Human Platelet-Rich Plasma-Derived Exosomes Promote the Proliferation of Schwann Cells Cultured in Vitro / 中国医学科学院学报

Objective To investigate the effect of human platelet-rich plasma-derived exosomes(PRP-exos)on the proliferation of Schwann cell(SC)cultured in vitro. Methods PRP-exos were extracted by polymerization-precipitation combined with ultracentrifugation.The morphology of PRP-exos was observed by transmission electron microscopy,and the concentration and particle size distribution of PRP-exos were determined by nanoparticle tracking analysis.Western blotting was employed to determine the expression of the marker proteins CD63,CD81,and CD9 on exosome surface and the platelet membrane glycoprotein CD41.The SCs of rats were isolated and cultured,and the expression of the SC marker S100β was detected by immunofluorescence staining.The fluorescently labeled PRP-exos were co-cultured with SCs in vitro for observation of their interaction.EdU assay was employed to detect the effect of PRP-exos on SC proliferation,and CCK-8 assay to detect the effects of PRP-exos at different concentrations(0,10,20,40,80,and 160 μg/ml)on SC proliferation. Results The extracted PRP-exos appeared as uniform saucer-shaped vesicles with the average particle size of(122.8±38.7)nm and the concentration of 3.5×1012 particles/ml.CD63,CD81,CD9,and CD41 were highly expressed on PRP-exos surface(P<0.001,P=0.025,P=0.004,and P=0.032).The isolated SCs expressed S100β,and PRP-exos could be taken up by SCs.PRP-exos of 40,80,and 160 μg/ml promoted the proliferation of SCs,and that of 40 μg/ml showed the best performance(all P<0.01). Conclusions High concentrations of PRP-exos can be extracted from PRP.PRP-exos can be taken up by SCs and promote the proliferation of SCs cultured in vitro.

Quantitative Evaluation of Sciatic Nerve Crush Injury with Conventional Ultrasound Combined with Shear-wave Elastography in Rabbit Models / 中国医学科学院学报

To explore the value of conventional ultrasound combined with shear-wave elastography in the quantitative evaluation of sciatic nerve crush injury in rabbit models. Forty healthy male New Zealand white rabbits were randomly divided into four groups (=10 in each group):three crush injury (CI) groups (2,4,and 8 weeks after crush) and control group (without injury). The thickness and stiffness of the crushed sciatic nerves and denervated triceps surae muscles were measured at different time points and compared with histopathologic parameters. Inter-reader variability was assessed with intraclass correlation coefficients. Compared with the control group,the inner diameters of the sciatic nerves significantly increased in the 2-week CI group [(1.65±0.34) mm (0.97±0.15) mm,=0.00] but recovered to the nearly normal level in the 8-week CI group [(1.12±0.18) mm (0.97±0.15) mm,=0.06];however,compared with control group [(8.75±1.02)kPa],the elastic modulus of the nerves increased significantly in all the CI groups [2-week:(14.77±2.53) kPa;4-week:(19.12±3.46) kPa;and 8-week:(28.39±5.26) kPa;all =0.00];pathologically,massive hyperplasia of collagen fibers were found in the nerve tissues. The thickness of denervated triceps surae muscle decreased gradually,and the elastic modulus decreased 2 weeks after injury but increased gradually in the following 6 weeks;pathologically,massive hyperplasia of collagen fibers and adipocytes infiltration were visible,along with decreased muscle wet-weight ratio and muscle fiber cross-sectional area. The inter-reader agreements were good. Conventional ultrasound combined with shear-wave elastography is feasible for the quantitative evaluation of the morphological and mechanical properties of crushed nerves and denervated muscles.

