This thesis aimed at the Bacillus natto TK-1 screened out from Natto.The lipopeptide was purified using Thin-Layer Chromatography(TLC),and investigated its anti-tumor activity.After acid precipitation and methanol extraction,the lipopeptide was separated on TLC.Then the authors get the monomer surfactin which molecular weight is 1036Da through the High performance liquid chromatography(HPLC),electro spray ionization-mass spectrometry(ESI-MS) and infrared(IR).MTT method was implied to testify the anti-tumor activity of the purified sample from TLC.The results indicated a concentration and time-dependent relationships.After 48h,their IC50 were 40 mg/L.The detection with inverted microscope fluorescence microscope displays that the surfactin will cause a series of Morphological changes to the cells.In TUNEL experiment,the authors noticed that surfactin has the ability to induce apoptosis,besides this inhibition shows an obvious time-dependent relationship.

The study was to develop cis-dichlorodiammineplatinum(DDP)-loaded formulations using a novel type of self-assembled compound composed of block copolymers synthesized by poly(?-glutamic acid)(?-PGA).For the potential of targeting liver cancer cells,D-galactose was conjugated on the prepared ?-PGA.In vitro,DDP can be released from the resulting conjugate in PBS:there was a burst release during the first 8 h,then followed by sustained release.DDP could be easily incorporated into poly(?-glutamic acid)-D-galactose esterifiable derivative through a covalent bond.The yield of DDP incorporation into the esterifiable derivative was 9.4%～10.2%.In vitro experiments conclusively established that the poly(?-glutamic acid)-D-galactose esterifiable derivative-Cisplatin Complex Compound(?-D+-DDP)was much less toxic to normal cell lines than DDP only.The surviving rate of cells treated with ?-D+-DDP compound is higher than those treated with free DDP.Also it has obvious antitumor efficiency on human liver tumor BEL-7402 cells.HE staining indicated that the ?-D+-DDP compound make the BEL-7402 apoptosis.These results indicated that the conjugation of DDP to the esterifiable derivative reduced its cytotoxicity activity,but retains its antitumor activity in vitro.In conclusion,the ?-D+-DDP compound could be used as a potential clinic antitumor drug.The ?-PGA obtained by fermentation can be used as a valuable drug carrier system.

DDP could be easily incorporated into poly (?-glutamic acid)-D-galactose esterifiable derivative through a covalent bond. The yield of DDP incorporation into the ?-PGA was 9.4%～10.2%. The DDP was released in the initial 8h in a burst manner,and thereafter in a sustained manner. The results that the conjugation of DDP to poly (?-glutamic acid)-D-galactose esterifiable derivative not only reduced the toxicity of the DDP but also enhanced antitumor activity and the targeting ability. The vivo experiments conclusively established that the ?-D+-DDP compound was much less toxic to animals than DDP alone. A direct evaluation showed that mice treated with ?-D+-DDP compound at a dose of 7.5 mg/kg displayed significant tumor regression. Furthermore,the implanted solid tumors disappeared completely from 35% of the H22 tumor-bearing mice after ?-D+-DDP compound administration. The aforementioned results of biodistributions of the prepared ?-D+-DDP compound in various organs in normal mice demonstrated that the ?-D+-DDP compound had a specific interaction with liver's parenchymal cells and H22 hepatocellular carcinoma tumor cells via ligand receptor recognition. In conclusion,the results indicated that the ?-D+-DDP compound prepared can effectively target the site of hepatoma tumor via the recognition and significantly reduce its size. The ?-D+-DDP compound was less toxic than the free DDP,and could effectively reduce xenografted H22 hepatocellular carcinoma cells in KM mice and prolong the survival of KM mice grafted with H22 hepatocellular carcinoma tumor cells. Therefore,the prepared ?-D+-DDP compound may be used as a potential drug delivery system for the targeted delivery to liver cancers or other liver diseases.

