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Objective To construct HaCaT cell lines stably expressing the wild type human GJB6 gene or its mutant by using a Tet-On lentiviral vector, and to lay an experimental foundation for studies on pathogenesis of hidrotic ectodermal dysplasia. Methods The wild-type human GJB6 gene and its mutant (A88V)were amplified by PCR, and then inserted into the Tet-on lentivirus plasmid to construct recombinant lentivirus vectors. The recombinants were identified by gene sequencing and enzymatic digestion. Cultured HaCaT cells were classified into three groups to be transfected with a negative control lentiviral vector (NC group), the lentivirus vector expressing the wild-type human GJB6 gene (WT group), or the lentivirus vector expressing the mutant human GJB6 gene (MU group). Puromycin was used to select HaCaT cell clones stably expressing the GJB6 gene which encodes the connexin 30 (Cx30)protein. The selected HaCaT cell clones were cultured with or without tetracycline for 48 hours, thereafter, real-time PCR(RT-PCR) was performed to detect GJB6 gene mRNA expression, Western-blot analysis to measure expressions of Cx30 and FLAG-tag proteins, and cell counting kit 8 (CCK8)assay to evaluate cellular proliferative activity. Results Enzymatic digestion and gene sequencing showed that recombinant lentivirus plasmids were successfully constructed. RT-PCR showed evidently increased mRNA expression of the GJB6 gene in stably transfected HaCaT cells. Moreover, the expression abundance of the GJB6 gene was 112.369 times higher in the WT group induced by tetracycline than in that without tetracycline treatment (P 0.05). Conclusion HaCaT cell lines which stably express the wild-type GJB6 gene or its mutant(A88V)are successfully constructed.
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Objective To screen for and identify targets of let-7a microRNA (miRNA) in A375 melanoma cells by using isobaric tags for relative and absolute quantitation (iTRAQ) technology,and to explore mechanisms underlying the tumor-suppressing effect of let-7a.Methods Cultured A375 cells were classified into two groups to be transfected with 100 nmol/L hsa-1et-7a mimics (hsa-let-7a mimics group) or negative control mimic (NC group).After 54-hour incubation,A375 cells were collected and total proteins were collected.iTRAQ technology was used to analyze and identify differentially expressed proteins,bioinformatic analysis was performed to assess let-7a candidate targets and their functions,and a dual-luciferase reporter system was utilized to verify let-7a targets.Results As mass spectrometry showed,a total of 327 differentially expressed proteins were identified in the hsa-1et-7a mimics group compared with the NC group,including 151 up-regulated proteins with iTRAQ ratio ＞ 1.2 and 176 down-regulated proteins with iTRAQ ratio ＜ 0.8.Of 176 down-regulated proteins,47 were predicted as miRNA targets by the miRWalk software.The dual-luciferase reporter system showed that the relative luciferase activity of the 3' untranslated region (UTR) of the wild-type HMGA2 and THOC2 genes were reduced by 64.3％ and 46.4％,respectively,in the hsa-1et-7a mimics group compared with the NC group.Conclusion A total of 47 candidate let-7a targets were screened out in A375 melanoma cells by using iTRAQ technology and bioinformatic analysis,and HMGA2 and THOC2 genes were identified as direct targets of let-7a.