Controlled study of ultrasound-guided acupotomy on spinal and articular diseases / 中国针灸

<p><b>UNLABELLED</b>OBJECTIVEL to study the visualization of acupotomy therapy and to summarize differences of interventional ultrasound combined with acupotomy on spinal and articular diseases.</p><p><b>METHODS</b>With randomized block design, 20 cases of shoulder periarthritis, 20 cases of knee arthritis, 20 cases of lumbar disc herniation and 20 cases of cervical disc herniation were divided into an ultrasound-guided group and regular group, 10 cases of each diseas in each group. The ultrasound-guided acupotomy and regular acupotomy were given for one time, respectively. Efficacy was observed after one week. Visual analogue scale (VAS) and fuction score were applied for efficacy assessment, including Wang's funtion evaluation table for cervical disc herniation, modified lumbago assessment by Japanese Orthopaedic Association for lumbar disc herniation, modified lumbago assessment arthritis and CMC shoulder funtion scale for shoulder periarthritis. The changes of scores of patients in two groups were observed before and after the treatment.</p><p><b>RESULTS</b>Each disease in each group achieved favorable efficacy; compared before and after treatment, differences of VAS and each function score all were statistically significant (allP<0.01). The efficacy of lumbar disc herniation and cerical disc herniation in two groups was not obviously different (both P>0.01). The efficacy of lumbar disc herniation and cervical disc herniation in two groups was not obviously different (bothP>0.05), while VAS and fuction scores improvements of shoulder periarthritis and knee +/-5.75 vs 75.72+/-8.56; knee arthritis: 90.40+/-7.35 vs 75.54+/-9.21, both P<0.05).</p><p><b>CONCLUSION</b>Whether ultrasound guidance is applied during acupotomy therapy made no obvious influence on efficacy of lumbar disc herniation and cervical disc herniation, but for shoulder periarthritis and knee arthritis better results could be acchieved with ultrasound guidance.</p>

Preliminary study on the visualization of ultrasound-guided acupotomy manipulation / 中国针灸

<p><b>OBJECTIVE</b>To evaluate the feasibility and safety of visualized acupotomy.</p><p><b>METHODS</b>The ultrasound-guided acupotomy was applied to 52 cases of soft tissue injury and osteoarthropathy. Visual Analog Scale (VAS) was adopted. VAS score was observed before and after treatment. The modified knee joint scale of hospital for special surgery (HSS) and Constant-Murley shoulder function scale were used to assess the joint function of patients with arthritis of knee and periarthritis of shoulder.</p><p><b>RESULTS</b>After treatment, VAS score was improved apparently as compared with that before treatment (6.560 +/- 0.893 vs 1.058 +/- 0.857, P < 0.05). The curative rate after one treatment was 48.1% (25/52) and the total effective rate was 98.1% (51/52). After treatment, HSS and Constant-Murley scores were improved apparently (both P < 0.05). In follow-up, the complications were not discovered, such as infection and nerve injury.</p><p><b>CONCLUSION</b>The ultrasound-guided acupotomy is the safe and effective therapy for soft tissue injury and osteoarthropathy and this therapy deserves to be promoted in clinical practice.</p>

Triphasic mechanical properties of osteoarthritis cartilage with different pathological grades / 医用生物力学

Objective To obtain the axial elastic modulus of rabbit articular cartilage based on the ultrasound swelling observation technique and the triphasic theory, and to explore the triphasic mechanical properties of the osteoarthritis cartilage with different pathological grades. Methods The articular cartilage samples from rabbit knees were assessed according to the different pathological grades. The swelling strains were recorded based on the high frequency transient ultrasound measurement technique. The axial elastic modulus of the cartilage samples was extracted based on the fixed charge density, the water volume fraction and the triphasic model. Correlation analysis was also made. Results There were both significant differences in axial elastic modulus between the normal cartilages and among the osteoarthritis cartilage with different pathological grades (P<0.05). The value of axial elastic modulus decreased with the increase of osteoarthritis grades. For normal cartilage samples, the average axial elastic modulus was (15.87±6.30) MPa. For osteoarthritis cartilages of grade 1, 2 and 3, the value of axial elastic modulus were (11.33±5.21), (9.15±5.68) and (6.05±4.99) MPa, respectively. Conclusions This study showed there are significant differences in triphasic mechanical properties of the articular cartilage with different osteoarthritis grades, which may provide some new thought for the quantitative assessment of osteoarthritis grade based on mechanical properties of cartilage.

