One of the main pathological features of airway inflammatory diseases is hypersecretion of airway mucus, which is manifested by goblet cell hyperplasia and mucociliary clearance dysfunction. In recent years, it has been found that the molecular structure of calcium activated chloride ion channels, transmenbrane protein 16A（TMEM16A）, is closely related to airway mucus hypersecretion.TMEM16A not only mediates ion transepithelial transport and hydration, but also participates in the regulation of mucin secretion. TMEM16A is highly expressed in airway epithelium of a variety of inflammatory diseases of upper and lower airway, such as asthma, cystic fibrosis, allergic rhinitis, chronic sinusitis and so on. Understanding the expression level and regulation mechanism of TMEM16A in different airway diseases and revealing its physiological function and pathological mechanism is critical for targeted disease treatment. This paper summarizes the research status of the discovery process, structural characteristics and regulatory mechanism of TMEM16A, and then summarizes the expression level of TMEM16A in asthma, cystic fibrosis, allergic rhinitis and chronic sinusitis ant related pathological mechanisms, clarifies the potential value of TMEM16A as a therapeutic target for the above four diseases, in order to guide treatment of airway inflammatory diseases.
Humans ; Asthma/metabolism* ; Anoctamin-1 ; Cystic Fibrosis/metabolism* ; Neoplasm Proteins/metabolism* ; Sinusitis/metabolism* ; Chloride Channels/metabolism* ; Rhinitis, Allergic/metabolism* ; Inflammation

ObjectiveTo explore the possible mechanism of Bimin Formula （鼻敏方） in treating lung-spleen qi deficiency syndrome of allergic rhinitis （AR） with high mucin secretion. MethodsThirty-four SD rats were randomly divided into a blank group （8 rats）， a model group （8 rats）， a low-dose Bimin Formula group （8 rats）， and a high-dose Bimin Formula group （10 rats）. Except for the blank group， the other groups were subjected to AR lung-spleen qi deficiency rat models induced by smoking， gavage of Ginkgo biloba leaf extract， and ovalbumin. After modeling， rats in the low- and high-dose Bimin Formula groups were given Bimin Formula concentrate （concentration of 2.16 g/ml） by gavage at doses of 1.08 g/100 g and 2.16 g/100 g， respectively， while rats in the model group were given 0.5 ml/100 g of normal saline by gavage， once daily for 28 days； the blank group was not intervened. Behavioral assessments were performed after intervention. ELISA was used to detect the levels of peripheral blood total immunoglobulin E （IgE）. HE staining was used to observe the pathological changes of nasal mucosa epithelium in rats， while immunohistochemistry was used to detect the expression of transmembrane protein 16A （TMEM16A） and mucin 5AC （MUC5AC） protein in nasal mucosa. Western Blot was used to detect the expression of nuclear factor kappa B （NF-κB） protein， and RT-PCR was used to detect the expression of TMEM16A， MUC5AC， and NF-κB mRNA in nasal mucosa. ResultsHE staining showed that the nasal mucosa epithelial cell structure in the blank group was intact without shedding， swelling， or necrosis； the nasal mucosa epithelial tissue of rats in the model group was thickened and partially shed， with infiltration of eosinophils and lymphocytes visible； the pathological changes in nasal mucosa tissue of rats in the high- and low-dose Bimin Formulagroups were improved， and more improvement was showen in the high-dose group. Compared with those in the blank group， the behavioral scores and peripheral blood total IgE levels of rats in the model group significantly increased， as well as the expression of TMEM16A， MUC5AC， and NF-κB proteins and mRNA in nasal mucosa （P＜0.05 or P＜0.01）. Compared with those in the model group， the behavioral scores and peripheral blood total IgE levels of rats in the high-dose Bimin Formula group decreased， and the expression of TMEM16A， MUC5AC， and NF-κB proteins and mRNA in nasal mucosaalso decreased （P＜0.05 or P＜0.01）； the behavioral scores and peripheral blood total IgE levels of rats in the low-dose Bimin Formula group were reduced， and the expression of TMEM16A and MUC5AC proteins and mRNA in nasal mucosa， as well as the expression of NF-κB protein decreased （P＜0.05 or P＜0.01）， but the difference in NF-κB mRNA expression was not statistically significant （P＞0.05）. Compared with the low-dose Bimin Formula group， the expression of NF-κB protein in the high-dose group decreased （P＜0.01）. ConclusionBimin Formula may improve the symptoms and high mucus secretion of AR lung-spleen qi deficiency by regulating the TMEM16A/NF-κB/MUC5AC signaling pathway in nasalmucosa.

