AIM:Neural stem cells can be induced to differentiate into various types of neural cells such as neurons and neuroglia cells,but the technique of depuration and cultivation does not consummate.This article determines the optimal culture technique of neural stem cells by different culture concentrations and passage methods. METHODS:Experiments were conducted from May to December 2006 at Laboratory of Transplantation Immunity of Sichuan University.①Clean pregnant female rats(embryonic age range from 12-16 days)and the disposition of animal met ethical standard.②The cerebral cortex of rat embryos were collected,and digested with trypsin and ethylenediamine tetraacetic acid mixture to obtain signal cell suspension.They were cultured in serum-free medium (DMEM/F12 medium containing B27,basic fibroblast growth factor and epidermal growth factor).The 3~(rd)passage cells were collected,and incubated at 1?10~7 L~(-1),1?10~8 L~(-1),1?10~9 L~(-1),1?10~(10)L~(-1),respectively.In addition,neural stem cells were collected 7-10 days after primary culture to harvest formative cell masses.Mechanical blow refers to soft blowing with haustorial tube from thick to thin after centrifugation,or sterile syringe with No.5 pinhead blowing cells when the blow was 5 times.Bubble production was avoided during the operation.Trypsin aspiration combined with mechanical blow refers to trypsin was added after centrifugation,at 37℃for 10 minutes,the neural stem cells were lightly blown with haustorial tube polished with flame or blown with sterile syringe with No.5 pinhead,and then fetal bovine serum was added to stop digestion.③The growth characteristic of the 3~(rd)passage cells at different culture concentration was observed and proliferation was measured at days 1,3,5 and 7 by MTT assay.The neural clone spheres of subcultured was counted to determine the optimal passage way.Immunofluorescence was carried out to detect nestin(special marker to neural stem cells),BrdU,neurone specific enolase,glial fibrillary acidic protein. RESULTS:①Growth characteristics and identification of rat embryonic neural stem cells in vitro:The dissociated neural stem cells from the cerebral cortex of rat embryos were continuously harvested and purified by suspension cultures to get the daughter cell clone.Nestin positive cells could be found in the neurospheres and after attachment they could differentiate into neurone specific enolase and glial fibrillary acidic protein positive cells,and immunofluorescence showed a great of BrdU-positive cells.②The effect of different incubated number of neural stem cells on proliferation:When the neural stem cells planted at the concentration of 1?10~9 L~(-1),the growth rate of the cells was the highest of all concentrations.The number of clone spheres exceeded others at concentration 1?10~7 L~(-1),1?10~8 L~(-1)and 1?10~10 L~(-1)(P

72 cases of treatments on insomnia were recorded in the medical books of Ming and Qing dynasties(A.D.1368～1911 years).The compatibility regularity of treating insomnia in these 72 cases were statistically analyzed with frequency and the R type cluster analysis methods. Combining the data and modern recognition, compatibility and medication regularity of treating insomnia was further analyzed.

Objective To observe the influence of needling the Eight Confluent Points plus Huo Xue Rong Luo granules on the levels of gamma-aminobutyric acid (GABA) and glycine (Gly) in patients with spastic paralysis after cerebral infarction. Method Sixty patients with spastic paralysis after cerebral infarction were randomized into a treatment group and a control group, 3 0 cases each. The control group was intervened by basic treatment, while the treatment group was by Huo Xue Rong Luo granules plus acupuncture at the Eight Confluent Points in addition to the basic treatment, for 6 treatment courses in total. Before the treatment, and after half-a-month, 1-month, and 3-month treatments, the Chinese Stroke Scale (CSS) and activities of daily living (ADL) were evaluated, the levels of GABA and Gly were detected by using high performance liquid chromatography (HPLC), and the therapeutic efficacies were compared. Result After half-a-month treatment, the CSS score dropped significantly in both groups (P<0.01), and the between-group difference was statistically significant (P<0.01);the ADL score (Barthel Index score) increased markedly in both groups (P<0.01), and the between-group difference was statistically significant (P<0.01); the level of serum GABA increased significantly in both groups (P<0.05), and the between-group difference was statistically significant (P<0.05);the level of serum Gly rose up significantly in both groups (P<0.01), and the between-group difference was statistically significant (P<0.05). After 1-month treatment, the CSS score dropped significantly in both groups compared to that before treatment (P<0.01), and the inter-group difference was statistically significant (P<0.01);the ADL score increased significantly in both groups compared to that before treatment (P<0.01), and the inter-group difference was statistically significant (P<0.01);the levels of serum GABA and Gly increased markedly in both groups compared to that before treatment (P<0.01), and the inter-group differences were statistically significant (P<0.01). After 3-month treatment, the CSS score dropped significantly in both groups compared to that before treatment (P<0.01), and the inter-group difference was statistically significant (P<0.01);the ADL score increased significantly in both groups compared to that before treatment (P<0.01), and the inter-group difference was statistically significant (P<0.01); the levels of serum GABA and Gly increased markedly in both groups compared to that before treatment (P<0.05), approaching the normal levels, and the inter-group differences were statistically insignificant (P>0.05). Conclusion Needling the Eight Confluent Points plus Huo Xue Rong Luo granules is effective in treating spastic paralysis after cerebral infarction, which is plausibly associated with the up-regulation of inhibitory neurotransmitters (GABA and Gly).

