Objective To investigate the roles of protein kinase C(PKC)β_2 and PPARα in the mRNA expression of vascular endothelial growth factor(VEGF)and vascular cell adhesion molecule-1(VCAM-1)in human umbilical vein endothelial cells(HUVECs)exposed to high glucose,and to explore their relationship.Methods The HUVECs were divided into eight groups:normal glucose(NG,5 mmoL/L D-glucose)group,high glucose(HG,25 mmol/L D-ucose)group,osmotic control(L,NG+20 mmol/L L-glucose)group,normal glucose transfected with empty vector(NN,NG+AdS-null)group,high glucose transfected with PKCβ_2(HB,HG+Ad5-PKCβ_2)group,high glucose plus fenofibrate(HF,HG+40 μmol/L fenofibrate)group,and HB plus fenofibrate (HBF,HB+40μmol/L fenofibrate)group.HUVECs were incubated with fenofibrate for 20 minutes as HF20 group.All cells in various groups were cultured for 6 days.The expressions of VEGF and VCAM-1 mRNA were detected by RT-PCR.PPARα protein expression was tested by Western blot.The expression and traaslocation of PKCβ_2 protein were observed by confocal laser scanning microscopo.Results (1)HG increased VEGF and VCAM-1 mRNA expressions,with 1.91 and 1.56 folds of NG group,respectively(both P<0.05).VEGF and VCAM-1 mRNA expressions in HB group further increased,with 2.59 and 2.07 folds of NG group,respectively(both P<0.05).Fenofibrate significantly decreased VEGF and VCAM-1 mRNA expressions,with 68%and 74%of HG group,respectively(both P<0.05).There were no significant difierences in the expressiong of VEGF,VCAM-1 mRNA between HF20 and HG groups.(2)The protein expression of PPARα decreased by 20%in HG group compared with NG group,and further decreased in HB group,being 78%of HG group.Compared with HG group,PPARαexpression increased by 13%in HF group(P<0.05).(3)HG induced PKCβ_2 translocation from cytosol to nucleus and quantitative analysis showed the ratio of plasma/nuclear fluorescence intensity in HG group decreased by 37% compared with NG group(P<0.05).The PKCβ_2 translocation was more obvious in HB group than in HG group.Conclusion Hiigll glucose stimulates VEGF and VCAM-1 mRNA expressions in HUVECs via PKCβ_2 activationdependent PPARα pathway.

Objective:To explore effects of trehalose as a cryopreserve agent on survival rate of fatty tissue after cryopreservation.Methods:The liposuction was used on the abdomen of adult female.After centrifugation and purification,adipose was randomized into the following three groups,the trehalose group,the fetal bovine serum (FBS)+ 10％DMSO group and the physiological saline group.The specimens were cryopreserved at-196 ℃ for 3 months and then the HE staining,glucose transfer method and CK method were used to detect the cell survival rate in each group.Results:The activity of adipose in the trehalose group and FBS+10％DMSO group adipose was higher than that in the physiological saline group (P＜0.05);while there was no significant difference between the trehalose group and FBS+10％DMSO group (P＞0.05).Conclusion:As cryoprotectant,trehalose could keep fat cell viability,and adipose tissue can be used for clinical transplantation after 3 months' freezing.

Objective To investigate the effects of Blastocyst MHC gene transfection to coronary on the survival time of mouse heart grafts and the mechanism. Methods Inbred male Balb/c mice and C57BL/6 mice were selected as donors and recipients respectively, to construct mouse cervical heart transplantation models. In the control group, the donor hearts were perfused using the 0～4 ℃ St. ThomasⅡ solution; in the cyclosporine A (CsA) group, the donor hearts were perfused as same as the control's and received intraperitoneal injection of CsA (5 rng·g-1·d-1) after surgery; in the transfection group, the donor hearts were perfused using St. Thomas Ⅱ solution with Blastocyst MHC gene plasmid; in the combined treatment group, the donor hearts were perfused using St. Thomas Ⅱ solution with Blastocyst MHC gene plasmid and received intraperitoneal injection of CsA (5 mg·g-1·d-1) after surgery. The survival time of transplanted heart allografts were observed, and their histopathological changes and the degrees of coronary intimal hyperplasia were estimated.Blastocyst MHC gene mRNA expression levels were detected by real-time fluorescence quantitative RT-PCR. Flow cytometry was applied in assessment of the levels of CD4+ CD25+ regulatory T cells (Treg) and CD3+ CD8+ T cells. Results The survival time in the CsA group, transfection group and combined treatment group was significantly longer than in the control group (P＜0.05) and that in the combined treatment group was the longest, up to (20. 50 ± 5. 61) days. On the postoperative day 1 and 3, Blastocyst MHC gene mRNA expression level in the transfection group was significantly higher than that in the control group (P＜0.05). On the postoperative day 7, the degrees of rejection and coronary intimal hyperplasia in the combined treatment group were the lightest. On the postoperative day 7 the number of Tregs in the CsA group and the combined treatment group was significantly increased as compared with that in the control group (P＜0.05), but that of CD3 + CD8+ T cells in the CsA group and the combined treatment group was less than that in the control group (P＜0.05). Conclusion Blastocyst MHC gene transfection in mouse transplanted cardiac allograft can extend its survival time through upregulation of Treg and downregulation of CD3 + CD8 + T cells in the mice. The combination of Blastocyst MHC gene and CsA may exert the synergic effects.

