BACKGROUND:It has been reported that mesenchymal stromal cells(MSCs)are capable of differentiating into cells of multilineage.Different methods and reagents have been used to induce the differentiation of MSCs,but most inducing systems contain serum and cytokines.OBJECTIVE:To investigate the gene expression of mash-1 and ngn-1 in differentiation of SD rat bone marrow-derived mesenchymal stem cells induced by salvia mitiorrhiza. DESIGN:Controlled experiment in vitro with repeated observation and measurement based on cells.SETTING:Department of Anatomy,School of Preclinical and Forensic Medicine,Sichuan University. MATERIALS:This study was performed in the Department of Anatomy,School of Preclinical and Forensic Medicine,Sichuan University from October 2004 to December 2005.SD male rats weighing 160-200 g were purchased from the Animal Center of Sichuan University.The experimental animals were disposed according to ethical criteria.Parenteral solution of salvia mitiorrhiza purchased from Tianyang Medicine Company Limited of Anhui(batch number:20050411).METHODS:The nucleated cells were separated from rat bone marrow through gradient centrifugation and cell adherent method,and then MSCs differentiated into neurcyte-like cells induced by salvia mitiorrhiza.Cells in the control group were cultured with salvia mitiorrhiza-free serum-free culture media.The expression of neuron specific enolase(NSE)and glial fibrillary acidic protein(GFAP)were detected by immunohistochemistry.RT-PCR was used to detect the mRNA of mash-1 and ngn-1. MAIN OUTCOME MEASURES:①mash-1 and ngn-1 expressions were detect by the RT-PCR method.②NSE and GFAP expressions were detected by immunohistochemistry.RESULTS:①The MSCs were well adherent to walls.Most of the cells transformed in dipolar-like or multipolar-like.Axon-like or dentrite-like process was developed and these processes synapse with each other.② RT-PCR showed that ngn-1 and mash-1 mRNA were negative before induction,but positive after induction.③Immunohistochemistry indicated that NSE and GFAP expressions were positive after induction but negative in the control group. CONCLUSION:MSCs can be induced to differentiate into neurocyte-like cells by salvia mitiorrhiza.

ObjectiveTo investigate the role of nuclear factor kappa B (NF-κB), epidermal growth factor (EGFR) and Mucin 1 (MUC1) in hepatolithiasis associated with intrahepatic cholangiocarcinoma. MethodsSpecimens were taken from 90 patients who underwent hepatectomies from August 1989 to June 2009 at the Second Affiliated Hospital of Sun Yat-sen University. The specimens were stained immunohistochemically for NF-κB, EGFR and MUC1. There were 33 patients who had hepatolithiasis associated with intrahepatic cholangiocarcinoma (the experiment group). 32 patients with hepatolithiasis served as the control group, and 25 patients with normal intrahepatic bile ducts taken at 1-2cm distal to benign hepatic neoplasm served as the blank group. The immunohistochemical staining were performed on tissue slices. Results NF-κB positive rate was 51.5％ (17/33), 25％(8/32) and 4％ 1/25) in the experiment group, the control group and the blank group respectively,P＜0. 01 ; EGFR positive rates were 57. 6％ (19/33), 31.3％ (10/32) and 0 (0/25) respectively,P＜0. 01; MUC1 positive rates were 54. 5％ (18/33), 28. 1 ％ (9/32), 0 (0/25) respectively,P＜0. 01. There were significant differences for high level expressions of EGFR and MUC1 among histopathologic grading, tumor invasion and metastasis. The survival rates of patients with EGFR and MUC1 expressed tumor were lower than of patients with non-expressed tumout (P＜0. 01). ConclusionsNF-κB is probably involved in the early stage of intrahepatic cholangiocarcinogenesis. Overexpressions of NF-κB, EGFR and MUC1 play coordinately and important roles during intrahepatic cholangiocarcinogenesis. High level expressions of EGFR and MUC1 are related to the malignant degree of cholangiocarcinoma and to worse prognosis.

Objective To explore the role of interleukin (IL)-1β in the pathogenesis of gout.Methods Real-time quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay(ELISA) were used to measure the expression of IL-1β mRNA in peripheral blood mononuclear cells (PBMCs) and IL-1β in plasma samples from 120 acute gouty (AG) arthritis,70 chronic gouty (CG) arthritis,80 intercritical gouty (IG) arthritis patients and 96 healthy control subjects respectively.Results The expression of PBMCs IL-1β mRNA and plasma concentration of IL-1β were both much higher in gout patients than those in controls (P ＜ 0.01,respectively).And the plasma levels of IL-1β mRNA and IL-1β significantly increased in the AG group compared with CG and IG groups (P ＜ 0.01,respectively) and much higher in the CG group than those in the IG group.Positive correlations existed between plasma concentration of IL-1β and the levels of white blood cell,neutrophil,monocyte,erythrocyte sedimentation rate,blood uric acid,globulin and PBMCs IL-1β mRNA (P ＜ 0.01,respectively) while negative correlation between plasma IL-1β and plasma level of apolipoprotein in gout patients (P ＜ 0.05).Conclusion Elevated plasma level of IL-1β may be involved in the pathogenesis of acute and chronic gouty arthritis.

