Value of miR-1271 and glypican-3 in evaluating the prognosis of patients with hepatocellular carcinoma after transcatheter arterial chemoembolization.

BACKGROUND: Hepatocellular carcinoma (HCC) is the third leading cause of cancer death, causing about 750000 deaths worldwide every year. Patients with advanced hepatocellular carcinoma will often only receive transcatheter arterial chemoembolization (TACE). Glypican-3 (GPC3) is one of the most promising serum markers for HCC. Abnormal expression of miRNAs may be involved in the occurrence and development of tumor. AIM: To explore the value of miR-1271 and GPC3 in evaluating the prognosis of patients with HCC after TACE. METHODS: From January 2016 to December 2018, 162 patients with advanced HCC who received TACE in our hospital were selected into the cancer group, and 162 patients who underwent physical examination during the same period were selected into the health group. The patients in the HCC group were treated with TACE. The changes of serum GPC3 and circulating miR-1271 in the HCC before and after TACE were analyzed. The expression of serum GPC3 was detected by enzyme-linked immunosorbent assay, and the expression of circulating miR-1271 was detected by real-time quantitative polymerase chain reaction. The methodological results of sensitivity, specificity, and accuracy of miR-1271 and GPC3 alone and joint detection of HCC were also evaluated. RESULTS: The level of serum GPC3 in patients with HCC was significantly higher than that in healthy controls. GPC3 levels were increased in both HCC patients and those treated with TACE compared with healthy controls. After TACE, the level of serum GPC3 was significantly lower than that before treatment (P < 0.05), and the level of circulating miR-1271 was significantly higher than that before treatment (P < 0.05). There were 112 cases (69.14%) with remission (complete remission + complete remission + stable disease) and 50 cases (30.86%) with relapse disease progression in HCC patients. After TACE, the miR-1271 level in patients with remission and relapse was lower than that in the healthy group, and the GPC3 level was higher than that in the healthy group, the differences were statistically significant (P < 0.05). The miR-1271 of relapsed patients was lower than that of remission patients, and the level of GPC3 was higher than that of remission patients, and the difference was statistically significant (P < 0.05). The sensitivity of combined detection of miR-1271 and GPC3 was significantly higher than that of single detection, and the difference was statistically significant (P < 0.05); while the specificity of the two combined detections was lower than that of the single detection; and the accuracy was slightly higher than that of single detection, but the difference was not statistically significant. CONCLUSION: The level of miR-1271 in patients with HCC was significantly increased and the level of GPC3 was decreased after TACE. Monitoring the levels of serum GPC3 and circulating miR-1271 has important clinical reference value for evaluating the prognosis of patients with HCC. The levels of serum GPC3 and circulating miR-1271 may help to determine tumor recurrence, evaluate survival status, and guide the next step of treatment.

Long noncoding RNA MEG3 silencing protects against hypoxia-induced pheochromocytoma-12 cell injury through inhibition of TIMP2 promoter methylation.

Hypoxia is a common pathological process caused by insufficient oxygen. Long noncoding RNAs (lncRNAs) have been proven to participate in this pathology. Hypoxia is reported to significantly reduce the secretion of tissue inhibitor of metalloproteinase 2 (TIMP2) and TIMP2 induces pheochromocytoma-12 (PC12) cell cycle arrest. Thus, our study aimed to explore the mechanism by which lncRNA maternally expressed gene 3 (MEG3) was implicated in hypoxia-induced PC12 cell injury through TIMP2 promoter methylation. To elucidate the potential biological significance of MEG3 and the regulatory mechanism between MEG3 and TIMP2, a hypoxia-induced PC12 cell injury model was generated. The hypoxia-exposed cells were subjected to a series of overexpression plasmids and short hairpin RNAs, followed by the measurement of levels of MEG3, TIMP2, lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD), reactive oxygen species (ROS), Bcl-2-associated X protein, B-cell lymphoma-2, and caspase-3, as well as the changes in MMP, cell proliferation, apoptosis, and cell cycle progression. On the basis of the findings, MEG3 was upregulated in hypoxia-injured PC12 cells. MEG3 recruited methylation proteins DNMT3a, DNMT3b, and MBD1 and accelerated TIMP2 promoter methylation, which in turn inhibited its expression. Moreover, PC12 cells following MEG3 silencing and TIMP2 overexpression exhibited significantly decreased levels of LDH, MDA, and ROS along with cell apoptosis, yet increased SOD and MMP levels, as well as cell cycle entry to the S phase and cell proliferation. In conclusion, MEG3 silencing suppresses hypoxia-induced PC12 cell injury by inhibiting TIMP2 promoter methylation. This study may provide novel therapeutic targets for hypoxia-induced injury.

Nicotinic Acetylcholine Receptor Alpha7 Subunit Mediates Vagus Nerve Stimulation-Induced Neuroprotection in Acute Permanent Cerebral Ischemia by a7nAchR/JAK2 Pathway.

