Objective To investigate the effect of CD147 monoclonal antibody (mAb) on the natural resistance to paclitaxel (TAX) in the human cervical cancer line (HCE1) multicellular spheroid (HCE1/MCS) model and if CD147 mAb can reverse the HCE1/MCS resistance to TAX. Methods HCE1/MCS was obtained by liquid overlay and rotating technique. HCE1/MCS morphological changes were observed before or after the interference of CD147 mAb. The effects of TAX on HCE1/MCS (including inhibition ratio, IC50 and index of multicellular resistance) before or after CD147 mAb treatment were determined by the method of WST-1 and the inhibition ratio curve was mapped. Cell cycle and apoptosis were detected by flow cytometer (FCM). The expression of CD147 and P-gp of both HCE1/MC and HCE1/MCS was detected by immunocytochemistry. Results HCE1/MCS was established successfully. CD147 mAb could inhibit HCE1/MCS from forming spheroids. CD147 mAb could enhance the sensitivity of HCE1/MCS to TAX. IC50 in different concentrations of CD147 mAb (5,10,20 μg/mL) HCE1/MCS group were (40.31±3.73), (32.43±1.56), and (30.69±1.01) μg/mL. CD147 mAb resulted in G1/G0 arrest in HCE1/MCS. CD147 mAb of low concentrations (0-10 μg/mL) caused a dose-dependent inhibition of HCE1/MCS (P<0.05). Combined with TAX, CD147 mAb could also induce HCE1/MCS cell cycle arrest in both G1/S and G2/M stage. The expression of CD147 and P-gp was consistent in HCE1/MCS groups. Conclusion CD147 plays an important role in muliticellular resistance of cervical cancer and inhibition of CD147 can synergistically reverse the multicellular drug resistance (MCR) in cervical cancer. The MCR of HCE1/MCS mediated by CD147 is related to P-gp.

Objective To explore the influence pulmonary function and inflammatory factors of symbicort combined with ambroxol hydrochloride in the treatment of acute exacerbation of chronic obstructive pulmonary disease( AECOPD) .Methods 86 patients with AECOPD were divided into control group 43 cases and treatment group 43 cases according to the computer random number table method.The control group was given intravenous injection of ambroxol hydrochloride 60 mg/time, 2 times/day, and the treatment group was given inhalation of symbicort 2 inhaling/times, 3 times/day on the basis of the control group.After 7 day treatment, the clinical efficacy and adverse reactions of two groups were observed.The percentage of forced vital capacity ( FVC%) , percentage of forced expiratory volume of 1 seconds( FEV1 %) , percentage of mid expiratory flow predicted value( MMF%) , percentage of max volume predicted value(MVV%) were measured by pulmonary function test apparatus, and the value of FEV1%/FVC% was calculated.The levels of serum tumor necrosis factor alpha(TNF-α), c-reactive protein(CRP) and interleukin 6(IL-6) were detected by ELISA.Results The total effective rate of treatment group and control group were 93.02%(40/43) and 74.42%(32/43), the difference was statistically significant(P <0.05).After treatment the FVC%(2.58 ±0.25),FEV1%(1.87 ±0.15),MMF%(70.24 ±5.86)and MVV%(72.43 ±4.35) in treatment group were higher than the control group (2.21 ±0.27),(1.68 ±0.16),(63.14 ±5.68)and 65.12 ±4.16), the difference was statistically significant(P<0.05).The levels of serum TNF-α(13.87 ±4.12)μg/mL,CRP(9.14 ±3.76)mg/mLand IL-6(76.07 ±10.14)pg/mL in treatment group were lower than the control group (16.56 ±6.48)μg/mL,(12.46 ±3.89)mg/mL and(89.55 ±11.24)pg/mL,the difference was statistically significant(P<0.05).The adverse reactions were mainly for heartburn, nausea, vomiting and skin rashes in the control group,and the adverse reactions were mainly for heart palpitations, headache, nausea, rashes, vomiting in the treatment group, the incidences of adverse reactions 23.26%(10/43) in treatment group compared to control group 18.60%(8/43) was no statistically significant difference.Conclusion The symbicort combined with ambroxol hydrochloride in the treatment of AECOPD have clinical curative effect, can improve the patient's lung function, reduce inflammation, have less adverse reactions.It was worthy of clinical popularization and application.