Objective To investigate the influence factors of serum CA125 level in patients with ovarian chocolate cysts,and to study the effect on serum CA125 level of interventional therapy.Methods A total of 103 patients with single unilateral chocolate cyst of ovary underwent interventional treatment.According the serum CA125 level before interventional therapy,the patients were divided into normal group (CA125≤35 U/ml) and abnormal group (35 U/ml＜CA125≤200 U/ml).The clinical indexes of patients and ultrasound characteristics of cyst were compared between the two groups.The changes of serum CA125 levels before and after interventional therapy were analyzed.Results The difference of the course of diseases,dysmenorrhea history,diameter of cysts had statistical difference between the two groups (all P＜0.05).There were no statistical differences of age,history of dilivery,abortion history,history of pelvic surgery,cyst location between the two groups (all P＞0.05).In abnormal group,the mean serum level of CA125 reduced at 3 months (P＜0.000 1) and 6 months (P ＜0.000 1) after interventional therapy.In the normal group,there was no significant difference of the mean serum level of CA125 before and after interventional therapy (all P＞0.05).Conclusion Serum CA125 level is influenced by dysmenorrhea history,course of disease,diameter of cysts.Ultrasound-guided interventional therapy has intervention effect on patients with abnormal serum CA125 level before interventional therapy.

OBJECTIVETo perform phenotypic diagnosis, genetic diagnosis and prenatal diagnosis of inherited coagulation factor XIII (FXIII)deficiency in a Chinese family also provide a review of inherited coagulation F XIII deficiency.

METHODSThe activity levels of F XIII (F XIII:C) of proband and family members were measured by clot solubility test and REA-chrom F XIII kit. The antigen levels of F XIII(FXIII:Ag) were measured by enzyme-linked immunosorbent assay. Thrombelastography (TEG) test was used to make a comprehensive evaluation of coagulation status in the proband. All 15 exons and exon-intron boundaries of the F13A1 gene were amplified by PCR, and DNA sequencing was performed then. The mutation identified in the proband was screened in the family members. Furthermore, the related literatures were reviewed to provide a profile of clinical manifestation, gene mutations, the relationship between the mutations and phenotype, and treatments of inherited coagulation F XIII deficient cases.

RESULTSThe clot solubility test was positive in the proband. The FXIII:Ag level of the proband was less than 1% and the FXIII:C level was below the lower limit of detection (<3%). Two compound heterozygous missense mutations (p.Arg662* and p.Trp665*) were identified in the proband. Family study showed that the two mutations were both inherited from the parents. The fetus also carried two compound heterozygous mutations, the same as the proband, and was diagnosed with severe F XIII deficiency. As reported in the literatures, most mutations were missense mutations and nonsense mutations, and no hot spot was found. The clinical pattern of F XIII deficiency varied among patients, with potentially fatal consequences from severe bleeding complications.

CONCLUSIONBetter understanding of F XIII biochemical properties and function and developing of FXIII laboratory assays and genetic detection could prevent missed diagnosis, and patients moght benefit from better care.

