The Role and Mechanism of Spinal NF-κB-CXCL1/CXCR2 in Rats with Nucleus Pulposus-induced Radicular Pain.

STUDY DESIGN: Experimental study of the role and mechanism of spinal NFκB-CXCL1/CXCR2 in rats with nucleus pulposus-induced radicular pain. OBJECTIVE: This study investigated the role and mechanism of spinal NFκB-CXCL1/CXCR2 in autologous nucleus pulposus-induced pain behavior in rats and to clarify the involvement and regulation of spinal NFκB as an upstream molecule of CXCL1 in autologous nucleus pulposus-induced radicular pain in rats. SUMMARY OF BACKGROUND DATA: The inflammatory response of nerve roots is an important mechanism for the occurrence of chronic pain. NFκB-CXCL1/CXCR2 pathway plays an important role in the development of radicular pain, but its regulatory mechanism in the model of radicular pain induced by autologous nucleus pulposus is still unclear. MATERIALS AND METHODS: We established a rat model of autologous medullary nucleus transplantation. We observed and recorded the changes in 50% mechanical withdrawal threshold and thermal withdrawal latency before and after the administration of CXCL1-neutralizing antibodies, CXCR2 inhibitor, and NFκB inhibitor in each group of rats and evaluated the expression of NFκB, CXCL1, and CXCR2 in the spinal dorsal horn using immunofluorescence and Western blot. To compare differences between groups in behavioral testing, analysis of variance was employed. Dunnett's method was used to compare differences at different time points within a group and between different groups at the same time point. A comparison of the relative concentration of protein, relative concentration of mRNA, and semiquantitative data from immunofluorescence staining was conducted utilizing one-way ANOVA and Dunnett's pairwise comparison. RESULTS: Autologous nucleus pulposus transplantation can induce radicular pain in rats and upregulate the expression of CXCL1, CXCR2, and NFκB in the spinal cord. CXCL1 is co-expressed with astrocytes, CXCR2 with neurons, and NFκB with both astrocytes and neurons. The application of CXCL1 neutralizing antibodies, CXCR2 inhibitors, and NFκB inhibitors can alleviate pain hypersensitivity induced by autologous nucleus pulposus transplantation in rats. Inhibitors of NFκB could downregulate the expression of CXCL1 and CXCR2. CONCLUSIONS: We found that spinal NFκB is involved in NP-induced radicular pain in rats through the activation of CXCL1/CXCR2, enriching the mechanism of medullary-derived radicular pain and providing a possible new target and theoretical basis for the development of more effective anti-inflammatory and analgesic drugs for patients with chronic pain following LDH.

Utility of intraoperative pathology consultations of whipple resection specimens and their impact on final margin status.

The resection margin status is a significant surgical prognostic factor for the long-term outcomes of patients undergoing pancreaticoduodenectomy (Whipple procedure). As a result, surgeons frequently rely on intraoperative consults (IOCs) involving frozen sections to evaluate margin clearance during these resections. Nevertheless, the impact of this practice on final margin status and long-term outcomes remains a topic of debate. This study aimed to assess the impact of IOCs on the clearance rate of resection margins following Whipple procedure and distal pancreatectomy. A retrospective database review of all patients who underwent Whipple procedure or distal pancreatectomy at our institution between 2018 and 2020 was performed to evaluate the utility of IOCs by gastrointestinal surgeons and its correlation with final postoperative surgical margin status. A significant variation in the frequency of IOC requests for margins among surgeons was noted. However, the use of frozen section analysis for intraoperative margin assessment was not significantly associated with the clearance rate of final post-operative margins. More frequent use of IOC did not result in higher final margin clearance rate, an important prognostic factor following Whipple procedure.

Osthole-Mediated Inhibition of Neurotoxicity Induced by Ropivacaine via Amplification of the Cyclic Adenosine Monophosphate Signaling Pathway.