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Objective To retrospectively analyze the clinical effect of 131 I in the treatment of hyperthyroid heart disease.Methods 269 cases who received nuclear medicine 131 I therapy for hyperthyroid heart disease were selected.Clinical laboratory and related examinations,including determination of serum thyroid hormones and antibod-ies (FT3 ,FT4 ,TSH,TRAb,TGAb and TPOAb),biochemical indicators,analysis of blood,electrocardiogram,thyroid ultrasonography,thyroid 131 I uptake rate determination,static imaging and color Doppler ultrasound of the heart were condutced.After diagnosis clear integrated touch technique,thyroid color Super and the thyroid static imaging deter-mine thyroid weight.131 I dose by following formula calculation determine:131 I dose =(each grams thyroid plans vol-ume)×thyroid weight (g)/thyroid 24h or highest 131 I rate (%),each grams thyroid plans volume for 2.96-4.44MBq,calculation 131 I dose,application SPSS 17.0 statistics software for statistics,used paired t test to analyze serum hormone levels of FT3 ,FT4 ,sTSH before treatment,3,6 and 12 months after treatment.Results After 131 I treatment 3,6,12 months,serum FT3 ,FT4 ,sTSH levels significantly declined compared with before treatment (t =36.03,23.88,17.81,45.01,24.85,13.95,49.97,25.66,10.28,all P <0.01).Of 269 patients received 131 I treat-ment,hyperthyroidism cured in 220 cases (81.8%),improved in 42 cases (15.6%),invalid in 7 cases (2.6%);hyperthyroidism heart recovered in 226 cases (84.0%),effective rate was 97.0%(261 /269).Conclusion The treatment of hyperthyroid heart disease as soon as possible is the key to control hyperthyroid,reduced thyroid hormone in the peripheral circulation,131I can be fast and effective treatment of hyperthyroidism,hyperthyroid abnormal ECG and cardiac anomalies symptom relief for time is the ideal treatment of hyperthyroid heart disease recovery as soon as possible,with normal thyroid function,hyperthyroid can return to normal or part of normal.
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Objective To assess the efficacy of topical application of Ephedrine and furacilin nasal drops combined with tetracaine in reducing complications of pack removal after nasal surgery.Methods 81caseshaving un-dergone nasal septum surgery were randomly divided into the experimental group and control group: the former were treated with Ephedrine and furacilin nasal drops combined with tetracaine and the blank control group.The occur-rence of complications at removing nasal fillers after nasal surgery was assessed.Results The hemorrhage and pain in the experimental group were significantly less than those in the blank control group (p <0.05).Conclusion top-ical application of Ephedrine and furacilin nasal drops combined with tetracaineis an effective method for reducing the pains of pack removal after nasal surgery.
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Objective Replacement of dexmedetomidine with propofol for maintaining the anes-thesia in elderly patients undergoing laparoscopic cholecystectomy.Methods Ninety patients,over 70 years old,undergoing laparoscopic cholecystectomy were randomly divided into 2 groups,propofol combined with remifentanil (group A),dexmedetomidine combined with remifentanil (group B),45 patients in each group.Group A was not treated with any preoperative medication,while group B was treated with loading dose of 0.5 μg/kg dexmedetomidine intravenously completed within 10 minutes. Induction methods were same in both groups,3 # or 4 # laryngeal mask were inserted after induction in both groups.Maintenance of anesthesia in group A treated with propofol 2.0-3.0 μg/ml + 4.5-5.5 ng/ml TCI;Maintenance of anesthesia in group B treated with dexmedetomidine 0.25 μg·kg-1·h-1 +remifentanil 4.5-5.5 ng/ml (TCI).HR,SBP,DBP,BIS were recorded at inserting the LMA (T1 ), beginning of the surgery (T2 ),dissociate the cholecyst (T3 ),withdrawal of the laparoscope (T4 ), extubate the LMA (T5 ).Postoperative recovery time,Steward awakening score and modified OAA/S score at extubation time were recorded.Results No significant difference was found between BIS val-ue of two groups at different time point.Compared with group A,HR at T1-T5 in group B were sig-nificantly lower,SBP,DBP were significantly decreased (P <0.05).There was no significant differ-ence between Steward awakening score and modified OAA/S score at recovery and extubation time in two groups.Conclusion Dexmedetomidine replacing propofol can be safely used in laparoscopic chole-cystectomy with less hemodynamic changes during maintenance of anesthesia in elderly patients.
