Objective:Expression of protein TSLP and selection of full human anti-TSLP single chain Fv ( scFv).Methods:The cDNA of TSLP was amplified.The amplified target gene and the expression vector pET 101/D-TOPO were ligated , and then transformed into E.coli BL21.The protein was induced to expression by IPTG and purified and identified.The biotinylated TSLP protein was used as antigen to select of human TSLP scFv from a constructed human scFv library by phage display .Results: The size of amplified cDNA of TSLP was about 423 bp,and that of expressed protein was about 26 kD.Dot blot and Western blot results showed that the expressed protein was correct.The constructed human scFv library was enriched for three rounds using biotinylated TSLP as antigen by phage display.ELISA results showed that 35% scFvs had binding activity with TSLP.The scFvs with good binding activity were selected and identified by Western blot and sequencing.Conclusion: The full human scFvs against for TSLP were selected suc-cessfully.

OBJECTIVE:To evaluate the effects of 3 kinds of serum containing blood-activating and stasis-eliminating TCM compound formulas on aggressive behavior,Janus kinase (JAK)/signal transduction and transcriptional activator (STAT) pathway of glioma U251 cells. METHODS:Rats were randomly divided into normal saline group (5 mL/kg),Taohong Siwu decoction group(5.7 g/kg),Xuefu Zhuyu decoction(8.5 g/kg)and Didang decoction(2.8 g/kg),calculated by crude drug,intragastrically administrated once a day,for 10 d. 10% drug-containing serum culture medium was prepared after 2 h of last administration. After 10% drug-containing serum culture medium intervening U251 cells for 1 week,Transwell method was conducted to detect the cell invasion rate, Western blot was adopted to detect the metal matrix protease 2 (MMP-2), MMP-9, phosphorylated JAK2 (p-JAK2),phosphorylated STAT3(p-STAT3)protein expression;and real-time fluorescence quantitative polymerase chain reaction method was used to detect MMP-2,MMP-9 mRNA expression. RESULTS:Compared with blank serum,Xuefu Zhuyu decoction drug-containing serum can reduce cell invasion rate (P<0.05),decrease the MMP-2,MMP-9 mRNA and its protein expression, and p-JAK2,p-STAT3 protein expression in cells (P<0.05 or P<0.01),while there was no significant difference in above-men-tioned indexes in Taohong Siwu decoction drug-containing serum group and Didang drug-containing serum group(P>0.05). CON-CLUSIONS:In the 3 kinds of blood-activating and stasis-eliminating TCM compound formulas,Xuefu Zhuyu decoction shows sig-nificant invasive effect on inhibiting U251 cells;the mechanism may be related to inhibiting the activation of JAK2/STAT3 signal pathway and decreasing MMP-2,MMP-9 gene and protein expressions.

Adult ; Female ; Heart Diseases ; etiology ; Humans ; Male ; Mitochondrial Diseases ; complications ; Young Adult

Objective To observe the effects of in vitro isolated Schwann cells co-cultured with chemically acellular nerve allografts on improving repair of large facial nerve defects. Methods A total of 30 Wistar rats were equally randomized into three groups, ie, experimental group, allograft group and autograft group. Nerve defect of 12 mm in length was made in the left inferior buccal branch of facial nerve and repaired with acellular nerve allograft implanted with Schwann cells, acellular nerve allograft and fresh tibial nerve autograft respectively. At the 5th month postoperatively, the function and morpholo-gy of the regenerated nerves were observed by electrophysiological method, methylene blue staining and transmission electron microscope. Results In experimental group, the recovery rate (operation side/normal side) of amplitude of nerve-muscle action potential was (35.8±2.5)%, the lantency recovery rate (normal side/operation side) (65.8±2.9)%, the number of the regenerated axon 1 570±188 and the myelin thickness (0.383±0.031) μm. The results in the experimental group were significantly supe-rior to those in the acellular nerve allograft group (P < 0.05), with similar results to fresh nerve autograft group (P > 0.05). Conclusion Transplantation of Schwarm cells in acellular nerve allograft can im-prove repair of large facial nerve defects.