Objective To observe the clinical efficacy of Huoxue Rongluo Particles combined with acupuncture in the eight confluent points of spastic cerebral infarction paralysis and its effects on Glu and Asp levels of serum. Methods Totally 60 patients were divided into two groups: the experimental group and the control group. Both groups received basic Western medicine treatment, and the experimental group received Huoxue Rongluo Particles additionally, one dose a day for two times orally taken; Acupuncture was on eight confluent acupoint, every two days. 15 d is a treatment course, with 6 courses in total. Clinical spasticity index (CSI) and TCM symptom scores before treatment and the treatment of half month, 1 month, 3 months were observed. The levels of Glu and ASP in serum were detected, and TCM clinical efficacy was observed. Results The CSI score, levels of Glu and Asp in serum and TCM symptom scores of the patients after half-month, one-month, and three-month treatment were lower than those before treatment (P<0.05, P<0.01). There was no statistical significance in CSI score after half month treatment between the two groups (t=0.329, P=0.743). The CSI score in the experimental group after one-month and three-month treatment was lower than the control group (t=-2.636, P=0.024; t=-4.213, P=0.021). There was statistical significance in the levels of Glu and Asp between the two groups after half-month and one-month treatment (P<0.05, P<0.01). There was no statistical significance in the levels of Glu and Asp in the two groups after three-month treatment (P>0.05). The TCM symptom scores in experimental group was lower than the control group after half-month, one-month and three-month treatment (P<0.05). The total TCM effective rate was 86.67% (26/30) in experimental group, and 80.00% (24/30) in the control group, with the experimental group better than the control group (P<0.05). Conclusion Huoxue Rongluo Particles combined with acupuncture eight confluence acupoints in the treatment of spastic cerebral infarction paralyzed patients can relieve spasm degree, improve TCM clinical symptoms, which mechanism may be related to reducing serum excitatory neurotransmitters.

Objectives To explore the correlation between the cerebrospinal fluid protein and facial paralysis in pa?tients with Guillain-Barre syndrome (GBS). Methods Clinical and biochemical data of 111 patients with GBS in depart?ment of neurology from January 2005 to September 2015 were retrospectively analyzed. According to facial paralysis, GBS patients were divided into the facial normal and paralysis groups. Their clinical and biochemical characteristics were compared between the two groups. According to level of cerebrospinal fluid protein, GBS patients were divided into cerebrospinal fluid protein normal, mild high and severe high groups. Incidences of facial paralysis were compared among these three groups. The correlation between the cerebrospinal fluid protein and facial paralysis was analyzed. Results There was no significant difference in gender, age, respiratory infection and other clinical symptoms (P>0.05), whereas there were statistically significant differences in cerebrospinal fluid protein, immunoglobulin G, and cerebrospinal fluid albumin/serum albumin ratio between the facial normal and paralysis groups (P<0.05). Among the three groups by differ?ent levels of cerebrospinal fluid protein, there were statistically significant differences in the incidence of facial paralysis (F=3.48,P=0.03). Cerebrospinal fluid protein was positively correlated with facial paralysis (r=0.288,P<0.01). Conclu? sions The incidence of facial paralysis is associated with the levels of cerebrospinal fluid protein. Thus, cerebrospinal flu?id protein may be helpful in monitoring of GBS patients with facial paralysis.