BACKGROUND The role of nicotinic acetylcholine receptor alpha7 subunit (a7nAchR) in the treatment of acute cerebral ischemia by VNS has not been thoroughly clarified to date. Therefore, this study aimed to investigate the specific role of a7nAchR and explore whether this process is involved in the mechanisms of VNS-induced neuroprotection in rats undergoing permanent middle cerebral artery occlusion (PMCAO) surgery. MATERIAL AND METHODS Rats received a7nAChR antagonist (A) or antagonist placebo injection for control (AC), followed by PMCAO and VNS treatment, whereas the a7nAChR agonist (P) was utilized singly without VNS treatment but only with PMCAO pretreatment. The rats were randomly divided into 6 groups: sham PMCAO, PMCAO, PMCAO+VNS, PMCAO+VNS+A, PMCAO+VNS+AC, and PMCAO+P. Neurological function and cerebral infarct volume were measured to evaluate the level of brain injury at 24 h after PMCAO or PMCAO-sham. Moreover, the related proteins levels of a7nAChR, p-JAK2, and p-STAT3 in the ischemic penumbra were assessed by Western blot analysis. RESULTS Rats pretreated with VNS had significantly improved neurological function and reduced cerebral infarct volume after PMCAO injury (p<0.05). In addition, VNS enhanced the levels of a7nAchR, p-JAK2, and p-STAT3 in the ischemic penumbra (p<0.05). However, inhibition of a7nAchR not only attenuated the beneficial neuroprotective effects induced by VNS, but also decreased levels of p-JAK2 and p-STAT3. Strikingly, pharmacological activation of a7nAchR can partially substitute for VNS-induced beneficial neurological protection. CONCLUSIONS These results suggest that a7nAchR is a pivotal mediator of VNS-induced neuroprotective effects on PMCAO injury, which may be related to suppressed inflammation via activation of the a7nAchR/JAK2 anti-inflammatory pathway.

Psychometric Properties of the Kessler 10 Scale in Chinese Parents of Children With Cancer.

BACKGROUND: The Kessler Psychological Distress Scale (K10) has been widely used in rating psychological distress in general and clinical populations. However, whether it can be used in parents of children with cancer is unknown. Still lacking is the evidence on its reliability and validity in culturally diverse groups. OBJECTIVE: The aim of this study was to translate the K10 into Mandarin Chinese and test its psychometric properties (especially the factor structure) of the Chinese version (C-K10) in parents of children with cancer. METHODS: By convenience sampling, 2 samples of parents of children with cancer (sample I, n = 206, and sample II, n = 103) were surveyed in Guangzhou, China. Sample I completed the C-K10, and the internal consistency reliability and exploratory factor analysis of the C-K10 were estimated. Sample II completed the C-K10, the State Subscale of State-Trait Anxiety Inventory, and the Zung Self-rating Depression Scale; confirmatory factor analysis and concurrent validity estimates were completed. RESULTS: The C-K10 demonstrated strong internal consistency reliability (Cronbach's α = .93). Both exploratory and confirmatory factor analyses supported a 2-factor structure (ie, anxiety and depression). The concurrent validity was moderate with Pearson correlations greater than 0.50 (P < .001). CONCLUSION: The C-K10 demonstrated very acceptable reliability and validity in screening psychological distress in Chinese parents of children with cancer. IMPLICATIONS FOR PRACTICE: This study provides evidence that the C-K10 is a valid tool that can be used in clinical settings to screen for psychological distress in Chinese parents of children with cancer.

[Surface electromyographic activities of submental muscles among stroke patients with dysphagia].

OBJECTIVE: To explore the swallowing functions of stroke patients with dysphagia. METHODS: A total of 41 subjects were recruited.There were 15 stroke patients with dysphagia, 12 stroke patients without swallowing disorders and 14 age-and gender-matched healthy controls.Surface electromyography (sEMG) was employed over the suprahyoid muscle group.Single swallow was applied twice with 5 and 10 ml of thin liquid barium as well as 5 and 10 ml of paste barium.The duration, average amplitude of sEMG and peak amplitude of submental muscle contraction were compared among three groups.Three-way analysis of variance (ANOVA) was performed. RESULTS: No significant differences existed in the general data among three groups (P > 0.05).However, all volumes, consistencies and durations [ (1.38 ± 0.21), (1.66 ± 0.30), (1.46 ± 0.24), (1.78 ± 0.28) s] were significantly longer for the group of dysphagia patients than for those without dysphagia and healthy subjects (P < 0.05).And the average amplitudes ( (16 ± 6), (15 ± 5), (20 ± 13), (19 ± 7) µV) were significantly smaller for the group of dysphagia patients than for those without dysphagia and healthy subjects (P < 0.05) while the peak amplitudes ((48 ± 23), (51 ± 23), (51 ± 31), (63 ± 32) µV) were significantly smaller for the group of dysphagia patients than for those without dysphagia and healthy subjects (P < 0.05). There were no significant differences between patients without dysphagia and those of healthy subjects (P > 0.05). CONCLUSION: As a simple and useful tool, sEMG is feasible for evaluating swallowing function and quantifying the strength of swallowing muscles in post-stroke patients with dysphagia.