Clinical value of contrast-enhanced ultrasound in the diagnosis of Budd-Chiari syndrome with inferior vena cava obstruction / 中国医学科学院学报

<p><b>OBJECTIVE</b>To explore the clinical value of contrast-enhanced ultrasound (CEUS) in the diagnosis of Budd-Chiari syndrome with inferior vena cava (IVC) obstruction.</p><p><b>METHOD</b>A total of 38 patients with Budd-Chiari syndrome with IVC obstruction were examined by CEUS before and after vascular interventional management, and the results were compared with angiographic findings.</p><p><b>RESULTS</b>The location and degree of IVC obstruction were clearly showed on CEUS. Enhancement was found in the occlusion site, and blood flow was narrowed at the stenosis site. The arrival time was earlier after treatment in the IVC obstruction, and it was positively correlated with the pressure of IVC( P< 0.05).</p><p><b>CONCLUSION</b>The location and type of the occlusion and stenosis in IVC can be accurately determined by CEUS. Therefore, CEUS can provide useful information for the selection of surgical procedures and post-operative effectiveness.</p>

Effect of Tbx1 knock-down on cardiac performance in zebrafish / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Tbx1 is the major candidate gene for DiGeorge syndrome (DGS). Similar to defects observed in DGS patients, the structures disrupted in Tbx1(-/-) animal models are derived from the neural crest cells during development. Although the morphological phenotypes of some Tbx1 knock-down animal models have been well described, analysis of the cardiac performance is limited. Therefore, myocardial performance was explored in Tbx1 morpholino injected zebrafish embryos.</p><p><b>METHODS</b>To elucidate these issues, Tbx1 specific morpholino was used to reduce the function of Tbx1 in zebrafish. The differentiation of the myocardial cells was observed using whole mount in situ hybridization. Heart rates were observed and recorded under the microscope from 24 to 72 hours post fertilization (hpf). The cardiac performance was analyzed by measuring ventricular shortening fraction and atrial shortening fraction.</p><p><b>RESULTS</b>Tbx1 morpholino injected embryos were characterized by defects in the pharyngeal arches, otic vesicle, aortic arches and thymus. In addition, Tbx1 knock down reduced the amount of pharyngeal neural crest cells in zebrafish. Abnormal cardiac morphology was visible in nearly 20% of the Tbx1 morpholino injected embryos. The hearts in these embryos did not loop or loop incompletely. Importantly, cardiac performance and heart rate were reduced in Tbx1 morpholino injected embryos.</p><p><b>CONCLUSIONS</b>Tbx1 might play an essential role in the development of pharyngeal neural crest cells in zebrafish. Cardiac performance is impaired by Tbx1 knock down in zebrafish.</p>

Effect of dihydrofolate reductase gene knock-down on the expression of heart and neural crest derivatives expressed transcript 2 in zebrafish cardiac development / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Folic acid is very important for embryonic development and dihydrofolate reductase is one of the key enzymes in the process of folic acid performing its biological function. Therefore, the dysfunction of dihydrofolate reductase can inhibit the function of folic acid and finally cause the developmental malformations. In this study, we observed the abnormal cardiac phenotypes in dihydrofolate reductase (DHFR) gene knock-down zebrafish embryos, investigated the effect of DHFR on the expression of heart and neural crest derivatives expressed transcript 2 (HAND2) and explored the possible mechanism of DHFR knock-down inducing zebrafish cardiac malformations.</p><p><b>METHODS</b>Morpholino oligonucleotides were microinjected into fertilized eggs to knock down the functions of DHFR or HAND2. Full length of HAND2 mRNA which was transcribed in vitro was microinjected into fertilized eggs to overexpress HAND2. The cardiac morphologies, the heart rates and the ventricular shortening fraction were observed and recorded under the microscope at 48 hours post fertilization. Whole-mount in situ hybridization and real-time PCR were performed to detect HAND2 expression.</p><p><b>RESULTS</b>DHFR or HAND2 knock-down caused the cardiac malformation in zebrafish. The expression of HAND2 was obviously reduced in DHFR knock-down embryos (P < 0.05). Microinjecting HAND2 mRNA into fertilized eggs can induce HAND2 overexpression. HAND2 overexpression rescued the cardiac malformation phenotypes of DHFR knock-down embryos.</p><p><b>CONCLUSIONS</b>DHFR plays a crucial role in cardiac development. The down-regulation of HAND2 caused by DHFR knock-down is the possible mechanism of DHFR knock-down inducing the cardiac malformation.</p>

