Objective:The recombinant human retinal pigment epithelium-derived factor(PEDF)protein to be obtained and the angiogenesis of the rPEDF to be identified.Methods: PEDF gene gene was amplified by PCR and cloned into pET32a,rPEDF protein was expressed in E.coli BL21 and confirmed by SDS-PAGE and Western blot.The rPEDF was purified by Ni-NTA on denature condition.The concentration of the rPEDF was determined by Bradford method.The angiogenesis of the rPEDF was determined by chick chorioallantoic membrane(CAM) method.Results: The expression plasmid pET32a-PEDF was constructed successfully.The rPEDF was expressed with stable efficiency in E.coli BL21.The results of the CAM experiment showed that the rPEDF had notable angiogenesis effect in the concentration 0.4、0.04 ng/ml,but had no effect in 4 ng/ml.Conclusion:The PEDF gene was cloned and expressed efficiently,the angiogenesis of the rPEDF to be identified and the activity was worked in certain range.The results can facilitate studying its function and spreading its application.

OBJECTIVETo purify caffeic acid tetramer (CAT) with macroporous resin on the basis of its fundamental physicochemical stability research.

METHODThe changes of CAT content were compared by HPLC method before and after the purification process, or while other conditions were altered.

RESULTLK001 was the best one among 7 kinds of macroporous resin in regard of purifying ability. The optimum absorbing technology was the solution concentration at 10 g x L(-1), pH at 4.5, and the flow rate at 3 BV x h(-1). The best eluting technology was 45% ethanol as eluting agent, pH at 5.0, eluting volume at 50 mL after applying super-purified water and 20% ethanol. The yield of product was 3. 6 percent, and the active compound CAT was 58 percent in the product.

CONCLUSIONMacroporous resin LK001 is effective in enriching CAT from the crude extracts, thus this method of purification is advisable.

Absorption ; Boraginaceae ; chemistry ; Caffeic Acids ; chemistry ; isolation & purification ; Chromatography, High Pressure Liquid ; Hydrogen-Ion Concentration ; Light ; Oxygen ; chemistry ; Porosity ; Resins, Plant ; chemistry ; Temperature ; Water ; chemistry

OBJECTIVETo explore the better therapy in the treatment of ganglion.

METHODSNinety cases of ganglion were randomized into a two-way quintuple puncture group, a common quintuple puncture group and a fire needling group, 30 cases in each one. In the two-way quintuple puncture group, the "9-in-1" multiple penetrating needling technique was used. In the common quintuple puncture group, the traditional "5-in-1" multiple penetrating needling technique was applied. In the fire needling group, the traditional multiple fire needling technique was adopted. The treatment was given once a day, 3 treatments made one session and the efficacy was analyzed statistically after 1 session treatment in the three groups.

RESULTSAll of the three therapeutic methods achieved the efficacy on ganglion. The curative rate was 96. 7% (29/30) in the two-way quintuple puncture group, which was better obviously than 66.7% (20/30) in the common quintuple puncture group and 60. 0% (18/30) in the fire needling group (both P<0. 01).

CONCLUSIONThe two-way quintuple puncture technique achieves the remarkably superior efficacy on ganglion as compared with the common quintuple puncture technique and fire needling technique.

Acupuncture Therapy ; Adolescent ; Adult ; Aged ; Female ; Ganglion Cysts ; therapy ; Humans ; Male ; Middle Aged ; Treatment Outcome ; Young Adult

In order to solve the problem of selection and in vivo delivery problem in siRNA treatment, hepatitis B virus (HBV) HBx gene which could be targeted by siRNA was studied. The siRNA expression plasmid which specific inhibits HBx expression was obtained by in vitro selection via a dual-luciferase plasmid including HBx-Fluc fusion protein expression domain. The selected siRNA expression plasmid was then encapsulated in PEG-modified cationic liposome, which was devoted into pharmacodynamic studies at both cellular and animal level. The results illustrated that the cationic liposome which encapsulated siRNA expression plasmid could effectively inhibit HBx gene expression both in vitro and in vivo.
Cations ; Gene Expression Regulation, Viral ; drug effects ; Hepatitis B virus ; genetics ; Liposomes ; chemistry ; Plasmids ; RNA, Small Interfering ; chemistry ; Trans-Activators ; genetics ; metabolism