Objective To examine neuroinflammation,oxidative damage and neuron loss in the contralateral parie-tal lobecortex of TBI model mice, and to investigate effects of berberine chloride on such secondary damage.Methods TBI model was established by a weight-drop hitting device and mice in berberine group were administered intragastrically with berberine chloride (50mg/kg.day) for 21 days.Immunofluorescence staining was used to assess activity of microglia and astrocyte.Immunohistochemistry was used to assess DNA oxidative damage, neuron loss and expression of COX-2 and iN-OS.Results Activation of microglia and astrocyte, expressions of COX-2 and iNOS and DNA oxidative damage were ob-viously increased by TBI,(19.82 ±1.88)and(16.96 ±1.69)、(13.79 ±4.32)and(8.67 ±0.96)、(27.86 ±5.38) and (16.00 ±7.59)、(31.92 ±6.57)and(24.79 ±2.78)respectively (P<0.01 or P<0.05).Activation of microglia and ex-pressions of COX-2 and iNOS were significantly suppressed by berberine ,(15.49 ±1.88)and(19.82 ±1.88)、(16.83 ± 7.89)and(27.86 ±5.38)、(26.25 ±2.41)and(31.92 ±6.57) respectively(P<0.01 or P<0.05).There was no differ-ence in neuron loss among three groups, (49.05 ±4.38),(48.56 ±3.56)and (47.75 ±4.14) respectively (P>0.05). Conclusions TBI can cause neuroinflammation and oxidative damage but not neuron loss in the contralateral parietal lobe cortex.Berberine chloride can significantly suppress neuroinflammtion in the contralateral parietal lobe cortex after TBI.

OBJECTIVE To investigate the genetic causes of deaf patients in a special educational school of Chifeng city, Inner Mongolia by SLC26A4 whole gene sequencing. This study focused on analyzing mutations of coding sequence of SLC26A4 gene and their relevant phenotype. METHODS DNA were extracted from peripheral blood of 134 deaf patients of Chifeng special educational school and 100 normal hearing controls in Northern China. SLC26A4 gene mutation was analyzed by direct sequencing for its 20 coding exons. All individuals found with SLC26A4 mutation were given temporal bone CT scan, and those with confirmed enlarged vestibular aqueduct and/or other malformation of inner ear were then given further ultrasound scan of thyroid and thyroid hormone assays. RESULTS The sequencing results revealed 32 cases carried SLC26A4 mutation. Twenty-nine cases underwent temporal bone CT scan. Twentycases were confirmed to have malformation of inner ear by CT scan (eighteen were EVA, one was EVA and other inner ear malformation and one was Mondini Syndrome). The shape and function of thyroid were confirmed to be normal by ultrasound scan of thyroid and thyroid hormone assays in nineteen of these 20 patients except one who had cystoid change in the right side of thyroid. Twelve types of novel variants of SLC26A4 gene were found. CONCLUSION Byscreening SLC26A4 gene coupled with temporal bone CT scan ,we could determine genetic cause related to this gene up to 14.93 % of deaf patients in special educational school of Chifeng city. SLC26A4 is another common gene besides GJB2 that cause deafness in this area. The discovery of novel variants of SLC26A4 gene makes the mutational and polymorphic spectrum more plentiful in Chinese population.

Objective To evaluate the efficacy of DSA-guided percutaneous transhepatic gallbladder catheter drainage (PTGCD) in treating aged patients with acute cholecystitis complicated by severe diseases. Methods The clinical data of 15 aged patients with acute cholecystitis or complicated by severe diseases, who were encountered at authors’ hospital in the past three years and were treated with PTGCD, were retrospectively analyzed. The clinical results were discussed. Results PTGCD was successfully accomplished with single procedure in all 15 patients. Abdominal pain was relieved within one to three days, and the abdominal symptoms and signs subsided or disappeared. Reexamination of routine blood test showed that the white blood cell count decreased to normal range in 1 - 2 weeks, and complete cure was achieved in some patients. Secondary surgery was carried out in some patients after the clinical condition was improved. During the follow-up period no complications occurred in all patients except one who developed biliary leakage after the catheter was retrieved two weeks after the treatment. Conclusion For the treatment of complicated acute cholecystitis in aged patients who are not suitable to receive surgery, DSA-guided percutaneous transhepatic gallbladder catheter drainage is an ideal therapeutic means as it can significantly relieve clinical symptoms.