Functional electrical stimulation increases neural stem/progenitor cell proliferation and neurogenesis in the subventricular zone of rats with stroke.

BACKGROUND: Functional electrical stimulation (FES) is known to promote the recovery of motor function in rats with ischemia and to upregulate the expression of growth factors which support brain neurogenesis. In this study, we investigated whether postischemic FES could improve functional outcomes and modulate neurogenesis in the subventricular zone (SVZ) after focal cerebral ischemia. METHODS: Adult male Sprague-Dawley rats with permanent middle cerebral artery occlusion (MCAO) were randomly assigned to the control group, the placebo stimulation group, and the FES group. The rats in each group were further assigned to one of four therapeutic periods (1, 3, 7, or 14 days). FES was delivered 48 hours after the MCAO procedure and divided into two 10-minute sessions on each day of treatment with a 10-minute rest between them. Two intraperitoneal injections of bromodeoxyuridine (BrdU) were given 4 hours apart every day beginning 48 hours after the MCAO. Neurogenesis was evaluated by immunofuorescence staining. Wnt-3 which is strongly implicated in the proliferation and differentiation of neural stem cells (NSCs) was investigated by Western blotting analysis. The data were subjected to one- way analysis of variance (ANOVA), followed by a Tukey/Kramer or Dunnett post hoc test. RESULTS: FES significantly increased the number of BrdU-positive cells and BrdU/glial fibrillary acidic protein double- positive neural progenitor cells in the SVZ on days 7 and 14 of the treatment (P < 0.05). The number of BrdU/doublecortin (DCX) double-positive migrating neuroblast cells in the ipsilateral SVZ on day 14 of the FES treatment group ((522.77 ± 33.32) cells/mm(2)) was significantly increased compared with the control group ((262.58 ± 35.11) cells/mm(2), P < 0.05) and the placebo group ((266.17 ± 47.98) cells/mm(2), P < 0.05). However, only a few BrdU/neuron-specific nuclear protein-positive cells were observed by day 14 of the treatment. At day 7, Wnt-3 was upregulated in the ipsilateral SVZs of the rats receiving FES ((0.44 ± 0.05)%) compared with those of the control group rats ((0.31 ± 0.02)%, P < 0.05) or the placebo group rats ((0.31 ± 0.04)%, P < 0.05). At day 14, the corresponding values were (0.56 ± 0.05)% in the FES group compared with those of the control group rats ((0.50 ± 0.06)%, P < 0.05) or the placebo group rats ((0.48 ± 0.06)%, P < 0.05). CONCLUSION: FES augments the proliferation, differentiation, and migration of NSCs and thus promotes neurogenesis, which may be related to the improvement of neurological outcomes.

A functional fetal HSD11B2[CA]n microsatellite polymorphism is associated with maternal serum cortisol concentrations in pregnant women.

BACKGROUND/AIMS: Cortisol plays an important role during pregnancy. It controls maternal glucose metabolism and fetal development. Cortisol metabolism is partially controlled by the 11b-HSD2. This enzyme is expressed in the kidney and human placenta. The activity of the enzyme is partially controlled by functional polymorphisms: the HSD11B2[CA]n microsatellite polymorphism. The impact of this functional gene polymorphism on cortisol metabolism and potential effects on the newborn's is unknown so far. METHODS: In the current prospective birth cohort study in southern Asia, we analyzed the association of the HSD11B2[CA]n microsatellite polymorphisms in 187 mothers and their newborn's on maternal and newborn's serum cortisol concentrations. RESULTS: Using multivariable regression analyses considering known confounding (gestational age, newborn's gender, the labor uterine contraction states and the timing during the day of blood taking), we showed that the fetal HSD11B2[CA]n microsatellite polymorphisms in the first intron was related to maternal cortisol concentration (R2=0.26, B=96.27, p=0.007), whereas as the newborn's cortisol concentrations were independent of fetal and maternal HSD11B2[CA]n microsatellite polymorphism. CONCLUSIONS: Our study showed for the first time that the fetal HSD11B2[CA]n microsatellite polymorphism of the HSD11B2 gene in healthy uncomplicated human pregnancy is associated with maternal cortisol concentration. This indicates that fetal genes controlling cortisol metabolism may affect maternal cortisol concentration and hence physiology in healthy pregnant women.

