BACKGROUND:As the main function cel of intervertebral disc, nucleus pulposus cel s are the focus of studying the degenerative mechanism;thereby, it is crucial to maintaining the physiological function of nucleus pulposus cel s in vitro and the stability of the cel phenotype.
OBJECTIVE:To study the excel ence of differential velocity adherent procedure in primary culture of nucleus pulposus cel s of rat intervertebral disc through comparison.
METHODS:Twenty male Wistar rats aged 4 weeks were enrol ed, and then nucleus pulposus cel s of intervertebral disc were isolated and cultured in vitro;cel passage culture was performed in different groups when the primary cel s were merged to 90%. Differential velocity adherent group cel s adhered for 30 minutes, and non-adherent cel s were aspirated and transferred to new culture dish after readjusting the concentration;the controls received no intervention. Passages 1 and 2 cel s in the differential velocity adherent group were isolated and purified by the same procedure. The morphology of three generations of cel s in the two groups was compared, the purity of the identification was detected by immunohistochemistry, the cel viability was detected by cel counting kit-8 and the cel growth curve was drawn.
RESULTS AND CONCLUSION:Inverted phase contrast microscope and hematoxylin-eosin staining revealed that the cel homogeneity of the differential velocity adherent group was significantly higher than that of the control group, and there were more kinds of fibroblast-like cel s in nucleus pulposus cel s in the control group. Identification and purity analysis of col agen type II showed that the cytoplasm of two groups were both stained brown, indicating that they were the nucleus pulposus chrondrocytes. The positive rate of differential velocity adherent group was significantly higher than that of the control group (P<0.05). The cel growth curve of cel counting kit-8 showed cel s in the two groups al passed by the latency phase within 2 days, then to the logarithmic phase of 3 days, and entered the lag phase, while the growth rate of the control group was more rapid during the latency and the early logarithmic phases. These findings suggest that differential velocity adherence method is a practical and effective procedure for the isolation and purification of primary cultured rat nucleus pulposus cel s. Through the primary culture, twice differential velocity adherence procedure, the passage 3 rat nucleus pulposus cel s are metabolic exuberant, consistent with the phenotype, the cel purity is higher, and the logarithmic growth phase can be used as the optimal time for studying the mechanism of intervertebral disc cel s.

Objective To evaluate the treatment efficacy,toxicities and prognostic factors of nasopharyngeal carcinoma ( NPC ) treated with intensity modulated radiation oncology ( IMRT ).Methods Between January 2006 and August 2008,300 patients with pathologically diagnosed NPC from 6 center received IMRT.The number of patients with stage Ⅰ,Ⅱ,Ⅲ and Ⅳa+b disease (UICC/AJCC 2002 staging system) were 6,45,141 and 108,respectively.The prescription doses were as follows:70-74 Gy/30f toplanning target volume of primary nasopharynx tumor ( PTVRλ),68-70 Gy/30f to planning target volume of positive lymphnode (ptvnd),60-64 Gy/30f to higher risk region (PTV1),50-54 Gy/30f to lower risk region (PTV2).Patients with stage Ⅲ and Ⅳa+b disease also received cisplatin-based chemotherapy.Cox method was used for Multivariate analysis.ResultsThe follow-up rate was 99.7％.The 4-year rate of local control,regional control,metastasis-free survival (DMFS),disease-free survival (DFS) and overall survival (OS) was 94.0％,95.5％,87.4％,80.8％,86.1％,respectively.Mucositis was the most severe acute toxicity,with 18.0％grade 1,48.7％grade 2,33.3％grade 3.No patient suffered from grade 4mucositis.Xerostomia was the most common late toxicity,with 12.0％ grade 0,75.7％ grade 1,12.3％grade 2.No grade 3-4 xerostomia was observed.There were 18,15 and 42 patients failed in local,regional and distant metastasis,respectively.Multivariate analysis showed that N stage was the only prognostic factor for OS (x2 =5.17,P=0.023),DMFS (x2 =6.91,P=0.009) and DFS (x2 =8.15,P=0.004) in these patients.ConclusionsIMRT can improve the treatment efficacy of NPC.The acute and late toxicities were tolerated.Distant metastasis becomes the main treatment failure.N stage is a significant prognostic factors.

