Objective To study the apoptosis of human leukemia K562 cells induced by snake venom of Agkistrodon Halys Pallas in Zhejiang Province.Methods IC_ 50 value and cytotoxity of K562 cell were detected by MTT method.Apoptotic cells were dyed by Hoechest 33258.Sub-G1 peak and cell cycle were detected by FCM.Protein expression of Bcl-2 gene was detected by FCM and western-blot method.Results The snake venom of Agkistrodon Halys Pallas in Zhejiang Province inhibited the growth of K562 cells,which appeared dose-dependent.The snake venom induced apoptosis of K562 cells.Meanwhile,protein expression of Bcl-2 was down-regulated.Conclusion Snake venom of Agkistrodon Halys Pallas in Zhejiang could induce apoptosis of human leukemia K562 cells.The mechanism may be related to down-regulation of Bcl-2 gene expression.

Objective To analyze the value of pulse sequential continuous cardiac output(PICCO) in chronic pulmonary heart disease with cardiogenic shock.Methods Sixty-eight patients with chronic pulmonary heart disease complicated with cardiogenic shock were randomly divided into study group(n=36) and control group(n=32).Both groups were treated with conventional therapy,while the control group underwent deep venous puncture to monitor the central venous pressure (CVP) changes.The study group underwent deep venous catheterization+ femoral artery catheterization monitoring + PICCO monitoring.The therapeutic effect and the changes of PICCO index of the two groups were observed.Results The time of administration of vasoactive drugs,hospital stay and mechanical ventilation were significantly lower in the study group than in the control group (P＜0.05).There was no significant difference in mortality rate between the two groups(P＞0.05).After treatment,cardiac output quantity (CI) in study group increased significantly and extravascular lung water index EVLWI and PVPI decreased significantly,the difference between the two groups were statistical significance (P＜0.05),ITBVI had no statistical significant difference betweent two groups(P＞0.05).CI in the survival group was significantly higher than the death group,PVPI and EVLWI were significantly lower than the death group (P＜0.05),and the two groups had no significant difference in ITBVI (P＞0.05).Conclusion PICCO can reflect the hemodynamic status of patients with chronic pulmonary heart disease complicated with cardiac shock,and it has important clinical value for guiding the treatment and prognosis of patients.

Objecitve To investigate the relationship between the level of pregnancy-assoiated plasma protein-A(PAPP-A) and high-sensitivity C-reactive protein(hs-CRP) in patiente with acute coronary syndrome(ACS) and the role of PAPP-A in predicting the stability of atherosclerotic plaques.Methods A total of 43 patients were selected as ACS group,25 patients as stable angina(SA) and 13 persons as control.ACS group ansisted of 20 patients with unstable angina(UA) and 23 patients with acute myocardial infarction(AMI).The peripheral venous blood samples of the both control and angina groups were drawn on the morning of the following day after hospitalization, and of the AMI group were drawn 24 hours after episode.The concentration of PAPP-A was detected by enzyme-linked immune sorbent assay(ELISA),and the concentration of C-reactive protein(CRP) was detected by scatter turbidimetry method.The concentration of PAPP-A and CRP in each group was compared.Results The plasma hs-CRP and PAPP-A levels in patients with AS increased significantly compared with those in UA and healthy volunteers; PAPP-A was significantly related with hs-CRP(r=0.308);using the method of multiple regression and correlation PAPP-A had linear relationship with hsCRP in all of the cases.Conclusion PAPP-A can play a role in evaluating the clinical stability of patients with coronary disease,and may serve as one of the biomarkers of vulnerable plaques.

Objective To investigate the value of prenatal ultrasound observation of subaortic conical connection in the diagnosis of complete transposition of great arteries(TGA).Methods Echocardiography findings of 22 cases with complete TGA confirmed by autopsy were retrospectively analyzed and summarized.Results All cases were shown subaortic conical connection on two-dimensional echocardiography,the two great arteries were parallel in 18 cases on left ventricular long-axis view,the character of normal right ventricular outflow tract surrounding the aortic root disappeared in 17 cases.There were 13 cases with ventricular septal defect and 2 cases with left ventricular outflow tract obstruction.Aorta and pulmonary artery were shown in right anterior and left posterior position among 20 cases,and in anterior and posterior position in 2 cases.Conclusions Prenatal ultrasound observation of subaortic conical connection is of great value in the diagnosis of complete TGA.