Folic acid antagonist methotrexate causes the development malformation of heart and down-regulates the BMP2b and HAS2 expressions in zebrafish / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the effect of methotrexate (MTX), a folic acid antagonist which can lead to folic acid deficient, on the cardiac development and on the expressions of BMP2b and HAS2 in zebrafish.</p><p><b>METHODS</b>The zebrafish embryos at 6-48 hrs post fertilization (hpf) were treated with various concentrations of MTX (0.5 x 10(-3), 1.0 x 10(-3) and 2.0 x 10(-3) M). At 48 hpf, the percentage of cardiac malformation and heart rate were recorded. The zebrafish embryos at 6-10 hpf treated with 1.5 x 10(-3) M MTX were used as the MTX treatment group. At 24 and 48 hpf the cardiac morphology was observed under a microscope. The expressions of BMP2b and HAS2 in zebrafish were detected by in situ antisense RNA hybridization and real-time PCR.</p><p><b>RESULTS</b>6-12 hpf, the early embryonic developmental stage, was a sensitive period that MTX affected cardiac formation of zebrafish. The retardant cardiac development and the evidently abnormal cardiac morphology was found in the MTX treatment group. The results of in situ antisense RNA hybridization showed that the expressions of BMP2b and HAS2 in the zebrafish heart were reduced in the MTX treatment group at 36 and 48 hpf. The real-time PCR results demonstrated that the BMP2b expression decreased at 12, 24, 36 and 48 hpf, and that the HAS2 expression decreased at 24, 36 and 48 hpf in the treatment group compared with the control group without MTX treatment.</p><p><b>CONCLUSIONS</b>The inhibition of folic acid function may affect cardiac development of early embryos, resulting in a retardant development and a morphological abnormality of the heart in zebrafish, possibly by down-regulating the expressions of BMP2b and HAS2.</p>

Effect of external retinoic acid on Tbx1 gene during zebrafish embryogenesis / 中华儿科杂志

