Meta-analysis of the effects of femtosecond laser-assisted cataract surgery on corneal endothelial cells / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To systematically evaluate the effects of femtosecond laser-assisted cataract surgery and conventional ultrasound cataract surgery(CUCS)on corneal endothelial cells.METHODS: Databases such as PubMed, Cochrane Library, Web of Science, Embase, CNKI, CBM, VIP and WanFang Data were searched for randomized controlled trials(RCT)from the establishment of the database to November 2021 on the effects of femtosecond laser cataract surgery and conventional ultrasound cataract surgery on corneal endothelial cells. Language is limited to Chinese or English. The literatures were evaluated by the Manual of Systematic Evaluation of Cochrane Interventions and the modified Jadad Scale. Stata 15.0 software was used for statistical analysis.RESULTS: A total of 13 RCT were included, including 1 446 eyes in the FLACS group and 1 472 eyes in the CUCS group. The Meta-analysis results showed that the cumulative dissipated energy(CDE)in FLACS group was obviously lower than that in CUCS group [WMD=-3.84, 95%CI (-6.30, -1.38), P=0.002]. The effective phacoemulsification time(EPT)in the FLACS group was obviously lower than that in the CUCS group [WMD=-3.03, 95%CI(-4.00, -2.05), P<0.001]. The density of corneal endothelial cells in both the FLACS group after surgery at 1 and 3mo was higher than that in CUCS group [WMD=121.76, 95%CI(79.31, 164.20), P<0.001; WMD=76.04, 95%CI(19.25, 132.82), P=0.009]; The thickness of the central cornea in the CUCS group was significantly thicker than that in the FLACS group at 1wk after the surgery [WMD=-9.89, 95%CI (-18.60, -1.18), P=0.026]; The incidence of postoperative corneal edema in the FLACS group was less than that in the CUCS group [RR=0.46, 95%CI(0.32, 0.66), P<0.001]. There were no differences in the percentage of hexagonal cells and coefficient of variation of corneal endothelial cells between the two groups.CONCLUSION: Compared with conventional ultrasound cataract surgery, femtosecond assisted cataract surgery can significantly reduce the phacoemulsification energy and the duration of the phacoemulsification energy, and significantly reduce the loss of corneal endothelial cells in the early postoperative period, while reducing the occurrence of postoperative corneal edema.

Neurological Diseases Responding Specifically to Traditional Chinese Medicine / 中国实验方剂学杂志

In recent years， the incidence of neurological diseases has been increasing year by year. To give full play to the advantages of traditional Chinese medicine （TCM） in the treatment of neurological disorders， identify the breakthrough point of integrating TCM with western medicine， and further standardize the clinical diagnosis and treatment of TCM， the China Association of Chinese Medicine organized neurologists in TCM and western medicine to carry out in-depth discussion on the neurological diseases responding specifically to TCM and integrated TCM and western medicine， such as stroke， headache， vertigo， multiple sclerosis， and epilepsy， aiming to formulate a well-recognized and integrated treatment protocol for TCM and western medicine and improve the efficacy of neurological disorders. Furthermore， the treatment suggestions of the corresponding diseases in TCM and western medicine were proposed to provide references for clinical practice and scientific research.

Optimized experimental method for determination of hydroxyproline in lung tissue of mice / 医学研究生学报

ObjectiveThere are many methods for the detection of hydroxyproline (HYP), but few of them are suitable for the detection of lung tissue in mice. We intend to establish an accurate and reliable method for measuring HYP levels based on mouse lung tissues to assess the degree of fibrosis development more effectively.MethodsBased on the alkali hydrolysis method, the effects of the concentration of alkali hydrolysate and hydrolysis time on the determination results of HYP level in mice lung tissue were compared; the effects of the changes of experimental conditions on the determination results of HYP standard were compared; and the results of the determination of HYP level in mice lung tissue under dry and wet conditions were compared on the basis of the above experimental results.ResultsThe optimum concentration of alkali hydrolysate is 2 mol/L and the optimum hydrolysis time is 20 min. The optimum pH value of citric acid buffer is 6.0-6.5. The optimum solvent for chloramine T is methanol, the optimum reaction time for chloramine T solution is 15 min, the optimum reaction time for perchloric acid solution is 5 min, and the optimum reaction time for 4-(dimethylamino) benzoyl toluene is 5 min. The optimum condition of aldehyde solution color development is that it is bathed in water at 85 for 3 minutes. Some related reagents are stored in suitable environment after preparation, and the experimental data will not be affected within 7 days. Dry lung tissue of mice can improve the detection level of HYP. The improved experimental protocol was applied to the bleomycin-induced mouse pulmonary fibrosis model, and the HYP measurement results were significantly higher than that of the original protocol.ConclusionAn accurate and reliable method for the determination of hydroxyproline in lung tissue of mice was established.