BACKGROUND: The transplantation of allogeneic cartilage has local immunological rejection, and it is necessary to further reduce the rejection to promote its application in clinic, thus it is significant to perform a series of experiments to induce local immune privilege.OBJECTIVE: To observe the in vivo growth of tissue engineered allogeneic cartilage reconstructed by chondrocytes transfected with recombinant retroviral vector pLNCX2-FasL.DESIGN: A randomized controlled observation.SETTING: Shanghai Jiao Tong University.MATERIALS: Thirty-six allogeneic New Zealand rabbits as recipients and 45 1-week-old chinchillas as donors, either sex,were purchased by the experimental animal center of Chinese Academy of Sciences. Amphotropic recombinant retrovirus coated cell line PT67 was purchased from Clontech Company; Dulbecco's modified eagle medium (DMEM), fetal bovine serum (FBS), G418 and Polybrene were bought from GIBCO BRL.METHODS: The experiment was carried out in original Shanghai Second Medical University from January 2000 to July 2005. The New Zealand rabbits were randomly divided into three groups: FasL-transfected group (n =12), untransfected group (n =12) and blank control group (n =12). The rabbit allogeneic cartilages were constructed by the compound of pLNCX2-FasL transfected chondrocytes and tissue engineered material of pluronic F-127. ① Gross observation and mass changes of the grafts: Corresponding materials were infused subcutaneously, the grafts were removed at 1, 2 and 3 months after transplantation for gross observation and the mass changes. ② Staining observation: The grafts were removed at 1, 2 and 3 months after transplantation, then prepared into sections, and observed by hematoxylin and eosin (HE), Safranin'O and Masson's trichrome stainings. ③ Antibody detection: Blood samples (1 mL) were collected at 1 and 2 months after transplantation, the chondrocytes of the chinchillas were lysed freezingly with lysis antigen as the mixed antigen, and separated by electrophoresis in agarose medium, then acted with serum of recipient to observe whether corresponding antibody generated. ④ Complement dependent cytotoxicity (CDC) test: The chondrocytes of chinchillas were prepared into cell suspension (2×109/L), and then seeded into 96-well plate, attached grew for 24 hours, then recipient serum was added for the CDC test, and the percentage of apoptotic cells was counted under microscope.MAIN OUTCOME MEASURES: ① Gross observation and mass changes of the grafts;② Histological changes; ③ Results of the antibody detection; ④ Percentage of apoptotic cells.RESULTS: All the 81 rabbits were involved in the analysis of results. ① Gross observation and mass changes of the grafts: Two weeks after inoculation, there were obvious nod formations at the inoculated sites, but no nod formed in the blank control group. The new cartilage tissues became smaller gradually and completely disappeared at 4 months in the untransfected group, whereas those in the FasL-transfected group became smaller, but still existed after 7 months. The masses of grafts in the FasL-transfected group were higher than those in the untransfected group (P ＜ 0.05). ②Histological observation: Plenty of lymphocytic infiltrations around cartilage tissue could be observed in the untransfected group, and obviously decreased in the FasL-transfected group. No lymphocyte was observed inside the chondrocytes.Masson's trichrome staining was performed, and it was observed under light microscope that the small white parts in the middle were immature chondrocytes, and there were green collagen around most of the mature chondrocytes. Safranin O staining showed strong positive reaction, suggested that there were rich glycosaminoglycan in matrix. ③ Antibody detection: The chondrocytes of the chinchillas were lysed freezingly with lysis antigen as the mixed antigen, then acted with serum of recipient, and the results showed that no corresponding antibody generated. ④ Percentage of apoptotic cells: The percentages of serum CDC apoptotic cells in the FasL can ransfected group, untransfected group and blank control group were 5%, 6% and 1%, which were all negative.CONCLUSION: Rabbit allogeneic chondrocytes transfected with recombinant retroviral vector pLNCX2-FasL can reconstruct tissue engineered cartilage, and can postpone the degeneration by 3 months.