Objective To study the effects of the serum Nesfatin-1 level change by type 2 diabetes in patients with Non-alcoholic fatty liver,and provides a reliable basis for its prysiological effects.Methods Selected 300 cases with type 2 diabetes mellitus in deoartment of endocrinology from January 2014 to January 2016 in Affiliated Hospital of Yan'an University as the object of this study.According to whether the patients were combined with non-alcoholic fatty liver,the patients were divided into T2DM with NAFLD group (150 cases) and T2DM without NAFLD group (150 cases).In addition,150 volunteers for healthy check-up in the hospital were selected as control group.Compared the serum Nesfatin-1 levels of three group patients,and monitored the insulin resistance index of two groups patients with type 2 diabetes.Results BMI,TG,ALT and INS of NGT(23.1±1.9,1.7±0.5,26.9±12.7 and 9.7±2.4,respectively) group and T2DM (22.1±2.5,2.0± 0.9,22.1 ± 10.5 and 11.2± 4.5,respectively) group were significantly lower than those of T2DM-NAFLD group (26.5 ± 3.8,3.0± 2.5,31.9 ± 11.5 and 14.2 ± 6.5;all P value were 0.00).FBG,HbAlc,LDL-C,HOMA-IR and Nesfatin-1 of T2DM group (8.4±3.1,8.8±2.7,3.4±1.0,4.5±2.9 and 6.9±3.0) and T2DM-NAFLD group (8.2±2.7,8.5± 1.9,3.7 ± 2.1,5.2 ± 2.7 and 5.2 ± 2.7) were significantly higher than those in serum NGT group (5.1 ± 0.5,5.4 ± 0.4,2.7 ±0.8,2.7±0.8 and 2.3± 0.7,all P value were 0.00).The serum Nesfatin-1 levels of T2DM patients and patients with T2DM-NAFLD had moderate correlation (r=-0.421,P＜0.05) by Pearson correlation analysis.BMI,TG and serum Nesfatin-1 levels were closely associated with the occurrence of fatty liver (r=-0.402 ～ 0.273;P=0.00 ～ 0.01).Conclusion Nesfatin-1 may be involved in type 2 diabetes mellitus and the occurrence of non-alcoholic fatty liver.

OBJECTIVETo validate the value of the Oxford classification of IgA nephropathy in predicting the renal outcome in Chinese population.

METHODSRetrospective study was done in patients with IgA nephropathy. All slides were re-assessed according to the Oxford classification of IgA nephropathy. The primary end point is doubling serum creatinine, or a 50% reduction in estimated glomerular filtration rate (eGFR), or end-stage renal disease. Pathologic predictors for the progression to the end point were determined by univariate and multivariate Cox regression.

RESULTSTotally 533 patients were enrolled in the study. During the follow-up (median: 39 months; range: 12-263 months), 5.07% of the patients reached the end point. While tubular atrophy and interstitial fibrosis and arterial/ arteriolar lesion were associated with the endpoint in univariate analysis, only the T score was predictive of the renal outcome in multivariate Cox regression. Combination of the patho- logic lesions had no impact on renal outcome.

CONCLUSIONAccording to the Oxford classification of IgA nephropathy, the degree of tubulointerstitial fibrosis is the only feature independently predictive of renal outcome.

Adolescent ; Adult ; Aged ; Female ; Follow-Up Studies ; Glomerulonephritis, IGA ; classification ; pathology ; Humans ; Kidney ; pathology ; Male ; Middle Aged ; Prognosis ; Retrospective Studies ; Young Adult

OBJECTIVETo construct the retroviral vector containing human micro-dystrophin gene and detect the expression of human micro-dystrophin in mdx mice bone marrow-derived mesenchymal stem cells (MSCs) after retrovirus infection.

METHODSRetroviral vector for micro-dystrophin gene was constructed and transferred into the packing cell PA317 mediated by Lipofectamine 2000. The retroviral supernatant containing the target genes were subsequently used to infect mdx mice MSCs. Micro-dystrophin expression was examined by methods of immunofluorescence staining and reverse transcriptase-polymerase chain reaction.

RESULTSMicro-dystrophin retroviral vector was successfully constructed and transferred into PA317 cells, and 48 h after infection with the recombinant retrovirus in mdx mice MSCs, 319 bp fragment could be detected by electrophoresis in the RT-PCR products. The red particles could be detected in some infected mdx mice MSCs with immunofluorescence staining. CONCLUSION mdx mice MSCs infected with retrovirus containing micro-dystrophin gene can express micro-dystrophin protein.

Animals ; Bone Marrow Cells ; cytology ; metabolism ; Dystrophin ; biosynthesis ; genetics ; Humans ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Mice ; Mice, Inbred mdx ; Muscular Dystrophy, Animal ; metabolism ; Retroviridae Infections ; Transfection

This study was purposed to explore the changes of possible angiogenetic factors other than VEGF after inhibition of NHE1 and their related mechanisms. The K562 cells were treated by NHE1 specific inhibitor cariporide, the angiogenesis factors after inhibition of NHE1 were screened by using protein chip, the IL-8 expression level after cariporide treatment was detected by real-time quantitative PCR; the K562 cells with stable interference of NHE1 were constructed, the IL-8 expression level after interference of NHE1 was detected by real-time quantitative PCR; the p38 phosphorylation level in K562 cells treated with cariporide was detected by Western blot. After treatment of K562 cells with p38 inhibitor SB203580, the IL-8 expression level was decreased by real-time quantitative PCR. The results of protein chip showed that IL-8 expression decreased after cariporide treatment. Real-time quantitative PCR confirmed this inhibitory effect. The p38 phosphorylation level increased after cariporide treatment. The down-regulation of IL-8 expression induced by cariporide treatment was partially restored after K562 cells were treated with p38 inhibitor SB203580. It is concluded that the inhibition of NHE1 can inhibit IL-8 expression through up-regulation of p38 phosphorylation.
Cation Transport Proteins ; antagonists & inhibitors ; Down-Regulation ; Guanidines ; pharmacology ; Humans ; Imidazoles ; pharmacology ; Interleukin-8 ; metabolism ; K562 Cells ; Phosphorylation ; drug effects ; Pyridines ; pharmacology ; Sodium-Hydrogen Exchanger 1 ; Sodium-Hydrogen Exchangers ; antagonists & inhibitors ; Sulfones ; pharmacology ; p38 Mitogen-Activated Protein Kinases ; metabolism