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Objective To optimize the concentration of a microRNA-21 (miR-21) inhibitor and a miR-494 mimic for the transfection of A375 human melanoma cells,and to estimate the effect of the miR-21 inbihitor and miR-494 mimic on the proliferation of A375 cells.Methods A miR-21 inbihitor and a miR-494 mimic were designed and constructed.To optimize the concentration of the miR-21 inbihitor and miR-494 mimic for transfection,six concentrations (70-250 nmol/L) of the inbihitor and mimic were transfected into A375 cells separately by using LipofectamineTM2000.Then,quantitative fluorescence-based PCR was performed to determine the expression of miR-21 and miR-494 in A375 cells.Some A375 cells were classified into five groups:Mock blank control group remaining untransfected,miR-21 inhibitor group transfected with the miR-21 inhibitor,miR-21 control group transfected with the miR-21 inhibitor negative control,miR-494 mimic group transfected with the miR-494 mimic,and miR-494 control group transfected with the miR-494 mimic negative control.Mter another 48-hour culture,the cells were collected for the analysis of cell apoptosis and cycle by using flow cytometry.Meanwhile,Cy5-labelled miR-494 mimic negative control was transfected into A375 cells for the evaluation of the transfection efficiency by using an inverted fluorescence microscope.Results miRNAs were successfully extracted from A375 cells.As quantitative PCR revealed,the A375 cells transfected with the miR-21 inhibitor at 120 nmol/L showed the lowest expression level (2-△△Ct) of miR-21 (average:0.80; range:0.65-0.92),and those transfected with the miR494 mimic at 250 nmol/L displayed the highest expression level of miR-494 (average:126.82; range:111.52-144.22).The transfection efficiency in A375 cells was higher than 90％.Compared with the corresponding negative control groups,the miR-21 inhibitor group and miR-494 mimic group showed increased apoptosis rate ((27.74 ± 1.39)％ vs.(12.93 ± 0.65)％,(34.30 ± 2.35)％ vs.(15.54 ± 1.02)％,both P ＜ 0.01),percentage of G1-phase cells ((61.61 ± 3.25)％ vs.(50.34 ± 5.62)％,(61.05 ± 3.17)％ vs.(49.95 ± 2.58)％,both P＜ 0.05),but decreased proliferation index ((38.39 ± 3.25)％ vs.(49.66 ± 5.62) ％,(38.95 ± 3.17)％ vs.(50.05 ± 2.58)％,both P ＜ 0.05).Conclusions Both the miR-21 inhibitor and miR-494 mimic can promote the G1-phase arrest and apoptosis in A375 cells,and miR-21 may act as a protooncogene accelerating the proliferation of A375 cells,while miR-494 may founction as a tumor suppressor inhibiting the proliferation of A375 cells.
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ObjectiveTo detect the mutation of GJB2 gene in a Chinese family with Vohwinkel syndrome.MethodsClinical data were collected from 5 patients with Vohwinkel syndrome in a family,and blood samples were obtained from the 5 patients and 4 unaffected individuals in the family as well as from 100 normal human controls.Genomic DNA was extracted and subjected to PCR for the amplification of the entire encoding and flanking sequences of GJB2 gene(1015 bp) followed by bidirectional sequencing with the ABI PRISM 3730 automatic DNA sequencer.Finally,sequence alignment was carried out by using the software Sequencher 4.10.1 Demo.ResultsA heterozygous missense mutation 196G→C in the GJB2 gene,which resulted in the substitution of aspartic acid by histidine at codon 66 (D66H) in the first extracellular domain of the protein,was observed in all the patients of this family,but in none of the 4 unaffected individuals in this family or the 100 normal human controls.ConclusionThe D66H missense mutation in the GJB2 gene may contribute to the occurrence of Vohwinkel syndrome in Chinese Han population.
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Objective To investigate the effects of NB-UVB on the expression of Gadd45α as well as cell apoptosis and cycle of human HaCaT keratinocytes.Methods Cultured HaCaT cells were exposed to various doses (100,200,400 mJ/cm2)of NB-UVB followed by an additional culture of 6,12 and 24 hours,respectively.Reverse transcription PCR and Western blot were performed to detect the mRNA and protein expression of Gadd45α respectively in HaCaT cells,cell counting kit 8 (CCK8)to measure the proliferation of cells,and flow cytometry to determine the cell cycle distribution of HaCaT cells before and after the exposure to NB-UVB.Results Gadd45α was expressed in HaCaT cells.After exposure to NB-UVB of the three doses,the mRNA and protein levels of Gadd45α increased at 6 hours and 12 hours,but declined at 24 hours,and significant changes were observed in HaCaT cells at the three time points after exposure to NB-UVB of the three doses (all P<0.05).The Gadd45α/β-actin mRNA ratio was 1.4360±0.6551.1.8633±0.0979,1.9266±0.1724 in HaCaT cells 12 hours after irradiation to NB-UVB of 100,200 and 400 mJ/cm2,respectively,significantly higher than that in unirradiated cells(0.6000±0.1276,all P<0.05).Also,increased Gadd45α/β-actin protein ratio was noted in HaCaT cells 12 hours after irradiation to NB-UVB of 100,200 and 400 mJ/cm2 compared with unirradiated cells (0.0773±0.0005,0.1936±0.0015,0.2373±0.0015 vs.0.0290±0.0010,all P<0.05).NB-UVB inhibited the proliferation of HaCaT cells in a time-and dose-dependent manner.Flow cytometry showed that irradiated HaCaT cells were blocked in G2 phase of the cell cycle.and the percentage of HaCaT cells in G2 phase was 13.53%±1.03%,17.77%±2.25%,30.03%±4.29%afler exposure to NB-UVB of 100,200 and 400 mJ/cm2,respectively,compared to 9.24%±0.97%in unirradiated cells (all P<0.05).Conclusions The expression of Gadd45α is increased in HaCaT cells after exposure to NB-UVB,and Gadd45α may be involved in the NB-UVB-induced suprression of cell proliferation of and cell cycle arrest in HaCaT cells.
