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AIM: This study was aimed at assessing the role of extracellular signal regulated kinase (ERK) in mechanical allodynia resulting from lumbar disc herniation (LDH) and exploring the osthole's anti-nociceptive effect on ERK activation. METHODS: Radicular pain was generated by applying nucleus pulposus (NP) to the L5 dorsal root ganglion (DRG). Allodynia was measured using Von Frey filaments to calculate the mechanical pain threshold. Phosphorylated ERK and total ERK protein in the lumbar spinal dorsal horn was detected by using the Western blot technique. Cyclooxygenase 2 (COX-2) mRNA was assessed by real-time reverse-transcription polymerase chain reaction. RESULTS: The application of NP to L5 DRG induced mechanical hypersensitivity which lasted for at least 28 days, and a significant increase of ERK phosphorylation in the ipsilateral spinal dorsal horn from postoperative day (POD) 1 to POD 21. ERK inhibitor attenuated NP-induced hyperalgesia compared to the dimethyl sulfoxide-(vehicle control) administered group (p < 0.05). Epidural treatment with osthole could ameliorate NP-evoked hyperalgesia by suppressing the activation of ERK rather than decreasing the expression of ERK protein. Osthole could also inhibit the increased expression of COX-2 mRNA in spinal dorsal horn, which was a known downstream effect of ERK signaling pathway. CONCLUSIONS: Our results suggest that ERK activation in the spinal dorsal horn plays a vital role in NP-evoked hyperalgesia. Osthole exerts analgesic effect on radicular inflammatory pain in LDH rat model, by down-regulating the mRNA expression of the target gene of COX-2 via inhibiting ERK activation in the spinal dorsal horn.
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Cnidium/química , Cumarinas/farmacología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Dolor/tratamiento farmacológico , Analgésicos/aislamiento & purificación , Analgésicos/farmacología , Animales , Western Blotting , Cumarinas/aislamiento & purificación , Ciclooxigenasa 2/genética , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/fisiopatología , Desplazamiento del Disco Intervertebral/complicaciones , Masculino , Núcleo Pulposo/metabolismo , Dolor/fisiopatología , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
INTRODUCTION: The systematic meta-analysis of randomized controlled trials (RCTs) evaluated the effects of intraoperative ulinastatin on early-postoperative recovery in patients undergoing cardiac surgery. METHODS: RCTs comparing intraoperative ulinastatin with placebo in cardiac surgery were searched through PubMed, Cochrane databases, Medline, SinoMed, and the China National Knowledge Infrastructure (1966 to May 20th, 2013). The primary endpoints included hospital mortality, postoperative complication rate, length of stay in intensive care unit, and extubation time. The physiological and biochemical parameters illustrating postoperative cardiac and pulmonary function as well as inflammation response were considered as secondary endpoints. RESULTS: Fifteen RCTs (509 patients) met the inclusion criteria. Ulinastatin did not affect hospital mortality, postoperative complication rate, or ICU length of stay but reduced extubation time. Ulinastatin also increased the oxygenation index on postoperative day 1 and reduced the plasma level of cardiac troponin-I. Additionally, ulinastatin inhibited the increased level of tumor necrosis factor-alpha, polymorphonuclear neutrophil elastase, interleukin-6, and interleukin-8 associated with cardiac surgery. CONCLUSION: Ulinastatin may be of value for the inhibition of postoperative increased inflammatory agents and most likely provided pulmonary protective effects in cardiac surgery. However, larger adequately powered RCTs are required to define the clinical effect of ulinastatin on postoperative outcomes in cardiac surgery.
