Objective:To construct a TPC-1 cell model that stably knocks out the HMGA2 by using CRISPR/Cas9 gene editing technology. Methods:Recombinant pLV[2gRNA]-EGFP:T2A:Puro- U6> {hHMGA2 [gRNA# A1]*}- U6>{hHMGA2 [gRNA#A2]*} of lentiviral plasmid vector was constructed: targeting HMGA2 Dual-gRNA sequence was designed, the synthesized Dual-gRNA fragment into pLV [2gRNA]-EGFP was cloned: T2A:Puro-U6 vector, extract a single clone for sequencing verification. the constructed recombinant plasmid vector with lentivirus was packed, and TPC-1 cells were infected, puromycin was used to obtain HMGA2 knock-out single clone, PCR and sequencing verification were performed, and real-time fluorescent quantitative qPCR was used to detect HMGA2 mRNA in cells Knockout efficiency. Results:After sequencing verification, pLV [2gRNA]-EGFP targeting HMGA2: T2A: Puro-U6>{hHMGA2 [gRNA#A1]*}-U6>{hHMGA2 [gRNA #A2]*} plasmid was successfully constructed; A single clone was picked for PCR identification and gene sequencing, TPC-1 cells were successfully obtained with HMGA2 gene completely knocked out; TPC-1 cells with HMGA2 knocked out were detected by real-time fluorescent quantitative qPCR, and they did not express HMGA2 mRNA.Conclusion:CRISPR/Cas9 gene editing technology enables us to construct a human papillary thyroid cancer cell line TPC-1 cell model with stable knockout of HMGA2.

Objective To investigate the relationship between apoptotic protein Bcl-2 and Bax in rat hippocampal neurons and cognitive dysfunction in rats with abnormal thyroid function.Methods Thirty healthy Wistar rats of 8-week-old SPF grade were randomly divided into three groups:(1) Normal control group (n=10);(2)hypothyroidism group (n=10);(3) hyperthyroidism group (n=10).All rats were sacrificed at the 4th week by heart blood sampling.The serum T3 and T4,TSH were measured.Morris water maze was used to train rats in each group for 6 days.At the end of the experiment,the hippocampus was taken from the rats,and HE staining was performed.The expression of apoptotic protein bcl-2 and Bax in rat hippocampal neurons was detected by immunohistochemistry.Results ①The escape latency of hyperthyroidism and hypothyroidism group was higher than that of the normal group at different time points (P＜0.05).In the test of the target area dwell time,the difference between the hyperthyroid group,the hypothyroid group and the normal group was statistically significant (P＜0.05).In the distance test of the target quadrant,The differences between the three groups were statistically significant (P＜0.05).In the number of passes through the target quadrant,the difference between hyperthyroidism group,hypothyroidism group and normal group was statistically significant (P＜0.05),but there is no significant difference between hyperthyroidism group and hypothyroid group (P＞0.05).②Hippocampus tissue HE staining:normal control group hippocampal neurons neatly arranged,the shape of the structure was complete and uniform,the nucleus was round or oval,nucleolus obvious,chromatin uniform level and more clear,nucleus round or oval,obviously,hyperthyroidism group,hypothyroidism group of neuronal structure loose,the number decreased,arranged disorder,deep nuclear staining,shrinkage,nucleolus disappeared,cytoplasm around the empty halo,cell spacing larger.③The positive cells expressing Bcl-2 and Bax protein in the hyperthyroidism group and the hypothyroidism group were increased compared with the normal control group (P＜0.05).Compareds with hyperthyroidism group,the expression of Bcl-2 positive cells was increased in hypothyroidism (P＜0.05).Conclusions ①The spatial learning and memory abilities of the rats with hypothyroidism and the hyperthyroid are lower than those in the normal control group.②The number of apoptotic protein positive cells in Bcl-2 and Bax neurons of hippocampus in rats with hypothyroidism and hyperthyroid increased,and the proportion of Bcl-2 and Bax was impaired,which indicates that apoptosis occurred in hippocampal neurons.This process may be one of the pathogenesis of cognitive impairment.