Objective:To investigate whether the overexpression of Oct4B1 gene induces epithelial mesenchymal transition in human colorectal cancer SW480 cells and its possible mechanism.Methods: Experimental group(SW480-Oct4B1):Transfection of SW480 cell lines in colorectal cancer with Oct4B1 overexpression plasmid;Control group(SW480-Oct4B1):negative control plasmid with G418 resistance.Stably transfected cell lines were obtained by G418 culture medium.The two groups were compared with:①Detection of Oct4B1 gene expression in stably transfected cell lines by RT-qPCR;②Scratches and Transwell assays were used to estimate migration and invasion;③Detection of EMT related markers E-cadherin,N-cadherin and Vimentin protein expression by Western blot assay;④Detection of Twist gene and protein expression by RT-PCR and Western blot assays.Results: The transient transfection was confirmed by RT-qPCR and the stable transfected cell lines were obtained from two groups of cells transfected with G418 culture medium.Compared with the control group:①RT-qPCR revealed increased expression of Oct4B1 gene in the experimental group(P<0.01);②Cell migration and invasion were significantly increased(P<0.01);③Epithelial marker:the expression of E-cadherin protein was significantly decreased (P<0.01),interstitial marker:the expression of N-cadherin and Vimentin protein was significantly increased (P<0.01);④Twist mRNA and protein expression were significantly increased(P<0.01).Conclusion: Overexpression of Oct4B1 gene can induce epithelial mesenchymal transition in human colorectal cancer SW480 cells,its molecular mechanism may be related to the promotion of Twist expression.

0.05),between the two groups,but in body mass index [(24.451?3.752) vs(22.468?2.434),P=0.030],previous history of pancreatitis(23.8% vs.7.5%,P=0.046) and bloody ascites(95.24% vs.46.24%,P0.05).MODS rate was higher in HL-group(52.4% vs.29%,P=0.04).The two groups had similar operation rates(66.7% vs 80.6%,P=0.178),hospitalization days [(25.476?14.383) vs(22.796?7.191),P=0.216] and mortality rate(28.6% vs.11.8%,P=0.069).Conclusions Hyperlipidemic severe acute pancreatitis has the characters of heavier weight habitus,history of frequent recurrence,high incidence of bloody ascites and prone to develop MOF.

Objective To investigate the effect of the ubiquitin-proteasome pathway inhibitor MG132 on the natural resistance to cisplatin in the human cervical cancer line (HCE1) muhicellular spheroid (HCE1/MCS) model and to probe it if MG132 could reverse the HCE1/MCS resistance to cisplatin, as well as the possible mechanism of drug resistance.Methods (1) HCE1/MCS was obtained using liquid overlay and rotating technique.(2)Four groups were established (MG132 group, cisplatin group, MG132 + cisplatin group, the control group).Cell viability were measured by trypan blue exclusion assay.Cell cycle and apoptosis were detected by flow cytometry.(3) The expression of nuclear factor (NF) kB of both HCE1 monolayer cells (HCEI/MC) and HCE1/MCS was detected by western blot, and the expression of B cell lymphoma/leukemia-2 (bcl-2) was detected by immunohistochemistry.Results (1) HCE1/MCS was established successfully.(2) Cell inhibited rate of HCE1/MC and HCE1/MCS was: in MG132 group, (11.67 ± 2.34) % vs (10.78 ± 1.17) % (P > 0.05) ; in MG132 + cisplatin group, (92.67 ± 2.52)% vs (91.33 ±2.18)% (P>0.05); in cisplatin group, (45.01±7.44)% vs (9.45±5.98)% (P<0.05).(3)The rate of apoptosis of HCE1/MC and HCE1/MCS were: in MG132 group, 8.14% and 5.97% ; in MG132 + cisplatin group, 99.01% and 95.22% ; in cisplatin group, 33.61% and 0.88%.(4)The expression level of NF-kB and the high expression rate of bcl-2 were: in HCE1/MCS of control group, 0.67 and 60% ; in HCE1/MCS of cisplatin group, 0.85 and 83% ; in HCE1/MCS of MG132 group, 0.39 and 20% ; in HCE1/MCS of MG132 + cisplatin group, 0.47 and 33%.Conclusions (1) HCE1/ MCS present natural resistance to cisplatin and may become a good model for the study of cervical cancer drug resistance in vitro.(2) MG132 could induce the inhibition and apoptosis of HCE1/MCS cells and partially reverse the natural resistance of HCE1/MCS to cisplatin, of which partially reverse the natural resistance may be in relation to the down-regulation of NF-kB and bcl-2 expression.