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Procedimientos Quirúrgicos Cardíacos/métodos , Glicoproteínas/administración & dosificación , Forma MB de la Creatina-Quinasa/sangre , Cuidados Críticos , Mortalidad Hospitalaria , Humanos , Inflamación , Interleucina-6/sangre , Interleucina-8/sangre , Periodo Intraoperatorio , Tiempo de Internación , Elastasa de Leucocito/sangre , Oxígeno/química , Complicaciones Posoperatorias/prevención & control , Periodo Posoperatorio , Ensayos Clínicos Controlados Aleatorios como Asunto , Resultado del Tratamiento , Troponina I/sangre , Inhibidores de Tripsina/química , Factor de Necrosis Tumoral alfa/sangreRESUMEN
One of the most treatable causes of lower back pain and associated sciatica is lumbar disc herniation (LDH), which is characterized by rupture of the hard outer wall (annulus fibrosis) in a lumbar intervertebral disc. In the current study, we aimed to: (1) develop and characterize a rat model of sciatica induced by LDH, while introducing a novel method of epidural catheterization; (2) use this model to evaluate the effect of osthole on pain due to LDH, and (3) gain insight into the mechanisms through which osthole affects sciatica induced by LDH. The results indicate that our newly developed rat model maintained mechanical allodynia for 28 days without reduction. Moreover, cyclooxygenase-2 (COX-2) and nitric oxide synthase (NOS) were overexpressed in the associated inflammatory response, which is consistent with clinical manifestations of the disease. We then used this model to study the effect and mechanisms through which osthole affected pain due to LDH. Our study suggests that osthole is capable of reversing hyperalgesia due to LDH, potentially through modulation of activity of COX-2 and NOS, two important proteins for the exacerbation of pain due to LDH. Finally, a molecular modeling simulation showed that osthole has unique binding capabilities to both NOS and COX-2. As the model-induced mechanical hyperalgesia response was consistent, and the position of the catheter tip and the extension/spreading of the drug in the epidural space were reliable, this study developed an improved model to study remedies for sciatic pain. Moreover, our studies demonstrate that osthole may be a feasible treatment for the reduction of pain due to hyperalgesia.
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Antiinflamatorios/uso terapéutico , Cumarinas/uso terapéutico , Modelos Animales de Enfermedad , Desplazamiento del Disco Intervertebral/complicaciones , Vértebras Lumbares , Ciática/etiología , Animales , Antiinflamatorios/farmacología , Cateterismo , Cumarinas/farmacología , Ciclooxigenasa 2/metabolismo , Inyecciones Epidurales , Desplazamiento del Disco Intervertebral/tratamiento farmacológico , Desplazamiento del Disco Intervertebral/enzimología , Masculino , Óxido Nítrico Sintasa/metabolismo , Dolor/tratamiento farmacológico , Dolor/enzimología , Ratas , Ratas Sprague-Dawley , Ciática/tratamiento farmacológico , Ciática/enzimologíaRESUMEN
BACKGROUND: Osthole (Ost), a natural coumarin derivative, has been shown to inhibit many pro-inflammatory mediators and block voltage-gated Na+ channels. During inflammation, acidosis is an important pain inducer which activates nociceptors by gating depolarizing cationic channels, such as acid-sensing ion channel 3 (ASIC3). The aim of this study was to examine the effects of Ost on nucleus pulposus-evoked nociceptive responses and ASIC3 over-expression in the rat dorsal root ganglion, and to investigate the possible mechanism. MATERIAL/METHODS: Radicular pain was generated with application of nucleus pulposus (NP) to nerve root. Mechanical allodynia was evaluated using von Frey filaments with logarithmically incremental rigidity to calculate the 50% probability thresholds for mechanical paw withdrawal. ASIC3 protein expression in dorsal root ganglions (DRGs) was assessed with Western blot and immunohistochemistry. Membrane potential (MP) shift of DRG neurons induced by ASIC3-sensitive acid (pH6.5) was determined by DiBAC(4) (3) fluorescence intensity (F.I.). RESULTS: The NP-evoked mechanical hyperalgesia model showed allodynia for 3 weeks, and ASIC3 expression was up-regulated in DRG neurons, reaching peak on Day 7. Epidural administration of Ost induced a remarkable and prolonged antinociceptive effect, accompanied by an inhibition of over-expressed ASIC3 protein and of abnormal shift of MP. Amiloride (Ami), an antagonist of ASIC3, strengthened the antinociceptive effect of Ost. CONCLUSIONS: Up-regulation of ASIC3 expression may be associated with NP-evoked mechanical hyperalgesia. A single epidural injection of Ost decreased ASIC3 expression in DGR neurons and the pain in the NP-evoked mechanical hyperalgesia model. Osthole may be of great benefit for preventing chronic pain status often seen in lumbar disc herniation (LDH).
