Effects of Platelet-Rich Plasma-Derived Exosomes on Proliferation and Migration of Tendon Stem/Progenitor Cell.

Objective To investigate the effects of platelet-rich plasma-derived exosomes (PRP-Exos) on the proliferation and migration of tendon stem/progenitor cell (TSPC). Methods PRP-Exos were extracted through the combination of polymer-based precipitation and ultracentrifugation.The morphology,concentration,and particle size of PRP-Exos were identified by transmission electron microscopy and nanoparticle tracking analysis.The expression levels of surface marker proteins on PRP-Exos and platelet membrane glycoproteins were determined by Western blot analysis.Rat TSPC was extracted and cultured,and the expression of surface marker molecules on TSPC was detected using flow cytometry and immunofluorescence staining.The proliferation of TSPC influenced by PRP-Exos was evaluated using CCK-8 assay and EdU assay.The effect of PRP-Exos on the migration of TSPC was evaluated by cell scratch assay and Transwell assay. Results The extracted PRP-Exos exhibit typical saucer-like structures,with a concentration of 4.9×1011 particles/mL,an average particle size of (132.2±56.8) nm,and surface expression of CD9,CD63 and CD41.The extracted TSPC expressed the CD44 protein.PRP-Exos can be taken up by TSPC,and after co-cultured for 48 h,concentrations of 50 and 100 µg/mL of PRP-Exos significantly promoted the proliferation of TSPC (both P<0.001),with no statistical difference between the two concentrations (P=0.283).Additionally,after co-cultured for 24 h,50 µg/mL of PRP-Exos significantly promoted the migration of TSPC (P<0.001). Conclusion Under in vitro culture conditions,PRP-Exos significantly promote the proliferation and migration of rat TSPC.

[Human Platelet-Rich Plasma-Derived Exosomes Promote the Proliferation of Schwann Cells Cultured in Vitro].

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 S100ß 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 S100ß,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.

A novel tissue engineered nerve graft constructed with autologous vein and nerve microtissue repairs a long-segment sciatic nerve defect.

Veins are easy to obtain, have low immunogenicity, and induce a relatively weak inflammatory response. Therefore, veins have the potential to be used as conduits for nerve regeneration. However, because of the presence of venous valves and the great elasticity of the venous wall, the vein is not conducive to nerve regeneration. In this study, a novel tissue engineered nerve graft was constructed by combining normal dissected nerve microtissue with an autologous vein graft for repairing 10-mm peripheral nerve defects in rats. Compared with rats given the vein graft alone, rats given the tissue engineered nerve graft had an improved sciatic static index, and a higher amplitude and shorter latency of compound muscle action potentials. Furthermore, rats implanted with the microtissue graft had a higher density and thickness of myelinated nerve fibers and reduced gastrocnemius muscle atrophy compared with rats implanted with the vein alone. However, the tissue engineered nerve graft had a lower ability to repair the defect than autogenous nerve transplantation. In summary, although the tissue engineered nerve graft constructed with autologous vein and nerve microtissue is not as effective as autologous nerve transplantation for repairing long-segment sciatic nerve defects, it may nonetheless have therapeutic potential for the clinical repair of long sciatic nerve defects. This study was approved by the Experimental Animal Ethics Committee of Chinese PLA General Hospital (approval No. 2016-x9-07) on September 7, 2016.

[Quantitative Evaluation of Sciatic Nerve Crush Injury with Conventional Ultrasound Combined with Shear-wave Elastography in Rabbit Models].

Objective To explore the value of conventional ultrasound combined with shear-wave elastography in the quantitative evaluation of sciatic nerve crush injury in rabbit models. Methods Forty healthy male New Zealand white rabbits were randomly divided into four groups (n=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. Results 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 vs. (0.97±0.15) mm,P=0.00] but recovered to the nearly normal level in the 8-week CI group [(1.12±0.18) mm vs. (0.97±0.15) mm,P=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 P=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. Conclusion 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.

A Multicenter Large-Sample Shear Wave Ultrasound Elastographic Study of the Achilles Tendon in Chinese Adults.

