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Ying Wang

Publications and source records attributed to Ying Wang.

At least 199 records · Page 11Linked to original sources
PubMed2006

[Effects of different donor cells and passages on the development of nuclear transfer porcine embryos].

This study was undertaken to systematically examine effects of different donor cells and passages on the development of nuclear transfer porcine embryos, so as to establish a preliminary procedure for porcine cloning. Porcine oocytes obtained from ovaries at slaughter were matured in vitro for 40-44h and then enucleated in manipulation medium containing 5 microg/mL cytochalasin B. Fibroblast cells (FC), oviduct epithelial cells (OEC), granulosa cells (GC) and cumulus cells(CC) after 3-9 passages in TCM + 10% FBS were treated by serum starvation (0.5% FBS for 2-9 days), 0.1 microg/mL aphidiconlin (APD) for 1 day and 0.5% FBS for 2-9 days or continue culturing in 10% FBS for 2-9 days, then, were transferred into enucleated oocytes by microinjection or electronic fusion (100 V/mm, 30 micros and 1 pulses). Reconstituted embryos were activated with a combination of calcium ionophore A23187 or electric pulse and 6-DMAP, and cultured for 6 days, to evaluate their cleavage and embryonic development. The cleavage rate of embryos reconstructed with FC and GC pretreated with 0.1 microg/mL APD + 0.5% FBS were significantly higher than that of serum starvation group and control group (P<0.01). There was significantly difference in the cleavage rate and embryonic development among embryos derived from GC, CC and FC, OEC pretreated with 0.1 microg/mL APD + 0.5% FBS. The cleavage rate of embryos reconstructed with GC by electrofusion was significantly higher than that by microinjection (P<0.05), but no difference was found in their proportion developing to blastocysts. 75% to 85% of GC at 3 and 6 passages, and FC at 6 and 10 passages had normal karyotype, which did not show significant difference among them (P>0.05). When GC at G3, G4, G5 and G6 of passages were used as donor cells, the cleavage rate and blastocyst rate was similar, moreover, FC at G6, G7, G8 and G9 of passages also resulted in a similar cleavage rate and blastocyst development. These results indicate that: (1) FC and GC can be cultured up to 9 passages and keep relatively stable karyotepe; (2) Using 0.1 microg/mL APD to treat donor cells prior to nuclear transfer can improve the efficiency of somatic cell nuclear transfer in buffalo but serum starvation is inefficient in our system; (3) Both of FC and GC cells can be used as the donor karyoplasts for nuclear transfer, and their efficiency are not influenced by the culture passages; (4) The development of reconstructed embryos by electrofusion is higher than that by microinjection, but there is no difference in the total efficiency between the two methods.

Adenine↗
PubMed2006

[Clinical study on haploid HLA-matched hematopoietic stem cell transplantation for treatment of malignant hematological disease].

BACKGROUND & OBJECTIVE: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the most effective method to treat malignant hematological disease. However the human leukocyte antigen (HLA)-matched sibling donors can be found for only 25%-30% patients. The probability of finding an unrelated matched donor is 1/50000-1/100000, or even lower. If we choose haploid HLA-matched hematopoietic stem cell transplantation instead, we may be able to find donors for 90% patients. Our present study was to explore the feasibility of allo-HSCT by using haploid HLA-matched donor in treatment for malignant hematological disease. METHODS: Twenty-five patients with malignant hematological disease received allo-HSCT with HLA 2 or 3 antigen mismatched related donors. All patients were treated with intensive immunosuppression, granulocyte colony stimulating factor (G-CSF) mobilization, antithymocyte globulin (ATG) and combination of bone marrow and peripheral blood stem cell transplantation. The conditioning regimen was intensified and prolonged by using the combination of cyclosporine (Cs) A, MMF, ATG and anti-CD25 antibody for graft-versus-host disease (GVHD) prophylaxis. RESULTS: All patients achieved sustained, full donortype engraftment. Acute GVHD occurred in 21 of 25 patients. Eight of them were grade I aGVHD, six grade II aGVHD, two grade III aGVHD and five grade IV aGVHD. The cumulative incidence of grade II-IV aGVHD was 48%,and grade III-IV aGVHD was 28.57%. Chronic GVHD was observed in 12 of 25 patients and none of them developed extensive cGVHD. Sixteen patients were alive and disease free, with 64.0+/- 2.98% 1 year disease-free survival rate. One year overall survival rate was 64.0+/-3.08%. Nine patients died, 1 from relapse and 8 from transplantation related mortality. CONCLUSIONS: Haploid HLA-matched allo-HSCT is a relatively efficient method for the treatment of patients with malignant hematological disease, who have no related matched donors. Nevertheless, strict administration should be carried out since it's a high risk approach.

Adolescent↗
PubMed2006

AKAPs competing peptide HT31 disrupts the inhibitory effect of PKA on RhoA activity.