OBJECTIVE: To explore the differentially expressed gene profile in cervical squamous cell carcinoma tissues with and without pelvic lymph node metastasis by microarray technique and to screen tumor metastasis-related genes. METHODS: Cancer tissue samples were collected from 4 cervical squamous cell carcinoma patients with pelvic lymph metastasis and 6 patients without metastasis before the treatment. Differentially expressed gene profile was identified between cervical squamous cell carcinoma tissues with and without pelvic lymph node metastasis by NimbleGen microarray. Fluorescent real-time quantitative PCR was employed to validate the accuracy of the microarray by detecting the expression of genes picked from the differentially expressed gene profile. We screened tumor metastasis-related genes through the gene bank, PUBMED and gene ontology analysis. RESULTS: With the application of microarray, 329 genes were identified as markedly differentially expressed genes between cervical squamous cell carcinoma tissues with and without pelvic lymph node metastasis. Real-time PCR was in accordance with microarray. Forty-four genes associated with tumor metastasis were identified from the differentially expressed gene profile. CONCLUSION Gene profile in cervical squamous cell carcinoma tissues associated with pelvic lymph node metastasis is preliminarily identified, indicating that lymph node metastasis of cervical squamous cell carcinoma is a process involving numerous genes.
Adult ; Aged ; Carcinoma, Squamous Cell ; genetics ; pathology ; Female ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Humans ; Lymphatic Metastasis ; Middle Aged ; Oligonucleotide Array Sequence Analysis ; Uterine Cervical Neoplasms ; genetics ; pathology

Objective:To explore the role and mechanism of long non-coding RNA KCNQ1 overlapping transcript 1 (LncRNA KCNQ1OT1) in the migration, proliferation and invasion of hepatocellular carcinoma (HCC).Methods:The experimental method was conducted. The expression levels of LncRNA KCNQ1OT1 in HCC tissues and normal liver tissues in the StarBase database were collected. The experimental methods including real-time quantitative PCR, cell transfection, scratch assay, CCK8 assay, Transwell assay, Western blot were used to determine the expression, migration, proliferation, invasion of LncRNA KCNQ1OT1 in HCC cells and its relationship with phosphatidylinositol 3-kinase/phosphorylated AKT Protein (PI3K /p-AKT) signaling pathways. Observation indicators: (1) expression of LncRNA KCNQ1OT1 in HCC tissues and normal liver tissues; (2) the migration of HepG2, SMCC-7721 and MHCC-97H HCC cells after LncRNA KCNQ1OT1 gene knockdown; (3) the proliferation and invasion of HepG2, SMCC-7721 and MHCC-97H HCC cells after LncRNA KCNQ1OT1 gene knockdown; (4) effects of LncRNA KCNQ1OT1 gene knockdown on PI3K/p-AKT signaling pathways. Measurement data with normal distribution were expressed as Mean± SD, and comparison between groups was analyzed using the t test. The Kaplan-Meier method was used to calculate survival rates and draw survival curves. Results:(1) Expression of LncRNA KCNQ1OT1 in HCC tissues and normal liver tissues. The expression levels of LncRNA KCNQ1OT1 in 374 HCC tissues and 50 normal liver tissues from StarBase database were 3.320±0.017 and 1.470±0.025, respectively, showing a significant difference ( t=5.24, P<0.05). Results