Neuroprotective effects of paeonol in a cell model of Parkinson disease / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the effects of paeonol on neuron cell model of Parkinson disease (PD).</p><p><b>METHODS</b>The cell model of Parkinson disease was induced by treatment of 1-Methyl-4-phenylpyridinium (MPP+) in PC12 cells, the PD model cells were treated with 1 μmol/L, 3 μmol/L or 9 μmol/L paeonol for 24h, respectively. Cell viability and LDH leakage were detected by MTT and lactate dehydrogenase (LDH) assay; the apoptosis of PC12 cells was assessed by Hoechst 33258 staining and flow cytometry; reactive oxygen species (ROS) production was detected by DCFH-DA method; and the ratio of Bax/Bcl-2 and activation of caspase-3 were determined by Western blotting.</p><p><b>RESULTS</b>MPP+ treatment significantly reduced cell viability, increased LDH leakage, enhanced the proportion of apoptotic cells and ROS production. In addition, MPP+ treatment dramatically increased the Bax/Bcl-2 ratio, and the activation of caspase-3. Compared to PD model group, paeonol treatment significantly enhanced cell viability, decreased LDH leakage, inhibited the proportion of apoptotic cells and ROS production, reduced the Bax/Bcl-2 ratio and the activated caspase-3 protein.</p><p><b>CONCLUSION</b>Paeonol can prevent PC12 cells from apoptosis induced by MPP+, and the mechanism may be associated with the down-regulation of ROS production, Bax/Bcl-2 ratio and Caspase-3 activation.</p>

Effects of gonadotropin-releasing hormone analog treatment on glucose and lipid metabolism in girls with idiopathic central precocious puberty / 中华实用儿科临床杂志

Objective To assess the effects of gonadotropin-releasing hormone analog on glucose and lipid metabolism in girls with idiopathic central precocious puberty(ICPP).Methods A total of 26 girls (aged 6-8 years,breast stage B2) with ICPP were followed up in Department of Endocrinology,Shenzhen Children's Hospital from Jan.2008 to Jun.2011.Those girls received triptorelin therapy for 12 months.Before and the end of the 6th month,the 12th month of the treatment,body mass index(BMI) was calculated,fasting plasma glucose(FPG),fasting plasma insulin(FPI),total cholesterol,triacylglycerols,high density lipoprotein cholesterol,low density lipoprotein cholesterol,apolipoprotein A,apolipoprotein B and estradiol(E2) were measured.Insulin sensitivity was estimated by homeostasis model assessment of insulin resistance(HOMA-IR).Twenty age-matched prepuberty girls were set as controls.Results 1.Before treatment,BMI,FPG,FPI and HOMA-IR in ICPP girls had no significant difference compared with the controls.2.After 6 months treatment of triptrolin,serum E2 concentration in ICPP girls declined from(30.5 ± 9.8) ng/L at the beginning of treatment to (11.2 ± 4.6) ng/L at the end of 6th month (P ＜ 0.01) ; The end of 12 th month of the treatment,FPG and FPI had no significant difference compared with that before of the treatment,but BMI increased from(16.46 ± 1.10) kg/m2 to(18.35 ± 1.30) kg/m2,the difference was significant(P ＜0.05),HOMA-IR increased from 1.24 ±0.30 to 2.08 ±0.40,the difference was significant(P ＜0.05).3.Lipid metabolism parameters remained unchangeable after 12 months of triptrolin treatment.Conclusion Triptorelin may lead to raise of BMI and HOMA-IR in girls with ICPP at 12 months after treatment.

