BACKGROUND:Previous studies have verified that mesenchymal stem cells could be transplanted into inflammatory bowel mucosa to repair inflammatory bowel tissue. OBJECTIVE:To observe the differential gene expression in large intestine before and after mesenchymal stem celltransplantation in repair of inflammatory bowel tissue of rats using microarray technology, and to primarily discover the main genes during mesenchymal stem celltransplantation, differentiation, and reparation in inflammatory colorectal tissue region. METHODS:Healthy Sprague-Dawley rats were randomly divided into two groups. Experimental rat models of inflammatory bowel disease were established using trinitrobenzene sulfonic acid via enema. At 24 hours after model establishment, green fluorescent protein-labeled mesenchymal stem cells were infused via the caudal vein. The control group was treated with physiological saline by enema, instead of trinitrobenzene sulfonic acid. At 28 days, large intestine was obtained from the experimental group and control group. Differential y expressed genes were screened in the experimental and control groups using microarray technique. RESULTS AND CONCLUSION:The microarray analysis results showed that there were 388 differential genes in the control and experimental groups (P<0.05, FC>2), in which 191 were up-expressed, and 197 were down-expressed. Al of these genes were mainly involved in inflammatory reaction, immune reaction and celldifferentiation. In the top 10 up-regulation and down-regulation differential genes (total y 20 genes), 3 genes were involved in inflammation, 3 genes were involved in immune reaction, and 2 genes were related to stem celldifferentiation. In the 388 genes, 33 were related to signaling pathways (P<0.05), 6 related to inflammation, 8 related to immunity, and 5 related to stem celldifferentiation. Results suggested that the main genes involved in mesenchymal stem cells in repair of inflammatory bowel tissue were primarily screened using gene expression microarray technique.

AIM: To determine proteins molecular weights in seed of Cortex Magnolia and identify its varieties by gel ecectrophorsis. METHODS: SDS-PAGE technique. RESULTS: There are distinct distinguish in SDS-PAGE. The molecular weights of the major proteins of the samples are analyzed by it. CONCLUSION: Cortex Magnolia can be discerned by gel electrophorsis and the molecular weights of them.

Objective To construct a lentiviral vector over-expressing β-catenin gene by multisite Gateway technology and confirm its effect.Methods By using multisite Gateway clone technique,the entry clone of pDown-Ctnnb1 was constructed using BP recombination reaction.Then,LR recombination reaction was performed among pUp-EF1A,pDown-Ctnnb1,pTail-IRES/DsRed-Express2 and pLV.Des3d.P/puro to generate an expression clone of pLV.EX3d.P/puro-EF1A＞Ctnnb1 ＞IRES/DsRed-Express2.In each step,PCR and sequencing analysis were used to verify the constructions.When it was verified that plasmids were transfected into 293T cells,PT-PCR was performed to determine the mRNA level of β-catenin gene.Results Both PCR and sequencing analysis revealed that β-catenin over-expression gene was inserted into the target site and the insertion sequence was perfectly corrected.The RT-PCR results showed that the expression of β-catenin gene was significantly upregulated.Conclusion The lenvivirus-mediate β-catenin over-expression gene was successfully constructed..

BACKGROUND:β-catenin is the most critical signaling molecule in the Wnt/β-catenin signaling pathway, which is involved in the regulation of cellproliferation, differentiation and tissue self-healing balance.
OBJECTIVE:To construct a stableβ-catenin over-expression lentivirus-mediated vector and to transfect mesenchymal stem cells line for investigating its effects on proliferation and migration of mesenchymal stem cells.
METHODS:Over-expression vector, PLV-EF1A-catenin-RFP, was constructed and transfected the 293T cellto infect mesenchymal stem cells, and positive cells were selected with puromycin. The up-regulated efficiency of targetingβ-catenin gene at mRNA level was detected by real-time quantitative PCR, the effect on proliferation of mesenchmal stem cellwas assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and growth curve, and the migration ability was detected by Transwel motility assay.
RESULTS AND CONCLUSION:The lentiviral vector targetingβ-catenin gene was constructed successful y, and a stable mesenchymal stem cellline that up-regulatedβ-catenin was established. Real-time quantitative PCR results showed that the expression ofβ-catenin gene was efficiently up-regulated by infecting PLV-EF1A-catenin-RFP (P<0.05). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and growth curve showed that celldoubling time was shortened after infected with pLV-EF1A-catenin-RFP (P<0.05), indicating that the over-expression of theβ-catenin gene successful y increased the proliferative capability of mesenchymal stem cells. The Transwel assay also showed similar increasing results on the migration ability (P<0.01). The lenvivirus-mediated over-expression of theβ-catenin gene can be used to increase the proliferation and migration abilities of the mesenchymal stem cells.