Asian Continental Ancestry Group ; Base Sequence ; DNA Mutational Analysis ; Enzyme-Linked Immunosorbent Assay ; Exons ; Factor XIII ; genetics ; Factor XIII Deficiency ; genetics ; Female ; Heterozygote ; Humans ; Introns ; Mutation, Missense ; Pedigree ; Phenotype ; Polymerase Chain Reaction ; Pregnancy ; Prenatal Diagnosis

Aim Toinvestigatetheanalgesiceffectsof epidural osthole application on the mechanical allodyn-ia and the ERK/MAPK signaling pathway and the expression of COX-2 mRNA in the spinal dorsal horn.Methods 125adultmaleSDratswererandomizedin-to five groups( n=25 each) :Blank, Sham, NP, Ost and vehicle. At postoperative day 6, 1mg/rat osthole 50 μl was injected epidurally into group Ost and the same volume of vehicle was given into group vehicle. The mechanical pain threshold was measured by 50%MWT at 1 day before operation and the 3 rd,6 th,7 th, 14 th,21 st day after operation. After the measurement of pain threshold on postoperative day 14 , the L4-6 segment of spinal dorsal horn was removed for determi-nation of the expression of ERK, pERK and COX-2 mRNAbyWesternblotandRT-PCR.Results Com-pared with blank group, the mechanical pain threshold was only down-regulated at day 1 after operation in sham group, the expression of pERK and COX-2 mR-NA in sham group showed no significant difference ( P>0. 05 ); the mechanical pain threshold was signifi-cantly down-regulated after operation in NP, Ost and vehicle groups( P<0. 05 ) and the expression of pERK and COX-2 mRNA was significantly increased ( P <0. 05). Compared with vehicle group, the pain thresh-old in Ost group was significantly increased after drug administration( P<0. 05 ) and the expression of pERK and COX-2 mRNA was significantly reduced ( P <0. 05 ) . The expression of ERK showed no significant difference among each group(P>0. 05). The correla-tion analysis on pERK1/2 and COX-2 mRNA revealed the Pearson correlation coefficient was 0 . 878 and 0 . 910 , suggesting a strong positive correlation between pERKandCOX-2mRNA.Conclusions Ostholead-ministrated in the early stage after surgery can alleviate the nucleus pulposus-induced radicular inflammatory pain probably by inhibiting the expression of pERK and COX-2 mRNA in spinal dorsal horn.

Objective To analyze the phenotype and genotype of three patients with yon Willebrand disease (vWD),and to explore its molecular pathogenesis.Methods Bleeding time (BT),APTT,ristocetin induced platelet aggregation (RIPA),von Willebrand factor (vWF):ristocetin cofactor (Rco)(vWF∶ Rco),vWF antigen (vWF∶ Ag),vWF activity (vWF∶ A) test,vWF collagen binding assay (vWF∶ CB) and multimer analysis were detected for phenotype diagnosis.The dynamic process of blood coagulation was evaluated by using the thrombelastography.Genomic DNA was extracted from the peripheral blood.The vWF gene mutation was detected by sequencing.Results APTT,BT were prolonged in the three probands.Plasma vWF∶ Rco,vWF∶ Ag,vWF∶ A and vWF∶ CB were decreased in different degrees.RIPA was reduced in probands B and C.vWF multimer analysis found the lost of the large molecular weight multimers in proband B,while basically normal in probands A and C.The dynamic process of blood coagulation of proband C presented obvious hypocoagulability by using the thrombelastography.Heterozygous missense mutation g.106782G ＞ T resulting in Cys1130Phe in exon 26,g.110988G ＞ A resulting in Gly1579Arg in exon 28 and g.110373C ＞T resulting in Arg1374Cys in exon 28 were found in the probands A,B and C,respectively.Conclusion Three probands were diagnosed as type 1,type 2A or type 2MvWD by phenotype detection.Heterozygous missense mutation Cys1130Phe,Gly1579Arg and Arg1374Cys induced vWD of three probands,respectively.

OBJECTIVETo study the molecular pathogenesis of protein C (PC) deficiency in a patient with pulmonary embolism and in his family members.

