Preparation of recombinant PTD-HSP27 and verification of its ability to penetrate the cell membrane of human lens epithelial cells and rabbit cor-nea / 中国病理生理杂志

AIM: To construct the prokaryotic expression system containing protein transduction domain (PTD) with heat shock protein 27 (HSP27) in order to prepare and purify the recombinant protein , and to verify whether the recombinant protein PTD-HSP27 has the ability to penetrate the human lens epithelial cell ( HLEC) membrane and the rabbit cornea.METHODS:The plasmid pKYB-PTD-HSPB1-6His was constructed by the technique of overlap extension PCR.The plasmid was transformed and PTD-HSP27 was purified through nickel affinity chromatography column and identi-fied by Western blotting.PTD-HSP27-6His was labeled with the fluorescein isothiocyanate (FITC).The penetrating ability of PTD-HSP27 into HLECs and rabbit cornea was tested .RESULTS:The recombinant PTD-HSP27 plasmid was success-fully cloned and effectively expressed .The correctness of the recombinant protein PTD-HSP27 was demonstrated .Fluores-cence microscopic examination showed that PTD-HSP27-FITC was internalized by HLECs .Fluorescent labeled PTD-HSP27 was then observed in the rabbit aqueous humor .CONCLUSION:The recombined gene PTD-HSPB1 was constructed by o-verlap extension PCR technique and the PTD-HSP27 fusion protein was prepared and purified by nickel affinity chromatog-raphy column.Using the technique of PTD-fusion protein, HSP27 was transduced into HLECs and passed through the cor-nea .

Construction of controlled expression system of class B G-protein coupled receptor PAC1 / 生物工程学报

PAC1 is the neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) preferring receptor, which belongs to class B G protein-coupled receptors (GPCR) family. PAC1 mediates the most effects of PACAP as neurotransmitter, neuroregulator and neuroprotectant, while its high expression has close relationship with some physiological and pathological processes such as nerve-injury and tumor. To further understand the function of PAC1, a cell line that expressed inducible PAC1 was constructed to achieve Doxycycline (Dox) dependent expression of PAC1 in CHO (Chinese hamster ovary) cell using the improved Tet (tetracycline)-on Advanced System. First, the PAC1-EYFP fusion gene composed of PAC1 gene and gene encoding EYFP (enhanced yellow fluorescent protein) was sub-cloned to the tetracycline response element pTRE-Tight vector to construct the recombinant vector pEYFP-PAC1-EYFP by double enzyme digestion. Second, the tetracycline regulation components pTet-On advanced vector and the response element pTRE-PAC1-EYFP vector were both introduced into CHO cells successively and the positive clones were screened with G418 and hygromycin respectively. Third, the controlled expression of PAC1-EYFP in CHO was induced by tetracycline analogues Dox in different concentrations and the different levels of receptor PAC1-EYFP were detected. The results of fluorescence analysis and western blotting show that the cell strain with Dox dependent expression of PAC1-EYFP named PAC1-Tet-CHO was obtained. Moreover, in PAC1-Tet-CHO cells the expression of PAC1-EYFP was induced by Dox in a dose-dependent manner. The inducible expression of PAC1 still was stable after sub-culturing for more than 10 passages. It was also found by MTT assay that the higher expression level of PAC1 endowed the cells with higher proliferative viabilities. The construction of controlled expression system of PAC1 will lay a foundation for the further research on PAC1 profiles.

Research progress in reprogramming induced pluripotent stem cells / 中国病理生理杂志

Induced pluripotent stem cells ( iPSCs) have been first induced from mouse fibroblasts since 2006, and the research on iPSCs has made great progress in the following years .iPS cell lines were established from different so-matic cells through DNA , RNA, protein, and small molecule compounds and various methods of transduction , making the induction of iPSCs more secure and effective , and more attractive prospect of clinical application .In this review , different somatic cell reprogramming , different levels of reprogramming , different transduction pathways , and prospect of application are discussed .