Objective To construct humanized monoclonal antibodies against CD 20 and check their affinity to CD 20 antigen and their anti-tumor activity.Methods Based on the computer model , human IgG1 candidates closest to rituximab in crystal structure were selected in the Protein Data Bank ( PDB) .With the selected human IgG 1 candidates as the frame , we modified and transplanted the complementarity determining region ( CDR) of rituximab .First,the target gene fragments were obtained by overlapping PCR.Then, the sequences of the light chains(L) and the heavy chains(H) were inserted in-to the pcDNA3.3 and pOptiVEC vectors.Next, the constructed clones were transfected into 293F cells through transient transfection.After a large-scale cell culture, the mAb was purified by affinity chromatography rProtein A column.The puri-ty and expression level of the humanized antibodies was tested by sodium dodecyl sulfate ( SDS)-polyacrylamide gelelectro-phoresis(PAGE).The affinity of the humanized antibodies to CD20 was assessed with Fortebio assay.Finally, the anti-tumor activity of the constructed antibodies was detected by checking the tumor growth inhibition of the nude mice transplan-ted with tumor .Results Three humanized monoclonal antibodies against CD 20 were expressed and purified successfully . In reducing SDS-PAGE, the antibodies exhibited two bands of approximately 25 ×103 and 55 ×103 , respectively.The band size of the antibodies matched the expected value.Fortebio assay revealed that the humanized antibodies could bind to CD20 with high affinity (rituximab:6.48 ×10 -9mol/L, L4H7:1.91 ×10 -9mol/L, L5H5:7.35 ×10 -10mol/L,and L5H7:1.91 ×10 -9mol/L).The tumor growth inhibition experiment showed that the anti-tumor activity of L5H7 mAb was better than that of rituximab .Conclusion Three humanized monoclonal antibodies against CD 20 have been successfully construc-ted and expressed.L5H7 mAb possesses high affinity for CD20 and a good ability to kill tumor cells.

Objective To observe the expression of Cathepsin S in the human abdominal aortic aneurysm (AAA) and angiotensin Ⅱ(AngⅡ) perfusion induced AAA lesions in apolipoprotein E-deficient(ApoE-/-) mice.Methods AAA specimens of clinical diagnosis patients(AAA group) and the normal blood vessels(control group)were collected.The AAA model was established by infused Ang Ⅱ 1 000ng · kg-1 · d-1 to ApoE-/-mice.Elastic fibersfracture was observed by elastic fiber staining and Cathepsin S expression by immunohistochemistry.Results The elastic fiber staining revealed that the elastic fibers fracture of the human and mouse AAA group increased significantly (P ＜ 0.05) ; Cathepsin S expression significantly increased in the human and mouse AAA group than the control group by immunohistochemistry(P ＜ 0.05).Conclusion Cathepsin S is activated in human and murine AAA lesions and may be involved in the pathogenesis of AAA.

Objective To evaluate the correlation of ADC values on 3.0 T MR with the pathological grades in pT1b clear cell renal cell carcinoma(CCRCC).Methods Conventional MR images,ADC values and Fuhrman pathological grading of pT1b CCRCC were performed in 30 patients.Grade Ⅰ and Ⅱ were defined as low-grade group; grade Ⅲ and IV were defined as high-grade group.The differences of ADC values among four different pathologic grades were compared with a one-way analysis of variance. The comparison of ADC values of two different grade groups was performed with t test,and the ROC curve was performed to evaluate the diagnostic efficacy of ADC value. Correlation between pathological gradings and ADC values was assessed with Spearman rank correlation analysis. Results ( 1 ) The mean ADC value of grading Ⅰ ( 10 patients ), Ⅱ ( 8 patients ),Ⅲ ( 7 patients ),IV ( 5 patients ) was ( 0.94 ± 0.11 ) ×10-3 mm2/s,(0.82 ±0.13) × 10-3 mm2/s,(0.68 ±0.09) × 10-3 mm2/s,(0.59 ±0.03) × 10-3 mm2/s,respectively.Significant differences of ADC values among the four grades were present (F =16.422,P =0.000 ).( 2 ) The mean ADC value of the low-grade group was significantly higher than that of the high-grade group(t =5.738,P =0.000).Sensitivity and specificity of diagnosing the low-grade group was 88.9％ and 83.3％ respectively.There was a negative correlation between pathological grading and ADC value ( r =-0.807,P ＜ 0.05). Conclusions The ADC values of pT1b CCRCC have close correlation with pathological gradings.They can be used to predict the degree of tumor malignancy preoperatively and guide surgical planning.

