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Objective To evaluate the role of mesenchymal stem cells (MSCs) derived from mouse bone and embryo dorsal aorta(DA) area in the treatment of irradiation induced lung injury of mouse model. Methods The mice were divided into four groups as normal control group, irradiation group,bone MSCs treatment group and DA MSCs treatment group. Immunohistochemical Analysis of lung tissue was observed after 9 months of treatment. Results Fibrosis and alveolar infiltration were scored in each group. The score for fibrosis and alveolar is 0. 17 in normal control group, 2 in irradiation group, 1 in bone MSCs treat group and 1.38 in DA MSCs treat group. Conclusion The extent of irradiation Induced Lung Injury could be reduced thorough the treatment of MSCs derived from mouse bone and embryos dorsal aorta ( DA ) area.
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Objective To study the probability of applying mesenchymal stem cells isolated from human umbilical cord blood (hUCB MSCs) to repair mouse skin wound in vivo. Methods hUCB MSCs isolated from full term delivery human umbilical cord blood were cultured and amplified in vitro.hUCB MSCs at passage 9 were labeled with BrdU (5-bromodeoxy-uridine) and grafted on the full-thickness skin loss wound created on the back of the severe combined immunodeficiency (SCID) mouse (treatment group), when a PBS control group was set. The wound healing rate was surveyed and compared at days 7 and 14 postoperatively. Meanwhile, the wound was biopsied at days 7, 14 and 28 after operation,and the expressions of BrdU antibody and K19 antibody were checked pathologically and immunohistochemically by HE staining, respectively. Results The wound in treatment group was healed more rapidly than that in control group (P ＜ 0.01 ). The pathological check of the biopsy sample showed that the epidermis was thicker, with more epidermal ridges in the treatment group, compared with control group.It was found that some BrdU positive cells were distributed successively on the hair follicle, the stratum basal and the spinosum layers, a few of which even expressed K19. Conclusion hUCB MSCs can be differentiated into skin tissue and cells and is possible to repair skin wound.
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Objective Non-invasive detection is the focus of intense research in diagnosis of acute renal allograft rejection currently. Urine protein is considered the cue to reflect the pathological changes in kidney disease. In this study, we explored the urine markers for early acute renal allograft rejection. Methods The urine protein of two patients with acute renal allograft rejection were examined by 2D gel electrophoresis and bioinformatics. We adopted pH 4-7 ready strip IPG and stained the gel with Sypro-Ruby. The digitized 2D maps of urine protein were quantitatively analyzed using 2D-analysis software packages. By analyzing the differential expressions of proteome between different time points (1, 2, 3 days before acute rejection and 7, 14, 21 days after acute rejection), 30 protein spots were selected and analyzed by MALDI-TOF-MS/MS. Results We obtained 2D gel electrophoresis maps of urine protein of the patients with acute renal allograft rejection, which are of good reproducibility and resolution. Sixteen protein spots were identified, resulting in thirteen corresponding proteins. Out of these proteins, we screened three proteins (alpha-1-antichymotrypsin, tumor rejection antigen gp96, Zn-Alpha-2-Glycoprotein) closely related to acute rejection. Conclusion The urine protein spots on 2D gel electrophoresis maps for the patients with acute renal allograft rejection were of obvious difference when detected at different time points of acute rejection. Alpha-1-antichymotrypsin, tumor rejection antigen gp96 and Zn-Alpha-2-Glycoprotein might be the candidate protein markers to diagnose acute renal allograft rejection after renal transplantation.
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Objective To screen the proteins interacting with FOXP3 in yeast two-hybrid system. Methods The "bait plasmid" pGBKT7 (named as pGBKT7-FOXP3) was constructed successfully. Using FOXP3 as bait, a human liver cDNA library was screened and the proteins interacting with FOXP3 were searched. The false positive clones were discarded by one to one yeast two-hybrid system, and the positive clones were sequenced and analyzed by bioinformatic methods. Results The bait plasmid pGBKT7-FOXP3 was constructed successfully and there was no self-activation or toxicity in AH109. Three proteins had been found in our system to be able to interact with FOXP3. They were tumor protein D52, splicing factor 3b subunit 1 and one hypothetical protein. Conclusion FOXP3 interacts with tumor protein D52, splicing factor 3b subunit 1 and one hypothetical protein, all of which may interfere in cell metabolism and function of T cell.
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Objective To construct a bait vector containing human Foxp3 gene in yeast two-hybrid system in order to screen the cDNA library of T lymphocyte. Methods RT-PCR was used to amplify the Foxp3 gene fragment from the peripheral blood mononuclear cells (PBMC) with the primers designed in accordance with the sequence in GenBank. The product was inserted into pMD18-T vector. After verified with restriction endonuclease digestion of EcoRⅠ and SalⅠ, the vector was inserted into the “bait plasmid” pGBKT7 (named as pGBKT7-Foxp3). After confirmation with restricted endonuclease digestion and sequence analysis, the plasmid was transformed into the yeast cell AH109, and its toxicity and transcriptional activation was tested by both the phenotype assay and the color assay. Results The amplified product of 1 203 bp was inserted into PMD18-T vector and proven correctly by double restriction enzyme digestion. Sequence analysis revealed that the fragment was correctly inserted into pGBKT7 with a right reading frame and its expression in yeast was verified. Conclusion The bait plasmid pGBKT7-Foxp3 constructed expresses correctly, and can not activate the transcription of reporter gene alone in yeast two-hybrid system
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Objective To explore the interaction between HT036(hypothetical protein HT036)and P311 by co-immunoprecipitation.Methods HA-tagged fusion protein(HA-HT036)expression vector was constructed,identified and transfected into human embryo kidney 293(HEK293)cells alone or with Myc-tagged fusion protein(Myc-P311)expression vector pCMV-Myc-p311.The interaction between P311 and HT036 was detected by co-immunoprecipitation.Results Double restriction enzyme digestion showed that pCMV-HA-HT036 was constructed correctly.When Myc-P311 was immunoprecipitated by anti-Myc antibody,HA-HT036 was identified by Western blotting with anti-HA antibody from immunoprecipitated complex.Conclusion The recombinant vector pCMV-HA-HT036 was constructed successfully.The interaction between HT036 and P311 could be identified by co-immunoprecipitation after co-expression of pCMV-HA-HT036 and pCMV-Myc-p311.The result provides an important basis for further study of the intracellular signal transduction of P311.
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Objective To express human HT036 protein in Escherichia coli(E.coli.)and identify it.Methods The cDNA sequence obtained by PCR was cloned into the prokaryotic expression plasmid pET30a(+).The target protein was expressed in E.coli..induced by IPTG and analyzed by Western blotting.Results The interest gene was identified by restriction endonucleases digestion and DNA sequencing.The protein was highly expressed in E.coli..Conclusion We successfully expressed the HT036 protein.