Objective:To explore changes of central retinal vascular caliber and fractal dimension (Df) and their cor‐relation with blood pressure in hypertensive population .Methods :A total of 2169 subjects>30 years old were en‐rolled in this cross‐sectional study .They were divided into hypertension group (n=819) and non‐hypertension group (n=1350) .Fundus photos were collected in all subjects ,and semi‐automatic software was used to quantitatively ana‐lyze central retinal vascular caliber and Df ,and they were compared between two groups .Results:Compared with non- hypertension group ,there were significant reductions in central retinal arteriolar equivalent [CRAE ,(135.2 ± 10.72) μm vs .(132.25 ± 11.56) μm] ,central retinal venular equivalent [CRVE ,(184.95 ± 16.29) μm vs . (182.52 ± 17.07)μm] and Df [ (1.38 ± 0.05) vs .(1.34 ± 0.05)] in hypertension group , P<0.01 all .After adjus‐ting for age and gender ,analysis of covariance indicated that CRAE and Df of hypertension group were still signifi‐cantly lower than those of non - hypertension group (P<0.01 both) .Linear correlation analysis indicated that sys‐tolic blood pressure (SBP) ,diastolic blood pressure (DBP) and pulse pressure (PP) were inversely correlated with CRAE and Df ( r= -0.340~ -0.174 , P<0.01 all) .After adjusting for cardiovascular risk factors ,multi‐factor linear regression analysis indicated that CRAE and Df were still inversely correlated with SBP ,DBP and PP (stand‐ardizedβ= -0.190~ -0.134 ,P<0.01 all) .Df of hypertension course >5 years group was significantly lower than that of ≤5 years group [ (1.33 ± 0.05) vs .(1.35 ± 0.05)] , P<0.01. Conclusion:CARE ,Df are significantly in‐versely correlated to SBP ,DBP and PP in hypertensive population ,while correlation of Df is most .

OBJECTIVETo detect mutations of human telomeric repeat binding factor 1 (TERF1) gene in 11 malignant hematopoietic cell lines, which have positive telomerase activity, and evaluate the significance of the mutations.

METHODSGenome structure of TERF1 was predicted by using biology information program, and verified by PCR and sequencing. Telomerase activity was detected by telomeric repeat amplification (TRAP)-ELISA. PCR and sequencing were used to detect mutation of each exon of TERF1 in 11 cell lines, including myelogenous leukemia cell lines K562, HL-60, U-937, NB4, THP-1, HEL and Dami; lymphoblastic leukemia cell lines 6T-CEM, Jurkat and Raji and MDS-RAEB cell line MUTZ-1. Five DNA samples from healthy volunteers were detected as normal controls.

RESULTSTERF1 gene has 10 exons and spans 38.6 kb. All the 11 cell lines showed positive telomerase activity. No mutation was found in all exons of TERF1 in the 11 cell lines. However, 4 variants were found in intron1, 2 and 8 near exon1, exon2 and exon9, respectively. The variants in MUTZ-1 was different from those in leukemia cell lines; but no difference was found between the variants in myelogenous and lymphoblastic leukemia cell lines.

CONCLUSIONTERF1 mutation is probably not among the main factors of the gene dysfunction in malignant hematopoietic diseases.

Base Sequence ; Cell Line, Tumor ; DNA Mutational Analysis ; Enzyme-Linked Immunosorbent Assay ; Exons ; genetics ; HL-60 Cells ; Hematologic Neoplasms ; genetics ; metabolism ; pathology ; Humans ; Jurkat Cells ; K562 Cells ; Mutation ; Polymerase Chain Reaction ; Telomerase ; metabolism ; Telomere-Binding Proteins ; genetics ; metabolism ; U937 Cells

To study the expression of tankyrase (a positive regulator of telomerase activity) in malignant hematopoietic cells and its relation with telomerase activity, the method of realtime quantitative PCR with fluorescence probe hybridization were used to measure expression of tankyrase and hTERT in myeloid leukemia cell lines K562, HL-60, U937, NB4, THP-1, HEL, Dami and T lymphocytic leukemia cell lines 6T-CEM, Jurkat and B-cell lymphoma cell line Raji. CD3(+), CD19(+) and CD33(+) cells separated from normal human mobilized peripheral blood by immunomagnetic bead system and 10 mononuclear cell samples separated from bone marrow of normal individuals were served as normal controls. The results indicated that the expression of tankyrase in malignant hematopoietic cell lines was significantly higher than that in normal controls (U = 19, P < 0.01). Its expression in myeloid leukemia cell lines is higher than in normal CD33(+) cells, the expression in T lymphocytic leukemia and B-cell lymphoma cell lines is higher than in CD3(+) and CD19(+) cells respectively. Its expression in myeloid malignant hematopoietic cell lines is significantly lower than in lymphocytic ones (0.0032 +/- 0.0010 vs. 0.012 +/- 0.0016, F = 23, P < 0.01). The expression of tankyrase correlated positively with hTERT (Spearman correlation coefficient is 0.395, P < 0.05). It is concluded that tankyrase is overexpressed in malignant hematopoietic cell lines, that may be one of the causes of high-produced telomerase activity in malignant hematopoietic diseases.
DNA-Binding Proteins ; HL-60 Cells ; Humans ; K562 Cells ; Leukemia ; enzymology ; Polymerase Chain Reaction ; Tankyrases ; genetics ; Telomerase ; genetics ; U937 Cells