<p><b>OBJECTIVE</b>DiGeorge/del22q11 syndrome is one of the most common genetic causes of outflow tract and aortic arch defects in human. DiGeorge/del22q11 is thought to involve an embryonic defect restricted to the pharyngeal arches and the corresponding pharyngeal pouches. Previous studies have evidenced that retinoic acid (RA) signaling is definitely indispensable for the development of the pharyngeal arches. Tbx1, one of the T-box containing genes, is proved to be the most attractive candidate gene for DiGeorge/del22q11 syndrome. However, the interaction between RA and Tbx1 has not been fully investigated. Exploring the interaction will contribute to discover the molecular pathways disrupted in DiGeorge/del22q11 syndrome, and will also be essential for understanding genetic basis for congenital heart disease. It now seems possible that genes and molecular pathways disrupted in DiGeorge syndrome will also account for some isolated cases of congenital heart disease. Accordingly, the present study aimed to extensively study the effects of external RA on the cardiac development and Tbx1 expression during zebrafish embryogenesis.</p><p><b>METHODS</b>The chemical genetics approach was applied by treating zebrafish embryos with 5 x 10(-8) mol/L RA and 10(-7) mol/L RA at 12.5 hour post fertilization (hpf). The expression patterns of Tbx1 were monitored by whole-mount in situ hybridization and quantitative real-time RT-PCR, respectively.</p><p><b>RESULTS</b>The zebrafish embryos treated with 5 x 10(-8) mol/L RA and 10(-7) mol/L RA for 1.5 h at 12.5 hpf exhibited selective defects of abnormal heart tube. The results of whole-mount in situ hybridization with Tbx1 RNA probe showed that Tbx1 was expressed in cardiac region, pharyngeal arches and otic vesicle during zebrafish embryogenesis. RA treatment led to a distinct spatio-temporal expression pattern for Tbx1 from that in wild type embryo. The real-time PCR analysis showed that Tbx1 expression levels were markedly reduced by RA treatment. Tbx1 expression in the pharyngeal arches and heart were obviously down regulated compared to the wild type embryos. In contrast to 5 x 10(-8) mol/L RA-treated groups, 10(-7) mol/L RA caused a more severe effect on the Tbx1 expression level.</p><p><b>CONCLUSION</b>These results suggested that there was a genetic link between RA and Tbx1 during development of zebrafish embryo. RA could produce an altered Tbx1 expression pattern in zebrafish. RA may regulate the Tbx1 expression in a dose-dependant manner. RA could represent a major epigenetic factor to cause abnormal expression of Tbx1, secondarily, disrupt the pharyngeal arch and heart development.</p>

Analysis on the cost and its related factors of clinically confirmeds severe acute respiratory syndrome cases in Beijing / 中华流行病学杂志

<p><b>OBJECTIVE</b>To explore the medical cost and its related factors of clinically confirmed severe acute respiratory syndrome (SARS) cases in Beijing.</p><p><b>METHODS</b>The average medical cost per person/day was calculated on 1272 SARS cases with complete hospitalization data from the Beijing SARS clinical database. Factors associated with medical expenditure were explored by both single factor and multi-factor analyses.</p><p><b>RESULTS</b>The medical costs of SARS patients in Beijing were 18 767.50 RMB/case and 632.02 RMB/day/case. Expenses on medication accounted for the highest proportion (0.66%). The increase in expenses on medication was significantly associated to the increase in total expenditure. Analyses by single factor, factor stratification as well as multi-factor analyses showed that: the expenditure increased significantly with the following factors of the patients: prolonged days of hospitalization, severe symptoms and signs, hospitalization at the early stage of disease onset, being medical staff themselves, with baseline diseases, being applied mechanical ventilation, and with medical insurance.</p><p><b>CONCLUSIONS</b>The medical expenditure on SARS patients at the hospital was higher than that on patients with common diseases which was associated to the following factors as: days of hospitalization, severity of disease, stage of the disease break-out, patient's occupation, baseline disease status, medical insurance status etc.</p>

Relationship between the severity, course, fatality of severe acute respiratory syndrome patients and the timing of hospitalization / 中华流行病学杂志