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Objective To investigate the expression of microRNA let-7a in formalin-fixed paraffinembedded (FFPE) melanoma tissue and its effect on the proliferation of A375 cell lines. Methods Tissue samples were obtained from 13 patients with malignant melanoma and 10 with congenital pigmented nevus,then fixed with formalin and embeded with paraffin. microRNAs were isolated from these samples and reversely transcripted to cDNA with stem-loop primer. Then, real time quantitative PGR was performed with Taqman MGB probe and matched primers to measure the expression of microRNA let-7a. cy3-Labeled negative control siRNA was transfected into A375 cells followed by the examination of transfection efficiency under fluorescense microscopy. Subsequently, hsa-let-7a mimics were transfected into A375 cells to observe their effect on the proliferation of these cells. Results microRNAs were successfully isolated from FFPE tissues, and quantitative PGR showed a significant reduction in the expression of microRNA let-7a in melanoma tissue compared with nevus tissue (t = 2.364, P ＜ 0.05). The transfection efficiency was found to be about 80% ～90%. After transfection with hsa-let-7a mimics, the proliferation of A375 cells was inhibited and the inhibition rate amounted to 32.7% at 24 hours. Conclusions The expression of microRNA-let-7a is attenuated in melanoma, and overexpression of let-7a can inhibit the proliferation of A375 cells, which implies that let-7a functions as a tumor suppressor gene in melanoma.
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Objective To investigate the effect of microRNA-let-7a on apoptosis in melanoma cell line A375 and its mechanism. Methods The expression of microRNA-let-7a was detected in A375 cells and melanocytes by real-time PCR. Then, microRNA-let-7a mimics was transfected into A375 cells followed by the measurement of apoptosis and caspase-3 protein expression by flow cytometry and Western blotting, respectively. Results The expression level of microRNA-let-7a was reduced by 0.462 folds in A375 cells compared with melanocytes. The apoptosis rate was 47.4% in A375 cells transfected with microRNA-let-7a mimics, significantly higher than that in untransfected A375 cells (16.9%). A significant decline was observed in the expression of caspase-3 protein in A375 cells after transfection. Conclusion microRNA-let-7a can promote the apoptosis and downregulate caspase-3 protein expression, in A375 human malignant melanoma cells.
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Objective To confirm the diagnosis and to localize the pathogenic gene of ectodermal dysplasia in a family SUffering from only hair and nail abnormalities.MethodsBlood samples were collected from 7 affected patients and 15 unafiected individuals in the family.Genomic DNA was extracted from blood samples by routine phenol-chloroform methods.The whole coding regions of candidate genes K16,K17,K6a,K6b and GJB6 were amplified by PCR followed by direct sequencing.Then,the gene mutation was further confirmed at mRNA level by RT-PCR.ResultsA heterozygous missense mutation 3 1G→A in the GJB6 gene.which leads to the substitution of glycine by arginine at codon 11(G11R)on the N-terminal of the protein,was detected in all the patients.but in none of the 15 normal individuals in this family.The mutation was also confirmed in the CDNA originating from the proband's skin biopsy.Conelusionn A missense mutation G31A.which has been shown previously to cause hidrotic ectodermal dysplasia(HED),is localized in the GJB6 gene of patients in this family.