Functional electrical stimulation increases neural stem/progenitor cell proliferation and neurogenesis in the subventricular zone of rats with stroke / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Functional electrical stimulation (FES) is known to promote the recovery of motor function in rats with ischemia and to upregulate the expression of growth factors which support brain neurogenesis. In this study, we investigated whether postischemic FES could improve functional outcomes and modulate neurogenesis in the subventricular zone (SVZ) after focal cerebral ischemia.</p><p><b>METHODS</b>Adult male Sprague-Dawley rats with permanent middle cerebral artery occlusion (MCAO) were randomly assigned to the control group, the placebo stimulation group, and the FES group. The rats in each group were further assigned to one of four therapeutic periods (1, 3, 7, or 14 days). FES was delivered 48 hours after the MCAO procedure and divided into two 10-minute sessions on each day of treatment with a 10-minute rest between them. Two intraperitoneal injections of bromodeoxyuridine (BrdU) were given 4 hours apart every day beginning 48 hours after the MCAO. Neurogenesis was evaluated by immunofuorescence staining. Wnt-3 which is strongly implicated in the proliferation and differentiation of neural stem cells (NSCs) was investigated by Western blotting analysis. The data were subjected to one- way analysis of variance (ANOVA), followed by a Tukey/Kramer or Dunnett post hoc test.</p><p><b>RESULTS</b>FES significantly increased the number of BrdU-positive cells and BrdU/glial fibrillary acidic protein double- positive neural progenitor cells in the SVZ on days 7 and 14 of the treatment (P < 0.05). The number of BrdU/doublecortin (DCX) double-positive migrating neuroblast cells in the ipsilateral SVZ on day 14 of the FES treatment group ((522.77 ± 33.32) cells/mm(2)) was significantly increased compared with the control group ((262.58 ± 35.11) cells/mm(2), P < 0.05) and the placebo group ((266.17 ± 47.98) cells/mm(2), P < 0.05). However, only a few BrdU/neuron-specific nuclear protein-positive cells were observed by day 14 of the treatment. At day 7, Wnt-3 was upregulated in the ipsilateral SVZs of the rats receiving FES ((0.44 ± 0.05)%) compared with those of the control group rats ((0.31 ± 0.02)%, P < 0.05) or the placebo group rats ((0.31 ± 0.04)%, P < 0.05). At day 14, the corresponding values were (0.56 ± 0.05)% in the FES group compared with those of the control group rats ((0.50 ± 0.06)%, P < 0.05) or the placebo group rats ((0.48 ± 0.06)%, P < 0.05).</p><p><b>CONCLUSION</b>FES augments the proliferation, differentiation, and migration of NSCs and thus promotes neurogenesis, which may be related to the improvement of neurological outcomes.</p>

[Cement leakages in percutaneous vertebroplasty:analysis of postoperative computed tomography].

OBJECTIVE: To evaluate the influencing factors of cement leakage in vertebroplasty for the treatment of osteoporosis vertebral compression fracture (OVCF) and vertebral metastases (VM). METHODS: Retrospective analysis was conducted for 653 vertebrae in 356 patients undergoing vertebroplasty at our hospital from May 2007 to January 2011. 251 cases had 438 vertebrae with painful OVCF while 105 cases had 215 vertebrae with VM. Pre-operative computed tomography (CT) was performed to determine the presence of cortical defects or osteolysis and within 3 days after PVP to observe the distribution of polymethylmethacrylate (PMMA) in vertebrae and whether leakage occurred. Volume of PMMA injected into each vertebral body and types of cement leakage were compared between the OVCF and VM groups by Z test or χ². The correlation between cortical defects and cement leakages around vertebrae was assessed with Pearson correlation coefficient. RESULTS: The successful rate of PVP was 100%. The mean volume of PMMA injected into each vertebra was (5.0 ± 2.0) ml and (4.0 ± 1.7) ml in the OVCF and VM groups respectively (P < 0.05). Asymptomatic PMMA leakage was demonstrated by CT in 93 vertebrae (21.2%) in the OVCF group and in 53 vertebrae (28.8%) in the VM group respectively (P < 0.05). Cement leakages into disk were found in 58 vertebrae in the OVCF group and 16 vertebrae in the VM group respectively (P = 0.025). Cement leakages into paravertebral vein were found in 12 vertebrae in the OVCF group and 26 vertebrae in the VM group respectively (P < 0.0001). Correlation was found between cortical defects and cement leakage into paravertebral soft tissues in the OVCF group (r = 0.14) or in the VM group (r = 0.27), between end-plate defects and cement leakage into disk in the OVCF group (r = 0.29) or in the VM group (r = 0.31). CONCLUSION: As a common occurrence in vertebroplasty, cement extravasation is well-tolerated in most patients. It occurs more frequently in the patients with VM than those with OVCF, especially in cases of leakage into paravertebral vein. Cement leakage into disc or paravertebral soft tissue is predisposed in vertebrae with end plate, cortical defects or osteolysis.

Effect of transcutaneous electrical nerve stimulation on symptomatic diabetic peripheral neuropathy: a meta-analysis of randomized controlled trials.

AIMS: To evaluate the effectiveness of transcutaneous electrical nerve stimulation (TENS) on diabetic peripheral neuropathy (DPN). METHODS: Randomized controlled trials (RCTs) comparing TENS with routine care, pharmacological interventions or placebo devices on patients with symptomatic DPN, were identified by electronic and manual searches. Studies were selected and available data were extracted independently by two investigators. Meta-analysis was performed by RevMan 4.2.8 software. RESULTS: Three RCTs involving 78 patients were included in this study. The reductions in mean pain score were significantly greater in TENS group than in placebo TENS group in 4 weeks and 6 weeks follow-up [4 weeks, SMD-5.37, 95% CI (-6.97, -3.77); 6 weeks, SMD-1.01, 95% CI (-2.01, -0.01)], but not in 12 weeks follow-up [SMD-1.65, 95% CI (-4.02, 0.73)]. TENS therapy was associated with significantly subjective improvement in overall neuropathic symptoms in 12 weeks follow-up [WMD-0.18, 95% CI (-0.32, -0.051)]. No TENS-related adverse events were registered in TENS group. CONCLUSIONS: TENS therapy may be an effective and safe strategy in treatment of symptomatic DPN. Due to small sample and short-term treatment duration, large multi-centre RCTs are needed to further evaluate the long-term effect of TENS on DPN.