Objective:To investigate the expression of immunoglobulin A (IgA) in human mesangial cells (HMCs).Methods:The HMCs were cultured.The subcellular location of IgA was detected by immunofluorescence staining;the transcripts of Ig α,Ig κ and Ig λ constant region were detected by reverse transcription-polymerase chain reaction (RT-PCR) and further analyzed by DNA sequencing.The expressions of Igαt and Ig λ were detected at transcription level by Western blot after the cytoplasmic protein extraction.The culture supernatant was collected to explore whether IgA could be secreted out of the cell and the protein was further analyzed by mass spectrometry after being purified by affinity chromatography with jacalin-sepharose.The results of DNA sequencing and mass spectrometry were aligned with the mRNA and amino acid sequences in the National Center of Biotechnology Information (NCBI) data-base.Results:By immunofluorescence staining,we detected the presence of IgA heavy chain Ig α,light chain,both Ig κ and Ig λ in expressions of transcripts of Ig α1,Ig α2,Ig κ and Ig λ in the HMCs and the alignment of the sequences of the RT-PCR products with those of the Ig Cα1,Ig Cα2,Ig κ and Ig λ mRNA in the NCBI database exhibited that the similarities were 99％,97％,98％ and 97％,respectively.Western blot showed Ig α and Ig λ expressions in the cell lysate and secretion of Ig α1 and Ig α2 heavy chains in cell culture supernatant.To further explore the protein that secreted into the supernatant,after supernatant affinity chromatography with jacalin-sepharose,the proteins were separated by sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) and the band approximating to 65 000 was cut and sent to mass spectrometry.The results were aligned with the amino acid sequences of Ig α1 and Ig α2 constant region in NCBI database,showing that amino acids between No.52 and No.104,amino acids between No.154 and No.221,amino acids between No.276 and No.327 from Ig Cα1 and amino acids between No.52 and No.113,amino acids between No.151 and No.204,amino acids between No.251 and No.314 from Ig Cα2 were the same with those derived from B cells.Conclusion:Our findings suggested that HMCs could synthesize and secret IgA.

Objective To study plasma coagulation factor Ⅶ effects of intracerebral hemorrhage in patients with craniocerebral injury.Methods 120 cases of patients with traumatic brain injury were treated,for patients admitted to hospital,the hospital after 24 hours,48 hours of live clotting enzyme activation part (APlT),peripheral venous blood specimen testing international standardization ratio (INR)and the activity of platelet and F Ⅶ were detected.According to the intracranial bleeding lesions was expanded or the emergence of a new bleeding lesions,and so on and so forth were divided into research group and the control group,43 patients in the research group,77 cases in the control group.Results The study showed that the two groups of patients with injury to the first CT time, subarachnoid hemorrhage,epidural hematoma,there were no significant difference between the indexes of subdural hematoma,patients in the study group lost 48h PPSB was (653.2 ±489.8)IU,platelet (180.7 ±63.5)mL,plasma (582.7 ±411.3)mL and red blood cells (612.3 ±490.1)mL,which were higher than those of the control group [(465.7 ±278.8)IU,(0.0 ±0.0)mL,(335.1 ±261.9)mL,(378.3 ±46.3)mL],there were statistifically significant differences between the two groups(t =2.399,2.388,2.582,3.231,P =0.020,0.022,0.010,0.001), the platelet and F Ⅶ of the research group were (101.43 ±41.85)×109 /L,(93.04 ±20.98)%,which were lower than those of the control group[(128.37 ±51.49)×109 /L,(107.67 ±20.25)%],there were statistifically significant differences between the two groups(t =2.583,2.893,P =0.010,0.004).Conclusion Lower levels of platelet activity and F Ⅶ of closely associated with intracranial hemorrhage in patients with craniocerebral injury,according to the clinical indicators to predict whether patients with intracranial hemorrhage,in order to for the treatment of patients with timely and accurate to ensure the patient's life and health.