Preparation and identification of recombinant PTD-maxadilan / 生物工程学报

In order to construct a novel fusion protein PTD-maxadilan (PTD-MAX) that can enter the blood-brain barrier (BBB) efficiently, a new gene encoding PTD-MAX was synthesized and cloned into the expression vector pKYB. The recombinant vector pKYB-PTD-MAX was transformed into Escherichia coli ER2566. The expression of fusion protein consisting of PTD-MAX, intein and chitin binding domain was induced by IPTG and the target PTD-MAX protein was purified using Intein Mediated Purification with an Affinity Chitin-binding Tag system. The molecular weight of PTD-MAX determined by the laser flight mass spectrometry was coherent with its theoretical value. The results of the experiment in vivo indicated that the recombinant PTD-MAX can permeate into BBS and inhibitory effects on the food intake were more significantly than maxadilan (P<0.05). The preparation of PTD-MAX lay the foundation for its further application.

Preparation and purification of advanced oxidation protein products / 中国组织工程研究

BACKGROUND: Advanced oxidation protein products (AOPPs) are a crucial pathogenic link to such long-term uremic complications in hemodialysis patients as immune system dysregulation, accelerated atherosclerosis, dialysis-related amyloldosis and so on. However, basic studies on AOPPs are relatively few, and one of the main reasons is the fact that it is difficult to obtain AOPPs with high pudty and biological activity.OBJECTIVE: To prepare, pudfy and indentify AOPPs, with the hope of searching for a method of preparing AOPPs with high purity and biological activity.DESIGN, TIME AND SETTING: A single sample observation was completed in the Clinical Biochemistry Section of Ecsomatics Department, Third Military Medical University of Chinese PLA from September to November in 2008. MATERIALS: Human serum albumin (HSA) was provided by Chengdu Rongsheng Company Ltd. Hitrap 26/60 sephacryl S-300 was purchased from GE Healthcare.METHODS: Hypochloric acid was used in the oxidation of purified HSA to prepare in vitro the AOPPs-modified HSA (AOPPs-HSA), which was then isolated by Hitrap 26/60 sephacryl S-300. Relative molecular mass was determined by sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE), native polyacrylamide gel electrophoresis (PAGE) and molecular weight standards. Structural features and biological activities were identified in the experiment of tumor necrosis factor α (TNF-α) release from monocytes.MAIN OUTCOME MEASURES: ①The purification and gel electrophoresis results of HSA. ②The purification and gel electrophoresis results of AOPPs. ③The dose-effect relationship between AOPPs-HSA and TNF-α release from monocytes. RESULTS: The relative molecular mass of AOPPs-HSA was 670 000 according to SDS-PAGE, native PAGE and molecular weight standards. Moreover, AOPPs-HSA could encourage the release of TNF-α from monocytes. The time effects showed that TNF-α release volume significantly increased after 6 hours of stimulation by AOPPs-HSA (1 g/L) and reached a peak at hour 12. CONCLUSION: Highly purified and bioactive AOPPs can be successfully prepared by the above-mentioned method, which builds a basis for further study on AOPPs.

Preparation and identification of recombinant maxadilan / 生物工程学报

To produce recombinant Maxadilan using gene engineering technology, the gene of recombinant Maxadilan which expressed in protocaryon were designed and synthesized according to the amino acid sequences of Maxadilan. The recombinant plasmid pKYB-MAX was constructed and transformed into host bacteria Escherichia coli strain ER2566. After the MAX-intein-CBD fusion protein was purified by chintin-affinity chromatography, the self-cleavage activity of the intein was induced by beta-mercaptoethanol and the recombinant Maxadilan was released from the chitin-bound intein tag. The molecular weight of peptides was determined by the laser flight mass spectrometry and the results was conformity with the theoretical value. The biological activity analysis showed that recombinant Maxadilan significantly enhanced the concentration of serum glucose.

