BACKGROUND:Studies have demonstrated that chitosan can indirectly promote the proliferation of fibroblast and the synthesis of collagen, Using chitosan and specific fibroblasts together to construct tissue-engineering materials for tendon repair may be an adequate way to promote healing and prevent adhesion during the healing process.OBJECTIVE: To observe the effect of water soluble chitosan (WSC) on the growth and genotype of 3T3TK fibroblasts.DESIGN: Controlled experiment based on observation.SETTING: Jiujiang University Medical College.MATERIALS: 3T3TK fibroblasts were provided by Cell Bank of Chinese Academy of Sciences. WSC (specification: 60 mesh, 30CPS, Jinan Haidebei Marine Bioengineering Co.,Ltd),DMEM (low sugar) (Gibco Company, USA), fetal bovine serum (FBS, Sijiqing Biological Engineering Institute, Hangzhou), penicillin, streptomycin (Biosharp Company, USA),trypsin (BioEev-Tech Scientific & Technical Co.,Ltd, Beijing).METHODS: This experiment was carried out in the Laboratory for Clinical Application of Biology, Center for Medial Scientific Research, Jiujiang University between November 2004 and April 2006. ①Cells in the log phase were digested with 2.5 g/L trypsin to produce single cell suspension and inoculated into a 96-well culture plate at a density of 6 000 cells /200 μL medium. Five groups were prepared, 5 holes per group. 200 μL cell suspension was added into each well.After 24 hours of cultivation, supernatant was discarded, 200 μL DMEM with 1, 0.1, 0.01 and 0.000 1 g/L chitosan was added respectively in the first four groups. The remaining group was taken as the negative control group, in which 200 μL DMEM medium without chitosan was added. The cell suspension was routinely cultured for 72 hours. The cell viability was measured by CellTilter- GloTM Luminescent Cell Viability Assay according to the manufacturer's protocol. Cells in the log phase were split and inoculated into 24-well culture plates. Four groups were prepared (5 holes per group). 1 mL DMEM medium supplemented with 1, 0.1, 0.01 g/L chitosan was added into the first 3 groups respectively, and the 4th group was set as control group. Hydroxyproline and alkaline phosphatase(ALP) kits were used for detecting the contents of collagen and ALP in the supernatant of fibroblasts.MAIN OUTCOME MEASURES : ①Effect of WSC on viability of fibroblasts cultured in vitro.②Contents of collagen and ALP in the cell culture supernatant of fibroblasts.RESULTS:①Detection results of viability of fibroblasts: There were no significant differences in viability of fibroblasts between each chitosan group and control group (P ＞ 0.05).②Contents of collagen and ALP in the cell culture supernatant of fibroblasts: Hydroxyproline content in the cell culture supernatant of fibroblasts of 1 g/L and 0.1 g/L groups was (7.598±0.770) and (8.366±0.308)mg/L, respectively, which was higher than that of control group [(11.269±0.707)mg/L,P ＜ 0.01]; Collagen content in the 1 g/L and 0.1 g/L groups was(56.703±5.748) and(62.437±2.301)mg/L, respectively, which was lower than that of control group [(84.099±5.280)mg/L,P ＜ 0.01]. With the concentration of chitosan increasing, the collagen content was decreased in a dose-dependent manner. There were no significant differences in ALP activity in the cell culture supernatant between each chitosan group and control group (P ＞0.05).CONCLUSION: WSC does not obviously inhibit the viability of 3T3TK fibroblasts, but can markedly reduce the content of secreted collagen. It is indicated that chitosan can be used as tissue engineering material for tendon repair, and inhibit adhesion formation during tendon repair.