Objective To observe the effects of transcutaneous electric acupoint stimulation(TEAS) at zusanli on fatigue after training.Methods 40 soldiers with exercise fatigue after marine training were divided into 2 groups.The soldiers in treatment group were treated with TEAS on Zusanli(ST36).Those in control group recovered naturally.Results The fatigue symptoms were improved,the consistency of the hemoglobin(HGB) were significantly increased,and the levels of lactic acid(LA) were significantly decreased in treatment group(P<0.01),which seemed better than that of the controls(P<0.05).Conclusion TEAS can facilitate the recovery from fatigue after training.

BACKGROUND:There is a linker between Hsp65 and Esat6 coding a segment of water repellent polypeptide.It is very gentle and easy to be folded,which is profitable to translate the keno-folding correctly between the two proteins and to make the space structure of fusional proteins consistent with those two native ones.Then,a correct structure is formed and the immunogenicity of the fusional proteins is improved.OBJECTIVE:This study was to clone the fusional genes Hsp65-Esat6 from mycobacterium tuberculosis(MTB)H37Rv, then to construct into a eukaryotic expressing vector which contains an enhanced green fluorescent protein(EGFP) reporter gene,and finally to identify the expression of Hsp65 protein and Esat6 protein by IHC methods.DESIGN:Single sample experiment.SETTING:Department of Microbiology ＆ Immunology,Medical College of Jinan University.MATERIALS:Plasmid pVAE was donated by Professor Cao from South China University of Technology.MTB H37Rv,E.coil DH5 α,expressing plasmid pEGFP-C1 and Hela cells were donated by Doctor Hu Ping.METHODS:The experiment was conducted at the Department of Microbiology ＆ Immunology and the Medical Experimental Center of Jinan University between September 2005 and June 2006.The whole-genome was extracted from MTB H37Rv by molecular cloning technique,and used it as template to amplify Hsp65 (no terminator) gene by polyrnerase chain reaction (PCR),to recombine it with pEGFP-C1 vector after purification to construct pEGHsp65(no terminator)recombination vector.pVAE vector was used as template to amplify Linker-Esat6 gene(with terminator)by PCR, and then to recombine it with pEGHSP65(no terminator)to construct an eukaryotic expressing vector pEGHsp65-Esat6 with the fusional genes Hsp65-Esat6 inside.Finally,genes Hsp65-Esat was checked by molecule biology methods such as PCR, restriction endonuclease and DNA sequencing.Hela cells were transfected with pEGHsp65-Esat6 and the expression of EGFP and the efficiency of transfection were observed.The expressions of Hsp65 protein and Esat6 protein were detected by IHC methods.MAIN OUTCOME MEASURES:①The size of pEGHsp65-Esat6.②The DNA sequencing result of the pEGFP-C1.③The expression of EGFP in the transfected Hela cells.④The IHC results of Hsp65 protein and Esat6 protein in Hela cells.RESULTS:①The size of pEGFP-C1 was 4.7 kb,that of pEGHsp65 was 6.4 kb,and that of pEGHsp65-Esat6 was 6.7 kb.There were differences between their speeds In agarose electrophoresis.②The results showed that it was the same as reported Hsp65 sequence and Esat6 sequence of MTB H37Rv.③After transfecting pEGHsp65-Esat6 for 24 hours,EGFP was found in 30% of Hela through Laser scanning confocal microscope. But there was no EGFP in non-transfected Hela.④Hsp65 protein and Esat6 protein with biological activities were detected in transfected Hela cells by IHC methods.CONCLUSION: Using Hsp65 and Esat6 as immunogens,we have successfully cloned and constructed a eukaryotic expressing vector which contain fusionaI genes Hsp65-Esat6 of MTB H37Rv and EGFP.

Objective To study the clinical and radiographic characteristics of complicated axis fractures combined with adjacent segment instability and explore reasonable surgical treatment strategy. Methods A retrospective study was performed on 21 patients with axis fractures treated from August 2003 to June 2009. There were 14 males and 7 females at mean age of 34 years. The treatment strategy was based on the fracture type and the stabilities of adjacent atlantoaxial joint and intervertebral C2/3.Treatment strategies included anterior C2/3 interbody discectomy and fusion, anterior cervical plate internal fixation, odontoid screw fixation, posterior C1-2 pedicle screw fixation, cervical lateral mass screw fixation or combined anteroposterior approach. Results All patients were immobilized in a hard collar for thee months and followed up for 6-36 months (average 12 months), which showed bony fusion and cervical stability, with no intraoperative surgery-related complications such as loosening, extrusion or breakage of fixation, vertebral artery injury, nerve damage, cerebrospinal fluid leakage or wound infection. Neurological recovery was observed in five patients. Conclusions For complicated atlas fractures, correct identification of fracture type and instability disturbance of adjacent atlantoaxial joint and C2/3 as well as active treatment can conduce to better effect.

