Objective To evaluate the effect of intrathecal ropivacaine on spinal histone deacetylase 1 (HDAC1) and HDAC2 expression in the rats with neuropathic pain (NP).Methods Thirty adult male Sprague-Dawley rats, weighing 220-250 g, in which intrathecal catheters were successfully placed, were randomly divided into 3 groups (n=10 each) using a random number table: sham operation group (group S), group NP, and NP + ropivacaine group (group R).NP was induced by chronic constriction injury (CCI) in anesthetized rats.Sciatic nerve was exposed and 4 loose ligatures were placed on the left sciatic nerve at 1 mm intervals with 4-0 chromic catgut.Starting from 7th day after CCI, 0.25％ ropivacaine 20 μl was injected intrathecally in group R, while the equal volume of normal saline was given instead of ropivacaine in S and CCI groups once a day for 7 consecutive days.At 1 day before CCI (T0) ,and 3, 7, 10, 14, 17 and 21 days after CCI (T1-6) , the mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured.After the pain threshold was measured at T4,3 rats from each group were sacrificed, and their lumbar enlargements were harvested for determination of the expression of HDAC1 and HDAC2 by Western blot.Results Compared with group S, the MWT was significantly decreased, and the TWL was shortened at T2-6, and the expression of HDAC 1 and HDAC2 was up-regulated at T4 in NP and R groups (P＜0.05).Compared with group NP, the MWT was significantly increased at T4, and the TWL was prolonged at T3.4, and the expression of HDAC1 and HDAC2 was downregulated at T4 in group R (P＜0.05).Conclusion The mechanism by which intrathecal ropivacaine alleviates NP may be related to inhibited up-regulation of spinal HDACI and HDAC2 expression in rats.

Objective To evaluate the role of histone deacetylase in the spinal cord in the maintenance of neuropathic pain (NP) in rats.Methods Twenty-seven male Sprague-Dawley rats,weighing 230-270 g,were randomly divided into 3 groups (n =9 each) using a random number table:sham operation group (group S),group NP,and NP + intrathecal Trichostatin A (TSA) group (group T).NP was induced by chronic constrictive injury.At 7 days after operation,5％ DMSO,5％DMSO and TSA 10 μg (10 μl) were injected intrathecally once a day for 3 consecutive days in S,NP and T groups,respectively.Mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 1 day before operation and 3,7,10,14 and 21 days after operation (T0-5).Results Compared with group S,MWT was significantly decreased and TWL was shortened at T2-5 in NP and T groups.Compared with group NP,MWT was significantly increased and TWL was prolonged at T3,4 in group T.Conclusion Histone deacetylase in the spinal cord is involved in the maintenance of neuropathic pain in rats.

Objective To analyze the effect of eukaryotic plasmids containing wild (sense) or anti- sense methylenetetrahydrofolate reductase (MTHFR) gene on cell viability and transcription level of tumor related genes in human gastric cancer cell line.Methods Human gastric cancer cell line MKN-45 was cultured.Recombinant plasmids containing wild MTHFR (W) or antisense MTHFR (A) gene, pCMV-W and pCMV-A,were constructed.Then pCMV-W,pCMV-A and pCMV blank plasmid were transfected into MKN45 cells respectively by using lipofect.Cell viability was analyzed by 3-(4,5-bime- thylthiazolyl-2)-2,5-diphenyhetrazolium dromide(MTT).The transcription levels of Dnmt 1,c-myc, p21~(WAF1) and hMLH1 genes were detected by real-time polymerase chain reaction(PCR).Results Cell vi- ability remarkably increased in those transfected with wild MTHFR (P＜0.01),which was contrary to those transfected with antisense MTHFR(P＜0.01).The expression of those tumor related genes mRNAs were all remarkably decreased in the MKN45-W cells in comparison with those in the MKN45-pCMV cells.No significant difference in the expressions of those tumor related genes mRNAs were found between the MKN45 cells transfected with pCMV-A and blank pCMV.Conclusion MTHFR influences cell viability and the expres- sion level of tumor related genes in human gastric cancer cell line MKN45.