Objective To access the effects of aldosterone (ALD) and its receptor antagonist- spironolactone (SPI) on the production of reactive oxygen species (ROS) and apoptosis in podocytes and to explore the possible mechanism involved. Methods Conditionally immortalized mouse podocytes were divided into control group, ALD group, SPI group and SPI +ALD group. The level of ROS production and the expression of nephrin protein were assayed by fluorescent spectrophotometry and indirect immunofluorescence technology. The apoptosis rate of podocytes was monitored by flow cytometry. The expression of bax and bcl-2 mRNA and protein was detected by RT-PCR and Western blotting methods. The anti-oxidant N-acetylcysteine (NAC)was applied to determine whether the effects of ALD on podocytes were mediated by ROS pathway.Results Compared with the control group, ALD significantly increased ROS production in podocytes (P＜0.05). SPI completely abolished the above-mentioned effect of ALD (P＜0.05). ALD induced the down-regulation of the expression of nephrin and the up-regulation of podocytes apoptosis (P＜0.05), which was accompanied with the elevated expression of bax mRNA and protein and the reduced expression of bcl-2 mRNA and protein (P＜0.05). SPI or NAC prevented the above-mentioned changes induced by ALD (P＜0.05). Conclusion ALD increases theexpression of pro-apoptotic factor (bax) but decreases the expression of anti-apoptotic factor (bcl-2)to induce podocytes apoptosis through the mineralocorticoid receptor (MR) possibly via the mechanisms involving ROS pathway.

Objective Cervical heterotopic heart transplantation model was established in different inbred strains of mice with modified cuff technique. Inbred male Balb/c mice and C57BL/6 mice were selected as donors and recipients, respectively. Mice were randomly assigned into four groups: control group (the donor hearts were perfused through coronary artery with 200 μl, 0℃～4℃ St. Thomas Ⅱ solution during 2 to 3 min, then they were immersed in it for 15 min), CsA group ( the donor hearts were perfused with the same method as for the control's and intraperitoneal injection of CsA 5 mg· g-1 · d -1 was given after surgery ), H2-B1 transfection group (the donor hearts were perfused through coronary artery with 200 μl, 0℃ -4℃ St. Thomas Ⅱ solution contained with 30 μg H2-Bl plasmid vectors during 2 to 3 min, then they were immersed in it for 15 min ), and H2-B1 + CsA group ( the donor hearts were perfused with St. Thomas Ⅱ solution contained H2-Bl gene plasmid and intraperitoneal injection of CsA was given after surgery as mentioned above. ). At 1,3 and 7 days after transplantation, three allografts were harvested at each time points in all of the groups, respectively, for pathological examination and analysis of CD40 expression with immunohistochemistry assays. The expression of Th1/Th2 cytokines were also determined with flow cytometry. The survival time of rest allografts were observed. Results Histological features for rejection were observed more apparent in the grafts of control group than those in other groups, especially those in H2-Bl + CsA group. The expression of CD40 in H2-Bl + CsA group and CsA group was lower significantly than that of the control group ( P ＜0.01 ), so was the expression of CD40 in the H2-Bl group as compare with that of the control group (P ＜0.05). No significant difference between H2-Bl group and CsA group (P ＞0.05 ) at 7 days was observed. The expression of IL-2, TNF-α (Th1 cytokines) in control group was much higher than that in other groups, and the expression of IL-4 ( Th2 cytokine) in control group was much lower ( P ＜0.05 ). The level of IL-4 in CsA group increased significantly at 3 days ( P ＜ 0.05 ), with a peak level at 7 days after transplantation (P＜0.01). The survival time of grafts was significantly prolonged in CsA group (P＜0.01), H2-Bl group (P＜0.05) and H2-Bl+CsA group(P＜0.01). Conclusion Treating the donor hearts with H2-Bl plasmid vectors at the time of transplantation may suppress rejection in the heart allografts and prolong the survival time through some presumed mechanisms such as preventing upregulation of CD40 expression, relucing the production of IL-2 and TNF-α, increasing the production of IL-4, and as a result, inducing immune tolerance, as well as improving the function of transplanted heart grafts.