Objective To study the distribution and clinicopathological characteristics between VEGF-C and pefitumoral lymph vessels density(PLVD) in breast cancer tissue, and to investigate the development and the mechanism of breast cancer-related lymphoedema (BCRL). Methods VEGF-C and VEGFR-3 were detected by using immunohistochemical technique for the detection of VEGF-C and its receptor VEGFR-3 in forty-seven breast cancer specimens. We measured the patients' circumferences of bilateral upper limbs to determine whether there was lymphoedema and made classification in the follow-ups. Results VEGF-C was positive in 33 out of 47 cases. PLVD significantly increased in VEGF-C positive groups (30.39±10. 46) than in negative groups (23.16±11.67) (P<0.05). VEGF-C semi-quantitative score was in a positive correlation with PLVD (r=0.334). The positive expression rate (42.55%) and semi-quantitative score (3.68±1.59) of VEGF-C increased in the lymph node positive group than in the negative group, PLVD increased in the lymph node positive group compared with that in negative group (32.12±10.29 vs. 24.82±11.06), P<0.05. The risk of lymphoedema increased in the VEGF-C negative group (5/14) compared with that in the positive group (3/33) (P<0.05). Conclusion VEGF-C has a high rate of positive expression in breast cancer, and is positively correlated with PLVD. High expression of VEGF-C can reduce the risk of BCRL in breast cancer.

Objective To investigate the role of C-Jun N-terminal kinase (JNK) in epithelial mesenchymal transition (EMT) induced by transforming growth factor β1 (TGF-β1) in rat peritoneal mesothelial cells(RPMCs). Methods RPMCs were harvested from the peritoneum of male Sprague-Dawley rats, then cultured in DMEM/F12 medium with 15% (V/V) FBS. After stimulation with TGF-β1, the expression of a-smooth muscle actin (α-SMA), E-cadherin and collagen I were detected in RPMCs. In some groups, the ceils were pretreated with SP600125, a specific inhibitor of JNK, for 4 hours before incubation with TGF-β1. The protein expression of phosphorylated JNK was detected by Western blotting. The mRNA and protein expression ofα-SMA, E-cadherin and collagen I were examined with RT-PCR and Western blotting, respectively.The intracellular distribution and expression of α-SMA was determined by indirect immunofluorescence. Results TGF-β1 could significantly increase the expression of α-SMA and collagen I, and decrease the expression of E-cadherin in RPMCs. TGF-α1 could stimulate the expression of phosphorylated JNK at 5 minutes with the peak at 10 minutes (P<0.01). The addition of SP600125 effectively inhibited TGF-β1-induced high expression of α-SMA and collagen I (P<0.05), and prevented TGF-β1-induced down-regulation of E-cadherin expression in RPMCs (P<0.05). The indirect immunofluorescence showed that the expression of intracellular α-SMA in RPMCs stimulated by TGF-β1 for 48 h increased significantly, which could be inhibited by SP600125. Conclusions JNK regulates epithelial mesenchymal transition induced by TGF-β1 in rat peritoneal mesothelial cells. JNK inhibitor may be used as a novel therapeutic agent for peritoneal fibrosis.

Object To observe the effects of the flavonoids extracted from Cuscuta chinensis Lam. (FC) on the ovarian endocrine functions in female rats exposed to psychologic stress. Methods Sound, light and electricity were combined into a stressful stimulus to induce dysfunction on ovarian endocrine functions in female rats; to observe the effects of FC on the ovarian endocrine functions in female rats exposed to psychologic stress, by determining the changes of serum E 2, P and ascorbic acid levels in adrenal gland, and by weighing the pituitaries, ovaries and uteruses. Results FC increased the serum E 2, P and the weight of pituitaries, ovaries and uteruses obviously, but did not increase ascorbic acid levels in adrenal gland in female rats exposed to psychologic stress. Conclusion FC improves the ovarian endocrine functions in female rats.