Objective To investigate the clinlic therapeutic result of detenial sigmoid neobladder after radical cystectomy.Methods 20 patients with bladder cancer(18 males and 2 females;age range from 33 to 76 years) were admitted and underwent radical cystectomy and detenial sigmoid neobladder.Results All the 20 patients were followed up for a mean of 18 months(range from 6 to 48 months).The blood Cr and BUN levels were both in the normal range without acidosis in all the cases.Only 1 had unilateral ureteral urine reflux during cystography.3 of the 20 cases were incontinent,but 2 of the 3 cases could control urination by being woken up at night.The capacity of the neobladder was 230 to 500ml with a mean of 330ml and the maximal pressure of the neobladder during filling was 16 to 50cmH_2O.Conclusion As a simple operation the detenial sigmoid neobladder can be performed easily with less complications and more reliable results.This operation may be generally applied in clinical practice.

Objective To study the genotypes of OXA-51-like carbepenemases in Aeinetobacter beumannii and its association with drug resistance. Methods The susceptibility of 174 Acinetobacter baumannii against ceftazidime, cefotriaxon, amikacin and ciprofloxacin were detected with disc diffusion method. The minimum inhibitory concentration (MIC) values for meropenem and imipenem were determined with an agar dilution method. VIM, IMP, OXA-23, OXA-24, OXA-51 and OXA-58 β-lactamase genes were determined by PCR. DNA sequencing and genotyping were performed against OXA-51 positivestrains. Results All 174 isolates were negative by PCR for genes OXA-24, OXA-58, IMP and VIM. OXA-23 and OXA-51were amplified in 15.5% (27/174) and 72.4% (126/174) isolates, respectively. Therewere 15.5% (27/174) isolates producing OXA-51-like and OXA-23 carbapenemase simultaneously. Among126 OXA-51-like carbapenemase producing strains, 82.5% (104/126)were OXA-66 genotype, whereas theremaining 17.5% (22/126) strains belong to other genotype. Eight novel OXA-51-like Genotype were foundin this study. Conclusions OXA-66 were the primary genotype of OXA-51-like carbapenemases in A.baumannii. OXA-66 were related to low-level carbapenems resistance and may be associated with resistanceof other drugs. We found new OXA-51-like genotype in clinic isolates of A. baumannii in this study.

Objective To study the method and clinical value of preservation of intercostobrachial nerve( ICBN) by fat dissolving meth-od during breast cancer operation. Methods The clinical data of 50 cases withⅠ~Ⅲa stage breast cancer from January 2013 to June 2013 were analyzed. Fifty patients were randomly divided into two groups,there were 26 patients in preservation group,whose ICBN were preserved by fat dissolving method during axillary lymph nodes dissection,and 24 patients in resection group,whose ICBN were not preserved by routine method during axillary lymph nodes dissection. Comparison of operation times,bleeding volume,the number of axillary lymph nodes dissection and upper arm sensory function of patients after operation between both groups was done. Results The mean time of operation was (102. 3 ± 15. 6) min in preservation group and(95. 6 ± 12. 4) min in resection group,while the number of axillary lymph nodes dissection was (19. 5 ± 8. 8 ) in preservation group and ( 19. 2 ± 9. 5 ) in resection group, with no significant difference between both groups (P>0. 05). Bleeding volume was (51. 2 ± 11. 5)mL in preservation group and (98. 5 ± 13. 4)mL in resection group,with significant differ-ences(P<0. 05). After postoperative one month,we observed upper arm sensory function of patients. It showed that 3 cases of sensory numb-ness or pain occurred in preservation group (11. 5%),20 cases of sensory abnormality occurred in resection group (83. 3%),mainly as sen-sory loss,numbness,pain or burning sensation,there was significant difference between both groups (P<0. 05). All patients were followed up half a year,patients with sensory abnormality in preservation group recovered,and recovery in resection group was not obvious,it still showed sensory abnormality in varying degrees. During the follow-up,no local recurrence or distant metastasis was found in both groups. Conclusion Preserving intercostobrachial nerve by fat dissolving method in breast cancer operation is based on conventional operation and made a few of improvements. It is simple and feasible. During the operation,we find that the axillary neurovascular is clearer,preservation of ICBN is easier. It does not affect the axillary lymph node dissection and operation time,while it can reduce incidence of postoperative sensory abnormality and improve the quality of life of patients,therefore it is worthy of clinical application.