Impact of transplant nephrectomy on panel reactive antibodies / 第三军医大学学报

Objective To observe the impact of transplant nephrectomy on panel reactive antibodies and a secondary renal transplantation.Methods Panel reactive antibodies in 15 patients with a failed renal transplant admitted in our hospital between 2004 to 2007 were measured before transplantation,before and 1 month,6 months,12 months after transplant nephrectomy,and the pathological changes were observed after transplant nephrectomy.Results Panel reactive antibodies was increasing after renal transplantation,and reached the highest level one month after transplant nephrectomy,then grandually got down.New HLA allosensitization sites were discovered after transplant nephrectomy.Large amount of C4d was stained in failed transplant.Conclusion Serum PRA increased after transplant nephrectomy.New HLA allosensitization sites were found,which may be useful in HLA matching.

Endothelial progenitor cells transplantation ameliorates the progression of progressive focal segmental glomerular sclerosis / 第三军医大学学报

Objective To investigate the therapeutic effect of endothelial progenitor cells(EPCs) in ameliorating rat progressive focal segmental glomerular sclerosis(FSGS) model induced by adriamycin.Methods Bone marrow mononuclear cells from male SD rats,after cultured by adherence method,were identified as EPCs.Female SD rats were divided into normal control group,adriamycin induced renal disease(ADR) group,EPCs transplantation group.ADR group and EPCs group underwent unilateral nephrectomy and received 5,3 mg/kg of adriamycin via tail vein 1 week and 2 weeks after operation,while the control group underwent sham operation and received 0.9% sodium chloride solution of equal volume.The whole body irradiation by 5 Gy X ray was done 1 week after the 2nd injection of adriamycin,then immediately 1?106 EPCs were transplanted via tail vein.The rats in control group and ADR group were only injected with 0.9% sodium chloride solution after whole body irradiation.The body weight and urine protein were measured before operation(0 week) and 4(1 week after EPCs transplantation),8,12 and 16 weeks after nephrectomy.Y chromatosome incorporation was detected with in situ hybridization at the 4th and 16th week.The histological and ultrastructural changes of kidney were evaluated at the 16th week.Results At the 4th and 16th weeks,Y chromatosome positive cells could be found incorporation in the area of glomerular and tubular epithelial cells.Since the 4th week,the weight of rats in both ADR group and EPC group became significantly less than that in control group and since the 8th week that in ADR group became less than that in EPC group(P

Effect of valsartan on P27 expression in mesangial cells of Thy1 glomerulonephritis rats / 第三军医大学学报

Objective To observe P27 expression and proliferation of mesangial cells of rats with Thy1 glomerulonephritis and to evaluate the interfering effect of valsartan, a specific angiotensin Ⅱ receptor antagonist, on Thy1 glomerulonephritis. Methods Rats were divided into normal control group, Thy1 glomerulonephritis group and valsartan treatment group. Following the onset of glomerulonephritis, renal morphological changes were observed on days 1, 3, 5 and 7, and proliferating cell nuclear antigen(PCNA) and P27 expression in glomerulus were measured by immunohistochemistry and Western blotting. Results High expression of P27 was found in quiescent mesangial cells of normal rats, but decreased expression in mesangial cells of rats with Thy1 glomerulonephritis, accompanying with the proliferation of mesangial cells. In valsartan treatment group, glomerular hypercellularity, mesangial matrix expansion and PCNA expression were significantly reduced as compared with those of the untreated rats with Thy1 glomerulonephritis from day 3 to day 7( P

Remission of severe nephrotic edema by hemodiafiltration and its effect on the prognosis of nephropathy / 中国实用内科杂志

Objective To evaluate the soothing effects of hemodiafiltration on patients with refractoriness nephrotic edema and its influence on prognosis of nephrotic syndrome.Methods All the 15 cases involved had undergone hemodiafiltration periodically during acme edema and administered with the same standard loading dose of prednisone for 8 weeks.Meanwhile,clinical and lab indexes (urine volume,Cr,Bun,urinary protein,albumin) were measured.Results All patients improved in urine volume and renal function gradually after the treatment of hemodiafiltration for 1 to 3 times.By the 8th week of post-hemodiafiltration,urinary protein 24 h declines obviously[(6.42?2.31)g/d to (0.87?1.24)g/d].Conclusion Hemodiafiltration is an effective treatment in relieving retention of water,increasing the remission rate of protein urine,recovering patients' renal function and improving their prognosis to nephritic syndrome patients with severe refractory edema.