OBJECTIVES: The changes in the viscoelasticity of the Achilles tendon are related to tendinopathy. Therefore, constructing a data model in the healthy population is essential to understanding the key factors affecting the viscoelasticity of the Achilles tendon. The purpose of our research was to obtain large sample data, construct a data model, and determine parameters that affect the elastic modulus of the Achilles tendon in healthy Chinese adults. METHODS: We designed a prospective multicenter clinical trial to evaluate the viscoelasticity of the Achilles tendon by using shear wave elastography. A total of 1165 healthy adult participants from 17 Chinese hospitals were recruited for the assessment. The necessary parameters (age, height, weight, and body mass index) were recorded. The elastic modulus (Young modulus) was obtained from the middle of the Achilles tendon and calculated with feet in naturally relaxed, dorsal, and plantar positions. The thickness and perimeter of the Achilles tendon were measured via cross section on the same site. A multiple linear regression was performed to find the key factors affecting the Young modulus of the Achilles tendon. RESULTS: The Young modulus of the left Achilles tendon in the natural relaxed position followed a normal distribution (P > .05) with a mean ± SD of 374.24 ± 106.12 kPa. The regression equations showed a positive correlation between the Young modulus and weight and a negative correlation between the Young modulus and the circumference or thickness of the left Achilles tendon (P < .05). CONCLUSIONS: The Young modulus of the Achilles tendon as measured by shear wave elastography is related to body weight as well as the perimeter or thickness of the tendon.

Evaluation of Elastic Stiffness in Healing Achilles Tendon After Surgical Repair of a Tendon Rupture Using In Vivo Ultrasound Shear Wave Elastography.

BACKGROUND There has been no published report assessing the mechanical properties of a repaired Achilles tendon after surgery using shear wave elastography (SWE). The aim of this study was to investigate the changes in mechanical properties of the healing Achilles tendon after surgical repair of a tendon rupture using ultrasound SWE and how these changes correlate with tendon function. MATERIAL AND METHODS Twenty-six patients who underwent surgical repair for Achilles tendon rupture were examined with ultrasound SWE coupled with a linear array transducer (4-15 MHz). The elasticity values of the repaired Achilles tendon in a longitudinal view were measured at 12, 24, and 48 weeks postoperatively. Functional outcomes were assessed with the American Orthopedic Foot and Ankle Society (AOFAS) rating system at 12, 24, and 48 weeks postoperatively. General linear regression analysis and correlation coefficients were used to analyze the relationship between elasticity and the AOFAS score. RESULTS There were significant differences with respect to the mean elasticity values and functional scores of the repaired Achilles tendon at 12, 24, and 48 weeks postoperatively (all P<0.05). Tendon function was positively correlated with the elasticity of the repaired Achilles tendon (P=0.0003). CONCLUSIONS Our findings suggest that SWE can provide biomechanical information for evaluating the mechanical properties of healing Achilles tendon and predict Achilles tendon function.

Low tristetraprolin expression promotes cell proliferation and predicts poor patients outcome in pancreatic cancer.

Tristetraprolin (also known as TTP, TIS11, ZFP36, and Nup475) is a well-characterized tumor suppressor that is down-regulated in several tumor types. In the current study, we found that TTP expression was markedly reduced in pancreatic cancer samples as compared to matched normal tissues. Low TTP level was associated with age (P=0.037), tumor size (P=0.008), tumor differentiation (P=0.004), postoperative T stage (pT stage, P<0.001), postoperative N stage (pN stage, P=0.008) and TNM stage (P<0.001). Moreover, low TTP expression predicted reduced survival rates and poor patient outcome. We also found that TTP impairs pancreatic cancer cell proliferation both in vivo and in vitro. Fluorescence Activated Cell Sorting (FACS) assay showed that TTP over-expression both increases apoptosis and decreases proliferation in pancreatic cancer cells. RNA-sequencing analysis showed that TTP over-expression downregulates several tumor-related factors, including Pim-1 and IL-6. Our findings indicate that TTP could serve as a potential prognostic indicator in pancreatic cancer.

Neuroprotective effects of ultrasound-guided nerve growth factor injections after sciatic nerve injury.

Nerve growth factor (NGF) plays an important role in promoting neuroregeneration after peripheral nerve injury. However, its effects are limited by its short half-life; it is therefore important to identify an effective mode of administration. High-frequency ultrasound (HFU) is increasingly used in the clinic for high-resolution visualization of tissues, and has been proposed as a method for identifying and evaluating peripheral nerve damage after injury. In addition, HFU is widely used for guiding needle placement when administering drugs to a specific site. We hypothesized that HFU guiding would optimize the neuroprotective effects of NGF on sciatic nerve injury in the rabbit. We performed behavioral, ultrasound, electrophysiological, histological, and immunohistochemical evaluation of HFU-guided NGF injections administered immediately after injury, or 14 days later, and compared this mode of administration with intramuscular NGF injections. Across all assessments, HFU-guided NGF injections gave consistently better outcomes than intramuscular NGF injections administered immediately or 14 days after injury, with immediate treatment also yielding better structural and functional results than when the treatment was delayed by 14 days. Our findings indicate that NGF should be administered as early as possible after peripheral nerve injury, and highlight the striking neuroprotective effects of HFU-guided NGF injections on peripheral nerve injury compared with intramuscular administration.