Objective To study the reasons and prevention of the hoarse voice after continuity-intact recurrent laryngeal nerve (RLN)operation without damage of RLN under naked eyes.Methods Data of 1871 patients undergoing thyroid surgery from Jan.2001 to Jan.2011 were retrospectly analyzed.919 patients had their RLN exposed,among whom 757 patients had bilateral RLN exposed.952 patients didn't have their RLN exposed in the surgery.Results A total of 1676 RLNs were exposed by the routine method and minimally invasive method.All the nerves were confirmed no damage and continuity intact under naked eyes.Hoarse voice occurred to 19patients after surgery,with the percentage of 2.12％ (19/897).The rate of hoarse voice in the non-exposed group was 5.46％ (52/952).The difference of horse voice between the RLN exposed group and non-exposed group had statistical significance(P ＜ 0.05).Conclusions RLN without damage under naked eyes and continuity intact doesn't mean no damage of electroneurophysiology.The rate of RLN damage in the exposed group was less than that in the non-exposed group.The major causes of hoarse voice may include misoperation,heat conduction and postoperative scar adhesion.The key to avoid RLN damage is prevention.

OBJECTIVETo observe the variations of intracellular localization and expression of importin 8 (IPO8) during osteoblast differentiation.

METHODSAlizarin red staining, immunocytochemistry and real-time PCR were employed to examine the changes in the intracellular localization and expression of IPO8 mRNA during induced osteogenic differentiation of human osteoblast-like SaOS-2 cells.

RESULTSNumerous red mineralized nodules were observed on day 10 in the induced cells with alizarin red staining. Immunocytochemical staining showed that IPO8 immunoreactivity was the strongest in the perinuclear cytoplasm of the cells. On day 3 of osteoblast differentiation, IPO8 immunoreactivity in the cell nuclei became stronger. On day 7, IPO8 was located mainly in the nuclei, and on day 10 the cells were osteocyte-like and IPO8 was distributed in the cytoplasm. Real-time PCR showed a significantly increased expression of OPN mRNA during osteoblast differentiation, and the expression level of IPO8 mRNA was the highest on day 3 and declined on days 7 and 10.

CONCLUSIONThe intracellular localization and expression level of IPO8 undergo significant changes during osteogenesis, indicating its role in regulating osteoblast differentiation.

Objective: To investigate the effect of nucleophosmin 1 (NPM1) mutant A on TGF-β1-induced K562 cell proliferation and AKT phosphorylation. Methods: K562 cells were infected with Ad5-NPM1 to create an NPM1 over-expression cell model. NPM1 levels were determined by ELISA and Western blot analysis. The levels of AKT and P-AKT were determined by Western blot. MTT was used to measure the proliferation of K562 cells. Results: NPM1 protein levels in K562 cells increased in an Ad5-NPM1-MOI-dependent manner. Cell proliferation and NPM1 levels in the supernatant were significantly increased in K562 cells infected with Ad5-NPM1-30 and Ad5-NPM1-100 compared to those infected with Ad5-vector-100 (P<0.01). Treatment with (10 ng/mL) TGF-β1 increased P-AKT levels, but not total AKT levels in K562 cells. TGF-β1-induced phosphorylation of AKT was significantly increased by infection of K562 cells with Ad5-NPM1-100. No significant differences were found in total AKT levels among all groups. TGF-β1 (10 ng/mL) treatment also in-creased the proliferation of K562 cells. TGF-β1-induced K562 cell proliferation was significantly increased by infection with Ad5-NPM1-100 (P<0.01). Conclusions: NPM1 improves TGF-β1-induced cell proliferation by up-regulating AKT phosphorylation levels.