Background: Ropivacaine is widely used for clinical anesthesia and postoperative analgesia. However, the neurotoxicity induced by ropivacaine in a concentration- and duration-dependent manner, and it is difficult to prevent neurotoxicity. Osthole inhibits phosphodiesterase-4 activity by binding to its catalytic site to prevent cAMP hydrolysis. The aim of this present study is to explore the precise molecular mechanism of osthole-mediated inhibition of neurotoxicity induced by ropivacaine. Methods: SH-SY5Y cell viability and apoptosis were measured in different concentration and duration. Protein concentration was determined in each signaling pathway. The molecular mechanism of osthole-mediated inhibition of ropivacaine-caused neurotoxicity was evaluated. Results: The study demonstrated that osthole inhibits SH-SY5Y cells neurotoxicity in a duration- and concentration-dependent manner. Moreover, ropivacaine significantly increased the expression of caspase-3 by promoting the phosphorylation of p38. Osthole-induced upregulation of cAMP activated cAMP-dependent signaling pathway, sequentially leading to elevated cyclic nucleotide response element-binding protein levels, which inhibits P38-dependent signaling and decreases apoptosis of SH-SY5Y. Conclusions: This study display the evidence confirmed the molecular mechanism by which osthole amplification of cAMP-dependent signaling pathway, and overexpression of cyclic nucleotide response element-binding protein inhibits P38-dependent signaling and decreases ropivacaine-induced SH-SY5Y apoptosis.

Protective Effect of Sufentanil on Myocardial Ischemia-Reperfusion Injury in Rats by Inhibiting Endoplasmic Reticulum Stress.

Objective: Sufentanil is the most common drug in clinical practice for the treatment of ischemic heart disease. This study is to investigate the protective mechanism of sufentanil on rat myocardial ischemia-reperfusion (I/R) injury. Methods: A rat I/R model was established by ligating the left anterior descending coronary artery. A total of 24 SD male rats were enrolled and divided randomly into the control group, I/R group, sufentanil group (SUF; 3 µg/kg), and diltiazem group (DLZ; 20 mg/kg; positive control). The rat hearts were subjected to 30 min of ischemia followed by 120 min of reperfusion. Subsequently, hemodynamics, pathological changes of myocardial tissue, serum biochemical parameters, oxidative stress factors, the level of serum inducible nitric oxide synthases (iNOS), interleukin-6 (IL-6), and other bioactive factors were analyzed in the rats. Result: Compared with the I/R group, sufentanil significantly improved cardiac action, myocardial fiber, and cardiomyocyte morphology and reduced inflammatory cell infiltration in rats in the SUF group. And the level of creatine kinase isoenzyme (CK-MB), troponin (cTn), lactate dehydrogenase (LDH), malondialdehyde (MDA), iNOS, and IL-6 was significantly declined in the serum of SUF group, while the activities of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) were significantly activated in the myocardial tissues. In addition, sufentanil also significantly decreased the protein expression of GRP78, CHOP, Caspase 12, and ATF6 in the myocardial tissue of the SUF group. Conclusion: Sufentanil has a significant protective activity on myocardial I/R injury in rats, the mechanism of which may be associated with the inhibition of endoplasmic reticulum stress and oxidative stress.

Osthole, a Coumadin Analog from Cnidium monnieri (L.) Cusson, Ameliorates Nucleus Pulposus-Induced Radicular Inflammatory Pain by Inhibiting the Activation of Extracellular Signal-Regulated Kinase in Rats.

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.

Does intraoperative ulinastatin improve postoperative clinical outcomes in patients undergoing cardiac surgery: a meta-analysis of randomized controlled trials.

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.

Modification of rat model of sciatica induced by lumber disc herniation and the anti-inflammatory effect of osthole given by epidural catheterization.

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.

Osthole, a herbal compound, alleviates nucleus pulposus-evoked nociceptive responses through the suppression of overexpression of acid-sensing ion channel 3 (ASIC3) in rat dorsal root ganglion.

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).

Ligand- and protein-based modeling studies of the inhibitors of human cytochrome P450 2D6 and a virtual screening for potential inhibitors from the Chinese herbal medicine, Scutellaria baicalensis (Huangqin,Baikal Skullcap).

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.

Autologous nucleus pulposus transplantation to lumbar 5 dorsal root ganglion after epineurium dissection in rats: a modified model of non-compressive lumbar herniated intervertebral disc.

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.

[Effects and mechanisms of osthole on sciatica induced by lumber disc herniation].

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.

Ginkgolide B promotes proliferation and functional activities of bone marrow-derived endothelial progenitor cells: involvement of Akt/eNOS and MAPK/p38 signaling pathways.