Isolation and characterization of a novel lectin with mitogenic activity from Pleurotus ferulae.

Lectins are the tools for the determination of sugar chain structure. Recently, lectin arrays have become a popular new technology; therefore, lectins with specific sugar-binding properties are required. The objective of the study was to isolate a novel lectin from Pleurotus ferulae mushrooms and characterize its various biological activities. A novel lectin was extracted with deionized water, precipitated from the aqueous extract using 75% saturated (NH4)2SO4, and subjected on DEAE-cellulose followed by affinity chromatography on sepharose-6B. The activity was tested using hemagglutination assays, and carbohydrate-binding specificity was determined by glycan microarray analysis. Its effects on the mitogenic activity of mouse splenocytes were determined by MTT assay. The novel lectin was adsorbed on ion-exchange chromatography DEAE-cellulose and shown as a band with the molecular mass of 17.5 kDa on a SDS-PAGE and as a single 35.0-kDa peak in gel filtration on Superdex G-75. The hemagglutinating activity of the lectin was inhibited by D-glucose, lactose, D-galactose, and galactosamine. The lectin was stable on 60°C. The hemagglutinating activity of lectin was reduced by 50% at 70°C. At 80°C, it was further reduced to 6.25% of its original activity. The hemagglutinating activity was the highest at pH 6-9. Moreover, its hemagglutinating activity was inhibited by Mg2+ and Ca2+ ions. The lectin isolated from P. ferulae in the current study possessed highly potent hemagglutinating and proliferative activities toward mouse splenocytes.

[Controlled study of ultrasound-guided acupotomy on spinal and articular diseases].

UNLABELLED: OBJECTIVEL to study the visualization of acupotomy therapy and to summarize differences of interventional ultrasound combined with acupotomy on spinal and articular diseases. METHODS: 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. RESULTS: 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). CONCLUSION: 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.

[Preliminary study on the visualization of ultrasound-guided acupotomy manipulation].

OBJECTIVE: To evaluate the feasibility and safety of visualized acupotomy. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Ultrasonic reflection coefficient and surface roughness index of OA articular cartilage: relation to pathological assessment.

BACKGROUND: Early diagnosis of osteoarthritis (OA) is essential for preventing further cartilage destruction and decreasing severe complications. The aims of this study are to explore the relationship between OA pathological grades and quantitative acoustic parameters and to provide more objective criteria for ultrasonic microscopic evaluation of the OA cartilage. METHODS: Articular cartilage samples were prepared from rabbit knees and scanned using ultrasound biomicroscopy (UBM). Three quantitative parameters, including the roughness index of the cartilage surface (URI), the reflection coefficients from the cartilage surface (R) and from the cartilage-bone interface (Rbone) were extracted. The osteoarthritis grades of these cartilage samples were qualitatively assessed by histology according to the grading standards of International Osteoarthritis Institute (OARSI). The relationship between these quantitative parameters and the osteoarthritis grades was explored. RESULTS: The results showed that URI increased with the OA grade. URI of the normal cartilage samples was significantly lower than the one of the OA cartilage samples. There was no significant difference in URI between the grade 1 cartilage samples and the grade 2 cartilage samples. The reflection coefficient of the cartilage surface reduced significantly with the development of OA (p < 0.05), while the reflection coefficient of the cartilage-bone interface increased with the increase of grade. CONCLUSION: High frequency ultrasound measurements can reflect the changes in the surface roughness index and the ultrasound reflection coefficients of the cartilage samples with different OA grades. This study may provide useful information for the quantitative ultrasonic diagnosis of early OA.

Effect of shRNA mediated down-regulation of Annexin A2 on biological behavior of human lung adencarcinoma cells A549.