Objective To investigate the influence of the purging fire and removing toxin method on chemokines and adhesion factors related to vascular endothelialitis injury induced by toxin of trimeresurus stejnegeri bite.Methods ① Animal experiment:50 healthy New Zealand white rabbits were chosen.According to random numbers generated by statistical software,they were divided into normal control group,model group,low,middle and high dose Sheshang capsule groups,10 in each group.Trimeresurus stejnegeri bite model was replicated by injecting 0.75 mL/kg snake venom into subcutaneous tissues of rabbits' right hind legs.And the same volume of normal saline was injected into the rabbit in the normal control group.After the model was established for 6 hours,the rabbits in low,middle and high dose Sheshang capsule groups received 174,348 and 522 mg· kg-1 · d-1 of Sheshang capsule solution respectively (the content of capsules was dissolved in normal saline to make liquid with 17.4,34.8 and 52.2 g/L Sheshang solution respectively,so the volume of gavage of each group was 10 mL· kg-1 · d-1);in the model and normal control groups,the same amount of normal saline was given by gavage,once daily for consecutive one week.24 hours after the last gavage,the blood of the rabbits was collected through an auricular border vein and the serum was separated by centrifuge ready for use.Meanwhile,the whole abdominal aorta segment of the rabbit was harvested and kept them in liquid nitrogen ready for use.② Cell experiment:human umbilical vascular endothelial cell (HUVEC) was cultured with MEM for 24 hours.The solution was replaced and according to the random number generated by statistical software,the cells were divided into blank control group,model group and low,middle,high dose Sheshang capsule medicinal serum groups,10 wells in each group.Trimeresurus stejnegeri toxin cell model was reproduced by addition of 5 mg/L snake venom into the cell culture medium.After 6-hour culture,the cells of model group and blank control group received 10％ normal rabbit serum,and the cells of low,middle and high dose Sheshang medicinal serum capsule groups received serum containing 5％,10％ and 15％ drug,respectively.After culture for 72 hours,the cells were collected and the total RNA was extracted.The real-time fluorescent quantitative polymerase chain reaction (qPCR) was used to detect the levels of mRNA of interleukin-8 (IL-8),monocyte chemoattractant protein-1 (MCP-1),intercellular adhesion molecule-1 (ICAM-1) and vascular endothelial cell adhesion molecule-1 (VCAM-1) in the vascular endothelial cells of rabbit aorta abdominalis and human umbilical vein,and the content of serum E-select element (CD62E) was measured by enzyme linked immunosorbent assay (ELISA).Results In model group,the expression levels of mRNA in IL-8,MCP-1,ICAM-1,VCAM-1 and the content of CD62E were all increased significantly in the endothelial cells of rabbit aorta abdominalis and HUVEC compared with those in control group [when the mRNA expression levels of IL-8,MCP-1,ICAM-1 and VCAM-1 in normal and blank control group were all being 1,the mRNA expression levels (2-△ △Ct) of the above mentioned inflammatory factors and adhesion molecule in animal model group were 3.96 ± 0.39,3.07 ± 0.27,3.71 ± 0.26,3.94 ± 0.26,and the mRNA expression levels (2-△ △Ct) of the above mentioned inflammatory factors and adhesion molecule in HUVEC model group were 3.53±0.70,2.24±0.48,3.13±0.44,2.80±0.13,respectively,all P ＜ 0.01].The content of CD62E in serum was increased significantly in model group compared with that in normal control group (μg/L:1.31 ± 0.22 vs.0.82 ± 0.13,P ＜ 0.01),the mRNA expression levels of IL-8,MCP-1,ICAM-1 and VCAM-1 were decreased significantly in low,middle,high dose Sheshang capsule groups compared with those in model group in endothelial cells of aorta abdominalis of rabbits and HUVEC [abdominal aorta:IL-8 mRNA (2-△ △Ct) were 1.13 ± 0.19,1.26 ± 0.16,1.27 ± 0.17 vs.3.96 ± 0.39,MCP-1 mRNA (2-△ △ Ct) were 1.79 ± 0.24,2.22 ± 0.38,1.76±0.19 vs.3.07±0.27,ICAM-1 mRNA (2 △△Ct) were 2.05±0.11,1.68±0.09,2.37±0.48 vs.3.71±0.26,VCAM-1 mRNA (2-△△Ct) were 1.59±0.08,1.40±0.11,1.84±0.11 vs.3.94±0.26;HUVEC:IL-8 mRNA (2-△△Ct) were 2.33±0.59,2.82±0.82,2.51±0.77 vs.3.53±0.70,MCP-1 mRNA (2-△△Ct) were 1.59±0.35,1.48±0.36,1.54±0.29 vs.2.24±0.48,ICAM-1 mRNA (2-△△Ct) were 1.46±0.38,1.77±0.65,1.73±0.50 vs.3.13±0.44,VCAM-1 mRNA (2-△△Ct) were 2.49±0.24,2.18±0.19,2.45±0.24 vs.2.80±0.13,all P ＜ 0.05].The contents of CD62E were decreased significantly in middle,high dose Sheshang capsule groups compared with the content in model group (μg/L:1.01 ±0.14,1.04±0.13 vs.1.31 ±0.22,all P ＜ 0.01),but there were no statistical significant differences among the three drug group (all P ＞ 0.05).Conclusion The therapy of purging fire and removing toxin can treat vascular endothelial injury by inhibiting the inflammatory response induced by Trimeresurus stejnegeri bites.