Objective To investigate the effect of high serum thyroid stimulating hormone (TSH) on malignant transformation of thyroid tissue in Hashimoto's thyroiditis (HT).Methods The data of 569 patients pathologically confirmed HT admitted from Jan.2009 to Dec.2013 were retrospectively analyzed.According to with or without papillary thyroid carcinoma (PTC),patients were divided into HT group and HT complicated with PTC group.Age,gender,preoperative TSH,free thyroxine,free tri-iodothyronine,thyroidperoxidaseantibody (TPOAb),antithyroglobulinanfibody (TGAb),and tumor multifocality were investigated.Results Among the 569 patients,306 patients had HT without PTC and 263 had HT with PTC.The avaerage age of patients complicated with PTC was (45.52±10.58) years,younger than that of patients without PTC (51.63+9.50) years (P＜0.05).In HT patients with or without PTC,31％ (81/263) and 22.5％ (69/306) of patients had high TSH levels,which was significantly different (P＜0.05).Among HT patients complicated with PTC,tumor multifocality was identified in 32％(26/81) of patients with high level of TSH and in 29％(52/182) of patients with normal level of TSH.The difference had no statistical significance (P＞0.05).Conclusion High preoperative TSH level may stimulate malignant transformation of thyroid tissue in HT patients.

Objective To elucidate the difference between methicillin-resistant Staphylococcus aureus (MRSA)and methicillin-sensitive S.aureus (MSSA)in terms of genotypes and distribution of virulence genes with the clinical strains isolated from Hohhot,and explore the relationship between the changing resistance of S.aureus and the virulence transition.Methods Pulsed field gel electrophoresis (PFGE)and multi locus sequence typing (MLST)methods were employed to do molecular typing for 30 MRSA strains and 30 MSSA strains isolated from inpatients in Hohhot,Inner Mongolia.PCR method was used to profile the distribution of virulence genes among these strains.Results PFGE typing results showed that 60 S.aureus strains were classified into 19 major types.MSSA strains belonged to 16 types,mainly types I and H.MRSA strains mainly belonged to types of K and M.Among the 20 strains with different PFGE types,MRSA strains were mainly identified as ST-239 type.but the prevalence of sec ,seg ,sei,sem,sen,seo,fnbB ,ebpS and cap 5 was higher in MSSA strains than in MRSA strains (P<0.05).Conclusions The clinical strains of S .aureus isolated from Hohhot showed diverse genotyping features.ST-239 was the major PFGE type of MRSA strains.The prevalence of virulence genes was higher in MSSA strains than in MRSA strains. Characteristic cluster is found for specific virulence genes.The results also suggest that acquisition of specific antibiotic resistance may be associated with change of specific virulence feature in S.aureus.

To evaluate the reforming effects of health service performance appraisal and distribution motivation system at our center,discuss some questions during the development and application of such an evaluation system and conduct comparative analysis of the policies and data before and after management pilot separation between revenue and expenditure.The performance evaluation system and distribution mechanism were established so as to boost the working efficiency of community health services and ensure their sustained and healthy developments.

Objective To investigate the clinical applicated value of simple transumbilical single-tunnel laparoscopic renal cyst decortication.Methods 7 patients with renal cysts were treated with simple single-tunnel laparoscopic renal cyst decortication.Results The operations of 7 patients were succeed.The mean operative time was 37 (28 ～ 90) min,and the mean time of pulling the drain tube out was 2 (1 ～ 4) d,and the mean int aoperative blood loss was 30 (25 ～ 80) ml,and the mean bed stay was 1.5 (1 ～ 3) d,and the mean hospital stay was 4 (3 ～ 8) d.In 7 patients,no recurrence of the cyst occurred during a follow-up of 1 ～ 6 months by color-ultrasonography,CT after operation.Conclusion The simple transumbilical single-tunnel laparoscopic renal cyst decortication has advantages of economic,minimal trauma,hairdressing,rapid recovery and better effect,therefore it maybe an ideal treatment choice for renal cysts.