Objective To explore the expression of HR and Her?2 in breast cancer primary tumor and axillary lymph node metastasis. Methods Four hundred and twenty?eight female patients with unilateral breast cancer combined with axillary lymph node metastasis treated in the Affiliated Suqian Hospital of Xuzhou Medical University from January 2011 to January 2016 were selected in this study. Immunohistochemistry was used to detect the expression of ER,PR,Her?2 and Ki67 in primary tumor and axillary lymph node metastasis. Results The positive rates of ER expression were 75. 9% ( 325/428 ) and 70. 3% ( 301/428 ) respectively in primary tumor and axillary lymph node metastasis. The positive rates of PR expression were 61. 4% ( 263/428) and 56. 1% ( 240/428 ) respectively in primary tumor and axillary lymph node metastasis. The rates of Her?2 overexpression were 20. 1% ( 86/428) in primary tumor and the positive rate of Her?2 in axillary lymph node metastasis was 22. 7%( 97/428 ) . The positive rates of Ki67 expression were 45. 6%( 195/428 ) and 39. 7%(170/428) respectively in primary tumor and axillary lymph node metastasis. The expression of ER,PR,Her?2 and Ki67 in primary and axillary lymph node metastasis showed no statistical significance ( P>0. 05 ) . The molecular typing of primary tumor and axillary lymph node metastasis were not consistent in 31 patients ( 31/428,7. 24%) ,including 14 cases of primary tumor Luminal A,9 cases of Her?2 overexpression in axillary lymph node metastasis and 5 cases of triple negative breast cancer. Primary tumor Luminal B was detected in 10 cases, while 6 cases of Her?2 overexpression in axillary lymph node metastasis and 4 cases of triple negative breast cancer. Primary tumor Her?2 was overexpressed in 4 cases,while 1 case of Luminal A,3 cases of Luminal B in axillary lymph node metastasis. There were 3 cases of primary tumor triple negative breast cancer,while 2 cases of Luminal B in axillary lymph node metastasis and 1 case of Her?2 overexpression. Conclusion The expressions of ER, PR, Her?2 and Ki67 in primary tumor and axillary lymph node metastasis of some breast cancer were different. Immunohistochemistry for primary tumor and axillary lymph node metastasis of stage II?III breast cancer patients should be routinely carried out. Based on molecular typing of primary tumor and axillary lymph node metastasis,individualized treatment plan can be developed,so that patients will benefit from it.

In order to explore the treatment of Her-2 positive breast cancer patients who failed in multi-line treatments, we retrospectively analyzed the clinical data of a patient with refractory Her-2 positive breast cancer.The patient was initially diagnosed as Her-2 positive advanced breast cancer.After six line treatment in the outer hospital, the patient′s condition was basically in a progressive state.The breast tumor was broken and purulent, the lung metastasis increased, and the patient′s quality of life was poor.The patient was admitted to Department of Breast Surgery of Affiliated Suqian Hospital of Xuzhou Medical University, after MDT discussion, we gave pyrrolotinib combined with capecitabine treatment, the chest wound healed gradually, the lung metastasis gradually reduced, and the quality of life was better.A retrospective analysis of this case showed that pyrrolidine combined with capecitabine may bring hope to Her-2 positive breast cancer patients who failed to receive multi-line therapies, especially those who failed to target therapy.

Objective To evaluate the efficacy and safety of gefitinib plus capecitabine in treatment of recurrent and metastatic triple negative breast cancer.Methods From Jan.2011 to Jun.,41 patients who have recurrent and metastatic triple negative breast cancer after treated by adjuvant chemotherapy were enrolled in this study They were divided into two groups according to their wishes.The 24 cases in the experimental group were treated with gefitinib plus capecitabine.The 17 cases in the control group were treated with capecitabine.The two groups were followed up for 12 months.They were treated until the disease progression or the toxicity could not be tolerated.Results The objective response rate (ORR) in the experimental group and the control group was 70.83％(17/24) vs 35.29％(6/17).The disease control rate (DCR) in the two groups was 91.67％ (22/24) vs 64.71％ (11/17).The difference between the two groups was statistically significant (P＜0.05).The incidence rate of adverse drug reactions in the two groups was similar (P＞0.05),and the reactions were tolerable.Conclusion Gefitinib plus capecitabine is an effective and safe treatment for recurrent and metastatic triple negative breast cancer with tolerable adverse reactions,and some patients were able to survive for more than 12 months.