OBJECTIVETo explore the antagonizing effect of celecoxib against the cytotoxicity of carboplatin in human esophageal cancer cells.

METHODSThe cell viability of cisplatin-resistant cell line EC109/CDDP and its parental cell line EC109 exposed to carboplatin alone or carboplatin plus celecoxib was determined by MTT assay. The expression of CTR1, caspase-3 activation and PARP cleavage in the exposed cells were examined by Western blotting. Caspase-3 activity and cell apoptosis after the exposure were detected with Caspase-3/7 assay and flow cytometry, respectively. The effect of celecoxib on carboplatin accumulation in the cells was measured using inductively coupled plasma mass spectrometry (ICP-MS).

RESULTSCelecoxib treatment significantly increased the IC50 of carboplatin, suppressed carboplatin-induced caspase-3 and PARP cleavage and caspase-3 activity in EC109 and EC109/CDDP cells. Celecoxib also inhibited carboplatin-induced apoptosis and suppressed intracellular carboplatin accumulation in both cell lines. A combined exposure to celecoxib and carboplatin did not cause significant changes in the protein expression of CTR1.

CONCLUSIONCelecoxib antagonizes the cytotoxic effect of carboplatin and inhibits carboplatin-induced apoptosis in human esophageal cancer cells by reducing intracellular carboplatin accumulation.

Apoptosis ; Blotting, Western ; Carboplatin ; antagonists & inhibitors ; Caspase 3 ; metabolism ; Celecoxib ; Cell Line, Tumor ; drug effects ; Cell Survival ; Drug Interactions ; Esophageal Neoplasms ; metabolism ; pathology ; Humans ; Pyrazoles ; pharmacology ; Sulfonamides ; pharmacology

Objective To investigate the value of fine-needle aspiration cytology (FNAC) combined with serum calcitonin in the diagnosis of medullary thyroid carcinoma (MTC).Methods Retrospective analysis of the serum calcitonin and FANC results in 17 cases of MTC confirmed by pathological examination.The diagnostic value of the two methods alone and in combination for MTC were compared.Results 13 of the 17 MTC patients (13/17,76.5％) underwent fine needle aspiration biopsy before surgery,11 cases (11/13,84.6％) diagnosed as thyroid malignant tumor,7 of whom were diagnosed or suspected of MTC (7/13,53.8％).Serum calcitonin levels increased in different degrees in 16 patients.The sensitivity of the two methods combined in the diagnosis of MTC was 92.3％ (12/13).Conclusions FNAC is an important diagnostic method for the evaluation of thyroid nodules,but it is not sensitive to the diagnosis of MTC.Serum calcitonin is highly sensitive but not universally available.Compared with FNAC alone,FNAC combined with the serum calcitonin examination may significantly improve the detection rate of MTC preoperative and reduce missed diagnosis.