Inhibition of MAPK signaling by eNOS gene transfer improves ventricular remodeling after myocardial infarction through reduction of inflammation.

Endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) may play an important role in attenuating cardiac remodeling and apoptosis after myocardial infarction. However, the anti-inflammation effects of eNOS in infarcted myocardium and the role of MAPK signaling in eNOS/NO mediated cardiac remodeling have not yet been elucidated. Adenovirus carrying Human eNOS gene was delivered locally into heart 4 days prior to induction of myocardial infarction (MI) by left anterior descending coronary artery ligation. Monocyte/macrophage infiltration was detected by ED-1 immunohistochemistry. Western blot was employed to examine the activation of MAPK. eNOS gene transfer significantly reduced myocardial infarct size and improved cardiac contractility as well as left ventricle (LV) diastolic function at 7 days after MI. In addition, eNOS gene transfer decreased monocyte/macrophage infiltration in the infarct region of the heart. Phosphorylation of MAPK after MI were also dramatically reduced by eNOS gene transfer. All the protective effects of eNOS were blocked by N(ω)-nitro-L-arginine methyl ester (L-NAME) administration, indicating a NO-mediated event. These results demonstrate that the eNOS/NO system provides cardiac protection after MI injury through inhibition of inflammation and suppression of MAPK signaling.

[Expression of PDCD5 in multiple myeloma and its relation with BCL-2].

OBJECTIVE: To determine the expression of apoptosis related gene PDCD5 in multiple myeloma (MM), and to analyze the relation between PDCD5 and BCL-2. METHODS: The expressions of PDCD5 and BCL-2 protein and mRNA were determined by immunohistochemical staining method, flow cytometry (FCM) and reverse transcription polymerase chain reaction (RT-PCR) method in bone marrow mononuclear cells. We also analyzed the relation between PDCD5 and BCL-2. RESULTS: Immunohistochemical staining showed that PDCD5 protein positive cell percentage, staining intensity index (SII) of PDCD5 protein, BCL-2 protein positive cell percentage, and SII of BCL-2 protein were (34.75 +/- 6.49)%, (281.16 +/- 75.33), (29.97 +/- 5.57)%, and (224.94 +/- 57.72) in the MM group and (52.98 +/- 5.84)%, (462.84 +/- 39.77), (5.56 +/- 1.95)%, and (27.84 +/- 9.75) in the control group (all P < 0.05). Results of FCM showed that PDCD5 protein positive percentage and mean fluorescence intensity of PDCD5 were (78.11 +/- 21.63)% and (61.73 +/- 11.04) in the MM group and (89.46 +/- 9.98)% and (353.04 +/- 123.26) in the control group (all P < 0.05). RT-PCR showed that relative expression of PDCD5 and BCL-2 mRNA were (0.33 +/ -0.07) and (0.33 +/- 0.08) in the MM group and (0.53 +/- 0.05) and (0.12 +/- 0.02) in the control group (all P < 0.05). The positive cell percentage of PDCD5 and BCL-2 protein was negative correlation (r = -0.86, P < 0.05); the expression of PDCD5 and BCL-2 mRNA was the same status (r = -0.90, P < 0.05). CONCLUSION: The expressions of PDCD5 protein and mRNA in MM patients are down-regulated, but the expressions of BCL-2 protein and mRNA are up-regulated. The mRNA and protein expression of PDCD5 and BCL-2 has negative correlation.

[Screening the effective target sequences of laryngeal carcinoma related gene LCRG1].

OBJECTIVE: To screen the effective target sequences of laryngeal carcinoma related gene LCRG1 using RNAi. METHODS: PCR site mutation method was used to reconstruct pSuper vector. Five pairs of siRNA sequences designed by siRNA software were annealed and inserted into the reconstructed pSuper vector. The reconstructed pSuper 362,398,432,789,903,and pSuper vectors were transfected into Hela cell lines and selected with the appropriate drugs to get resistant and pool cells, respectively. The colonies were identified by RT-PCR or real-time RT-PCR analysis. The silence effects were observed by cloning formation analysis. RESULTS: pSuper vector was reconstructed to restore Bgl II restriction enzyme sites using PCR mutation. The RT-PCR or real-time RT-PCR Results of pool clones showed 362, 398, and 432 pool clones all had better effects of LCRG1 gene-silence, especially 362 pool clones. The expression level of LCRG1 mRNA of selected 362 group anti-puromycin clones A2 and A5 was decreased. The Results of clone forming efficiency revealed that the cellular proliferation in A2 of 362 group was significantly higher than that of the vector and control Hela cells (P<0.05). CONCLUSION: The reconstructed pSuper vector is successfully constructed. The 362 group has better gene silence and has 2 effective 362 group anti-clones, suggesting that methodology has important values in studYing the function and molecular mechanism of LCRG1.