Objective:To investigate the reasonable approach and surgical indication for acute necrotizing pancreatitis(ANP)by analyzing the factors that affecting the mortality of ANP.Methods:One hundred and twelve patients with ANP were retrospectively divided into two groups-the dead and the survivors.Some parameters were analyzed statistically to reveal what's the reason for death.Results:The average age,sex ratio and onset of illness were similar between two groups.And the ratio of early shock,early adult respiratory distress syndrome (ARDS),high temperature,leukocytosis and high blood glucose between two groups were also similar between two groups(P＞0.05,respectively).The important factors that affecting the mortality were:①severity of pancreatic necrosis,②improper surgical approach,③incorrect surgical indication.Conclusion:The patiets with mild or moderate ANP should mainly receive conservative treatment for 48～72 hours.The early shock and ARDS should be redressed before surgical intervention.If the operation is unavoidable,the swelling pancreas should be dissected fully,which will provide sufficient drainage after operation,and duodenostomy should be performed during operation.

AIM: To investigate the expression of neurok inin-2 receptor (NK-2R) in normal pancreas and chronic pancreatitis (CP) tissues. The relation of expres sion of NK-2R with pain in CP was also evaluated. METHODS: CP tis sues were ob tained from 18 men and 7 women undergoing pancreatic head resection as a result of CP. Normal human pancreatic tissues were obtained from 20 patients (11 males/ 9 females). Real-time quantitative reverse transcription polymerase chain reacti on was used to determine the mRNA expression of NK-2R, Western blot analysis was used to determine its protein expression level, and immunohistochemistry was us ed to localize expression site of NK-2R protein. NK-2R mRNA level and pain were also analysed whether correlation exists. RESULTS: NK-2R mRNA and p rotein level were enhanced expressed in CP tissue samples compared with normal pancreas. Ove rexpression of NK-2R was related to the intensity ( r=0 59, P

AIM: To study the expression and the role of neurokinin-1 receptor(NK-1R) and neurokinin-2 receptor (NK-2R) in distal ileum during acute necrotizing pancreatitis(ANP).METHODS: 130 adult Sprague-Dawley rats were divided into 2 groups: the rats in ANP group( n=80 ) were induced by the retrograde intraductal infusion of 5% sodium taurocholate. The rats in sham operation control group ( n=50 ) received laparotomy only. Reverse transcription polymerase chain reaction (RT-PCR) was used to determine the mRNA expression of NK-1R and NK-2R, Western blot was used to investigate expression level of NK-1R.RESULTS: NK-1R and NK-2R mRNA levels were enhanced in the distal ileum of ANP rats compared with controls. Western blot revealed stronger NK-1R immunoreactivity exited in intestinal mucosa in ANP rats. The overexpression of NK-1R was associated with mucosal pathological score( r=0.77,P

Objective To investigate the role of substance P (SP) and its neurokinin-1 receptor (NK-1R) in the pathophysiologic process of Crohn′s disease. Methods In 23 surgical patients of Crohn′s disease and 24 healthy controls, reverse transcription polymerase chain reaction (RT-PCR) was used to determine the mRNA expression of NK-1R, Western blot analysis was used to determine NK-1R protein expression levels, and immunohistochemistry was used to localize expression of NK-1R. Results Compared with normal gut NK-1R mRNA and NK-1R protein in Crohn′s disease were overexpressed. In Crohn′s disease moderate to strong intestinal NK-1R immunoreactivity was found in the lamina propria mononuclear cells, lymphoid follicles, and the surface and crypt epithelium, lymphoid follicles, submucosal vessels, smooth muscle and myoenteric plexus neurones. Conclusions In cases of Crohn′s disease, overexpression of NK-1R may disturb neuropeptides loop balance, and may be involved in the pathophysiological change in this disease.