Objective:To establish clear cell renal cell carcinoma(ccRCC)cell lines from clinical ccRCC specimens of Han nationality in China and to characterize the biological features.Methods:From 2005 to 2007,fresh surgical samples of ccRCC were obtained from 43 patients;the samples included primary tumor in situ,osseous metastasis,lymph node metastasis,and cancerous embolus.The samples were cultured in vitro using explant-culture method within 30-60 rain after surgery.Analysis on cell growth and colony-forming efficiency was recorded for the lines which were passaged for over 50 generations.Chromosome examination,pathological examination and tumorigenesis in NOD-SCID mice were used to determine their malignancy.Flow cytometry was used to determine expression of CA9 and CD133.Results:Most of the primary cells could only be passaged for less than 5 generations;5 lines could be serially passaged for over 5 passages,3 lines for over 10 passages,and only 2 lines could be stably passaged.One line,named RCC05-TXJ,was from osseous metastatic ccRCC and had been serially passaged for 110 generations in 21 months;the average doubling time was 19.2 h,average chromosome number was 75,and colony forming efficiency was 41%.Another line,named RCC05-ZYJ,was from primary ccRCC specimen and had been serially passaged for 160 generations in 18 months;the average doubling time was 16.5 h,average chromosome number was 55,and the colony forming efficiency was 37%.Immunohistological analysis demonstrated that both lines expressed CA9 and CD133.Flow cytometry analysis found that expression levels of CA9 and CD133 increased with the passages.Both RCC05-ZYJ and RCC05-TXJ lines were able to form tumor and to metastasize in NOD-SCID mice;however,their metastatic ability was obviously different. Conclusion:We have established 2 ccRCC cell lines with different metastatic potentials from the clinical ccRCC specimens of Han nationality in China.The ratio of tumor stem cells increases with the passages.

Objective To investigate effects of ulinastatin preconditioning on cerebral ischemia-reperfusion injury in patients undergoing operation on aorta with deep hypothermic circulatory arrest.Methods 30 patients aged 30-50 with national institutes of health stroke scale(NIHSS) ＜ 10 undergoing operation on aorta with deep hypothermic circulatory arrest,were randomly divided into 2 groups(n =15):normal saline control group(group C),ulinastatin preconditioning group(group U).In group U,ulinastatin 20 000U/kg was infused via central vein at 500-1000 U · kg-1 · min-1 from after tracheal intubation,until 10 min before ascending aortic cross-clamping.In group C,same volume normal saline was infused instead of ulinastatin.Blood samples were taken from internal carotid vein at 5 min before the beginning of deep hypothermic circulatory arrest(T1),15 min after the beginning of deep hypothermic circulatory arres(T2)and 15 min after the end of deep hypothermic circulatory arrest(T3)for determination of plasma concentrations of S-100β,CK-BB,Glutamate(Glu) 、TNF-α、IL-1 、IL-10、MDA,SOD and TGF-β1.Cerebral funcition was evaluated and scored using NIHSS at 2 day after operation.Results Plasma concentrations of S-100β,CK-BB,Glu,TNF-o、IL-1 and MDA were lower,the levels of SOD,IL-10 and TGF-β1 were higher,and the NIHSS score was lower in group U (P ＜ 0.05).Conclusion Ulinastatin preconditioning can lighten cerebral ischemia-reperfusion injury in patients undergoing operation on aorta with deep hypothermic circulatory arrest.The mechanism is involved in inhibit the formn of reactive oxygen free radical.

Objective To evaluate the role of Fas/FasL signaling pathway in ulinastatin postconditioning-induced attenuation of apoptosis in the myocardial cells of patients undergoing cardiac valve replacement with cardiopulmonary bypass (CPB).Methods Forty patients of both sexes,aged 21-59 yr,of ASA physical status Ⅱ or Ⅲ (NYHA class Ⅱ or Ⅲ),scheduled for elective cardiac valve replacement with CPB,were randomly divided into 2 groups (n =20 each):control group (group C),and ulinastatin postconditioning group (group U).In group U,ulinastatin 10 000 U/kg was perfused via the aortic root at 4 000-5 000 U·kg-1 ·min-1 starting from 5 min before aortic unclamping.In group C,the equal volume of normal saline was infused instead of ulinastatin.Myocardial specimens were taken from the right auricle at 45 min after aortic unclamping for determination of Fas,Fas ligand (FasL),caspase-8,Bcl-2 and Bax expression and cell apoptosis.The ratio of Bcl-2 expression to/Bax expression (Bcl-2/Bax) and apoptotic index were calculated.Results Fas,FasL,caspase-8 and Bax expression and apoptotic index were significantly lower,and Bcl-2 expression and Bcl-2/Bax were higher in group U than in group C.Conclusion Ulinastatin postconditioning attenuates apoptosis in the myocardial cells through inhibiting Fas/FasL signaling pathway in the patients undergoing cardiac valve replacement with CPB.