Screening the effective target sequences of laryngeal carcinoma related gene LCRG1 / 中南大学学报(医学版)

OBJECTIVE@#To screen the effective target sequences of laryngeal carcinoma related gene LCRG1 using RNAi.@*METHODS@#PCR site mutation method was used to reconstruct pSuper vector. Five pairs of siRNA sequences designed by siRNA software were annealed and inserted into the reconstructed pSuper vector. The reconstructed pSuper 362,398,432,789,903,and pSuper vectors were transfected into Hela cell lines and selected with the appropriate drugs to get resistant and pool cells, respectively. The colonies were identified by RT-PCR or real-time RT-PCR analysis. The silence effects were observed by cloning formation analysis.@*RESULTS@#pSuper vector was reconstructed to restore Bgl II restriction enzyme sites using PCR mutation. The RT-PCR or real-time RT-PCR Results of pool clones showed 362, 398, and 432 pool clones all had better effects of LCRG1 gene-silence, especially 362 pool clones. The expression level of LCRG1 mRNA of selected 362 group anti-puromycin clones A2 and A5 was decreased. The Results of clone forming efficiency revealed that the cellular proliferation in A2 of 362 group was significantly higher than that of the vector and control Hela cells (P<0.05).@*CONCLUSION@#The reconstructed pSuper vector is successfully constructed. The 362 group has better gene silence and has 2 effective 362 group anti-clones, suggesting that methodology has important values in studYing the function and molecular mechanism of LCRG1.

Expression of PDCD5 in multiple myeloma and its relation with BCL-2 / 中南大学学报(医学版)

OBJECTIVE@#To determine the expression of apoptosis related gene PDCD5 in multiple myeloma (MM), and to analyze the relation between PDCD5 and BCL-2.@*METHODS@#The expressions of PDCD5 and BCL-2 protein and mRNA were determined by immunohistochemical staining method, flow cytometry (FCM) and reverse transcription polymerase chain reaction (RT-PCR) method in bone marrow mononuclear cells. We also analyzed the relation between PDCD5 and BCL-2.@*RESULTS@#Immunohistochemical staining showed that PDCD5 protein positive cell percentage, staining intensity index (SII) of PDCD5 protein, BCL-2 protein positive cell percentage, and SII of BCL-2 protein were (34.75 +/- 6.49)%, (281.16 +/- 75.33), (29.97 +/- 5.57)%, and (224.94 +/- 57.72) in the MM group and (52.98 +/- 5.84)%, (462.84 +/- 39.77), (5.56 +/- 1.95)%, and (27.84 +/- 9.75) in the control group (all P < 0.05). Results of FCM showed that PDCD5 protein positive percentage and mean fluorescence intensity of PDCD5 were (78.11 +/- 21.63)% and (61.73 +/- 11.04) in the MM group and (89.46 +/- 9.98)% and (353.04 +/- 123.26) in the control group (all P < 0.05). RT-PCR showed that relative expression of PDCD5 and BCL-2 mRNA were (0.33 +/ -0.07) and (0.33 +/- 0.08) in the MM group and (0.53 +/- 0.05) and (0.12 +/- 0.02) in the control group (all P < 0.05). The positive cell percentage of PDCD5 and BCL-2 protein was negative correlation (r = -0.86, P < 0.05); the expression of PDCD5 and BCL-2 mRNA was the same status (r = -0.90, P < 0.05).@*CONCLUSION@#The expressions of PDCD5 protein and mRNA in MM patients are down-regulated, but the expressions of BCL-2 protein and mRNA are up-regulated. The mRNA and protein expression of PDCD5 and BCL-2 has negative correlation.

[3 polymorphisms of gene GP IIb and the function of GP IIb T13959 G in the platelet transfusion refractoriness].

OBJECTIVE: To investigate the relationship among 3 polymorphisms of GP IIb and the function of GP IIb T13959 G in the platelet transfusion refractoriness(PTR). METHODS: The 26th exon, the 30th exon and the 21st intron of gene GP IIb in 110 individuals were amplified by polymerase chain reaction (PCR), and the PCR products were analyzed with single-strand conformation polymorphism(SSCP) and sequenced to investigate whether there was linkage among the polymorphisms of the gene. Human platelet antigen-3 (HPA-3) gene frequency was detected by Fok I enzyme in 147 patients with hematologic diseases, and was compared with that in 110 normal individuals. Forty-four patients who received apheresis platelet transfusion repeatedly were randomly divided into the HPA-3 homotype group and the control group. The antibodies of the platelet were detected after 3 times of platelet transfusion. RESULTS: There were polymorphisms of gene GP IIb in the 26th, 30th exon and the 21st intron, and the mutations were: T changed into G in 13,959 th of the 26th exon; C changed into T in 16,997 th of the 30th exon; the 9 bps deletion occurred in 11,996-12,004 th of the 21st intron. The 3 polymorphisms had synchronization in the individuals. The results of Fok I enzyme indicated that the frequency of HPA-3a was 83.6% (92/110)and 81.9%(119/147), and that of HPA-3b was 16.4%(18/110) and 19.1%(28/147) in the normal individuals and the patients respectively. There was no significant difference between the patients and normal individuals (P>0.05). After the platelet transfusion, the antibodies of all the cases of the homotype platelet transfusion were negative, but the antibodies in 2 cases of the control group were positive, and there was antibody to HPA-3a in one of the antibodies positive cases. CONCLUSION: (1)There is close linkage among the polymorphisms of gene GP IIb, which is T->G in 13 959 th of the 26th exon, C->T in 16,997 th of the 30th exon, and the 9 bps deletion in 11,996-12,004 th in the 21st intron. (2)The gene frequency of HPA-3a/3b is similar in the normal individuals and patients with hematologic diseases. (3) HPA-3 system may be one of the reasons for PTR in Chinese.

