt/PI3K pathways were determined by RT-PCR, Western blot and immunocytochemical methods. Meanwhile, mRNA expressions of some proto-oncogenes were assayed by RT-PCR method. Our data showed that HDO (0.01, 0.1 and 1 μmol · L(-1)) inhibited 30 ng · mL(-1) PDGF-induced VSMCs proliferation in a dose-dependent manner, blocked the G1/S transition and inhibited the level of key regulatory proteins and some proto-oncogenes (P < 0.05). The results showed that HDO may decrease the key regulatory proteins expression, hence suppress the transcription of proto-oncogene and G1/S transition, finally inhibiting VSMCs proliferation.

Objective To investigate the mechanisms of fluconazole resistance in clinical and experimental induced isolates of C.glabrata.Methods Efflux of rhodamine 6G was performed to evaluate the effects of efflux pumps.The expression levels of transporter genes CDR1,CDR2,SNQ2 and ERG11 were examined by real-time RT-PCR.Meanwhile,sequence of PDR1 was determined by PCR based DNA sequencing.Results Efflux pumps of all fluconazole-resistant isolates had stronger effects than that of susceptible isolates,consistently with significant upregulation of CDR1,but no obvious difference was found in CDR2 or SNQ2.Also,no notable change in the expression level of ERG11 between susceptible and resistant isolates.PDR1 mutations existed in both clinical and experimental induced isolates of C.glabrata,among which P927S,L543P and S947L haven't been reported previously.Conclusion Mutations of PDR1 were induced by fluconazole both in vivo andin vitro,which will result in overexpression of CDR1 and strengthen the effect of efflux pump.

Objective To assess the application value of REP-PCR in genotyping of candida glabrata strains in clinical pratice. MethodsFrom 2009 to 2010, thirty-eight candida glabrata strains were isolated from Shanghai Ruijin Hospitals, Shanghai Renji Hospital, Shanghai Huashan Hospital, Anhui Medical University Hospital, Shenzhen People's Hospital. Six loci in housekeeping genes (FKS, LEU2,NMT1, TRP1, UGP1 and URA3 ) were amplified and sequenced. The sequences were compared with the MIST database and allele profile and sequence type (ST) were obtained. With primers Ca21, Ca22 and Com21 used to amplify the adjacent variable gene regions,the amplicons were analyzed through electrophoresis to generate different REP-PCR types. Finally, the results of these two genotyping methods were compared. ResultsFor REP-PCR, Ca22-Com21 has the best genotyping effect. REP-PCR and MLST have the same genotyping results. Five REP-PCR types were found in 38 candida glabrsta isolates. Type A,B, C, D and E strains from REP-PCR were genotyped as ST 7, 3, 19, 45 and new type respectively byMIST. REP-PCR saves time compared with MIST. Conclusions REP-PCR offers a simple and rapid method for molecular typing, with similar discriminatory power with MIST. Therefore, REP-PCR can be the preferred choice in laboratory, especially for a large number of isolates.

Objective·To prepare chitosan-gelatin porous microspheres by high voltage electrostatic method combined with freeze-drying and ionic cross-linking method and investigate the factors that influence the formation of porous medium.Methods·Porous chitosan microspheres and chitosangelatin porous microspheres were prepared using high voltage electrostatic method combined with freeze-drying and ionic cross-linking method,with sodium tripolyphosphate (STPP) as crosslinking agent.Factors that affect the porous structure and pore size of porous microspheres were compared,such as different chitosan-gelatin ratio,freezing temperature,curing time with saturated STPP in 85％ ethanol solution.The morphology,surface and internal structure,particle size of the porous chitosan microspheres and chitosan-gelatin porous microspheres were observe by using light microscope,scanning electron microscope and hematoxylin-eosin staining.Results·Microspheres prepared by freeze-drying with an electrostatic and ionic cross-linking method have open,interconnected and highly macroporous,with good spherical surface.Saturated STPP ethanol solution (85％ ethanol) was chosen as the crosslinking agent to prevent destruction of the porous structure.The order of freeze and crosslinking,cross-linking time and the second freezing temperature,can influence the pore size of porous microspheres.Gelatin and chitosan can form polyelectrolyte complexes,and can also be used as porogen in porous structure.Conclusion·The preparation of porous chitosan-gelatin microspheres via this method has a large pore size (diameter 100-200 μm),suitable for cell growth and the migration.

Objective To evaluate the role of C-type lectin domain family 2,member B (CLEC2B) gene in the pathogenesis of vitiligo.Methods Real time fluorescence-based PCR was performed to detect the expression of CLEC2B mRNA in the peripheral blood and lesional skin of 37 patients with vitiligo as well as in the peripheral blood and normal skin of 40 healthy controls.Data were statistically analyzed by t test and chisquare test.Results Among the 37 patients,23 had progressive vitiligo,14 stable vitiligo,31 vitiligo vulgaris,6 segmental vitiligo.The expression level of CLEC2B mRNA was significantly higher in vitiligo lesions than in the control skin (1.21 ± 0.03 vs.1.00,t =4.432,P ＜ 0.05),but was of no significant difference in peripheral blood between the patients and healthy controls (1.02 ± 0.05 vs.1.00,t =1.435,P ＞ 0.05).Increased expression of CLEC2B mRNA was noted in lesions of vulgaris vitiligo compared with those of segmental vitiligo (1.21 ± 0.03 vs.1.02 ± 0.01,t =5.330,P ＜ 0.05),as well as in lesions of progressive vitiligo compared with those of stable vitiligo (1.25 ± 0.05 vs.1.08 ± 0.03,t =3.046,P ＜ 0.05).No significant difference was observed in the expression of CLEC2B mRNA among lesions of vitiligo with different courses (P ＞ 0.05).Conclusion The differential expression of CLEC2B mRNA may take part in the pathogenesis of vitiligo.

Objective To investigate the clinical efficacy and safety of carotid endarterectomy (CEA)and carotid artery stenting(CAS)for the treatment of carotid artery stenosis in the elderly.Methods Clinical data of 116 elderly patients aged over 65 years with carotid artery stenosis were retrospectively analyzed.Of 116 patients,73 patients underwent CAS(the CAS group) and 32 received CEA(the CEA group).The success rate,30-day perioperative complications and follow-up results were compared between the two groups.Results There was no significant difference in the success rate (96.8％ vs.100.0％,P ＞ 0.05),30-day perioperative complications,such as bradycardia (6.25％ vs.4.5％,x2 =0.228,P=0.663),acute myocardial infarction(0.0 vs.1.4％,x2 =0.432,P=0.511),transient hypotension(6.3％ vs.8.1％,x2 =0.114,P =0.735),ischemic stroke(6.3％ vs.6.8％,x2 =0.009,P =0.923),and cerebral hyperperfusion syndrome (18.8 ％ vs.10.8％,x2 =0.009,P =0.923),between the CEA and CAS groups.The incidence of persistent hypotension was lower in the CEA group than in the CAS group(3.1％ vs.17.6％,x2 =4.398,P=0.036).No significant difference was found in carotid artery restenosis(moderate:6.3％ vs.8.1％,x2 =0.114,P =0.735;severe:3.1 ％ vs.2.7％,x2=0.014,P=0.905)and ipsilateral stroke(3.1％ vs.5.4％,x2 =0.279,P=0.598)between the CEA and CAS groups at one-year fellow-up.Conclusions Both CEA and CAS have good effieacies in treating carotid artery stenosis in the elderly,while the incidence of persistent hypotension is higher with CAS than with CEA.