Study of immune-associated genes in the aged rats by cDNA microarray / 中国免疫学杂志

Objective: To screen the changes of immune-associate genes expression which is related with the ageing using cDNA microar-ray.Methods:The mRNA from the spleens of young and aged rats were extracted respectively and reversely transcribed to cDNAs with the incorporation of different fluorescent-labeled dUTP as the hybridization probes. The mixed probes were hybridized to the cDNA microarray that contains the cDNA products of 416 immune-associated genes. After high-stringent washing, the cDNA microarray was scanned for the fluorescent signals and showed the gene expression differences between the young and the aged. Some biochemical assays were used to confirm the physiological differences between the young rats and the aged rats. Results: Among the examined genes, 13 down-regulated genes were identified. These genes correlated with immuned response, cell proliferation, cell differentiation and DNA/RNA repair. Only one gene which encoded ?-amylase was much higher in the aged than that in the young. Conclusion: Further analysis of the differenially expressed immune-associated genes based on cDNA microarray will be helpful for understanding the molecular mechanism of the ageing.

Molecular cloning, fusion expression and bioactivity of pro-nattokinase gene / 中国病理生理杂志

AIM: To construct engineered E.coli strains which can express nattokinase with fibrinolysis activity using gene engineering technology. METHODS: The pro-nattokinase (pro-NK) gene was amplified by PCR and inserted into expression vector pET3c. The recombined plasmid pENK which expressed the fusion protein of pro-NK and 22 amino acid peptide was then transferred into lysogenic host strains BL21(DE3)pLysS - and BL21(DE3)pLysS +. Both SDS-PAGE and the fibrin plate assay were used to examine the expression and the activity of the target protein. RESULTS: SDS-PAGE assay showed the fused gene encoding 42 kD fusion protein was expressed in both expression strains pENK-(DE3)pLysS - and pENK-(DE3)pLysS +, and the fibrin plate assay indicated that the expression product had fibrinolysis activity. pENK-(DE3)pLysS - exhibited the basal expression of the target gene, while fusion protein was only induced by IPTG in pENK-(DE3)pLysS +. Basal expression of the fused toxic gene in pENK-(DE3)pLysS - led to bacteriolysis and hollow lawns. CONCLUSION: A pro-NK fusion protein with fibrinolysis activity is successfully expressed in E.coli , which lay a foundation for the exploitation of nattokinase.

Expression, purification and biological assay of recombinant hEGF-hbFGF(78-154aa)fusion protein / 中国病理生理杂志

AIM: To construct a recombinant hEGF-hbFGF(78-154aa)fusion protein, which not only has the heparin-binding ability, but also promotes the growth of the cells, and to express the fusion protein in E. coli expression system with high expression level.METHODS: hEGF gene was joined with 231 bp fragment coding hbFGF(78-154aa) and expressed in E. coli. The fusion protein was purified using affinity chromatography of heparin-Hyper D and analyzed with western blot. The pI value and the biological activity were both assayed.RESULTS: The fusion protein was expressed in a high expression level of about 30% of the total cell protein, as estimated by SDS-PAGE. Western analysis results showed that the antigenicity of fusion protein was similar to hEGF. Fusion protein could not only bind heparin but also promote the growth of 3T3 cell. The pI value of fusion protein was 5.2.CONCLUSION: The recombinant hEGF-hbFGF(78-154aa) fusion protein possessed the characteristics of both hEGF and hbFGF. This new-designed protein would become a good object for the research on the relationship between the structure and the function of the growth factor.