3 polymorphisms of gene GP IIb and the function of GP IIb T13959 G in the platelet transfusion refractoriness / 中南大学学报(医学版)

OBJECTIVE@#To investigate the relationship among 3 polymorphisms of GP IIb and the function of GP IIb T13959 G in the platelet transfusion refractoriness(PTR).@*METHODS@#The 26th exon, the 30th exon and the 21st intron of gene GP IIb in 110 individuals were amplified by polymerase chain reaction (PCR), and the PCR products were analyzed with single-strand conformation polymorphism(SSCP) and sequenced to investigate whether there was linkage among the polymorphisms of the gene. Human platelet antigen-3 (HPA-3) gene frequency was detected by Fok I enzyme in 147 patients with hematologic diseases, and was compared with that in 110 normal individuals. Forty-four patients who received apheresis platelet transfusion repeatedly were randomly divided into the HPA-3 homotype group and the control group. The antibodies of the platelet were detected after 3 times of platelet transfusion.@*RESULTS@#There were polymorphisms of gene GP IIb in the 26th, 30th exon and the 21st intron, and the mutations were: T changed into G in 13,959 th of the 26th exon; C changed into T in 16,997 th of the 30th exon; the 9 bps deletion occurred in 11,996-12,004 th of the 21st intron. The 3 polymorphisms had synchronization in the individuals. The results of Fok I enzyme indicated that the frequency of HPA-3a was 83.6% (92/110)and 81.9%(119/147), and that of HPA-3b was 16.4%(18/110) and 19.1%(28/147) in the normal individuals and the patients respectively. There was no significant difference between the patients and normal individuals (P>0.05). After the platelet transfusion, the antibodies of all the cases of the homotype platelet transfusion were negative, but the antibodies in 2 cases of the control group were positive, and there was antibody to HPA-3a in one of the antibodies positive cases.@*CONCLUSION@#(1)There is close linkage among the polymorphisms of gene GP IIb, which is T->G in 13 959 th of the 26th exon, C->T in 16,997 th of the 30th exon, and the 9 bps deletion in 11,996-12,004 th in the 21st intron. (2)The gene frequency of HPA-3a/3b is similar in the normal individuals and patients with hematologic diseases. (3) HPA-3 system may be one of the reasons for PTR in Chinese.

[In vitro differentiation into megakaryocytes and generation of platelets from CD34+ cells of umbilical cord blood].

OBJECTIVE: To induce hematopoietic progenitor/stem cells of umbilical cord blood to differentiate into mature megakaryocytes and platelets in vitro and to investigate the mechanism of production of platelets. METHODS: The CD34+ cells were sorted from umbilical cord blood by magnetic activated cell sorting (MACS) and then cultured in vitro with optimized medium to be differentiated into mature megakaryocytes and platelets. The cultured cells and the platelet-like particles were isolated from the culture and were checked by the fluorescence-activated cell sorter (FACS), immunohistochemistry assays, light microscope,electron microscope and platelet aggregation tests. RESULTS: The cultured megakaryocytes were detected with proplatelets and both the cultured cells and the platelet-sized particles were found to have the same structure with the normal megakaryocytes and platelets by light and electron microscope. The immunohistochemistry assays revealed the cultured cells expressed GP II b III a with a positivity of 95% which was a special antigen for platelets and megakaryocytes. Culture-derived platelet-sized particles aggregated in response to thrombin as the plasma derived-platelets did. The cultured platelets had the same positivity of CD41 as the platelets from platelet rich plasma. CONCLUSION: The hematopoietic progenitor/stem cells can be induced to differentiate into purified and mature megakaryocytes and platelets. It provides a practical way to study the mechanism of platelets production.

[Effects of botulinum toxin guided by electric stimulation on spasticity in ankle plantar flexor of children with cerebral palsy: a randomized trial].