METHODSAnticoagulated blood samples were collected from the proband and his family members to detect PC, PS and AT activities. PC antigen level was measured using ELISA. The genomic DNA was extracted to amplify all the 9 exons and their flanking sequences of PC gene using PCR, and the PCR products were sequenced. The mutated exons identified were amplified and sequenced for the other family members.

RESULTSThe proband and his parents and sister were identified as carriers of PC gene mutation, which led to type II PC deficiency. Sequencing of the proband's PC gene showed two heterozygous point mutations in exon 3 (G5540A) and exon 7 (C10230T) to cause compound heterozygous mutations of PC E29K and PC R147W, which were inherited from his father and mother, respectively. His sister was a heterozygote of PC R147W.

CONCLUSIONThe proband is a compourd heterozygous mutations carrier of PC E29K and PC147W. PC E29K is a novel PC mutation, and PC R147W is a reported PC gene mutation seen in patients with type II hereditary PC deficiency and recurrent thrombosis.

Adolescent ; Base Sequence ; Heterozygote ; Humans ; Male ; Molecular Sequence Data ; Pedigree ; Point Mutation ; Protein C ; genetics ; Protein C Deficiency ; complications ; genetics ; pathology ; Pulmonary Embolism ; etiology ; genetics

Objective To analyze the phenotype and genotype of a Chinese family with inherited hypofibrinogenemia,and to investigate its molecular mechanism.Methods Peripheral blood was collected from seven people of this family and then plasma was separated.Activated partial thromboplastin time ( APTT),prothrombin time ( PT),thrombin time ( TT),reptilase time ( RT),the activities of antithrombin( AT∶ A ),protein C ( PC ∶ A ) and protein S ( PS ∶ A ) were tested.The activity and antigen of plasma fibrinogen were analyzed by Clauss method and immunoturbidimetry method,respectively.The fibrinogen peptide chain of the proband was semiquantitatively assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).Thrombin generation test was performed by calibrated automated thromhogram.The dynamic process of blood coagulation was evaluated by the thrombelastography (TEG).Genomic DNA was extracted from the peripheral blood.The sequences of all the exons and exon-intron boundaries of the three fibrinogen genes FGA,FGB and FGG were amplified by polymerase chain reaction ( PCR ) and analyzed by direct sequen(c)ing.Results The activity and the antigen levels of the proband' s plasma fibrinogen were reduced to 0.48 g/L and 0.68 g/L,respectively.TT prolonged to 29.2 s and RT prolonged to 75.8 s.The assays of SDS-PAGE showed no abnormal molecular weight of fibrinogen.Peak height of thrombin generation was reduced to 249.93 nmol/L and endogenous thrombin potential was reduced to 1007.0 nmol · L-1 · min.Hypocoagulability state of the whole blood was found by TEG test.The coagulation index was - 8.6.The proband was diagnosed as inherited hypofibrinogenemia by phenotype analysis.Two mutations (Gln143Pro and g.4642delC) were found in the proband's fibrinogen Aa-chain gene,Gln143Pro came from her mother and g.4642delC came form her father.Conclusion Compound Heterozygous Mutations (Gln143Pro and g.4642delC ) of fibrinogen Aa-chain causes the proband congenital hypofibrinogenemia.