ObjectiveTo analyze the diagnosis and treatment of patients with delayed traumatic intracranial hematoma.MethodsClinical data of 50 patients with delayed traumatic intracranial hematoma were retrospectively analyzed.According to the Glasgow Outcome criteria,the prognosis of patients recovery results were assessed.ResultsIn 50 patients after treatment,a good recovery in 29 cases,moderate disability in 11 cases,5 cases of severe disability,vegetative state survival in 3 cases,2 deaths.ConclusionDelayed traumatic head injury caused by intracranial hematoma should be early diagnozed and timely treatment can improve the prognosis of patients and improve survival.

BACKGROUNDAlpha 2A adrenergic receptor (AR) is a subtype of α2 AR belonging to G protein-coupled receptors, and exerts a variety of biological effects. Recent studies have demonstrated that the α2A AR activation was closely related with inflammatory reaction. The present study aimed to investigate the influence of α2A AR antagonist, yohimbine, on the severity of endotoxin-induced acute lung injury in rats.

METHODSA total of 72 male Sprague-Dawley rats were randomly divided into three groups: control group, lipopolysaccharide (LPS) group and LPS + yohimbine group. Rats were intratracheally administrated with normal saline or LPS (300 µg), and the rats in the LPS + yohimbine group were treated with additional yohimbine (2 mg/kg, i.p) soon after LPS administration. Six, 24 and 48 hours after treatment, arterial blood gas analysis was carried out, and optical microscopy was performed to evaluate pathological changes in the lung, and lung injury score was assessed. The count of white blood cells in bronchoalveolar lavage fluid (BALF) was determined. The levels of norepinephrine, tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 in BALF were measured with enzyme-linked immunosorbent assay. Immunocytochemistry was performed for the detection of α2A AR on inflammatory cells in BALF.

RESULTSWhen compared with the control group, the oxygenation index in the LPS group was significantly decreased, and white blood cell count, the lung histopathological scores, levels of norepinephrine and IL-6 as well as α2A AR expression on inflammatory cells in the BALF were dramatically increased at different time points, and the concentrations of TNF-α and IL-1β were also increased except at 48 hours after LPS administration. The oxygenation index decreased while white blood cell count in BALF and the lung histopathological scores were obviously increased in the LPS + yohimbine group. The level of norepinephrine in BALF was increased at each time interval in the LPS + yohimbine group, and so did the levels of TNF-α, IL-1β and IL-6 at 6 and 48 hours after LPS administration respectively. When compared with the LPS group, the oxygenation index, white blood cell count, the lung histopathological scores and the level of IL-6 in the LPS + yohimbine group were significantly improved at each time interval, and the concentrations of TNF-α and IL-1β were also lower at 24 hours of LPS administration (all P < 0.05). Correlation analysis indicated the level of norepinephrine was related to the levels of TNF-α, IL-1β and IL-6 in the BALF and the lung histopathological scores (r = 0.703, r = 0.595, r = 0.487 and r = 0.688, respectively, P < 0.001) and the intensity scores of immunoreactivity to α2A AR on inflammatory cells were also associated with the levels of TNF-α, IL-1β and IL-6 as well as the lung histopathologial scores (r = 0.803, r = 0.978, r = 0.716 and r = 0.808, respectively, P < 0.001).

CONCLUSIONSYohimbine can inhibit TNF-α, IL-1β and IL-6 overproduction and relieve the severity of pulmonary inflammation induced by endotoxin, which is maybe mediated by blockade of α2A AR on inflammatory cells.

Acute Lung Injury ; chemically induced ; drug therapy ; Adrenergic alpha-2 Receptor Antagonists ; therapeutic use ; Animals ; Bronchoalveolar Lavage Fluid ; chemistry ; Enzyme-Linked Immunosorbent Assay ; Immunohistochemistry ; Interleukin-1beta ; metabolism ; Interleukin-6 ; metabolism ; Lipopolysaccharides ; toxicity ; Male ; Norepinephrine ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptors, Adrenergic, alpha-2 ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism ; Yohimbine ; therapeutic use