Objective To investigate the effect of Hp infection on the inflammatory factors, gastrointestinal hormones and insulin resistance in patients with type 2diabetes mellitus.Methods A total of 86patients with type 2diabetes mellitus treated in a hospital from May 2014to October 2016were enrolled in the study.All patients underwent 13C urea breath test after admission, and according to the test results, patients were divided into the observation group, which was type 2diabetes mellitus with Hp infection (n=46) and the control group, which was type 2diabetes mellitus without Hp infection (n=40) .Inflammatory factors such as highsensitivity C reactive protein (hs-CRP) , serum interleukin 6 (IL-6) , tumor necrosis factorα (TNF-α) , gastrointestinal hormones such as serum gastrin (GAS) , somatostatin (SS) and fasting blood glucose (FPG) , fasting insulin (FINS) , insulin resistance index (HOMA-IR) were compared between the two groups, and the correlation of the observation indicators with Hp infection was explored.Results Levels of hs-CRP, IL-6, TNF-αand GAS, FPG, FINS and HOMA-IR in the observation group were significantly higher than those in the control group, while SS was significantly lower than that in the control group, the difference was statistically significant (P<0.05) .Further Sperman correlation analysis showed that hs-CRP, IL-6, TNF-α, GAS, FINS and HO-MA-IR were positively correlated with Hp infection (r=0.452, 0.350, 0.398, 0.389, 0.421, 0.568, P<0.05) , and SS was negatively correlated with Hp infection (r=-0.401, P<0.05) .Conclusion Hp infection is closely related to inflammatory reactions, gastrointestinal hormone disorders and insulin resistance in patients with type 2diabetes mellitus, and taking reasonable preventive measures is the key to delay the progression and deterioration of type 2diabetes mellitus.

Objective To investigate the relationship between obesity and esophageal high resolutionmanometry ,24‐hour pH monitoring and gastroscopic results of patients with gastroesophageal reflux disease (GERD) .Methods A total of 196 patients with GERD(DeMeester score>14 .72) were selected and divided into normal weight group (18 .5 kg/m2 < BMI < 24 kg/m2 ) , overweight group (24 kg/m2 ≤BMI<28 kg/m2 ) and obese group (BMI≥28 kg/m2 ) according to body mass index (BMI) . Esophageal high resolution manometry ,gastroscopy and 24‐hour pH monitoring were performed with DeMeester score calculated . The classification of esophagitis was according to Los Angeles standard . Normal distributed measurement data were compared by analysis of variance .Non normal distributed measurement data were repesent as M(P25 ,P75) ,and were compared by rank sum test .Chi square test was for count data comparison .Results Compared with normal weight group and overweight group , abdominal length of low esophageal sphincter (LES) of obese group was shorter (1 .90 cm ,0 .85 cm to 2 .45 cm ;2 .85 cm ,2 .23 cm to 3 .20 cm ;2 .50 cm ,1 .98 cm to 3 .0 cm ) , and the differences w ere statistically significant (Z=19 .913 ,P<0 .01) .But there was no significant difference in pressure ,total length of LES and distal esophagus amplitude (all P>0 .05) .The percent total time pH≤4 of obesity group was 15 .42% (10 .31% to 21 .49% ) ,percent supine time pH≤4 was 14 .21% (5 .75% to 34 .98% ) and percent upright time pH≤4 was 14 .25% (8 .19% to 18 .13% ) .The reflux episodes (106 .50 ,67 .00 to 145 .75) and the longest duration of reflux episodes (28 .10 min ,10 .90 min to 47 .93 min) were more than those of normal group (9 .74% ,5 .35% to 15 .96% ;7 .31% ,3 .25% to 11 .80% ;8 .45% ,5 .43% to 17 .48% ;72 .50 ,53 .00 to 100 .50;15 .80 min ,9 .90 min to 21 .28 min) and overweight group (11 .36% , 6 .74% to 15 .87% ;7 .74% ,2 .36% to 15 .05% ;11 .27% ,3 .37% to 14 .73% ;85 .50 ,58 .75 to 117 .75;21 .40 min ,11 .50 min to 39 .90 min) ,and the differences were statistically significant (Z=7 .054 ,11 .181 , 6 .429 ,6 .452 ,8 .246 ,all P<0 .05) .The incidences of hiatus hernia and reflux esophagitis of the obese group (both 56 .67% (17/30)) were both higher than those of normal weight group (36 .46% (35/96) and 30 .21% (29/96)) and overweight group (30 .00% (21/70) and 27 .14% (19/70)) ,and the differences were statistically significant (χ2=6 .439 and 9 .000 ,both P<0 .05) .However ,there was no statistically significant difference among the three groups in the incidence and severe degree of asthma as an extra esophageal appearance (all P>0 .05) .There was no statistically significant difference in the incidence of Barrett′s esophagus among three groups (all P>0 .05) .Conclusions Compared with that of normal weight group and overweight group of patiento with GERD ,abdominal length of LES of obesity group was shorter .With an increase in BMI , acid exposure and the incidences of reflux esophagitis and hiatal hernia also increased .