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Cumarinas/farmacología , Ganglios Espinales/patología , Disco Intervertebral/patología , Proteínas del Tejido Nervioso/metabolismo , Nocicepción/efectos de los fármacos , Canales de Sodio/metabolismo , Canales Iónicos Sensibles al Ácido , Analgésicos/farmacología , Animales , Western Blotting , Cumarinas/química , Cumarinas/uso terapéutico , Técnica del Anticuerpo Fluorescente , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Concentración de Iones de Hidrógeno/efectos de los fármacos , Hiperalgesia/complicaciones , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/patología , Hiperalgesia/fisiopatología , Disco Intervertebral/efectos de los fármacos , Masculino , Potenciales de la Membrana/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Dolor/complicaciones , Dolor/tratamiento farmacológico , Dolor/patología , Dolor/fisiopatología , Umbral del Dolor/efectos de los fármacos , Fitoterapia , Preparaciones de Plantas/química , Preparaciones de Plantas/farmacología , Preparaciones de Plantas/uso terapéutico , Ratas , Ratas Sprague-DawleyRESUMEN
We have previously examined the binding patterns of various substrates to human cytochrome P450 2D6 (CYP2D6) using a series of molecular modeling methods. In this study, we further explored the binding modes of various types of inhibitors to CYP2D6 using a combination of ligand- and protein-based modeling approaches. Firstly, we developed and validated a pharmacophore model for CYP2D6 inhibitors, which consisted of two hydrophobic features and one hydrogen bond acceptor feature. Secondly, we constructed and validated a quantitative structure-activity relationship (QSAR) model for CYP2D6 inhibitors which gave a poor to moderate prediction accuracy. Thirdly, a panel of CYP2D6 inhibitors were subject to molecular docking into the active site of wild-type and mutated CYP2D6 enzyme. We demonstrated that 8 residues in the active site (Leu213, Glu216, Ser217, Gln244, Asp301, Ser304, Ala305, and Phe483) played an important role in the binding to the inhibitors via hydrogen bond formation and/or π-π stacking interaction. Apparent changes in the binding modes of the inhibitors have been observed with Phe120Ile, Glu216Asp, Asp301Glu mutations in CYP2D6. Finally, we screened for potential binders/inhibitors from the Chinese herbal medicine Scutellaria baicalensis (Huangqin, Baikal Skullcap) using the established pharmacophore model for CYP2D6 inhibitors and molecular docking approach. Overall, 18 out of 40 compounds from S. baicalensis were mapped to the pharmacophore model of CYP2D6 inhibitors and most herbal compounds from S. baicalensis could be docked into the active site of CYP2D6. Our study has provided insights into the molecular mechanisms of interaction of synthetic and herbal compounds with human CYP2D6 and further benchmarking studies are needed to validate our modeling and virtual screening results.