Objective To investigate the development of rat heart and the expressions of TGF-?1,SMAD4 and Bax protein to detect the location and mechanism of action in different developmental periods of rat heart. Methods Histology and immunohistochemistry of rat embryonic hearts from day11 to day19(E11～E19) in paraffin-embedded were used to analyze the heart development and TGF-?1,SMAD4 and Bax protein expressions. Results The muscular part of interventricular septum appeared on E12.5,and the partition of the ventricle finished on E16.The positive expression of TGF-?1 can be seen in the rat embryonic heart during E11～E19.The positive staining was increased to E15 and then declined significantly.The expression of SMAD4 was enhanced gradually and the positive signals were strong on E17,and a spatial difference was found in the expression on E13.The expression peaks of the Bax protein appeared on E15 then subsided to a stable.Conclusion The critical period of cardiac muscle cell differentiation and heart moulding was E15.TGF-?1,Smad4 and Bax protein play important roles during the development of rat embryonic heart.

Objective To explore the value of anatomical liver resection in the treatment of bilateral intrahepatic biliary lithiasis. Methods We collected the clinical data of 32 patients with bilateral intrahepatic biliary lithiasis who received anatomical liver resection and 25 patients who recevied non-anatomical liver resection from May 2010 to May 2012 in our hospital and Sun Yat-sen Memorial Hospital. We comapred the therapeutic efficacy of these two operative modalities in the diagnosis and treatment of bilateral intrahepatic biliary lithiasis. Results The intraoperative blood loss was 436 ±48.162 mL in patients who received anatomical liver resection, and was significantly less than that in the control group (763 ± 37.645ml) ( < 0.05 ) . Postoperative liver function status:for patients who received anatomical liver resection,on the third day after operation,the total bilirubin and AST were significantly lower than those in the control group ( <0.05), and the postoperative hospitalization time was also shorter than that of the control group ( <0.05 ) . The two groups in the operation time, postoperative complications and residual calculus had no significant difference ( >0.05 ), but there 1 patients died of liver failure in the control group. Conclusions Anatomical liver resection is a favorable method to completely remove the lesions under the premise of retaining the residual liver function as much as possible. The rate of remnant biliary lithiasis and recurrence is lower and the recovery is quicker in these patients after anatomical liver resection. Thus, anatomical liver resection is worthy of promotion.

Objective To evaluate the efficacy of destruction of dorsal root ganglia with local doxorubicin injection guided by CT for postherpetic neuralgia involving thoracic back region. Methods One hundred and fifty patients suffering from postherpetic neuralgia in thoracic back region were randomly divided into 3 groups ( n = 50 each): group A oral medicine; B and C groups undergoing local injection under the guidance of C-arm and CT respectively + oral medicine. Three spinal segments severely affected by herpes virus were chosen for paravertebral puncture. 1% lidocaine 1 ml was injected at each segment as test dose. Fifteen minutes later doxorubicin 3.3 mg and betamethasone compound 4.7 mg were injected at each segment if no side-effect occurred. All 3 groups were given oral medicine according to the intensity of pain after local injection. The number of patients who exited from the study because of the side effects of oral medicine was recorded. VAS, sleep interference score (SIS) and a short form of McGill pain questionnaire (SF-MPQ) were used to evaluate the efficacy of the treatment the day before (baseline), 24 h, 1 week, 1, 3 and 6 months after local injection. The dosage of oxycodone extended-release tablets and gabapentin was recorded, and also the incidence of pneumothorax within 12 h after local injection. Results The exit rate, VAS, SIS and SF-MPQ scores, dosage of oxycodone extended-release tablet and gabapentin were significantly lower in B and C groups than in group A, but there was no significant difference between the 2 groups. The incidence of pneumothorax was 10% in group B but no pneumothorax developed in group C.Conclusion Destruction of dorsal root ganglia with local doxorubicin injection guided by CT is more effective for the treatment of postherpetic neuralgia.