of gene expression profile interactive analysis showed that the 30-month disease-free survival rates of HCC patients with high and low expression levels of LncRNA KCNQ1OT1 were 41% and 55%, respectively, with a significant difference ( χ2=6.209, P<0.05). The relative expression of LncRNA KCNQ1OT1 in HepG2, SMCC-7721and MHCC-97H cells were 1.470±0.042, 3.300±0.032, 4.040±0.031, respectively, versus 1.000±0.022 in normal liver cells (LO2), showing significant differences ( t=17.66, 95.40, 114.20, P<0.05). (2) The migration of HepG2, SMCC-7721 and MHCC-97H HCC cells after LncRNA KCNQ1OT1 gene knockdown. ① Results of cell transfection showed that the relative expression levels of LncRNA KCNQ1OT1 in HepG2, SMCC-7721 and MHCC-97H cells after LncRNA KCNQ1OT1 gene knockdown were 0.350±0.016, 0.310±0.020, 0.380±0.018, respectively, versus 1.000±0.021, 1.000±0.018, 1.000±0.019 in the negative control cells, showing significant differences ( t=23.40, 28.15, 22.32, P<0.05). ② Results of scratch assay showed that the healing rates of HepG2, SMCC-7721, MHCC-97H cells after LncRNA KCNQ1OT1 gene knockdown were 85.0%±1.9%, 75.0%±1.8%, 90.0%±1.7%, respectively, versus 100.0%±2.0%, 95.0%±1.8%, 72.0%±1.7% of the negative control cells, showing significant differences ( t=31.35, 47.36, 38.42, P<0.05). ③ Results of Transwell assay showed that the vertical migration rates of HepG2, SMCC-7721, MHCC-97H cells after LncRNA KCNQ1OT1 gene knockdown were 195±10, 205±12, 85±8, respectively, versus 520±11, 430±7, 405±20 of the negative control cells, showing significant differences between them ( t=922.30, 458.20, 708.40, P<0.05). (3) The proliferation and invasion of HepG2, SMCC-7721 and MHCC-97H HCC cells after LncRNA KCNQ1OT1 gene knockdown. ① Results of CCK8 assay showed that 72-hour optical densities of HepG2, SMCC-7721, MHCC-97H cells after LncRNA KCNQ1OT1 gene knockdown were 1.370±0.018, 1.240±0.016, 1.360±0.020, respectively, versus 0.900±0.023, 1.740±0.032, 1.230±0.025 of the negative control cells, with significant differences ( t=10.79, 12.00, 7.56, P<0.05). ② Results of Transwell assay showed that the invasion numbers of HepG2, SMCC-7721, MHCC-97H cells after LncRNA KCNQ1OT1 gene knockdown were 186±12, 155±7, 75±9, respectively, versus 505±1, 245±8, 300±15 of the negative control cells, showing significant differences ( t=955.90, 163.40, 530.90, P<0.05). (4) Effects of LncRNA KCNQ1OT1 gene knockdown on PI3K/p-AKT signaling pathways. Resluts of Western blot showed that the relative repression levels of PI3K in HepG2, SMCC-7721, MHCC-97H cells after LncRNA KCNQ1OT1 gene knockdown were 0.447±0.009, 0.430±0.012, 0.354±0.006, respectively, versus 0.820±0.017, 0.850±0.012, 0.531±0.001 of the negative control cells, showing significant differences ( t=18.94, 25.72, 27.46, P<0.05). The relative repression levels of p-AKT in HepG2, SMCC-7721, MHCC-97H cells after LncRNA KCNQ1OT1 gene knockdown were 0.343±0.015, 0.410±0.012, 0.579±0.006, respectively, versus 0.546±0.012, 0.620±0.012, 0.830±0.012 of the negative control cells, showing significant differences ( t=10.78, 12.86, 19.02, P<0.05). Conclusions:LncRNA KCNQ1OT1 plays an important role in the occurrence and development of HCC. LncRNA KCNQ1OT1 gene knockdown can inhibit the PI3K/AKT signaling pathways, so it can significantly inhibit the proliferation, migration and invasion of HCC cells.