Objective To investigate the alterations of invariant nature killer T( iNKT) cells in peripheral blood samples from patients with rheumatoid arthritis ( RA) and to clarify the correlation between the percentage of iNKT cells and the ratio of IFN-γ/IL-4 in order to further understand the significance of iNKT cells in the development of RA.Methods Peripheral blood mononuclear cells ( PBMCs) were isola-ted from 70 patients with RA and 40 healthy subjects.Among them, thirty patients in the stage of inactive RA were involved in a follow-up study.Fluorescence activated cell sorting ( FACS) was used to detect the percentage of iNKT cells.PBMCs were cultured in vitro for analysis of cytokine production.The dynamic changes of iNKT cells in percentages were analyzed by FACS.MILLIPLEX MAP Human Cytokine/Chemo-kine kit was used to measure the secretion of IFN-γand IL-4 in serum samples and culture media of PBMCs. The expression of IFN-γand IL-4 in iNKT cells at mRNA level were analyzed by RT-PCR.Results Com-pared with the healthy subjects, the patients with active RA showed the delayed proliferation of iNKT cells and the decreased percentages and proliferation rates of iNKT cells (P<0.05).The percentages and prolif-eration rates of iNKT cells in patients with active RA were significantly lower than those in patients with inac-tive RA (P<0.05).No statistical significant differences with iNKT cells were found between healthy sub-jects and patients with inactive RA (P>0.05).The ratios of IFN-γ/IL-4 in serum samples and culture media of PBMCs were increased in patients with active RA as compared with those in patients with inactive RA and healthy subjects (P<0.05).No statistical significant differences with the ratios of IFN-γ/IL-4 were observed between healthy subjects and patients with inactive RA (P>0.05).Compared with healthy subjects and patients with inactive RA, patients with active RA showed increased transcriptional level of IFN-γand decreased transcriptional level of IL-4.No significant differences with the expression of IFN-γand IL-4 in iNKT cells at mRNA level were observed between healthy subjects and patients with inactive RA.The per-centage of iNKT cells was negatively related to the IFN-γ/IL-4 ratio in patients with RA (P<0.05).Con-clusion Decreased percentage and impaired function of iNKT cells were detected in patients with RA. iNKT cells were closely related to the development and disease activity of RA.