〔Abstract〕 The paper introduces the informatization construction status of biobank in Peking University People's Hospital, including construction scope, basic infrastructure, steps as well as informatization management system application status, elaborates the application effect.The informatization construction of biobank could shorten the time of operation and ensure the quality of the samples.

Objective To explore the key molecular targets and possible mechanisms of Aidi injection in the treatment of ovarian cancer using network pharmacology and cell experiments.Methods TCMSP database was used to screen the active ingredients and tar-gets of Aidi injection,and the abnormal expressed genes of ovarian cancer were screened,and the possible targets of Aidi injection in o-varian cancer were obtained after intersection analysis.Then,protein-protein interaction analysis,drug-compact-target network con-struction and enrichment analysis of possible targets were performed.The target was further screened,and the key genes related to the prognosis of ovarian cancer were experimentally verified.After treated with 50mg/ml Aidi injection,the cell proliferation ability was ob-served by CCK-8 assay,and the expression of core target genes was detected by real-time quantitative polymerase chain reaction.Results A total of 13 possible targets of Aidi injection in ovarian cancer were screened.These targets were mainly enriched in signaling pathways closely related to the occurrence and development of tumors,such as apoptosis,platinum resistance and interleukin-17.Among the 13 genes,claudin 4(CLDN4),secretory leukocyte peptidase inhibitor(SLPI)and baculoviral IAP repeat containing 5(BIRC5)were associated with the prognosis of ovarian cancer.Cell experiments showed that Aidi injection significantly inhibited the proliferation of ovari-an cancer cell,promoted the expression of BIRC5,a protective target of ovarian cancer,while significantly decreased the levels of ovarian cancer risk factors CLDN4 and SLPI.Conclusion Aidi injection may achieve multi-component,multi-target and multi-pathway anti-ovarian cancer and combination chemotherapy by affecting the expression of CLDN4,SLPI and BIRC5.

Objective To observe the change of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression during 3T3-L1 adipocyte differentiation as well as other reference gene expressions.Methods The mRNA expressions of several common reference genes were detected by real time-PCR on day 0,1,3,5,and 7 of 3T3-L1 adipocyte differentiation.Western blot was used to confirm the protein expressions of three common reference genes.Results (1) GAPDH and transferrin receptor(TFRC) mRNA expressions were significantly increased during adipocyte differentiation.GAPDH mRNA level was increased by 5.7,7.6,22.0,and 24.5 folds on day 1,3,5,and 7 after induction of adipocyte differentiation,but no apparent changes of β-actin,α-tubulin,peptidylprolyl isomerase A (PIPA),and 18S mRNA expressions were detected.The expression changes of key transcript factors for adipocyte differentiation such as PPARγ2,C/EBPα,and C/EBPβ were under-estimated by real time-PCR if GAPDH was chosen as the reference gene.Western blotting results showed that the GAPDH protein level increased gradually during adipocyte differentiation,especially on day 5 and 7 after adipocyte differentiation.There were no obvious changes of β-actin and α-tubulin protein expressions.(2) Berberine significantly inhibited mRNA and protein expressions of GAPDH in the process of adipocyte differentiation.GAPDH mRNA levels were reduced by 68.1％ and 66.3％ on day 5 and 7 after induction of adipocyte differentiation,but with no significant change in other reference genes.Conclusion It is not suitable for GAPDH to be used as an endogenous reference gene during 3T3-L1 adipocyte differentiation.