Bone marrow-derived, circulating endothelial progenitor cells (EPCs) contribute to neovascularization in various diseases, and represent a very interesting alternative cell source for enhancing vasculogenesis in regenerative medicine. In this study, we investigated the effects of Ginkgolide B (GB) on proliferation and differentiation of EPCs, and the involved signaling pathway in vitro. EPC proliferation, migration, adhesion and angiogenesis activities were assessed with the WST-8 assay, Transwell chamber assay, cell counting and angiogenesis kit, respectively. Apoptosis was detected with annexin V and propidium iodide staining. The protein expression of angiogenesis-related makers was detected by Western blot, and related gene expression was determined by real-time polymerase chain reaction (RT-PCR). The results showed that GB promoted the proliferation and endothelial gene expression, and markedly enhanced vascular endothelial growth factor-induced migration response and the capability to incorporate into the vascular networks in EPCs. GB protected EPCs from H2O2-induced cell death. GB induced the phosphorylation of eNOS, Akt and p38, which in turn promoted cell proliferation and function. In conclusion, the present study demonstrates that GB, at a near medical applied dose, increases the number and functional activities of EPCs with involvement of Akt/endothelial nitric oxide synthase and mitogen-activated protein kinase (MAPK)/p38 signal pathways. These findings raise the intriguing possibility that GB may play an important role in the protection and revascularization of blood vessels.

An improved processing method for breast whole-mount serial sections for three-dimensional histopathology imaging.

To develop and validate improved processing methods for producing diagnostic-quality, whole-mount serial sections for 3-dimensional imaging of whole-breast histopathologic studies, we subjected 4-mm-thick whole-specimen slices to a 38-hour microwave-assisted protocol. Morphologic features, antigenicity, and tissue shrinkage were evaluated. A schedule using the tissue processor was optimized by evaluating the serial section yield for 3 schedules. The microwave-based processing schedule is adequate for producing diagnostic-quality whole-mount breast serial sections of an area up to 6,000 mm(2) and is compatible with a variety of immunohistochemical stains. A mean +/- SE total tissue shrinkage of 8.4% +/- 0.2% resulted. For the tissue processor, optimal results are obtained using a 59-hour schedule. Total fixation and processing time for whole-mount serial breast sections has been reduced from 21 days to 38 hours, with microwave assistance, and to 59 hours without. No adverse effects of microwaves on morphologic features, antigenicity, or gross tissue dimensions were observed.

Intermixed normal tissue within prostate cancer: effect on MR imaging measurements of apparent diffusion coefficient and T2--sparse versus dense cancers.

PURPOSE: To investigate differences in apparent diffusion coefficient (ADC) and T2 values between dense and sparse regions in prostate cancer. MATERIALS AND METHODS: Eighteen patients (median age, 61 years; range, 44-72 years) gave informed consent for this retrospective Research Ethics Board-approved study. Prior to radical prostatectomy, ADC (b value, 600 sec/mm(2)) and T2 maps were obtained by using 1.5-T magnetic resonance (MR) imaging. Twenty-eight peripheral zone (PZ) tumors were reviewed by using whole-mount histologic findings, and regions assessed to contain primarily (>60%) normal PZ tissue were delineated. Tumors were categorized as "sparse" if more than 50% of their cross-sectional areas were these primarily normal PZ regions and were considered "dense" otherwise. Normal PZ tissue was outlined separately on the same section. Tumor and normal tissue outlines were transferred to corresponding ADC and T2 maps, and median values were calculated. Values were compared by using multiple regression analysis. Matched-pair tumor-to-normal tissue differences and log(2)-transformed ratios were assessed by using nonparametric tests. RESULTS: Thirty-six percent (10 of 28) of tumors were sparse; 64% (18 of 28) were dense. For both overall and intrapatient comparisons, dense tumors had significantly lower ADC and T2 values than normal PZ tissue (P < .05), but no significant differences were observed between sparse tumors and normal tissue. Log(2)-transformed tumor-to-normal tissue ratios were significantly less than zero for dense tumors for both ADC and T2 (P < .01) measurements but not for sparse tumors. Both matched-pair differences and log(2)-transformed ratios were significantly different between sparse and dense tumors (P < .01). ADC and T2 values were moderately correlated (Pearson correlation coefficient range, r = 0.770-0.804). CONCLUSION: Sparse prostate tumors have similar ADC and T2 values to those of normal PZ tissue. This may limit MR imaging detection and the assessment of tumor volume of some cancers.

[Analysis of factors leading to prescheduled analgesic withdrawal in patients with postoperative epidural analgesia].

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.

[Antisense hTERT inhibits gene expression and functional activity of telomerase in leukemia cell lines].

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.

[Effects of antisense human telomerase reverse-transcript protein subunit (hTERT) gene on biological characteristics of hepatoblastoma cell line in vitro].

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.

[Antisense human telomerase reverse transcriptase inhibits leukemia cell proliferation in vitro].

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.

[Construction and identification of eukaryotic expression vector for sense and antisense human telomerase reverse transcriptase].

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.