Objective To evaluate the effects of ulinastatin postconditioning and combination of ulinastatin preconditioning and postconditioning on myocardial apoptosis in patients undergoing cardiac valve replacement with cardiopulmonary bypass (CPB).Methods Eighty New York Heart Association (NYHA) class Ⅱ or Ⅲ patients of both sexes,aged 21-59 years,scheduled for cardiac valve replacement with CPB,were randomly divided into four groups (n =20 each):normal saline control group (group C),ulinastatin preconditioning group (group U1),ulinastatin postconditioning group (group U2) and ulinastatin preconditioning plus postconditioning group (group U3).In group U1,uinastatin 20000 U/kg was infused via the central vein at 500-1000 U·kg-1 · min-1 after endotracheal intubation until 10 minutes before blocking the ascending aorta.In group U2,ulinastatin 10000 U/kg was infused via the aortic root at 4000-5000 U· kg-1 · min-1 at 5-7 minutes before opening the aorta.In group U3,ulinastatin preconditioning and postconditioning were performed as described in groups U1 and U2.In group C,the same volume of normal saline was infused instead of ulinastatin.Blood samples were taken from the radial artery at 10 minutes before blocking the ascending aorta,40 minutes after blocking the ascending aorta,45 minutes after opening the aorta and at the end of operation for determination of plasma concentrations of tumor necrosis factor-alpha (TNF-α) and soluble tumor necrosis factor receptor 1 (sTNF-R1).Myocardial tissues were obtained from the right atrial appendage at 45 minutes after opening the aorta for determination of the expression of TNF-α,bcl-2,bax,caspase-3,and apoptosis.The bcl-2/bax ratio and apoptotic index were calculated.Results Plasma concentrations of TNF-α and sTNF-R1 and the expression of TNF-α,bax,caspase-3 and apoptotic index were lower and the expression of bcl-2 and bcl-2/bax ratio were higher in groups U1,U2 and U3 than in group C and they were lower in group U3 than in groups U1 and U2 (P ＜ 0.05).Conclusion Ulinastatin postconditioning can inhibit myocardial apoptosis in patients undergoing cardiac valve replacement with CPB,and the efficacy of combination of ulinastatin preconditioning and postconditioning is stronger than that of ulinastatin postconditioning.The mechanism is involved in balancing the expression of bax and bcl-2 and down-regulating the expression of TNF-α and its receptor.

Objective To evaluate the effects of ulinastatin on renal ischemia-reperfusion injury in patients undergoing operation on aorta with deep hypothermic circulatory arrest (DHCA ) .Methods Thirty patients ,aged 30-50 yr ,of ASA physical status Ⅲ or Ⅳ (NYHA Ⅱ or Ⅲ) ,scheduled for elective operation on aorta with DHCA ,were randomly divided into 2 groups ( n=15 each) using a random number table :control group (group C ) and ulinastatin group (group U ) .In group U ,ulinastatin 20 000 U/kg was infused via the central vein at 500-1 000 U·kg-1 ·min-1 from the time immediately after tracheal intubation until 10 min before ascending aortic cross-clamping .In group C ,the equal volume of normal saline was infused instead of ulinastatin .At 5 min before the beginning of DHCA (T1 ) and 15 min after the end of DHCA (T2 ) ,blood samples were taken from the extracorporeal circulation for determination of polymorphonuclear leukocyte counts , and plasma levels of intercellular adhesion molecule-1 , tumor necrosis factor-α, iterleukin-6 (IL-6 ) IL-8 , IL-10 , malondialdehyde , myeloperoxidase ,atrial natriuretic peptide ,cystatin C ,and creatinine .Results The polymorphonuclear leukocyte counts and plasma levels of intercellular adhesion molecule-1 , tumor necrosis factor-α, IL-6 , IL-8 , malondialdehyde , myeloperoxidase , cystatin C , and creatinine were significantly lower , and the plasma concentrations of IL-10 and atrial natriuretic peptide were higher in group U than in group C ( P< 0.05 ) . Conclusion Ulinastatin can attenuate renal ischemia-reperfusion injury in patients undergoing operation on aorta with DHCA and inhibition of inflammatory responses is involved in the mechanism .