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A case of granular parakeratosis is reported. A 31-year-old woman presented with a 23-year history of pruritic erythema and erosion in the left axilla. On examination, there was a ring-like annular erythematous patch sized 8 cm×10 cm in the left axilla. Bright mauve, cone-shaped, millet-like papules were observed in the center of the lesion, some confluenced into plaques. Erythema was present in the pedlesional region along with mild erosion, exudation and small numbers of grain-sized pustules. Scar formed in some perilesional areas. No lesions were noted at any other intertriginous regions. Fungal microscopy of lesion secretions was negative. Histological examination of biopsy specimens from the center of the left axilla revealed psoriasiform hyperplasia of epidermis and thickened stratum comeum with hyperkeratosis and parakeratosis. Most cells in the stratum comenm retained nuclei and contained numerous basophilic granules. Granular layer could be noted under the parakeratotic cells with cytoplasm vacuolization of some cells. There was a perivascular, mixed inflammatory infiltration predominated by lymphocytes and hemangiectasis in the dermis. A diagnosis of granular parakeratosis was made.
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OBJECTIVE@#To explore the better management of the stoma in endoscopic dacryocystorhinostomy.@*METHOD@#To review the 102 chronic dacryocystitis patients (109 eyes), who underwent the endoscopic dacryocystorhinostomy surgery with silver clips used to maintain the stoma. They were given combined therapy after the surgery, and were followed up for a period range from 3-73 months.@*RESULT@#99/109 eyes (91%) were cured, 5/109 eyes (4.5%) were improved, and the total effective rate reached to 104/109 (95.5%).@*CONCLUSION@#The application of silver clip in endoscopy dacryocystorhinostomy surgery and combined therapy after the surgery can effectively prevent the stoma stenosis or atresia.
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Adolescente , Adulto , Idoso , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Dacriocistite , Cirurgia Geral , Dacriocistorinostomia , Métodos , Endoscopia , Resultado do TratamentoRESUMO
Objective To detect gene mutations in a family of congenital atrichia with papular lesions (APL). Methods Polymerase chain reaction and DNA sequencing were used to search for mutations in the HR gene (a causative gene of APL), the CJB6 gene, and the CDSN gene. Results No mutation was found in these three genes except for single nucleotide polymorphisms (SNPs) in the HR and CDSN genes. Conclusion No mutation is identified in the HR, CJB6 or CDSN gene in this family affected by congenital APL.
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Objective To target the disease gene of disseminated superficial form of porokeratosis (DSP) in a six-generation of a Chinese family including a total of 254 family members in Shandong province. Methods The clinical data and the peripheral blood samples were collected in the pedigree members. The genomic DNA was extracted from the blood samples. A genome-wide scan was performed using 382 pairs of primers labelled with fluorescent stain. The primers were designed for human autosomes. The sequencing results were analyzed by the software of Genescan and Genotype. Linkage analysis was processed by Linkage software package to define the region of disease gene. For fine targeting the disease gene, other 10 micro-satellite markers for the above region were set up for further fine sequencing. Results We obtained the maximum two-point LOD scores of 3.06 at micro-satellite marker D12S78 (recombination fraction ? = 0.00). After fine mapping, the DSP gene is located within a 38.5 cM region between markers D12S326 and D12S79. Conclusion The DSP gene is mapped to chromosome 12q21.2~24.2.
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Objective To study the relation between vascular endothelial growth factor(VEGF) and the degree of coronary artery stenosis as well as the formation of collateral circulation in patients with acute myocardial infarction (AMI). Methods Seventy-six cases of AMI had undertaken selective coronary angiography on the third to seventh day after admission and also at six months afterwards in order to define the degree of coronary artery stenosis and the presence of collateral circulation. A blood sample of 5 mL was taken in each patient before the first coronary angiography to test for the level of VEGF by enzyme linked immunosorbend assay. Results The level of VEGF in patients with coronary artery stenosis less than 50%, 50%~75% and greater than 75% were (97.6?17.3) ng/L, (241.6?28.9) ng/L and (391.7?48.4) ng/L respectively (P