Objective To investigate the effects of different doses of alcohol on the synthesis of testosterone and the expression of androgen binding protein(ABP)mRNA in rat testis.Methods Forty male Wistar rats were randomly divided into 4 groups(10 rats each group)and received either distilled water(control group)or alcohol(alcohol-fed groups)for 5 months.Alcohol was administered by garage with a single daily dose : 5 g/kg(large dose group),2.5 g/kg(middle dose group)and 0.5 g/kg(small dose group).Testosterone content was measured by ELISA.mRNA levels of peripheral-type benzodiazepine receptors(PBR),PPARct and ABP were assayed by RT-PCR.Results Compared with control group:(1)ethanol feeding with daily doses of 5 g/kg,2.5 g/kg and 0.5 g/kg significantly decreased testosterone levels by 31.13%(P0.05)respectively,indicating that ethanol might impair testosterone synthesis;(2) mRNA levels of PBR were decreased in all three ethanol-treated groups(all P

OBJECTIVETo study the effect of ginsenoside on apoptosis of human leukemia-60 (HL-60) cells and its mechanism.

METHODSMTT cytotoxicity assay was used to determine the growth inhibition activity of ginsenoside (100, 50, 25, 12.5, 6.25, 3.125 and 1.5625 μmol/L) on HL-60 cells. The apoptosis of HL-60 cells after treatment with ginsenoside (0,5,10 and 20 μmol/L) was determined by Annexin V-FITC/PI staining and flow cytometry. The cleavage of total proteins by caspase-8, caspase-9 and caspase-3 was evaluated by Western blot. The cleavage of caspase-3 protein was detected by Western blot after treatment with 10 μmol/L ginsenoside and caspase-8 and 9 inhibitors.

RESULTSGinsenoside had potent cytotoxicity on HL-60 cells, with an IC50 value of 7.3±1.2 μmol/L. After treatment with ginsenoside (0, 5, 10 and 20 μmol/L) for 48 hours, the apoptotic rate displayed a dose dependency, as shown by flow cytometry, with significant differences between the groups (F=12.67, P<0.01). Western blot showed that there were caspase-9 and caspase-3 cleavage bands, but without caspase-8 cleavage band. The specific inhibitor of caspase-9 Z-LEHD-FMK could block the caspase-3 cleavage induced by 10 μmol/L ginsenoside, but the specific inhibitor of caspase-8 Z-IETD-FMK did not have this effect.

CONCLUSIONSGinsenoside can induce apoptosis of HL-60 cells, which may be related to a mitochondria-dependent pathway.

Apoptosis ; drug effects ; Caspase 9 ; physiology ; Caspase Inhibitors ; pharmacology ; Ginsenosides ; pharmacology ; HL-60 Cells ; Humans

AIM:To explore whether the angiotensin II type 1 receptor autoantibodies(AT1-AA)induces islet β-cell apoptosis and whether autophagy is involved in the process.METHODS:The INS-1 cells treated with AT1-AA at 10-6mol/L for 24 h,and then the apoptosis was analyzed by flow cytometry,Western blot and Hoechst 33258 staining.In addition,the expression of autophagy-related proteins such as LC3 and beclin 1 were determined by Western blot.The effects of AT1-AA on the apoptosis,autophagy and viability of INS-1 cells with or without 3-methyladenine(3-MA;a com-mon autophagy inhibitor)or telmisartan(an angiotensin Ⅱ type 1 receptor blocker)pretreatment, were detected by flow cytometry,Western blot and CCK-8 assay.RESULTS: Treatment with AT1-AA at 10 -6mol/L for 24 h significantly re-duced the cell viability(P<0.05).Compared with the negative IgG control group,the apoptotic cells increased after incu-bation with AT1-AA for 12 h,24 h and 36 h,respectively(P<0.05).Moreover,the protein levels of LC3 and beclin 1 also increased gradually with the prolongation of treatment time,and the elevation of apoptosis and autophagy were blocked by telmisartan.After pretreatment with 3-MA, the apoptotic rate of the cells was obviously decreased compared with the cells treated with AT1-AA alone.CONCLUSION: AT1-AA induces the apoptosis of INS-1 islet βcells by upregulating autophagy via the angiotensin Ⅱtype 1 receptor pathway.

Cystic fibrosis (CF) is a multisystem disease mainly caused by pathogenic mutation of the cystic fibrosis transmembrane conduction regulator gene.In recent years, with the deepened understanding of the disease and the popularization of gene detection technology, an increasing number of children are diagnosed with CF in China.Lung involvement is reported to affect the prognosis of the disease.Lung involvement is closely related to the airway, and good airway management can prolong the life of children.In this paper, the selection of airway clearance techniques and inhaled drugs were expounded, so as to improve the long-term airway management of children with CF in China.