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Inhibidores del Citocromo P-450 CYP2D6 , Medicamentos Herbarios Chinos/química , Inhibidores Enzimáticos/aislamiento & purificación , Inhibidores Enzimáticos/farmacología , Ensayos Analíticos de Alto Rendimiento , Scutellaria baicalensis/química , Dominio Catalítico/efectos de los fármacos , Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP2D6/metabolismo , Inhibidores Enzimáticos/química , Humanos , Ligandos , Modelos Moleculares , Relación Estructura-ActividadRESUMEN
BACKGROUND: Nucleus pulposus of intervertebral discs has proinflammatory characteristics that play a key role in neuropathic pain in lumbar herniated intervertebral disc. One of the most commonly used animal models (the traditional model) of non-compressive lumbar herniated intervertebral disc is created by L4-L5 hemilaminectomy and the application of autologous nucleus pulposus to cover the left L4 and L5 nerve roots in rats. However, such procedures have the disadvantages of excessive trauma and low success rate. We proposed a modified model of non-compressive lumbar herniated intervertebral disc in which only the left L5 dorsal root ganglion is exposed and transplanted with autologous nucleus pulposus following incision of epineurium. We aimed to compare the modified model with the traditional one with regard to trauma and success rate. METHODS: Thirty Sprague-Dawley male rats were randomized into three groups: sham operation group (n = 6), traditional group (n = 12), and modified group (n = 12). The amount of blood loss and operative time for each group were analyzed. The paw withdrawal threshold of the left hind limb to mechanical stimuli and paw withdrawal latency to heat stimuli were examined from the day before surgery to day 35 after surgery. RESULTS: Compared with the traditional group, the modified group had shorter operative time, smaller amount of blood loss, and higher success rate (91.7% versus 58.3%, P < 0.05). There was no decrease in paw withdrawal latency in any group. The sham operation group had no decrease in postoperative paw withdrawal threshold, whereas the modified and traditional groups had significant reduction in paw withdrawal threshold after surgery (mechanical hyperalgesia). CONCLUSIONS: Transplantation of nucleus pulposus onto the L5 dorsal root ganglion following incision of epineurium in rats established an improved animal model of non-compressive lumbar herniated intervertebral disc with less trauma and more stable pain ethology.
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Ganglios Espinales/patología , Degeneración del Disco Intervertebral/patología , Desplazamiento del Disco Intervertebral/patología , Animales , Modelos Animales de Enfermedad , Masculino , Distribución Aleatoria , Ratas , Ratas Sprague-DawleyRESUMEN
OBJECTIVE: To study the effects of osthole on sciatica induced by lumber disc herniation and its mechanisms. METHODS: 54 male SD rats were randomly divided into 6 groups. Model (M) group (n = 12): Autologous nucleus pulposus was harvested from the tail and applied to the L5 dorsal root ganglion (DRG) and epidural space. Epidural catheterization was performed. Control(C) group (n = 12): On the basis of nucleus pulposus group, 50 microL tween-80 was administered epidurally on the day 6th after surgery. T2 (n = 6), T6 (n = 12), T13 (n = 6) and T20 (n = 6) group: 50 microL 2% osthole was administered epidurally on the 2th, 6th, 13th day and 20th after surgery respectively. General behaviors were observed and 50% paw withdrawal threshold (50% PWT) was measured 1 day before surgery, on the 1st, 3th, 7th,14th, 21th, 28th day after surgery, immediately before and 1 hour after osthole or tween-80 administration in each group. On the 7th day after surgery, the left L5 DRGs were obtained for detecting the expression of NOS and COX-2 in M, C and T6 group with 6 rats. RESULTS: No lameness or autophagy was oberserved. 50% PWT decreased after surgery (P < 0.05). In T2 and T6 group, 50% PWT after osthole administration were significantly higher than those of M group and C group (P < 0.05), which recovered to the same level as 1 day before surgery (P > 0.05). In T13 and T20 group, 50% PWT 1 hour after osthole administration were significantly higher than those of M group and C group (P < 0.05), which recovered to the same level as 1 day before surgery (P > 0.05), but on days after 1 hour after administration, there was no significant difference when 50% PWT compared with M group or nucleus C group (P > 0.05). NOS positive cells and COX-2 positive cells were no significant difference when M group compared with C group (P > 0.05). But these positive cells in T6 group were significantly lower than those of M group and C group (P < 0.05). CONCLUSION: 50 microL 2% osthole could completely inhibit the mechanical allodynia in the rat model of sciatica induced by lumbar disc herniation when it was administered epidurally on 2 or 6 day after surgery. But when administered on 13 or 20 day after surgery, its analgesic effect was transient. The effect of 50 microL 2% osthole epidural administration on day 6 after surgery on the rat model of sciatica induced by lumbar disc herniation may relate to inhibition of the expression of COX-2 and NOS in DRG.