MicroRNA expression profile and pathogenetic initial study in essential hypertension / 中华心血管病杂志

<p><b>OBJECTIVE</b>To study the differential microRNAs expression between patients with essential hypertension and healthy controls.</p><p><b>METHODS</b>Whole blood from 15 hypertensive patients and 5 controls healthies were separated into plasma at 3000 rpm for 10 minutes. MicroRNAs were harvested using kit, and stored at -80°C. MicroRNAs profiling were performed using Exiqon microRCURY(TM) LNA microRNAs array, and were quantitative RT-PCR for the differential microRNAs expression. In addition, we used a set of plasma samples from 24 hypertensive patients and 22 healthy donors to independently validate the expression of these signature microRNAs.</p><p><b>RESULTS</b>MicroRNAs expression profile was found to be differentially in the essential hypertensive patients compared with the healthy donors. Of 1700 microRNAs detected on the microarray, 46 microRNAs were found to be differentially expressed in the essential hypertensive patient, 27 microRNAs were collected in Sanger microRNAs data-bank, the function of remaining 19 microRNAs were unknown. In the 27 microRNAs, 9 microRNAs were up-regulated in the hypertension patient samples, while 18 known microRNAs were down-regulated. MiR-296-5p (Fold change 0.47, P = 0.013) and miR-133b (Fold change 0.57, P = 0.033) were consistently down-regulated in the patient plasma, whereas let-7e (Fold change 1.62, P = 0.009) and hcmv-miR-UL112 (Fold change 2.72, P = 0.004), one human cytomegalovirus encoded microRNAs, were up-regulated in the patient samples. The microRNAs expression was independently validated using another sample. We showed that MHC class I polypeptide-related chain B (MHC class I polypeptide-related chain B, MICB) and Interferon regulatory factor 1 (Interferon regulatory factor 1, IRF1) were functional targets of hcmv-miR-UL112 by fluorescent reporter assays.</p><p><b>CONCLUSIONS</b>The hypertensive patients have distinct microRNAs expression Profile. Hcmv-miR-UL112 may have important implications toward pathogenesis of essential hypertension.</p>

Culture and characterization of and lentiviral vectors mediated glial cell derived neurotrophic factor expression in mesenchymal stem cells from human umbilical cord blood / 中国医学科学院学报

<p><b>OBJECTIVE</b>To isolate and culture mesenchymal stem cells from umbilical cord blood (UCB-MSCs), study its biological characterization in vitro, transfect UCB-MSCs using lentiviral vectors encoding glial cell derived neurotrophic factor (GDNF) gene, evaluate the biological function change of UCB-MSCs, and detect GDNF expression level in vitro.</p><p><b>METHODS</b>We isolated monocyte by Ficoll density gradient, separated two kinds of adherent cells through different trypsin digestion time, and detected the cells surface markers by fluorescence activated cell sorting when it was proliferated for P7 passages. At the same time, we sub-cloned GDNF gene into lentiviral vectors and packaged lentiviral supernatant through three plasmids co-transfection method, then transfected the UCB-MSCs using lentiviral vectors encoding GDNF at different multiplicity of infection, and evaluated the change of biological function by observing the ability of proliferation and differentiation, morphology, and the cells surface markers. We detected the GDNF mRNA and protein expression level by using real-time polymerase chain reaction (real-time PCR) and enzyme-link immunosorbent assay (ELISA).</p><p><b>RESULTS</b>The UCB-MSCs were successfully isolated and cultured in vitro, and induced it to differentiate into fat cells. FACS results showed that the UCB-MSCs expressed CD90, CD73, and CD105 positively, and CD14, CD34, CD45, CD19, HLA-DR, Stro-1, and CD106 negatively. Real-time PCR and ELISA showed that the expressions of GDNF protein and mRNA were correlated with the copy number of transfected cells: high copy number of transfected cells were associated with high GDNF expression. The biological characterization of UCB-MSCs did not obviously change after sub-cloning with GDNF.</p><p><b>CONCLUSIONS</b>UCB-MSCs was successfully isolated and cultured in vitro. By transfecting UCB-MSCs with GDNF gene-containing lentiviral vectors, the secretion of GDNF protein and mRNA expression level can be controlled by the copy number of transfected cells, and thus make it constantly express GDNF at high level.</p>