Objective Gastrokines 2(GKN2) protein expression is detected in gastric cancer tissues, the distal gastric mucosa tissues and the adjacent normal gastric tissues; the effect of GKN2 overexpression is displayed on proliferation,migration and invasion in human gastric cancer cell line MKN28. Methods Immunohistochemistry was performed to observe GKN2 expression in gastric cancinoma tissues,adjacent gastric mucosa tissues and distal gastric mucosa tissues. Then,the GKN2 gene eukaryotic expression vector Xhol GKN3SP-hGKN2-TEV-SBP Xhol was transfected into human gastric cancinoma MKN28 cells. Western blot was used to evaluate the expression of GKN2 in MKN28 cells. Cell viability,migration and invasion ability were investigated by MTT,Transwell migra-tion assay and Transwell invasion assay. Results GKN2 expression in gastric cancinoma tissues is lower in com-parison with those in the adjacent normal gastric tissues or distal gastric mucosa tissues respectively (P< 0.05).The viability of MKN28 cells was significantly inhibited after transfected by GKN2 vs untransfected cells (P<0.05). GKN2 overexpression decreased the cells' number passing through both the membrane and matrigel, compared to those of untransfected cells(P < 0.05). Conclusions Downregulation of GKN2 protein expression is associated with the occurrence of gastric cancer; GKN2 overexpression can suppress the prolifera-tion, migration and invasion of MKN28 cells.

Objective To analyze the characteristics and limitations of MRI diagnosis of Guillian Barre syndrome. Methods Fifteen patients with Guillian Barre syndrome (14 in acute stage, 1 in chronic stage) were performed MRI plain scans and contrast-enhanced (CE) T1-weighted images (T1WI) with fat saturation to observe the relation between MRI manifestations and clinical characteristic in peripheral nerves in the vertebral canal. Results Eight patients in acute stage appeared cauda equina nerve thickening to a varying degree by MRI plain scan and the involved nerves showed isointensity on T1WI and isointensity or slight hypointensity on T2WI. In the chronic patient, the cauda equina and the spinal nerve at all the levels of the spinal cord showed obvious thickening. CE T1WI indicated partial spinal and canda equina nerves thickening to a varying degree below T8 level in 14 acute stage patients with 2 accompanied by partial cervical nerves thickening and 2 by partial cranial nerves thickening, in the chronic patient, T1WI showed enhancement and thickening of all the spinal nerves, cauda equina and partial cranial nerves. Every patient had a symptom of weakness in lower limb, including 9 paralyses in both limbs. Enhanced MRI displayed pathologic changes in the cauda equina in all the patients, while 7 complained of weakness in both upper limbs with only 3 (43%) defined on MRI and 6 suffered from functional disorder of the cranial nerves with 3 (50%) defined on MRI. Conclusions MRI is a sensitive method to demonstrate the pathological changes of the cauda equina in GBs;however, under-diagnosis can often happen in involvements of the cervical and cranial nerves.

OBJECTIVETo assess the diagnostic value of double balloon endoscopy (DBE) for obscure gastrointestinal bleeding (OGIB) METHODS: The data of 103 OGIB patients who underwent DBE from January 2007 to September 2010 in the First Affiliated Hospital, Zhejiang University School of Medicine were retrospectively analyzed.

RESULTSDBE was successfully performed in all 103 patients without complications. Of 103 patients, 66(64.1 %) had positive DBE findings and 28 had surgery procedures(27.2 %). Ninety-four patients finally acquired positive diagnosis, including small intestine tumor(31.1 %), angiodysplasia(22.3 %), exulceratio simplex(9.7 %), Crohn's disease(6.8 %), diverticulum(4.9 %), abdominal purpure(4.9 %), etc. Lesions occurred more frequently in proximal small intestine than in distal small intestine (56.3 % Compared with 30.1 %, P<0.001).

CONCLUSIONDBE is a safe, effective and reliable procedure for the diagnosis of OGIB.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Capsule Endoscopy ; methods ; Female ; Gastrointestinal Hemorrhage ; diagnosis ; Humans ; Male ; Middle Aged ; Retrospective Studies ; Young Adult