UNLABELLEDOBJECTIVE To explore the changes of bone gla protein (BGP) and tartrate resistant acid phosphatase (TRACP) in patients with stage 3 -4 chronic kidney disease (CKD) before and after treatment, to study their correlation with interleukin-17 (IL-17) and regulatory T cells (Treg), and the effects of Yishen Jiangzhuo Granule (YJG) on the bone metabolism.

METHODSFifty-three patients with stage 3-4 CKD were randomly assigned to the treatment group and the control group using random digit table. The following parameters in blood were detected: Treg (CD4+ CD25+ CD127lo) using tri-chrism fluorescent labeling by flow cytometry; levels of TRACP, BGP, and IL-17 by double antibody sandwich ELISA. The hemoglobin (HGB) content was detected using Beckman-Coulter heme/analysis. The urinary contents of creatinine (UCr) were determined using reversed HPLC. The blood contents of calcium (Ca), phosphate (P), blood urea nitrogen (BUN), serum creatinine (SCr), and plasma albumin (ALB) were determined using automatic biochemical analyzer. Then the calcium-phosphate (Ca x P) product was calculated on the basis of blood contents of Ca and P. The clearance rate of endogenous creatinine (CCr) was calculated on the basis of blood BUN and SCr contents.

RESULTS(1) There was no obvious change in CD4+ CD25+ CD127lo in the two groups before and after treatment (P > 0.05). Compared with before treatment in the same group, there were statistical difference in the levels of CD4+ and TRACP in the two groups, as well as the IL-17 level in the control group (P < 0.01, P < 0.05). But compared with the healthy group, statistical difference was shown in each index (except CD4+) (P < 0.01). Compared with the control group after treatment, there was no statistical difference in each index of the treatment group after treatment (P > 0.05). Compared with before treatment in the same group, the levels of Hb, ALB, and CCr increased (P < 0.05, P < 0.01), and the SCr level decreased in the two groups after treatment (P < 0.05). Compared with the control group after treatment, the SCr level decreased and the CCr level increased more obviously in the treatment group (P < 0.05). There was no correlation among the levels of IL-17, TRACP, BGP, and Treg between before and after treatment in the two groups.

CONCLUSIONSYJG could improve the kidney function and delay the progression of micro-inflammation of stage 3 -4 CKD patients. It could not improve the level of CD4+ CD25+ CD127lo. It also showed no effects on bone metabolism. The CD4+ T cells were differentiated to Th17 cells in stage 3-4 CKD patients. Their immunity was in a state of anergy but continually activated. The inflammatory factors in patients with stage 3-4 CKD play important roles in inducing the activation of osteoclasts.

Acid Phosphatase ; metabolism ; Adult ; Aged, 80 and over ; Bone and Bones ; metabolism ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Humans ; Inflammation ; Interleukin-17 ; metabolism ; Isoenzymes ; metabolism ; Male ; Middle Aged ; Osteocalcin ; metabolism ; Renal Insufficiency, Chronic ; drug therapy ; immunology ; metabolism ; T-Lymphocytes, Regulatory ; immunology ; Tartrate-Resistant Acid Phosphatase

Background and purpose:Tumor budding is a poor prognostic factor in colorectal cancer. In this study, we studied the tumor budding by counting the actual number in 10 high power fields and evaluated itsclinical application in predicting lymph node metastasis of T1 colorectal cancer.Methods:Tissue specimens from 307 patients with histologically conifrmed T1 colorectal cancer were enrolled. The clinicopathological characteristics including tumor budding were evaluated for their predictive value in lymph node metastasis. A formula was created to calculate the risk score for prediction of lymph node metastasis which was validated by 14 new cases.Results:In the multivariate analysis, it showed that tumor grade, lymphovascular invasion and the number of tumor budding were signiifcantly associated with lymph node metastasis. The probability of lymph node metastasis was calculated using the following equations:Z=1.571×(lymphovascular state: invasion, 1; no invasion, 0)+2.661×(tumor grade: high grade, 1; low grade, 0)+0.024×(budding counts)-3.885; Probability=1/1+e-Z. The high scores were correlated with the lymph node metastasis in the validations.Conclusion:We can accurately assess the risk of lymph node metastasis by counting the number of tumor budding in 10 high power fields. Therefore tumor budding could potentially assist treatment decision making in T1 colorectal cancer patients with high-risk lymph node metastasis.