Objective To observe the function of immature CD8α+ dentritic cells (DCs) in vitro. Methods The bone marrow and spleen of C57BL/6(H-2b) and Balb/c (H-2d) mice were got to prepare immature CD8α+ DCs and spleen lymphocytes,and treated by mytomycin. MTT test was used.MLR group, MLR plus variable density syngeneic CD8α+ DC group, MLR plus variable density allogeneic CD8α+ DC group,MLR plus variable density CD8α+ DC supernatant group,CD8α+ DC plus syngeneic T cell group and negative control group were established. MLR group was set up by responder cell ratio of 0.2,0.5,0.8,1.0,to build the MLR plus syngeneic and allogeneic CD8α+ DC experimental groups. Culture supernatant from different density (1 × 105/ml - 5 × 106/ml) of CD8α+DCs was added into MLR to build CD8α+ DC supernatant group. CD8α+ DCs were co-cultured with syngeneic T cells to build CD8α+ DCs plus syngeneic T cells group. 2 × 105/well responder cells served as the negative control group. ELISA was used to detect the concentrations of IFN-γ and IL-10 in the DCs could both suppress MLR (P＜0. 05), and the difference was not statistically significant (P＞0. 05). When CD8α+ DCs were increased, the suppressive effect was enhanced. When CD8α+ DC/responder cell ratio ＞0. 2, the inhibitory effect could be observed, and this effect reached the peak when the ratio was 1.0. The CD8α+ DCs had weak ability to stimulate syngeneic lymphocyte proliferation in vitro, and certain stimulating effect could be seen only when CD8α+ DC/responder cell ratio ＞2 (P＜0. 05). Its culture supernatant also showed suppressive effect (P＜0. 05), and the supernatant with a cell density of 5 × 105/ml showed the maximum effect. IL-10 concentration in the concentration was 1.0 ± 1.2 pg/ml. Conclusion The in vitro function of immature CD8α+ DCs was immunosuppression/tolerance,and they could secret high level of IL-10. The CD8α+ DCs and their culture supernatant could suppress MLR in vitro.

OBJECTIVETo evaluate the fidelity of multiple displacement amplification (MDA) from small number of cells (1-10 cells) by 10K 2.0 SNP mapping array.

METHODSA fibroblast cell line (Tri-18; GM02732, 47, XY, +18) was used as the template, and 6 groups were set up in the study. Groups A and B were positive and negative control, respectively; groups C-F were experimental groups involving the MDA products from 1, 2, 5 and 10 cells respectively. In combination of single nucleotide polymorphism (SNP) array, the product of each group was assessed based on the genome coverage, loss of heterozygosity (LOH) rate and allele dropout (ADO) rate.

RESULTSThe nonspecific product of negative control presented an average call rate of 3.2%. The genome coverage of the MDA product increased from 86.4% to 96.4% with the increasing number of template from 1 to 10 cells, while the LOH rate and ADO rate decreased significantly (P<0.05).

CONCLUSIONMDA is a highly efficient and reliable method for whole genome amplification. The fidelity of MDA will be improved significantly with the increasing number of template cells. 10K 2.0 SNP mapping array is a quick, accurate and comprehensive method to evaluate the fidelity of amplified DNA products, but the ADO SNPs should be distinguished from those of preferential amplification from the LOH loci to avoid errors.

Cell Line ; Cells ; cytology ; DNA ; genetics ; Humans ; Loss of Heterozygosity ; Nucleic Acid Amplification Techniques ; methods ; Polymorphism, Single Nucleotide ; Templates, Genetic

OBJECTIVETo screen the point mutation of the low-density lipoprotein receptor (LDL-R) gene in Chinese familial hypercholesterolemia (FH) patients, characterize the relationship between the genotype and the phenotype and discuss the molecular pathological mechanism of FH.

METHODSA patient with clinical phenotype of homozygous FH and her parents were investigated for mutations in the promoter and all eighteen exons of the LDL-R gene. Screening was carried out using Touch-down PCR and direct DNA sequencing; multiple alignment analysis by DNASIS 2.5 was used to find base alteration, and the LDL-R gene mutation database was searched to identify the alteration. In addition, the apolipoprotein B gene (apo B) was screened for known mutations (R3500Q) that cause familial defective apo B100 (FDB) by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).

RESULTSTwo new heterozygous mutations in exons 4 and 9 of the LDL-R gene were identified in the proband (C122Y and T383I) as well as her parents. Both of the mutations have not been published in the LDL-R gene mutation database. No mutation of apo B100 (R3500Q) was observed.

CONCLUSIONTwo new mutations (C112Y and T383I) were found in the LDL-R gene, which may result in FH and may be particularly pathogenetic genotypes in Chinese people.

Adult ; Apolipoproteins B ; genetics ; Asian Continental Ancestry Group ; Child ; China ; Female ; Heterozygote ; Homozygote ; Humans ; Hyperlipoproteinemia Type II ; genetics ; Male ; Mutation ; Receptors, LDL ; genetics