<p><b>OBJECTIVE</b>To study the relationship of timing of hospitalization and the severity, course, fatality of severe acute respiratory syndrome (SARS) patients.</p><p><b>METHOD</b>1291 hospital records of clinically diagnosed SARS patients with complete data gathered from "2003 Beijing SARS Clinical Database" were analyzed.</p><p><b>RESULTS</b>SARS cases were categorized into four groups, according to the time of hospitalization after onset of the disease: within 3 days, during day 4 to day 7, during day 8 to day 14 and after day 14. The numbers of cases for each group were 568, 496, 177 and 50 respectively. Data showed that from group 1 to 4, the prevalence rates of major symptoms on the first day of hospitalization were: (1) 9.7%, 16.5%, 23.1% and 24.0% for "feeling chest pain" (P < 0.001), (2) 7.4%, 13.7%, 19.2% and 22.0% for "suffering from breathing obstruction" (P < 0.001), (3) 32.8%, 44.8%, 59.9% and 48.0%, for "coughing" (P < 0.001) and (4) 14.1%, 22.4%, 27.1% and 18.0% for "coughing up phlegm" (P = 0.0002), respectively. The rates of high respiratory frequency (>or= 24 bits/min.) were 11.1%, 15.5%, 22.8% and 25.5% (P < 0.001). The rates of abnormal chest X-ray were 80.3%, 89.0%, 92.3% and 88.9%, respectively (P = 0.002). The average numbers of abnormal lung field (the lung were divided into 6 fields) were 1.7, 1.9, 2.5 and 2.6 (P < 0.001); The numbers of cases receiving continuous oxygen supply treatment were 33.6%, 50.0%, 53.7% and 74.0% (P < 0.001), and the numbers of cases receiving glucocorticosteroids treatment were 28.2%, 35.9%, 53.7% and 62.0% (P < 0.001), respectively. With cases having had chronic baseline diseases prior to SARS infection, the age-standardized fatality rates were 14.9%, 11.7%, 50.0% and 33.9% (P < 0.001), and the average courses of the disease were 30.3, 34.2, 42.9 and 47.5 days (P < 0.001), respectively. In cases without chronic baseline diseases, the age-standardized fatality rates were 5.3%, 9.8%, 9.2% and 8.3% (P = 0.101), and the average courses for each group were 32.4, 35.3, 40.9 and 47.6 days (P < 0.001), respectively.</p><p><b>CONCLUSION</b>Delayed hospitalization would cause the situation of SARS patient to deteriorate, losing the best chance for treatment and increase case fatality. In terms of control program on SARS, emphasize should be paid on decreasing the panic of patients to the disease so as to get early hospitalization.</p>

Preparation and functional study of an adenovirus vector expressing human plasminogen kringle 5 gene / 生物工程学报

Tumor angiogenesis plays a pivotal role in the progress of tumor. Among the various endogenous angiogenic inhibitors discovered, the human plasminogen kringle 5 (K5) has been demonstrated to be a potential inhibitor of the proliferation and migration of vascular endothelial cells in vitro. The replication-incompetent adenovirus (Ad) vector Adeno-X-CMV-K5 (Ad-K5) (where CMV is cytomegalovirus) was constructed and its antiangiogenic effect was tested on vascular endothelial cell and tumor cell. For the construction, the K5 cDNA was fused in-frame with human plasminogen signal sequence and inserted into the eukaryotic expression vector pcDNA3 to form pcDNA3K5. The recombinant plasmid was subcloned into the shuttle plasmid pShuttle under the control of the constitutive CMV immediate-early promoter. The plasmid carrying the cDNA for K5 (pShuttleKS) was then recombined with the Adeno-X viral DNA and transformed into E. coli DH5alpha. The resultant recombinant plasmid pAd-K5 was transfected into human embryonic kidney (HEK) 293 cells with liposome. The adenovirus expressing human plasminogen kringle 5 (Ad-K5) was successfully packaged and propagated in 293 cells, as detected by the cytopathic effect (CPE) on the cells, and the viral titer in the supernatant was 5 x 10(8) pfu/mL by plaque assay. Both human umbilical vein endothelial cell line ECV304 and human breast carcinoma cell line MDA-MB-231 were infected with Ad-K5 and Ad-LacZ, which was used the negative control, and assayed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Compared with uninfected control and Ad-LacZ infected control, Ad-K5 infected group at 80 MOI (multiplicity of infection) significantly inhibited ECV304 proliferation; the difference between uninfected control and Ad-LacZ infected control was not significant. In contrast, there was no significant difference in the proliferation of MDA-MB-231 among all the treatments. In addition, the Ad-K5 at 100 MOI inhibited the differentiation and tube formation of ECV304 on ECMatrix gel. These results suggested that the recombinant replication-defective Adenovirus expressing human plasminogen kringle 5 inhibited the proliferation, differentiation and tube formation of ECV304 and had no effect on the proliferation of MDA-MB-231. Adenovirus mediated human plasminogen kringle 5 gene therapy may be a potential treatment of cancer through angiogenesis inhibition.