Synthesization, expression, purification and activity assay of hIGF-1 / 中国病理生理杂志

AIM: To express the synthesized human insul in like growth factor I (hIGF-1) gene in E.coli with high expression level a nd explore the way to increase the efficiency of factor Xa cleavage. METHODS: The gene of hIGF-1 was designed and synthesized accordi n g to the preference of E.coli. A fusion protein with a recognized site of fa ctor Xa between CBD (cellulose binding domain) and hIGF-1 was expressed and puri fied by cellulose affinity chromatography. MTT method was used to assay the bioa ctivity of CBD-IGF fusion protein. hIGF-1 was released by factor Xa. In order to improve the sensitivity of fusion protein to factor Xa, the short flexible pept ide (Gly-Thr-Gly- Gly-Gly-Ser-Gly) was added before the recognized site of fac tor Xa. RESULTS: SDS-PAGE results indicated that the CBD-IGF fusion prot ein was expressed and purified . Biological assay results indicated CBD-IGF fusi on protein could promote the growth of NIH3T3 cell. The short flexible peptide (Gly-Thr-Gly-Gly-Gly-Ser-Gly), which was added before the recognized site of f actor Xa, improved the sensitivity of fusion protein to factor Xa. CONCLUSION: CBD-IGF fusion protein with bioactivite are expresse d and purified. The amio acid sequences changes between the site recognize of fa ctor Xa can help to improve the cleavage efficiency of Factor Xa.

Effect of Liuwei Dihuang Wan on the expression of genes in aged rats by cDNA microarray / 中国病理生理杂志

AIM: To study the effect of Liuwei Dihuang Wan (LWDHW) on the expression of immune-associated genes in the aged rats using cDNA microarray. METHODS: Forty 20-months-old rats were divided into two groups equally. One group was treated with LWDHW for 5 weeks,another was untreated as control. Some biochemical assays were used to confirm the physiological differences between two groups. The mRNA from the spleens of twenty 4-months-old young rats were extracted and divided equally into two parts. Each part was conjoined with mRNA from drug treated aged rats and that from untreated control aged rats,respectively,and two experimental combinations for reverse transcription and cDNA microarray were formed. By comparing two groups of data from two combinations,the effects of LWDHW on the expression of the associated genes were evaluated. RESULTS: 13 down-regulated genes and one up-regulated gene were identified in the untreated control old rats,whose expression did not change obviously in treated old rats compared with young rats. The expressions of another two sequence were up-regulated distinctly in treated old rats compared with young rats. CONCLUSIONS: 16 immune-associated genes expressions were affected markedly by LWDHW. It will be helpful for understanding the molecular mechanism of LWDHW in ageing.

Differential diagnosis of diffuse glomerular nodular changes / 第三军医大学学报

ObjectiveTo explore the clinical and pathological characteristics in diagnosing the renal diseases with diffuse glomerular nodular changes. MethodsThe clinical and pathological data of 127 patients whose renal pathological feature was mainly diffuse glomerular nodular changes were retrospectively analyzed. ResultsThe most common diagnosis was diabetic nephropathy and renal amyloidosis, and sometimes light chain deposit diseases and Mix cryoglobulinemia. ConclusionDiffuse glomerular nodular changes occurred in many diseases. We should make differential diagnosis based on the clinical symptoms, the results of laboratory examinations and the renal pathological characteristics.

Treatment of hepatitis C after kidney transplantation with combined interferon-? and Ribovirin / 第三军医大学学报

ObjectiveTo investigate the treatment and safety of interferon ? plus Ribovirin for chronic hepatitis C after kidney transplantation. MethodsFive patients with chronic hepatitis C after kidney transplantation were administered with interferon ? (50 ?g) subcutaneously once a week, plus Ribovirin (600 mg) orally once daily. The levels of HCV-RNA, ALT and serum creatinine in patients’ serum were monitored monthly. ResultsFour in 5 patients presented normal ALT and negative HCV-RNA in serum 12 weeks after treatment, and obtained sustained viral response 24 weeks after interferon ? plus Ribovirin therapy. During treatment, renal graft rejection did not occur. The most frequent side-effects were the decrease of leukocyte and hemoglobin, myalgia and fever, but did not influence the course of treatment. ConclusionCombination of interferon ? with Ribovirin can be a valid therapeutic option in renal transplant recipients with hepatitis C, and shows no influence on the renal function.