Objective To investigate the role of phosphatidylinositol 3-kinase (PI3K)/protein-serine-threonine kinases (Akt) signal pathway in ulinastatin postconditioning-induced attenuation of apoptosis in myocardial cells in patients undergoing cardiac valve replacement with cardiopulmonary bypass (CPB).Methods Forty NYHA class and ASA physical status Ⅱ or Ⅲ patients of both sexes,aged 21-59 yr,scheduled for cardiac valve replacement with CPB,were randomly divided into 2 groups (n =20 each):normal saline control group (group C) and ulinastatin postconditioning group (group U).In group U,ulinastatin 10 000 U/kg was perfused via the aortic root at 4000-5000 U· kg-1 · min-1 starting from 5 min before aortic unclamping.In group C,the equal volume of normal saline was given instead of ulinastatin.Myocardial specimens were taken from the right auricle at 45 min after aortic unclamping for determination of the expression of Akt,phosphorylated Akt (p-Akt),cytochrome c,caspase-9,Bcl-2 and Bax,and cell apoptosis.Bcl-2/Bax ratio and apoptotic index were calculated.Results The expression of p-Akt and Bcl-2 and Bcl-2/Bax ratio were significantly higher,and the expression of cytochrome c,caspase-9 and Bax and apoptotic index were lower in group U than in group C (P ＜ 0.05).Conclusion Ulinastatin postconditioning attenuates apoptosis in myocardial cells in patients undergoing cardiac valve replacement with CPB through activating PI3K/Akt signal pathway.

Objective: To examine the associations of fibroblast growth factor 19 (FGF19), its co-receptor KLB gene and its receptor FGFR4 with susceptibility to sarcopenia, so as to provide insights into elucidation of sarcopenia pathogenesis and formulation of precision interventions for sarcopenia. Methods: A case-control study was conducted. Patients with sarcopenia at ages of 60 years and older included in the Zhejiang Provincial Elderly Health Surveillance Cohorts were selected as the sarcopenia group, and normal residents at ages of 60 years and older were served as controls. Subjects' demographics were collected using questionnaire surveys, and the height, body weight, appendicular skeletal muscle mass and grip strength were measured. Genomic DNA was extracted from blood samples for multiplex PCR targeted capture. The associations between the KLB gene single-nucleotide polymorphisms (SNPs) and susceptibility to sarcopenia were evaluated using multivariable logistic regression models. Results: There were 200 cases in the sarcopenia group, including 91 men and 109 women, and 180 cases in the control group, including 70 men and 110 women. All SNPs satisfied the Hardy-Weinberg equilibrium, and the minor allele frequencies were all > 0.05. There were no significant differences in the distribution of SNPs between the sarcopenia and control groups (all P>0.05). Multivariable logistic regression analysis showed that the SNP rs2687968 locus in the KLB gene was significantly associated with the susceptibility to sarcopenia among the elderly men (superdominant model), and individuals carrying the AC allele had a 2.332-fold higher risk of sarcopenia than those carrying the AA/CC allele (95%CI: 1.882-3.313). Conclusions KLB gene may correlate with the susceptibility to sarcopenia among the elderly men.

Objective: To examine the effect of dietary behaviors on handgrip strength loss among the elderly, so as to provide insights into the prevention of handgrip strength loss. Methods : Based on the health surveillance cohort among the elderly in Zhejiang Province, two villages or communities were randomly sampled from each of Shaoxing and Zhoushan cities using a multi-stage cluster sampling method, and all residents that had lived in local areas for one year and longer and had an age of 60 years and older were enrolled. Participants' demographics, dietary behaviors, smoking, drinking, and exercise were collected through questionnaire surveys, and the height, body weight and handgrip strength were measured. The handgrip strength loss was diagnosed according the 2019 Consensus Update on Sarcopenia Diagnosis and Treatment proposed by Asian Working Group for Sarcopenia, and the effect of dietary behaviors on handgrip strength loss was examined using a multivariable logistic regression model. Results: A total of 1 265 residents were enrolled, with a mean age of (70.67±7.30) years, and including 565 men (44.66%) and 700 women (55.34%). The overall prevalence of handgrip strength loss was 42.85% among the participants, and the prevalence was 40.35% in men and 44.86% in women, respectively. Multivariable logistic regression analysis showed that nut intake for 1 to 3 times a week (OR=0.180, 95%CI: 0.088-0.367) and for 4 to 6 times a week (OR=0.241, 95%CI: 0.113-0.514) led to a reduced risk of handgrip strength loss among the elderly, and intake of sugary drinks for 4 to 6 times a week led to an increased risk of handgrip strength loss among the elderly (OR=2.298, 95%CI: 1.120-4.714) after adjustment for age, body mass index, educational level and exercise. Conclusion Intake of nuts and sugary drinks may affect the development of handgrip strength loss among the elderly.