Objective To compare the efficacy of dexmedetomidine versus remifentanil in combination with sevoflurane for gynecological laparoscopy. Methods Forty ASA Ⅰ or Ⅱ patients aged 18-64 yr with body mass index of 18-30 kg/m2 undergoing gynecological laparoscopy were randomly assigned to one of two groups ( n =20 each): dexmedetomidine group (group D) and remifentanil group (group R). Starting from 5 min before induction of anesthesia, dexmedetomidine was infused at 0.05 μg · kg - 1 · min- 1 in group D and remifentanil at 0.1 μg· kg- 1· min-1 in group R for 10 min, then dexmedetomidine infusion rate was increased to 0. 3 μg· kg-1 · h-1 and remifentanil infusion rate was increased to 0.15 μg· kg-1 · min-1 . Anesthesia was induced with propofol 1.5-2.0 mg/kg and fentanyl 2 μg/kg. Tracheal intubation was facilitated with cis-atracurium 0.15 mg/kg. Anesthesia was maintained with sevoflurane and fentanyl 1 μg/kg and intermittent iv boluses of cis-atracurium. Narcotrend index was maintained at 40-50. Blood sample was taken from external jugular vein for blood gas analysis and determination of serum concentrations of corticosteroid, norepinephrine and epinephrine before administration, at 5 min after intubation, at 10 min of aeroperitoneum and at 5 min after extubation. The pH value and concentrations of lactic acid and glucose were recorded. The time for recovery of spontaneous breathing, eye-opening time, extubation time, orientation time and perioperative side-effects were recorded. Numeric rating scale was used to assess the intensity of pain during 2 h after operation. The analgesics used were also recorded. Results The serum concentrations of norepinephrine and epinephrine were significanfly lower at 10 min of aeroperitoneum, the time for recovery of spontaneous breathing was shorter, eye-opening time longer and the incidence of shivering and nausea and vomiting lower, the percentage of patients requiring rescue opioids lower in group D than in group R ( P ＜ 0.05). Conclusion The efficacy of dexmedetomidine combined with sevoflurane anesthesia is better than remifentanil combined with sevoflurane anesthesia for gynecological laparoscopy.

Objective To identify the clinical features, the molecular diagnosis and the molecular mechanism of three unrelated factor X deficiency families. Methods Three probands were male and the diagnosis was validated by coagulant parameters. The F X coagulation activity ( F X∶ C ) and antigen (FX∶ Ag) were tested by clotting test and ELISA method. The cross-corrected test was used to rule out the inhibitor of FX in plasma. Thrombin generation test was evaluated. The antigen and the molecule weight of the FX in plasma were measured with western blotting. Gene mutations were analyzed in the probands and their family members with PCR and DNA sequencing. FX expression plasmids were constructed and transientby being transfected into 293T cells. FX: C and FX: Ag of the expression products were tested. Results APTT and PT in proband 1 were obviously prolonged, 113.4 s and 62.3 s, respectively. And there was no inhibitor in plasma. The thrombin generation was lower compared to normal reference. APTT and PT in proband 2 were 56. 5 s and 28.7 s. There was no inhibitor in the plasma. The thrombin generation was 1 101.5 nmol · min. APTT and PT in proband 3 were 117.3 s and 44. 3 s. The thrombin generation was 782.5 nmol · min. FX∶ C and FX∶ Ag in proband 1 were 1.4% and 3.6%, with a homozygous mutation in FX gene (Ser425→Pro). In vitro expression of the mutation showed a normal synthesis in the cell but secretion dysfuntion. In proband 2 F X: C and F X: Ag were 2. 2% and 5. 5%, with two heterozygous mutations in FX gene (Ala-29→Pro and Phe324→Leu). The Ala-29 → Pro mutation led to significantly reduced expressions of FX in both cell lysate and cell culture supernatants compared to wild-type plasmid,(41.32 ±5.21 )% and(6. 30 ± 1.84)% respectively. However Phe324→Leu mutation almost did not affect the FX synthesis. FX: C and FX: Ag in proband 3 were 2. 2% and 35%, with two heterozygous mutations in FX gene( Ala235→Thr and Arg347→Cys). The expressions of these two mutant FX proteins in cell lysate were similar to those of wild-type but obviously lower in the supernatant. Conclusions Five mutations of F X gene are found in this study. These mutations (Ser425Pro, Phe324Leu, Ala235Thr and Arg347Cys)can not affect F X protein synthesis. However Ala-29Pro mutation can reduce F X protein synthesis and cause secretion dysfunction.