ObjectiveTo investigate the effects of paravertebral injection of doxorubicin on pain behavior and ultrastructure of dorsal root ganglion ( DRG ) neurons in rats with neuropathic pain.MethodsOne hundred male SD rats,aged 3 months,weighing 200-250 g,were studied.The spared nerve injury (SNI) model was established by ligation and separation of tibial and common fibular nerves.The rats were randomly divided into 5 groups ( n =20 each):SNI group and different concentrations of doxorubicin groups (groups A1,A2,A3,A4 ).The animals were anesthetized with intrapetitaneal 10％ chioral hydrate 0.3 ml/100 g.The L4,6 intervertebral foramina was exposed.Normal saline and 0.25％,0.50％,0.75％ and 1.00％ doxorubicin 5 μl were injected into the intervertebral foramina in groups SNI,A1,A2,A3,and A4 respectively.The paw withdrawal latency to the radiant heat stimulation (PWL) and paw withdrawal duration to acetone (PWD) were measured before administration and on day 1-28 after administration.Three rats were chosen in each group on day 28 after administration and DRG were removed to observe the ultrastructure of large and small neurons with electron microscope.ResultsCompared with group SNI,PWL was significantly prolonged and PWD was significantly shortened in groups A1,A2,A3,A4 in a concentration manner ( P ＜ 0.05).Nucleus condensation,mitochondrial swelling,increased lysosomes,chromatin condensation,fibrosis of intercellular substances,and cell necrosis were observed in small neurons.Mitochondrial swelling and increased fibers wcre observed in large neurons and most of large neurons survived.ConclusionParavertebral injection of doxorubicin can reduce neuropathic pain and result in damage to large and small DRG neurons,and small neurons in the DRG are more susceptible and vulnerable to doxorubicin than large neurons in rats.

Objective To evaluate the histologic changes of dorsal root ganglion (DRG) and pain threshold following implantation of iodine-125(125I) seeds in the lumbar intervertebral foramina in rats.Methods Thirtysix adult male SD rats aged 6 months weighing 220-250 g were randomly assigned into 3 groups (n =12 each):group sham operation (group A) and groups low and high radioactive 125I seeds (groups B and C).125I seeds of 0.4 and 0.8 mCi were implanted in L4,5 and L5,6 intervertebral foramina in groups B and C respectively.Paw withdrawal threshold to von Frey filament stimulation (MWT) and paw withdrawal latency to thermal nociceptive stimuli (TWL) were measured 2 days before (T1,baseline) and at 2,7,14,30 and 60 days after implantation (T2-6) in 6 animals in each group.The remaining 6 animals in each group were sacrificed at 14 and 60 days after 125I seeds implantation for microscopic examination of L5 DRG.Results MWT and TWL were significantly decreased after 125I seeds implantation at T4 compared with the baseline values at T1 in both groups,and returned to the baseline in group B and were significantly increased in group C at T6.The neurons were severely injured in DRG in groups B and C in a time-and dose-dependent manner.Conclusion Implantation of 125I seeds in intervertebral foramen can induce radiation resection of DRG.

Objective To investigate the association of TLR4 gene polymorphisms with susceptibility to primary gouty arthritis (GA) in Chinese Han population.Methods A total of 459 patients with GA and 459 healthy control subjects were enrolled into this study.All the genotyping assays of TLR4 gene polymorphisms loci [rs4986790 (Asp299Gly),rs4986791 (Thr399Ile),rs2 1 49356T＞G] were measured using TaqMan probes that specifically target the alternate alleles.Results All the subjects were found to be homozygous for the wild-type TLR4 alleles (Asp/Asp,Thr/Thr) on TLR4 Asp299Gly and Thr399Ile genotyping.There was no significant deviation from HWE both in GA and controlsin the rs2149356 (x2=0.778,1.295; P＞0.05,respectively).Significant differences were observed between the GA and control groups with respect to genotype and allele frequencies of TLR4 gene rs2149356 (x2=16.23,17.08; P＜0.01,respectively),and TT genotype was the risk factor for gout (adjusted OR=2.09).Conclusion The TLR4 gene rs2149356 SNP may be associated with GA susceptibility,and TT genotype may be the risk factor for developing gout.

Objective To evaluate the effect of pulsed radiofrequency application to dorsal root ganglions (DRGs) on neuronal Navl.8 expression in a rat model of inflammatory pain.Methods Twenty-four adult male Sprague-Dawley rats,aged 6-8 weeks,weighing 200-250 g,were randomly divided into 4 groups (n =6 each) using a random number table:control group (group C),inflammatory pain group (group IP),pulsed radiofrequency group (group PR),and inflammatory pain + pulsed radiofrequency group (group IP+PR).2.5％ formalin 100 μl was injected into the plantar surface of the right hindpaw to induce inflammatory pain.Pulsed radiofrequency was applied on L4,5 DRGs at 42 ℃ for 180 s starting from 4 days after formalin injection.At 1 day before formalin injection (T0),and 1,3,5 and 7 days after formalin injection (T1-4),the mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured in the right hindpaw.After the last measurement of pain threshold,the rats were sacrificed,and the DRGs of the L4.5 were removed for determination of Navl.8 mRNA expression (by real-time reverse transcriptase-polymerase chain reaction) and Nav1.8 protein expression (by Western blot).Results Compared with group C,the MWT was significantly decreased,and the TWL was shortened at T1-4,and the expression of Navl.8 protein and mRNA was up-regulated in IP and IP+PR groups (P＜0.05).Compared with group IP,the MWT was significantly increased,and the TWL was prolonged at T1.4,and the expression of Navl.8 protein and mRNA was down-regulated in group IP+PR (P＜0.05).Conclusion The mechanism bv which pulsed radiofrequency application to DRGs reduces inflammatory pain is probably related to down-regulation of neuronal Nav1.8 expression in rats.