In the previous study, we found that Annexin A2 was significantly up-regulated in lung cancer and could induce related-antigen in lung cancer patients' serum. To further study the function of Annexin A2, the short hairpin RNA plasmid targeting Annexin A2 was constructed in vitro and transfected into human lung adencarcinoma A549 cells. Knocking down Annexin A2 expression by shRNA, the mRNA level of Annexin A2 was investigated by semi-quantitative RT-PCR. The expression of Annexin A2 protein was examined by Western Blotting and Immuocytochemistry. MTT assay and Transwell chamber model were used to evaluate proliferation and invasion of A549 cells in vitro. The concentration of matrix metalloproteinase-2 (MMP-2) and cathepsin B (CB) in the supernatant was evaluated by ELISA. At 48 h after transfection, the expression of Annexin A2 mRNA and protein was down-regulated significantly, respectively (p < 0.05).The proliferation and invasion capability of A549 cells also decreased significantly (p < 0.05). The concentration of MMP-2 and CB was down-regulated obviously, respectively (p < 0.05). This study implies that Annexin A2 might play an important role in the progression and invasion of human lung cancer cells, and could promote progression of lung cancer by regulating the expression of MMP-2 and CB.

[Efficacy and safety of focused ultrasound ablation in treatment of submucosal uterine fibroids].

OBJECTIVE: To evaluate the efficacy and safety of focused ultrasound ablation in the treatment of submucosal fibroids which broke into uterine cavity less than 50%. METHODS: From Oct. 2006 to Sept. 2009, 66 patients with 69 submucosal fibroids broke into uterine cavity less than 50% diagnosed by MRI in Chinese People's Liberation Army General Hospital were enrolled in this study. They were treated by ultrasound-guided focused ultrasound ablation in the outpatient department, which using the contrast enhanced ultrasonography to assess the efficacy after ablation immediately, to measure reduction of fibroids volume and record adverse effect before and after ultrasound ablation. At 3, 6, 12 and 24 months after treatment, ablation outcome and fibroids volumes were evaluated by contrast ultrasound. The changes of clinical symptom were evaluated by the symptom severity score (SSS) of the uterine fibroid quality-of-life instrument (UFS-QOL). RESULTS: The average volume of fibroids in 66 patients with 68 submucosal fibroids were (151 ± 134) cm(3) before treatment and (114 ± 104) cm(3) no enhanced regional after treatment. The ablation rate of target fibroids was (77 ± 16)%. All patients completed this treatment successfully, they were followed up for 6 - 44 months, the median follow-up time was 24 months. No serious complication was observed. However, there were 52% (34/66) patients presented vaginal discharge after ablation, it disappeared gradually after 3 to 4 menstrual cycles. The SSS and the menstrual period symptom scores were significantly lower than that before ablation at the follow-up of 3, 6, 12 and 24 months, the rates were 20.9%, 38.0%, 45.1%, 47.1% and 42.0%, 63.8%, 64.2%, 68.8%, which all reached statistical difference (P < 0.05). The necrotic fibroids were absorbed gradually, the reduction rates of fibroid volume were 44.7%, 66.0%, 77.7% and 89.8%. CONCLUSION: It was safe and efficacy that focused ultrasound ablation was used in treatment of submucosal fibroids which broke into the uterine less than 50%.

[Clinical value of contrast-enhanced ultrasound in the diagnosis of Budd-Chiari syndrome with inferior vena cava obstruction].

OBJECTIVE: To explore the clinical value of contrast-enhanced ultrasound (CEUS) in the diagnosis of Budd-Chiari syndrome with inferior vena cava (IVC) obstruction. METHOD: 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. RESULTS: 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). CONCLUSION: 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.

Effect of Tbx1 knock-down on cardiac performance in zebrafish.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

[Influence of CRX gene on development of photoreceptors in zebrafish].

OBJECTIVE: To study the development and opsin expression of zebrafish photoreceptors after CRX gene knock-down. METHODS: It was a experimental study. CRX-MOs was microinjected into the zygote of zebrafish. Seventy-two and 96 hours post fertilization, we prepared the eye histological slides, electron microscope samples and whole mount in-situ hybridizations to study the development and opsin expression. of photoreceptors. RESULTS: There were no obvious histological changes in optical microscope observation after CRX-MOs microinjection. Outer segments were developed in control and blank groups, but no obvious outer segment was found in CRX group by electron microscope observation. Opsin Rho, SWS1 and SWS2 were expressed in control and blank groups and the expression of these genes was obviously decreased in CRX group. CONCLUSION: CRX gene can influence the development and opsins expression of photoreceptors outer segments in zebrafish.

[Effect of external retinoic acid on Tbx1 gene during zebrafish embryogenesis].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Effect of dihydrofolate reductase gene knock-down on the expression of heart and neural crest derivatives expressed transcript 2 in zebrafish cardiac development.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

[Folic acid antagonist methotrexate causes the development malformation of heart and down-regulates the BMP2b and HAS2 expressions in zebrafish].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.