Inhibitory effect of siRNA targeting gene Aqp1 on K562 cells / 中国实验血液学杂志

The objective of this study was to establish an RNAi approach that can specifically target aqp1 gene sequence in vitro, and to assess the inhibitory effect of this siRNA on K562 cells. The siRNA targeting aqp1-mRNA was designed and transfected into K562 cells by using Lipofectamine(TM) 2000 reagent. Phase-contrast microscopy was used to analyze morphology changes of K562 cells. Cell viability was determined by MTT assay, and flow cytometry and DNA ladder analysis were carried out to identify siRNA-induced apoptosis. The expression levels of aqp1-mRNA at different transfection time were detected by RT-PCR. The results showed that the siRNA was successful by established. The transfected K562 cells displayed the significant apoptosis. The aqp1-siRNAs could obviously inhibit the activity of K562 cells. Cellular DNA fragmentation was observed in the siRNA group after transfection for 48 hours, the apoptosis rates at 24, 48 and 72 hours after transfection were 24.2%, 36.1% and 42.9% respectively. The aqp1-mRNA expression in the cells treated by aqp1-siRNA for 24, 48 and 72 hours were significantly reduced by 33%, 46% and 57% respectively. It is concluded that the aqp1-siRNA can efficiently and specifically inhibited the proliferation and inducing apoptosis of K562 cells. Gene aqp1 can be a potential target point for therapy of malignant tumor.

Signal pathway in apoptosis of K562 cells induced by STI571 / 中国实验血液学杂志

This study was aimed to investigate the effect of tyrosine kinase inhibitor (STI571) on growth and proliferation of K562 cells by using microarray method, the changes of gene expression in the process of K562 cell apoptosis induced by STI571 and the mechanism of K562 cell apoptosis. The gene microarray probes were prepared by RD-PCR technique, then the microarray of gene expression map was constructed; the morphologic changes of K562 cells were observed under phase-contrast microscopy before and after treatment with STI571; the apoptosis of K562 cells treated with STI571 was assayed by MTT method; the expression level of genes was analyzed by self-made microarray. The results indicated that after the treatment of STI571 for 24 hours, in K562 cells appeared major morphological changes, which included nuclear shrinkage, membrane bleb and scattered apoptotic bodies. DNA gel electrophoresis also showed that the typical "DNA ladder" phenomena existed in the treated group. After hybridization, detection and analysis with microarray method, expression of 9 genes significantly down-regulated and expression of 4 genes up-regulated. These differentially expressed genes included cell cycle related genes, cell metabolizing pathway related genes, signal transduction and transcription regulation related genes and antiapoptosis genes. It is concluded that STI571 can effectively inhibit the K562 cell growth and induce K562 cell apoptosis. The genes screened from this microarray offer new information for exploration of pathogenesis of K562 cell malignant transformation and shows abundant potential targets for the treatment of CML.

Effects of reperfusion arrhythmia on myocardial apoptosis and left ventricular remodeling in patients with acute myocardial infarction / 中华心血管病杂志