[ ABSTRACT] AIM:To investigate the effects of Chaihu Shugan powder ( CSP) on lipid metabolism and the pro-teins involved in adenosine 5’-monophosphate-activated protein kinase (AMPK)/sirtuin 1 (SIRT1) pathway in the liver tissues of the rats with non-alcoholic fatty liver disease (NAFLD).METHODS: Sprague-Dawley rats were randomly di-vided into normal control ( NC) group, with HFD ( HFD) group and CSP group.The NAFLD models were established by feeding with HFD for 16 weeks in the rats.The rats in CSP group were intragastrically administered with CSP extracts (9.6 g· kg-1 · d-1 ) , and blood and liver samples were collected 16 weeks later.Serum and liver levels of total cholesterol ( TC) and triglyceride ( TG) , and serum levels of alanine aminotransferase ( ALT) and aspartate aminotransferase ( AST) were measured using an automatic biochemical analyzer.The histological changes of liver tissues were observed with HE staining, while the lipid deposition was observed with Oil Red O staining.The ultrastructural changes of the liver tissues were observed under transmission electron microscope.Moreover, the protein levels of AMPK, phosphorylated AMPK (pAMPK), SIRT1 and uncoupling protein 2 (UCP2) in the liver were detected by Western blot.RESULTS:The results of HE staining, Oil Red O staining and electron microscopy demonstrated that NAFLD rat model was successfully estab-lished.Compared with NC group, the serum and liver levels of TC and TG, and serum level of AST in model group were markedly elevated ( P<0.01) .Moreover, the protein levels of pAMPK and SIRT1 in HFD group were markedly reduced (P<0.01), whereas UCP2 level was elevated (P<0.01).Furthermore, liver levels of TC and TG, and serum level of AST in GSP group were markedly reduced as compared with HFD group ( P<0.05 ) .The protein levels of pAMPK and SIRT1 were elevated ( P<0.05 ) , whereas the UCP2 level was reduced as compared with HFD group ( P<0.01 ) .The protein level of AMPK between the 3 groups had no significant difference.CONCLUSION: CSP attenuates hepatic lipid disorder and hepatic lipid deposition in NAFLD rats induced by feeding with HFD for 16 weeks, which is associated with the activation of AMPK/SIRT1 pathway.

Objective To analyse and compare the effects and safety of early use (in emergency room, intravenous loading followed by infusion) with bolus injection during primary PCI of tirofiban, on post-procedural TIMI flow and 30d clinical outcomes. Methods Seven hundred and seven patients with acute STEMI treated by primary PCI in Ruijin hospital were retrospectively and enrolled screened. Among them, 86 patients with single bolus intra-coronary injection of tirofiban (25 μg/kg) during the procedure were served as observation group. Baseline, angiographic, PCI features and rate of major adverse cardiac events (MACE) at 30 d follow-up were compared with those received early intravenous infusion of tirofiban (10ug/kg bolus followed by 0.15μg/(kg·min) intravenous infusion)(control group, n=239). Results Compared with control group, patients in observation group were older[(63.8±11.4) vs. (57.9±8.8), P=0.01], had higher prevalence of hypertension (58.6%vs. 51.0%, P=0.005), multivessel disease (57.0%vs. 34.3%, P<0.001), and female in gender (40.7%vs. 25.1%, P=0.006). Post-procedural TIMI flow in culprit vessel and TMP grade were comparable between the two groups (P=0.66 and P=0.48, respectively). Reduction in TIMI minimal bleeding events were found in the observation group (2.3%vs. 9.6%, P=0.03). MACE free survival rate at 30d clinical follow-up was similar between the two groups (P=0.48). Conclusions Single bolus intra-coronary injection of tirofiban exerts similar effects in post-procedural TIMI flow, TMP grade in culprit vessel and 30d clinical outcomes compared with early use in emergency room with intra-venous loading and infusion, nevertheless, intra-coronary injection resulted in significantly reduced TIMI minimal bleeding events. Prospective, randomized clinical study is mandatory to prove our current results.