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Analgésicos/farmacología , Cumarinas/farmacología , Ganglios Espinales/metabolismo , Desplazamiento del Disco Intervertebral/complicaciones , Ciática/tratamiento farmacológico , Analgésicos/administración & dosificación , Animales , Conducta Animal , Cumarinas/administración & dosificación , Ciclooxigenasa 2/metabolismo , Modelos Animales de Enfermedad , Ganglios Espinales/cirugía , Inyecciones Epidurales , Vértebras Lumbares/cirugía , Masculino , Óxido Nítrico Sintasa/metabolismo , Dimensión del Dolor/métodos , Umbral del Dolor/efectos de los fármacos , Umbral del Dolor/fisiología , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Ciática/etiología , Ciática/metabolismo , Raíces Nerviosas Espinales/lesionesRESUMEN
OBJECTIVE: To analyze the factors leading to prescheduled analgesic withdrawal in patients with postoperative epidural analgesia. METHODS: A retrospective study of 4876 patients with postoperative epidural analgesia was conducted and the effect of analgesia and incidence of prescheduled analgesic withdrawal were recorded. The factors precipitating the occurrences of analgesic withdrawal and complications were analyzed. RESULTS: Early analgesic withdrawal occurred in 113 cases (2.3%), among which 74 (0.5%) were due to factors irrelevant to analgesic complications. Analgesia-related complications occurred in 578 patients, but only 39 (0.7%) of them needed discontinuation of the analgesics. CONCLUSION: Prescheduled analgesic withdrawal is predominantly due to technical inadequacies rather than complications arising from the analgesics, and improvement of the operation skills for postoperative analgesia may reduce early analgesia discontinuation and enhance the patients' satisfaction.
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Analgesia Epidural , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Analgesia Epidural/estadística & datos numéricos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Complicaciones Posoperatorias , Periodo Posoperatorio , Estudios Retrospectivos , Factores de Tiempo , Adulto JovenRESUMEN
OBJECTIVE: To explore the inhibitory effects of gene expression and functional activity of telomerase in leukemia cell lines by in vitro antisense hTERT treatment. METHODS: An antisense hTERT eukaryotic expression vector was constructed by using gene recombination technique, targeting the 5' end mRNA sequence of the telomerase catalytic subunit. The vector expression in leukemia cell lines (HL60 and K562) was achieved by transfection using the SuperFect transfection reagent (Qiagen). After transfection, ectopic expression of the telomerase catalytic subunit was analyzed by quantitative fluorescence real-time RT-PCR, and cellular apoptosis and cell cycle parameters were evaluated by flow cytometry respectively. RESULTS: An antisense pcDNA-hTERT eukaryotic expression vector was successfully constructed. Leukemia cell lines transfected with antisense hTERT constructed displayed a significant inhibition of gene expression of telomerase and its activity in vitro, as compared with the result of the control groups (without transfection and vector control). CONCLUSION: In-vitro antisense hTERT expression may down-regulate the gene expression and biological activity of telomerase in leukemia cells, suggesting a possibility of gene therapy against human malignancy through the telomerase-targeted molecular mechanism.