<p><b>OBJECTIVE</b>To observe plasma soluble Fas/APO-1 concentration in patients with reperfusion arrhythmia immediately after coronary reperfusion in patients with acute myocardial infarction (AMI) and to investigate the impact of reperfusion arrhythmia on left ventricular (LV) remodeling in AMI patients. To observe the relationship between cardiomyocytes apoptosis with reperfusion arrhythmia in patients with acute myocardial infarction (AMI), and investigate the impact of reperfusion arrhythmia on left ventricular (LV) remodeling in patients with AMI.</p><p><b>METHODS</b>One hundred and fifty-six patients with AMI who received reperfusion therapy were selected as subjects. Fifty-eight patients underwent reperfusion arrhythmia within 24 hour after coronary reperfusion treatment (RA group). Ninety-eight patients did not occurred reperfusion arrhythmia (Non-RA group). Strepavidin-biotin ELISA was used to determine the soluble Fas/APO-1 plasma concentration at baseline, 7 day (d) and 2 - 4 week (W). All patients were followed up with scheduled evaluations of LV function and morphology with left ventriculography for 1 year.</p><p><b>RESULTS</b>1. It was later that the coronary reperfusion occurred in patients of RA group than that of Non-RA group, and the left anterior descending was more frequent infarct related artery (60.3%) than of Non-RA group (36.9%, P < 0.05). 2. The Fas/APO-1 levels in patients of RA group higher than those of Non-RA group at baseline [(13.82 +/- 4.36) microg/L vs (8.19 +/- 3.56) microg/L, P < 0.01]. 3. The highest level of Fas/APO-1 was on 7 d after AMI and the plasma levels of Fas/APO-1 in 2 - 4 W were slightly lower than those in 7 d in the two groups [RA group: (10.91 +/- 3.65) microg/L vs (14.26 +/- 4.98) microg/L, P < 0.05; Non-RA group: (4.69 +/- 1.87) microg/L vs (12.19 +/- 3.25) microg/L, P < 0.01]. However, the Fas/APO-1 level of 2 - 4 W in RA group was slightly higher than the level in Non-RA group [(10.91 +/- 3.65) microg/L vs (4.69 +/- 1.87) microg/L, P < 0.01]. 4. There was on difference between two groups in left ventricular ejection fraction (LVEF) and the left ventricular end-diastolic dimension (LVEDD) one week after AMI [LVEF: (47.7 +/- 9.6)% vs (49.2 +/- 8.9)%, P > 0.05; LVEDD: (59.7 +/- 10.3) mm vs (57.4 +/- 12.4) mm, P > 0.05]. 5. In the Non-RA group, the LVEF significantly increased from 1 W phase to the 1-year phase [from (49.2 +/- 8.9)% to (59.5 +/- 9.2)%, P < 0.05], but unchanged in the 58 patients without reperfusion arrhythmia [from (47.7 +/- 9.6)% to (49.9 +/- 10.1)%, P > 0.05]. The LVEF of Non-RA group was slightly higher than that of RA group at 1 year [(59.5 +/- 9.2)% vs (49.9 +/- 10.1)%, P < 0.05]. The LVEDD had no significant difference between two groups, but there was downtrend in the Non-RA group at 1 year after AMI.</p><p><b>CONCLUSION</b>Reperfusion arrhythmia was related with cardiomyocytes apoptosis in patients with AMI, and might influence left ventricular function and promote LV remodeling.</p>

Serum autoantibodies against the cardiac beta(1)-adrenergic receptor in patients with chronic heart failure: clinical characteristics and response to carvedilol / 中华心血管病杂志

<p><b>OBJECTIVE</b>To detect the serum autoantibodies against the cardiac beta(1)-adrenergic receptor and observe the clinical characteristics and response to carvedilol use in patients with chronic heart failure (CHF).</p><p><b>METHODS</b>Cardiac function was examined by echocardiography and levels of autoantibodies against cardiac beta(1)-adrenergic receptor were detected in 65 patients with CHF by means of enzyme linked immune assay. Carvedilol was added on ACEI, diuretics and digitalis regimen for a target dose of 50 mg/d. All patients were followed up for 6 months.</p><p><b>RESULTS</b>Autoantibodies against cardiac beta(1)-adrenergic receptor were detected in 30 patients (group 1) and not detected in the remaining 35 patients (group 2). The achieved target dose of carvedilol was significantly higher in group 1 than that in group 2 [(36.25 +/- 14.31) mg/d vs. (25.97 +/- 8.83) mg/d, P < 0.01]. Heart rate was significantly higher in group 1 compared to group 2 [(94.19 +/- 14.46) beats/min vs. (86.56 +/- 15.88) beats/min, P < 0.05] before treatment and heart rate and blood pressure of both groups decreased significantly (P < 0.01) and there was no significant difference between two group (P > 0.05) after 6 months treatment. LVEDD and LVESD were significantly larger while LVEF significantly lower in group 1 patients than those in group 2 patients (all P < 0.05) before treatment and LVEDD and LVESD decreased and LVEF increased significantly in both groups (all P < 0.01 vs. before treatment) and there was on significant difference in LVEDD, LVESD and LVEF between two groups (all P > 0.05) post 6 months treatment. Moreover, average titer of autoantibodies against the cardiac beta(1)-adrenergic receptors significantly decreased after 6 months treatment (1:119.35 vs. 1:72.21, P < 0.01).</p><p><b>CONCLUSION</b>The detection of autoantibodies against the cardiac beta(1)-adrenergic receptors is related to severer cardiac dysfunction and autoantibodies title decrease was found with improved cardiac function after standard therapy (ACEI, digitalis, betablocker) in patients with CHF.</p>