Purpose To investigate the clinical pathology significance of epithelial cellular adhesion molecule (EpCAM, CD326) expression in colorectal carcinoma cells. Methods Flow cytometry and immunofluorescence assay were used to detect EpCAM expression in 55 cases of fresh colorectal cancer tissues and adjacent normal mucosa. The percentage of positive cells (PPC) and mean fluorescence index (MFI) were calculated. Correlation of EpCAM expression with DNA ploidy change and its value were investigated in the early diagnosis of colorectal cancer. Results The values of PPC and MFI were significantly higher in colorectal carcinoma tissue than that in the normal colorectal tissue [(PPC: (83.48 ± 7.07)％ vs (43.56±5.29)％, t =39.22, P＜0.001. MFI: 28.90(19.60-45.89) vs4.89(3.79-6.28), Z=-6.45, P＜0.001) ]. There were also significant differences (P＜0.01) in the values of PPC and MFI between invasive type and ulcer types, between well-and moderately-differentiated and poorly-differentiated cancers, between Dukes stages A + B and C + D, (between pTNM stages I+ II and III + IV, between pTl + T2 and pT3 + T4, and between pNO and pNl. DNA content analysis showed that DNA polyploid was detected in 39 of 55 colorectal carcinoma (70.90％ ). The DNA index (DI) and ploid were correlated with differentiation degree and Dukes stages, but uncorrelated with lymph node metastasis. At the same time, in the EpCAM positive cases, DI was increased with increased expression of EpCAM (r = 0.668, P =0.000) and the proliferation index of cells in S phase ratio(Sphase fraction, SPF) (r1 =0.664, P1 =0.000, r2 =0.651, P2 = 0.000 ). Conclusion EpCAM expression is obviously upregulated in colorectal carcinoma, and it is closely correlated with the invasion, metastasis and proliferation of tumor cells. The combination of EpCAM expression and DNA content analysis provides references to the early diagnosis and prognosis evaluation in colorectal carcinoma.

OBJECTIVETo identify the exosomes-like vesicles from the plasma and study their biologic characteristics and regulatory effect.

METHODSThe exosomes-like vesicles were purified from healthy donors plasma with a series of high-speed centrifugations and ultrafiltration. Morphology was identified by transmission electron microscopy and biologic characteristics by Western blot and flow cytometry. CD4(+)T cells and CD4(+)CD25(+)CD127low Treg cells were purified from peripheral blood mononuclear cells (PBMCs) by Magnetic cell sorting. After exosomes-like vesicles cultured with CD4(+)T cells or CD4(+)CD25(+)CD127low Treg cells, cell proliferation and apoptosis were assayed. Phosphorylated β-catenin level in Wnt signaling by phosflow.

RESULTSExosomes-like vesicles from plasma were similar to previously described exosomes in shapes and size and expressed exosome marker proteins CD63 and CD81 as well as the MHC-II molecule, costimulatory molecules CD86 etc. After co-cultured with CD4(+) T cells, exosomes-like vesicles inhibited the proliferation of the T cells in a dose-dependent manner. After Treg cells cultured with exosomes-like vesicles for 14 days, the survival rate of the Treg cells was 57.07%, while that of the control Treg was 30.91%. Frizzled receptors 2, 3, 4and LRP6 gene mRNA expressed (the relative gray value was 48.50, 34.84, 23.85, 49.73) in the Treg cells by RT-PCR, and Wnt molecular expressed in exosomes-like vesicles. After Treg cells co-cultured with exosomes-like vesicles, the MFI of phosphorylated β-catenin decreased (from 20.06 ± 2.99 to 12.41 ± 2.08), and the expression of Bcl-2 mRNA was upregulated significantly (the relative gray value from 0.45 to 84.97).

CONCLUSIONSExosomes-like vesicles existed in human plasma and express immune regulatory molecules. They can suppress the proliferation of activated CD4(+) T cells induce their apoptosis and pro-long the survival of natural Treg cells via Wnt signaling pathway.

CD4-Positive T-Lymphocytes ; immunology ; Cells, Cultured ; Exosomes ; Flow Cytometry ; Humans ; Immunologic Factors ; Interleukin-2 Receptor alpha Subunit ; Leukocytes, Mononuclear ; immunology ; T-Lymphocytes, Regulatory ; immunology