Objective: To evaluate the short-term and long-term outcomes after laparoscopic surgery compared with traditional laparotomy in cases of stage ⅠA2-ⅡA2 cervical cancer. Methods: We conducted a retrospective study on the clinical data of 1 863 patients diagnosed as FIGO stages ⅠA2-ⅡA2 cervical cancer in 6 third-grade class-A hospitals in Guangxi province between January 2007 and May 2014. One thousand and seventy-one received laparoscopy, and 792 received laparotomy. T-test, U-test and χ² test were used to compare the short-term and long-term outcomes. The short-term outcomes included surgical related outcomes and operative complications, and the long-term outcomes included quality of life (pelvic floor functions and sexual functions), survival and recurrence. Pelvic floor function and sexual function were assessed with the International Consultation on Incontinence Quesonnaire Female Lower Urinary tract(ICIQ-FLUTS) and the Female Sexual Function Inventory (FSFI), respectively. Survival rates were estimated by Kaplan-Meier analysis. The survival curves were compared with Log-rank test. Cox regression analysis was used to evaluaterisk factors for prognosis. Results: (1)The short-term outcomes : There were significant difference in operative time([(257±69) vs(238±56)min], estimated blood loss[(358±314) vs(707±431)ml], anus exhausting time[(2.5±0.9) vs (2.9±0.8)d], preserved days of catheter[(15±7) vs(18±9)d], and post-operative length of stay[(19±16) vs (30±21)d] between the laparoscopic surgery group and the opensurgery group(P<0.05). There was no significant difference in lymph nodes yielded[(21±9) vs (21±11)], left parametrial width[(2.5±0.8) vs (2.7±0.7)cm], right parametrial width [(2.6±0.3) vs (2.7±0.2)cm], vaginal cuff length[(2.4±0.7) vs (2.2±0.7)cm] between the laparoscopic surgery group and the opensurgery group(P>0.05). The intra-operative complications occurred in 8.1%(87/1 071)in the laparoscopic surgery group and in 10.7%(85/792)in the open surgery group(P>0.05). However, the complications of vascular injury in the laparoscopic surgery group[2.6%(28/1 071)]was lower than that in the open surgery group[7.7%(61/792), P<0.001]. The laparoscopic surgery exhibited lower post- operative complication rate [33.8%(362/1 071)vs 40.2%(318/792), P<0.05] and poorer wound healing rate [0.7%(7/1 071)vs 4.0%(32/792), P<0.05]. (2)The long-term outcomes(Hierarchical analysis): The overall incontinence in ICIQ-FLUTS questionnaire in nerve-sparing laparoscopic group [28.4%(67/236)] was lower than that in the open surgery group [35.9%(71/198), P=0.004] . However, There was no significant difference in degree of incontinence between the two groups(P>0.05). The overall sexual dysfunction in FSFI questionnaire after 12 months of postoperative in the nerve-sparing laparoscopic group [47.0%(111/236)]was lower than that in the open surgery group [58.6%(116/198), P=0.001], and the six different dimension scores in the laparoscopic surgery group were higher than that in the open surgery group (P<0.05). The recurrence rate was 3.5%(35/1 007)in the laparoscopicsurgery group and 4.7%(35/740)in the open surgery group(P>0.05). The 5-year OS was 94.0% for the laparoscopic surgery group and 90.2% for the open surgery group(P>0.05), and the 5-year DFS was 93.9% for the laparoscopic surgery group and 89.1% for the open surgery group(P>0.05). (3) Prognostic fators: In univariate analysis, tumor dimension, clinical stage, deep stromal invasion, LVSI, and retroperitoneal lymph node metastasis signficantly affected 5-year OS and 5-year DFS(P<0.05); In multivariate analyses, LVSI, deep stromal invasion and LN metastasis were independent prognostic factors(P<0.05). Conclusions Laparoscopy can reduceestimated blood loss, accelerate postoperative recovery and improve the quality of life after surgery compared to laparotomy, and it ensures the same oncological results as open surgery. Laparoscopic approach is a safe and effective treatment for early-stage cervical cancer.

Ischemic stroke is a major public health problem worldwide. Although the circadian clock is involved in the process of ischemic stroke, the exact mechanism of the circadian clock in regulating angiogenesis after cerebral infarction remains unclear. In the present study, we determined that environmental circadian disruption (ECD) increased the stroke severity and impaired angiogenesis in the rat middle cerebral artery occlusion model, by measuring the infarct volume, neurological tests, and angiogenesis-related protein. We further report that Bmal1 plays an irreplaceable role in angiogenesis. Overexpression of Bmal1 promoted tube-forming, migration, and wound healing, and upregulated the vascular endothelial growth factor (VEGF) and Notch pathway protein levels. This promoting effect was reversed by the Notch pathway inhibitor DAPT, according to the results of angiogenesis capacity and VEGF pathway protein level. In conclusion, our study reveals the intervention of ECD in angiogenesis in ischemic stroke and further identifies the exact mechanism by which Bmal1 regulates angiogenesis through the VEGF-Notch1 pathway.
Rats ; Animals ; Vascular Endothelial Growth Factor A/pharmacology* ; Brain Ischemia/metabolism* ; Ischemic Stroke ; Signal Transduction ; ARNTL Transcription Factors/pharmacology* ; Neovascularization, Physiologic/physiology*