Preparation and application of oligo microarrays for hepatitis virus detection and genotyping / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To prepare oligo microarrays for hepatitis virus detection and genotyping.</p><p><b>METHODS</b>By analyzing the DNA or cDNA of HBV, HDV and 4 different genotypes of HCV with the BLAST program, a group of specific sequences for the candidate probes was specified. Array Designer 3.0 software was applied to analyze the candidates to select probes with high specificity, identical length and similar melting temperature (Tm). Altogether 16, 8 and 68 oligonucleotide probes were designed for diagnosis of HBV, HDV, and genotyping HCV. Following the synthesizing and purification, oligo probes were deposited on oligonucleotide chips as microarrays for hepatitis virus detection and genotyping. The samples were labeled by RD-PCR method. Hybridization results were analyzed to cross out those probes with low specificity and sensitivity, and those with signal to noise ratios (SNR) less than 4.0.</p><p><b>RESULTS</b>Two types of gene chips were successfully developed: microarrays for HBV and HDV simultaneous detection and for HCV genotyping.</p><p><b>CONCLUSION</b>Using oligo probes to construct gene chips for clinical diagnosis of hepatitis virus is a simple and effective method. It may be widely used in detecting hepatitis viruses and their genotyping in clinical settings.</p>

Determination of scopolamine and atropine in Flos Daturae by RP-HPLC / 中国中药杂志

<p><b>OBJECTIVE</b>To develop a quantitative analytical procedure of scopolamine and atropine in Flos Daturae using RP-HPLC.</p><p><b>METHOD</b>The two alkaloids were separated on a Hypersil BDS C18 column (4.6 mm x 250 mm, 5 microm) with a mobile phase of 0.02 mol x L(-1) sodium acetate buffer (containing 0.02% triethanolamine and the pH was adjusted to 6.0 with acetic acid)-methanol (60:40) and a detection wavelength of 215 nm. The flow rate was 1.0 mL x min(-1) and the column temperature was maintained at room temperature.</p><p><b>RESULT</b>The mean recovery was (99.6 +/- 1.8)% for scopolamine and (100.4 +/- 1.5)% for atropine.</p><p><b>CONCLUSION</b>This method was simple, accurate and sensitive.</p>

Equivalent cardioprotective effect of "half-conditioning" and post-conditioning in a canine model of myocardial ischemia and reperfusion / 中华心血管病杂志

<p><b>OBJECTIVE</b>To study the effects of "half-conditioning", a modified postconditioning process, on myocardial injury induced by severe myocardial ischemia/reperfusion (I/R) in anesthetized dogs.</p><p><b>METHODS</b>Mongrel dogs of both sexes were subjected to 40 min ischemia (coronary blood flow reduced by 80% via controlled coronary stenosis). At the end of ischemia, dogs were randomly received one of the following treatments: (1) control, reperfusion for 3 h (n = 7); (2) post-conditioning, three cycles of ischemia 30 s followed by reperfusion for 30 s and then reperfusion for 3 h (n = 7); (3) half-conditioning, coronary blood flow recovered to 50% for 2 min, then 80% for 2 min, thereafter 100% for 3 h (n = 7). Electrocardiogram (ECG), arterial blood pressure and left ventricular pressure were monitored throughout the experiment. Plasma creatine kinase (CK) and lactate dehydrogenase (LDH) activity were measured spectrophotometrically. Myocardial necrosis was defined by TTC-staining.</p><p><b>RESULTS</b>Compared with control animals, arrhythmia incidence, LVEDP at 2 and 3 h reperfusion, CK and LDH were significantly reduced in animals received post-conditioning and half-conditioning treatments, infarct size as a percentage (%) of the area at risk was also significantly reduced by post-conditioning and half-conditioning treatments. No differences were observed in the post-conditioning and half-conditioning groups.</p><p><b>CONCLUSION</b>Half-conditioning exerts the same cardioprotective effects on post-ischemic hearts as postconditioning.</p>