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Apoptosis , Proteínas de Unión al ADN/biosíntesis , ARN sin Sentido/genética , Telomerasa/metabolismo , Transfección , Ciclo Celular , Proteínas de Unión al ADN/genética , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Vectores Genéticos , Células HL-60 , Células HeLa , Humanos , Células K562 , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Telomerasa/biosíntesis , Telomerasa/genéticaRESUMEN
OBJECTIVE: Telomerase, a complex of ribose and nucleoprotein, is a specific marker of tumor, which expresses in 98% infinite cell lines and 90% malignant tumor organizations and whose function is to maintain the length of telomere. Human telomerase reverse-transcript protein subunit (hTERT) is the key element and rate-limiting factor of telomerase activity. Our study was to investigate the effects of antisense hTERT gene on biological characteristics of hepatoblastoma cell line in vitro. METHODS: The sense and antisense hTERT eukaryotic expression vectors that we had constructed before were transfected into hepatoblastoma cell line HepG2 by using the SuperFect transfection reagent (Qiagen) according to the manufacturer's instructions, then the HepG2-s and HepG2-as of G418-resistant colonies were obtained with G418 and identified for the presence of hTERT insert by PCR with T7 and pcDNA3.1/BGH reverse primers. After that, we have detected the endogenous hTERT mRNA expression and telomerase activity by quantitative real-time RT-PCR and TRAP-silver staining assay in cells from each group. Meanwhile, MTT cellular proliferation assay, soft agar colony formation assay and flow cytometry were employed to analyze if the proliferation capacity of liver cancer cells was affected in vitro and the tumor cells could be induced to apoptosis by antisense hTERT. RESULTS: Antisense hTERT significantly down-regulated the endogenous hTERT mRNA expression (15.35 +/- 1.72/HepG2-as, 43.8 +/- 2.89/HepG2-s, 45.2 +/- 3.46/HepG2) (n = 10, t = 7.61, P < 0.01) and telomerase activity in HepG2, compared to blank control and sense hTERT. After 20 passages of three group cells, a 7-day cell growth curve and the numbers (size) of soft agar colony formation showed the proliferation and the anchorage-independent growth in HepG2-as were significantly suppressed (50.6 +/- 4.8/HepG2-as, 113.52 +/- 8.15/HepG2-s, 119.12 +/- 10.82/HepG2) (n = 10, t = 4.54, P < 0.01 and n = 10, t = 3.96, P < 0.01), compared to HepG2 and HepG2-s. However there was a significant increase in apoptosis percentage of HepG2-as by flow cytometry (n = 10, t = 9.24, P < 0.01 and n = 10, t = 8.37, P < 0.01), compared to control group. CONCLUSIONS: Antisense hTERT could significantly suppress the hepatoblastoma cell growth and reverse its malignant phenotypes in vitro and cause the increase in apoptosis percentage of HepG2, thus it might be applied in malignant tumor gene therapy through the telomerase-targeted molecular mechanism.
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ARN sin Sentido/genética , Telomerasa/genética , División Celular/genética , Línea Celular Tumoral , Proteínas de Unión al ADN , Hepatoblastoma/genética , Hepatoblastoma/patología , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
OBJECTIVE: To study the inhibitory effect of antisense human telomerase reverse transcriptase (hTERT) on leukemia cell proliferation in vitro. METHODS: Sense and antisense hTERT eukaryotic expression vectors previously constructed were transfected into leukemia cell line HL(60) using SuperFect transfection reagent (Qiagen) to obtain HL(60)-s and HL(60)-as, and the G418-resistant colonies were identified for the presence of hTERT insert by PCR with T7 and pcDNA3.1/BGH reverse primers. Endogenous hTERT mRNA expression and telomerase activity were then detected by quantitative real-time RT-PCR and telomerase associated protein -silver staining in each cell line. MTT cellular proliferation assay, soft agar colony formation assay and flow cytometry were also employed to analyze the changes in proliferation capacity of leukemia cell in vitro and apoptosis of the tumor cells induced by antisense hTERT. RESULTS: Antisense hTERT remarkably reduced endogenous hTERT mRNA expression (P<0.01) and down-regulated telomerase activity in HL(60), as compared with the blank control and sense hTERT. After 25 passages of the 3 cell lines, a 7-day cell growth curve and the numbers (size) of soft agar colony formation showed that the proliferation rates and the anchorage-independent growth ability of HL(60)-as cells were significantly decreased in comparison with HL(60) and HL(60)-s cells, but a significant increase in apoptosis of HL(60)-as cells occurred as determined by flow cytometry. CONCLUSIONS: Antisense hTERT can obviously inhibit leukemia cell growth and proliferation in vitro, and this telomerase-targeted molecular biotherapy may be achieved by apoptosis pathway through down-regulation of hTERT mRNA and telomerase activity.