Background and purpose:Metastatic colorectal cancer (mCRC) patients with K-ras mutation won’t benefit in the anti-epidermal growth factor receptor (EGFR) treatments. Thus K-ras mutation analysis is mandatory before this treatment. There is controversy that K-ras mutation analysis should be performed on primaries or related metastases. The aim of our study was to evaluate the concordance of K-ras status between primary and related metastases tumors, thus investigate the validity and rigorousness of clinical K-ras testing. Methods:Seventy-six patients with confirmed mCRC treated in Fudan University Shanghai Cancer Center were enrolled. After DNA extraction and PCR amplification, tumor specimens with paired primary tumors and related metastatic sites were put into sequencing analysis. And the K-ras mutation status in exon 2 was assessed. Results: K-ras mutation was detected in 31 out of 76 primary tumours (40.8%) and also 40.8%of the metastatic sites. But discordance was found between primary tumor and metastasis in 15 cases (19.7%):8 primary tumors had a K-ras mutation with a wild-type metastasis, meanwhile 7 primary tumors were wild type with a K-ras-mutated metastasis. Conclusion:Our study indicated that quite a few mCRC cases have different K-ras status between primary tumors and related metastatic sites, and it’s not very rigorous to choose the anti-EGFR treatments merely according to the primary tumor-K-ras mutation.Further study and consultation are needed on this problem.

Objective To investigate the detection rate of contrast-enhanced ultrasound (CEUS) for iso-echo liver cancers, and to evaluate the application of CEUS in treating iso-echo liver cancers with radiofrequency ablation (RFA). Methods A total of 27 patients with liver cancers (31 lesions in total) were enrolled in this study. Of the 27 patients, hepatocellular carcinoma (HCC) was seen in 11 (11 lesions) and hepatic metastasis in 16 (20 lesions). In all patients the diagnosis was detected by contrast-enhanced CT or contrast-enhanced MRI and was clinically confirmed. Under real-time CEUS guidance, RFA was carried out for all the 31 lesions. The ablated extent was evaluated by CEUS, and CEUS findings were used to guide the further treatment. Results All 31 lesions were detected by CEUS and were treated with RFA. No serious complications occurred after RFA. Both CEUS and CECT performed one month after RFA showed that complete tumor necrosis was seen in all the 31 lesions. After RFA the serum AFP levels in all 11 HCC patients fell to normal range, and the serum CEA, CA-199 as well as CA-153 levels in patients with hepatic metastasis decreased significantly(P<0.01). Conclusion CEUS can markedly increase the detection rate of iso-echo liver cancers. Besides, CEUS plays an important role in guiding the performance of RFA for iso - echo liver lesions.

Objective To examine expression of PTEN gene in ovarian cancer cisplatin-sensitive cell line OV2008 cells and cisplatin-resistant cell line C13K cells,and evaluate the effect of wild-type PTEN gene on reversing cisplatin-resistance of C13K cells and underlying mechanisms.Methods The expression of PTEN mRNA and protein in OV2008 and C13K cells were detected by semi-quantitative RT-PCR and western blot.Recombinant eukaryotic expression plasmid containing human wild-type PTEN gene was transfected into C13K cells by lipofectamine 2000.The expression of PTEN mRNA was monitored by RT- PCR and the expression of PTEN,protein kinase B(AKT),phospho-AKT(p-AKT)protein were analyzed by western blot in PTEN transfected and untransfected C13K cells.Proliferation and chemosensitivity of cells to cisplatin were measured by methyl thiazolyl tetrazolium(MTT),and cell apoptosis was detected by flow cytometry after treatment with cisplatin.Results(1)The expression of PTEN mRNA and protein(1.02 ?0.05,1.02?0.07)in OV2008 cells were significantly higher than those in C13K cells,which were 0.45 ?0.03 and 0.55?0.03 respectively(P