Effect of serum autoantibodies against the M2 muscarinic acetylcholine receptors from patients with heart failure on L-Type Ca2+ channel activity in guinea pig cardiac myocytes / 中华心血管病杂志

<p><b>OBJECTIVE</b>To investigate the effect of serum autoantibodies against the human M(2) muscarinic acetylcholine receptors (M(2)-receptors, Abs) from patients with congestive heart failure on L-Type Ca(2+) channel activity in guinea pig cardiac myocytes.</p><p><b>METHOD</b>Using whole cell patch-clamp technique, we quantitatively measured the ionic intensity and density of L-Type Ca(2+) channel (I(Ca-L)).</p><p><b>RESULTS</b>The M(2)-receptors agonist (carbachol) could decrease the I(Ca-L) peak intensity and density stimulated by isoprenaline from (2111.65 +/- 203.13) pA and (18.83 +/- 1.14) pA/pF to (1230.87 +/- 208.14) pA (P < 0.01) and (10.72 +/- 1.06) pA/pF (P < 0.01). The serum Abs could also decrease I(Ca-L) peak intensity and density [from (1995.21 +/- 195.13) pA and (18.13 +/- 1.03) pA/pF to (636.42 +/- 110.07) pA (P < 0.01) and (5.54 +/- 0.81) pA/pF, P < 0.01]. The M(2)-receptors antagonist, atropine was able to block these effects of carbachol and Abs.</p><p><b>CONCLUSIONS</b>The circulating serum autoantibodies against the M(2)-receptors has similar effect as M(2)-receptors agonist on decreasing the isoprenaline stimulated I(Ca-L) in guinea pig cardiac myocytes and possess negative inotropic effect. These results further suggest that serum autoantibodies against the human M(2) muscarinic acetylcholine receptors may participate in the pathophysiological processes in patients with heart failure.</p>

Removing Murine Embryonic Stem Cells From the Differentiating Cell Culture By Using Magnetic Activated Cell Sorting / 中国生物工程杂志

Objective:To remove murine embryonic stem cells(mESC)from the differentiating cell culture and purify the differentiated cells by Magnetic Activated Cell Sorting(MACS).Methods:Neural differentiation of mESC was induced by a 5-stage method.The specific cell surface marker,SSEA-1,was used to identify ES cells in the differentiating cells.The optimal dilutions of mouse anti mouse SSEA-1 IgM primary antibody and FITC conjugated goat anti mouse secondary antibody were determined before the flow cytometry test.The incubation time and incubation temperature of primary antibody were all optimized to make the cytometry test accurate.After the optimization,stage 4 cells were dissociated into single cell suspension,incubated with antibody of SSEA-1 and microbeads conjugated goat anti mouse IgM,and then sorted through the magnetic field.The rate of SSEA-1 positive cells in pre-and post-separation groups was assessed by flow cytometry,and the viability of cells was evaluated by trypan blue staining counting under light microscopy.Results:The proportion of SSEA-1 positive cells in the separated cells can be reduced from(7.19?1.36)% to(1.34?0.80)%.The survival rate of sorted cells was more than 92%,similar to that of pre-separation cells.Conclusions:The MACS system we used can effectively remove mESC from the differentiated cells.The sorted cells will be well provided for the subsequent studies about transplantation therapy.

Construction of OTX1 Lentiviral Vector and Overexpression Research / 中国生物工程杂志

OTX1 gene is one of the pivotal transcriptional factors involved in the neurogenesis.In order to overexpress the OTX1 gene in distinct cell types and find out its contribution to the proliferation and differentiation of stem cells in vitro,OTX1 cDNA was subcloned into lentiviral vectors.The resulting constructions pDUETOTX1,pDUETGFPOTX1 and pDUETGFP were packaged in 293 cells producing viral particles to transduce 293T cells,SY5Y cells,mouse embryonic stem cells and E15 neural stem cells.It was proved that the transferred OTX1 gene was located in the nuclei of the transduced cells in stead of plasma.Lentivirus is an ideal vector delivering gene to different cells.The overexpression of OTX1 in transduced 293T cells were validated by Western blot and immunofluorescence.