ObjectiveTo investigate the effects of ulinastatin postconditioning and combining ulinastatin postconditioning with pretreatment on myocardial inflammatory response in patients undergoing cardiac valve replacement under CPB.MethodsEighty NYHA class Ⅱ or Ⅲ patients of both sexes aged 21-59 yr undergoing cardiac valve replacement under CPB were randomly divided into 4 groups ( n =20 each): group control (group C) ; group ulinastatin pretreatment ( group U1 ) ; group ulinastatin postconditioning (group U2 ) and group ulinastatin pretreatment and postconditioning combined (group U3 ).Ulinastatin 20 000 U/kg was infused via central vein at 500-1000 U·kg-1 ·min-1 after tracheal intubation until 10 min before cross-clamping of ascending aorta in groups U1 and U3.Ulinastatin 10 000 U/kg was infused into root of aorta at 4000-5000 U· kg- 1 · min- 1 at 5-7 min before declamping of aorta in groups U2 and U3.Blood samples were obtained from radial artery before cross clamping of ascending aorta,at 40 min after aortic cross-clamping,at 45 min after declamping of aorta (T3) and at the end of operation for polymorphonuclear leukocyte (PMN) count,routine analysis of blood and determination of plasma concentrations of IL-10,TNF-α,IL-1 and IL-6 (by ELISA).Myocardial specimens were obtained at 45 min after declamping of aorta for determination of IL-1β and IL-6 expression by immune-histochemistry.Results Ulinastatin pretreatment and/or postconditioning significantly increased plasma IL-10 concentration and decreased plasma IL-1,IL-6,TNF-α concentrations and PMN count and myocardial IL-1β and IL-6 expression in groups U1,U2 and U3 as compared with group C.Plasma IL-10 concentration was significantly higher and plasma IL-1,IL-6 and TNF-α concentrations,PMN count and myocardial IL-1β and IL-6 expression were lower in group U3 than in groups U1 and U2.ConclusionUlinastatin postconditioning can inhibit myocardial imflammatory response in patients undergoing valve replacement under CPB.The protective effect can be augmented by combining ulinastatin postconditioning with pretreatment.

Objective To evaluate the effects of ulinastatin postconditioning and combination of ulinastatin preconditioning and postconditioning on myocardial apoptosis in patients undergoing cardiac valve replacement with cardiopulmonary bypass (CPB).Methods Eighty NYHA class Ⅱ or Ⅲ patients of both sexes,aged 21-59,scheduled for cardiac valve replacement with CPB,were randomly divided into 4 groups ( n =20 each):normal saline control group ( group C ),ulinastatin preconditioning group ( group U1 ),ulinastatin postconditioning group (group U2 ) and ulinastatin preconditioning plus postconditioning group(group U3 ).In group U1,uinastatin 20 000U/kg was infused via central vein at 500-1000 U·kg-1 ·min-1 from after tracheal intubation until 10 min before ascending aortic cross-clamping.In group U2,ulinastatin 10 000 U/kg was perfused via aortic root at 4000-5000 U· kg-1 · min-1 at 5-7 min before aortic unclamping.In group U3,ulinastatin preconditioning and postconditioning were performed as described in groups U1 and U2.In group C same volume normal saline was infused instead of ulinastatin.Blood samples were taken from radial artery at 10 min before ascending aortic cross-clamping,40 min after ascending aortic cross-clamping,45 min after aortic unclamping and the end of operation for determination of plasma concentrations of TNF-α and soluble tumor necrosis factor receptor 1 (sTNF-R1).Myocardial tissues were obtained from right atrial appendage at 45 min after aortic unclamping for determination the expression of TNF-d,Bcl-2,Bax and caspase-3 and apoptosis.The Bcl-2/Bax ratio and apoptotic index were calculated.Results Plasma concentrations of TNF-α and sTNF-R1 and the expression of TNF-α,Bax,caspase-3 and apoptotic index were lower,the expression of Bcl-2 and Bcl-2/Bax ratio were higher in groups U1,U2 and U3 thah group C and in group U3 than groups U1,U2 ( P ＜ 0.05 ).Conclusion Ulinastatin postconditioning can inhibit myocardial apoptosis in patients undergoing cardiac valve replacement with CPB,and efficacy of combination of ulinastatin preconditioning and postconditioning is stronger than that of ulinastatin postconditioning.The mechanism is involved in balancing the expression of Bax and Bcl-2 and down-regulating the expression of TNF-α and its receptor.