OBJECTIVE: To compare the effects of botulinum toxin A (BTX-A) injection guided by electric stimulation combined with physiotherapy, with physiotherapy only on the spasticity of the ankle plantar flexor in children with cerebral palsy (CP). METHODS: After signing the informed consent, 43 children with CP, aged 52.4 +/- 13.2 months (35 to 82 months), were randomly assigned into 2 groups, (1) BTX-A group (n = 23) treated with BTX-A injection guided by electric stimulation and (2) physiotherapy alone group (n = 20). Children in BTX-A group received injection of HengLi BTX-A in the ankle plantar flexors. A maximum dose of 12 units of BTX-A per kilogram body weight and maximumly 10 units of BTX-A per site were administered. Localization technique was the use of electrical stimulation guidance. Physiotherapy and ankle-foot orthosis were applied to children at 72 hours after injection in BTX-A group and at the time of being recruited into physiotherapy group. Ten days after entering into the study, the program was applied by the parents. Demographic data, including age, gender, number of the spastic lower limbs, affected side (left or right) were recorded. Clinical assessments included the range of passive movement (PROM) measured by goniometer while children maintained the knee extended, modified Ashworth scale (MAS), composite spasticity scale (CSS), D and E dimensions of the Gross Motor Function Measure (GMFM), and walking velocity (WV) was determined before treatment and at 2 weeks, 1, 2, and 3 months after treatment. RESULTS: No statistically significant differences were found in age, gender, number of the spastic lower limbs, affected side, as well as clinical assessments (PROM, MAS, CSS, GMFM and WV) before treatment between the 2 groups (P > 0.05). All the children showed a reduction of spasticity (PROM, MAS and CSS) after 2 weeks, 1, 2, and 3 months of treatment (P < 0.05). When compared with the baseline findings, the improvement of standing and walking (GMFM), walking velocity were statistically significant after 2 weeks, 1, 2, and 3 months of treatment (P < 0.05). Furthermore, the differences of PROM, MAS and CSS between the 2 groups at 2 weeks, 1, 2, and 3 months examination were also statistically significant (after 3 months of treatment: t(PROM) = 6.48, t(MAS) = 9.74, t(CSS) = 9.59; P < 0.05). The difference in GMFM between the 2 groups was statistically significant (t(1M) = 2.20, t(2M) = 3.26, t(3M) = 4.13; P < 0.05) at 1, 2, and 3 months after treatment. The difference of WV between the 2 groups was statistically significant (t(2M) = 2.12, t(3M) = 2.57; P < 0.05) at 2 and 3 months after treatment. CONCLUSION: BTX-A injection guided by electrical stimulation in combination with physiotherapy was more effective than physiotherapy alone in terms of reducing spasticity and improving functional performance in standing, walking, walking pattern and velocity on spasticity in ankle plantar flexors of ambulant children with CP.

[Effects of functional electrical stimulation on the improvement of motor function of patients with acute stroke: a randomized controlled trial].

OBJECTIVE: To investigate the effects of functional electrical stimulation (FES) on the improvement of motor and walking ability of the lower extremities of the patients with acute stroke. METHODS: Forty-six patients with stroke (including cerebral infarction and hemorrhage), aged 71 +/- 8 (45 - 84), hospitalized within 2 weeks (9 +/- 4 days) after the onset, matched with one another in the baseline measurements before treatment, were assigned randomly into 3 groups: FES group (n = 13), receiving standard rehabilitation combined with FES 30 minutes per day, 5 days per week for 3 weeks, placebo stimulation group (n = 15) receiving standard rehabilitation combined with the installment of the FES apparatus, operated in the same manner as mentioned above, however, without real electric stimulation, and control group (n = 13), receiving standard rehabilitation alone. The score of the composite spasticity scale (CSS) was measured, electromyography was conducted to measure the maximum isometric voluntary contraction (MIVC) of the ankle dorsi-flexors and plantar-flexors, and walking ability by the test of timed "Up and Go" before treatment, weekly during the 3-week treatment, and 8 weeks after the onset of stroke. RESULTS: After 3 weeks of treatment, the percentage of CSS score of the FES group was 31% +/- 35%, significantly lower than those of the placebo and control groups (50% +/- 88% and 65% +/- 65% respectively, both P < 0.05); the ankle dorsiflexion torque of MIVC of the FES group was 9 Nm +/- 5 Nm, significantly higher than those of the placebo and control groups (5 Nm +/- 3 Nm and 4 Nm +/- 5 Nm respectively, both P < 0.05), and the electromyogram co-contraction ratio of the FES group was 8% +/- 5%, significantly lower than those of the placebo and control groups (27% +/- 26% and 28% +/- 19% respectively, both P < 0.05). The time needed to recover the walking ability after the stroke onset of the FES group was 18 +/- 8 days, shorter by 2 approximately 3 days than those of the placebo and control groups (20 +/- 7 and 21 +/- 8 days respectively). The percentage of the patients able to walk with the help of a stick 3 weeks after treatment of the FES group was 85%, significantly higher than those of the placebo and control groups (60% and 46% respectively, both P < 0.05). 84.6% of the patients of the FES group returned home, a percentage significantly higher than those of the placebo and control groups (53% and 46% respectively, both P < 0.05). CONCLUSION: FES, plus standard rehabilitation, is effective in improving the motor and walking ability of the patients with acute stroke, to the degree that most patients are recovered to be able to return home.