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Elementos sin Sentido (Genética)/farmacología , Leucemia/tratamiento farmacológico , Telomerasa/antagonistas & inhibidores , Apoptosis/efectos de los fármacos , División Celular/efectos de los fármacos , Proteínas de Unión al ADN , Células HL-60 , Humanos , Leucemia/patología , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Telomerasa/genéticaRESUMEN
OBJECTIVE: To construct eukaryotic expression vectors for sense and antisense human telomerase reverse transcriptase (hTERT), to facilitate further study of the regulation of telomerase activity and gene therapy of malignant tumors. METHODS: According to the published hTERT cDNA sequence in Genbank, a pair of primers containing the sites for given restrictive endonuclease at both ends were designed and synthesized. Reverse transcriptional PCR (RT-PCR) of the total RNA extracted from HeLa cell line was performed, the product of which was cloned into pGEM-T vector by using TA cloning and then subcloned into a highly efficient eukaryotic expression vector pcDNA3.1 (+/-). The recombinants were finally sequenced and identified by restrictive endonuclease digestion. RESULTS: A 210-bp DNA fragment was amplified by PCR as expected. The sense and antisense hTERT eukaryotic expression vector were successfully constructed and identified by double restrictive endonuclease digestion. Sequence analysis of the inserted target fragment revealed the same sequence as that of partial hTERT cDNA published in Genbank. CONCLUSION: The sense and antisense hTERT eukaryotic expression vector has been successfully constructed.
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ADN sin Sentido/análisis , ADN Complementario/análisis , Vectores Genéticos , Telomerasa/genética , Proteínas de Unión al ADN , Terapia Genética , Humanos , Reacción en Cadena de la PolimerasaRESUMEN
OBJECTIVE: Kawasaki disease (KD) is an acute febrile vasculitic syndrome of unknown etiology that preferentially affects coronary artery. It has been suggested that proinflammatory cytokines like tumor necrosis factor alpha (TNF-alpha) and interleukin-10 (IL-10) are key players during acute KD. Recently, the polymorphisms relative to major transcriptional start site of TNF-alpha and IL-10 gene were shown to influence the level of TNF-alpha and IL-10 production in vitro. This study was aimed to investigate the genetic association of TNF-alpha and IL-10 promoter polymorphisms in juvenile patients of Han nationality with KD, and to investigate the possible associations with clinical manifestations of the disease. METHODS: Four polymorphism sites of TNF-alpha and IL-10 gene promoter regions from 96 children with KD were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). One hundred and sixty age-matched normal children of the Han nationality were used as control. All patients accepted Doppler echocardiography examination in order to differentiate coronary artery lesions. RESULTS: There was significant difference in allele frequencies of -308 (A/G) site of the TNF-alpha gene between children of the Han nationality and those of Japanese and Caucasian in America. There were significant differences in the allele frequencies of -1082 (G/A), -819 (C/T) and -592 (A/C) of IL-10 gene between children of the Han nationality and their British Counterparts (P < 0.01). There was no significant difference in allele frequencies of -308 (A/G) site of TNF-alpha gene between children with KD and normal controls. There was no significant difference in the haplotypes and the allele frequencies of the above three sites of IL-10 between the two groups. However, when clinical features were examined, the genotype frequency of TNF-alpha-308A was significantly higher in IVIG-resistant KD patients than that of TNF-alpha-308G genotype (67% vs 5%, chi(c)(2) = 90.48, P < 0.01). The genotype of TNF-alpha-308A was closely associated with IVIG-resistant KD (P < 0.01, relative risk 42.25, 95% confidence interval 15.81-112.88). The haplotype frequency of IL-10 -1082A/-819T/-592A was also higher in patients with coronary artery lesion (CAL) caused by KD than those of Non-ATA haplotype (52% vs 20%, chi(2) = 18.36, P < 0.01). The haplotypes of IL-10 -1082A/-819T/-592A was significantly associated with CAL caused by KD (P < 0.01, relative risk 4.26, 95% confidence interval 2.20-8.25). CONCLUSION: The genotype of TNF-alpha-308A is one of the important factors that probably influence the therapeutic effect of KD. The haplotypes (-1082/-819/-592) of IL-10 gene promoter might be related to the pathogenesis of coronary artery complication of KD and -1082A/-819T/-592A haplotypes might be regarded as a genetic marker of risk factor for coronary artery lesion in KD.