Advances of Lentiviral Vectors Design and Application / 中国生物工程杂志

As an efficient gene transfer vehicle lentiviral vector has been widely used in therapy research. Comparing with other retrovirus vectors, lentiviral vectors have the unique ablility of transfecting nondividing cells and terminal differentiated cells. In addition, lentiviral vectors can accommodate two or more promoters and can carry larger foreign gene insertions. Now the new generation of lentiviral vectors encoding transcriptional control sequence provides effective means for the regulation of foreign gene expression. The development of lentiviral vectors and its application in the gene therapy field were summarized.

Effect of the positive sera of autoantibodies against the human beta1-adrenoceptor from patients with congestive heart failure on activity of L-type Ca2+ channel in guinea pig cardiac myocytes / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the effect of the positive sera of autoantibodies against the human beta1-adrenoceptor from patients with congestive heart failure on activity of L-Type Ca2+ channel in guinea pig cardiac myocytes.</p><p><b>METHOD</b>Using whole cell patch-clamp technique, we quantitatively researched the ionic intensity and density of L-type Ca2+ channel (ICa-L).</p><p><b>RESULTS</b>The beta-adrenocepter agonist isoprenaline increased the ICa-L peak intensity and density from (997.09 +/- 227.5) pA and (8.20 +/- 0.86) pA/pF to (2241.01 +/- 348.5) pA and (18.98 +/- 1.18) pA/pF, respectively (P < 0.01). The positive sera of autoantibodies against the beta1-adrenoceptor could also increase ICa-L peak intensity and density from (963.57 +/- 207.56) pA and (8.14 +/- 0.72) pA/pF to (1382.41 +/- 241.36) pA and (11.70 +/- 1.03) pA/pF (P < 0.01). Esmolol, a beta1-adrenoceptor antagonist blocked these effects of isoprenaline and autoantibodies.</p><p><b>CONCLUSIONS</b>Human cardiac positive sera of autoantibodies against the beta1-adrenoceptor has an isoproterenol-like effect on cardiac myocytes receptor. It may participate in the pathophysiologic process of cardiac myocytes.</p>

Study of autoantibodies against the G-protein-coupled beta 2- and alpha 1-adrenergic and AT1 receptors in patients with primary hypertension / 中国医学科学院学报

<p><b>OBJECTIVE</b>To determine whether autoantibodies against the cardiac G-protein-coupled beta 2- and alpha 1-adrenergic and AT1 receptors are related to patients with primary hypertension.</p><p><b>METHODS</b>Synthetic peptides corresponding to amino acid sequences of the second extracellular loops of the beta 2- and alpha 1-adrenergic and AT1 receptors were respectively used as antigens to screen sera from patients with hypertensive heart diseases (n = 50) as well as simple hypertension (n = 40) and healthy blood donors (n = 40) using ELISA test.</p><p><b>RESULTS</b>The positive ratio of autoantibodies against beta 2 and alpha 1 and AT1 receptors in patients with hypertensive heart diseases were significantly higher than patients with simple hypertension and healthy donors. The geometric mean titers of autoantibodies against beta 2- and alpha 1-adrenergic and AT1 receptors had no difference between the patients with hypertensive heart diseases and the patients with simple hypertension, but the geometric mean titers of two groups were higher than healthy donors. In the patients with hypertensive heart diseases, 81.0% of the patients with autoantibodies against beta 2-adrenergic receptor had autoantibodies against alpha 1-adrenergic receptor and 76.2% had autoantibodies against AT1 receptors. The percent of the autoantibodies against three receptors in patients with hypertensive heart diseases were 52.4%.</p><p><b>CONCLUSIONS</b>Autoantibodies against beta 2- and alpha 1-adrenergic and AT1 receptors play an important role in the pathophysiological changes of primary hypertension, and may participate myocardial and vessel remodeling.</p>