A-kinase can inhibit RhoA activation through phosphorylating ser188 of RhoA. AKAP is a novel protein that can target PKA to different subcellular compartment. Evidence has been presented that PKA anchorage by AKAP is important for the kinase to exert its function. This study analyzed the role of PKA anchorage in PKA-induced antagonism against RhoA activity and function. The cells transfected with pcDNA HT31wt/mut were treated with LPA and/or CPT-cAMP. The amount of GTP-RhoA and phosphorylation of RhoA was detected by Western blotting with specific antibodies. The formation of stress fiber was visualized under fluorescent microscope. The gene expression activity was analyzed by luciferase reporter gene assay. The motility and the anchorage-independent growth assays were carried out with stably transfected cells expressing the AKAP inhibitory peptide HT31. The results showed that HT31 not only blocked the PKA-induced phosphorylation of RhoA but also prevented the PKA-induced inhibition on RhoA activation. The disruption of PKA anchorage abolished its inhibition on the LPA-induced expression of reporter gene SRE-luciferase. The ability of PKA to antagonize the LPA-induced stress fiber formation was partly impaired upon the disruption of the PKA anchorage. The control of PKA on migration and the proliferation excited by LPA disappeared in stably transfected cells highly expressing HT31. The results revealed that PKA anchorage was necessary for the kinase to exert its inhibitory effect on RhoA activation and RhoA-dependent biological activities.

Adaptor Proteins, Signal Transducing↗
PubMed2006

[Study of determination method of trace elements in white jade snail].

The contents of six trace elements, Ca, Mg, Fe, Zn, Cu and Co, in white jade snail were determined by ICP-AES and AAS using nitrifying method of high pressure nitrifying pot. The results of two analysis methods were compared, showing no obvious differences. The recoveries were 92.4%-103.0%, and the relative standard deviations were less than 2.98%. The experiment proved that white jade snail contains rich useful trace elements, and is a kind of green food with high nutrition.

Animals↗
PubMed2006

[Expression of G-6-PD mRNA in children with G-6-PD deficiency].

OBJECTIVE: To detect the level of glucose-6-phosphate dehydrogenase (G-6-PD) mRNA expression in children with G-6-PD deficiency and their lineal family members in order to explore possible mechanisms of the disease at the transcriptional level. METHODS: RNA was extracted from peripheral blood of 41 children with G-6-PD deficiency and of their lineal family members (29 father lineages and 40 mother lineages). cDNA was then harvested using the reverse transcription method. G-6-PD mRNA expression was detected by quantitative real-time PCR (QRT-PCR). The detection results of G-6-PD mRNA expression in three groups (Patient, Father lineage and Mother lineage) were compared using Statistical Software SPSS 10.0 system. RESULTS: The mean G-6-PD mRNA value in the Patient, Father lineage and Mother lineage groups were 0.57 +/- 0.19, 0.74 +/- 0.21 and 0.67 +/- 0.21 respectively. The G-6-PD mRNA value in the Patient group was significantly lower than both Father lineage (t = -3.18, P < 0.01) and Mother lineage groups (t = -2.54, P < 0.05). CONCLUSIONS: The expression of G-6-PD mRNA decreased in children with G-6-PD deficiency, suggesting that the pathogenesis of this disorder relates to the varying levels of gene transcription.

Child↗
PubMed2006

[Identification of the rice (Oryza sativa L.) mutant msp1-4 and expression analysis of its UDT1 and GAMYB genes].

A rice male-sterile mutant msp1-4 (MULTIPLE SPOROCYTE) with japonica cultivar '9522' background, was obtained in M(3) population treated with (60)Co gamma-ray. Results of genetic analysis indicated that the male-sterile phenotype was controlled by a single recessive locus. To map this locus, an F(2) population was constructed from the cross between the msp1-4 (japonica) and 'LongTeFu B' (indica). This locus was mapped between the two InDel markers, WY-4 and WY-8, with physical distance of 247 kb. A deletion with 10 base pairs between 758 bp and 767 bp in MSP1 open reading frame was confirmed by sequence analysis, which led to pre-termination of MSP1 translation. Phenotype analysis of msp1-4 indicated that it was similar to the msp1 mutant. To insight the expression change of rice anther developmental genes in this mutant, semi-quantitative RT-PCR analysis was carried out. The results showed that the expression level of rice UDT1 and GAMYB were reduced in msp1-4, implying that UDT1 and GAMYB are possibly the downstream genes of MSP1 gene in rice pollen development.

Flowers↗
PubMed2006

[Cloning and expression of HLA-A*0201-BSP].

High-yield production of HLA-A*0201 heavy chain is a prerequisite to the preparation of HLA-A2 tetramer. The present study was aimed to construct the expression vector of recombinant HLA-A*0201-BSP fusion gene for preparing HLA-A2 tetramers. The extracellular region HLA*0201 was cloned by RT-PCR from HLA-A2(+) donor, and a 15-amino acid biotin-protein ligase (BirA) substrate peptide (BSP) for BirA-dependent biotinylation was added to the COOH-terminus of HLA-A*0201 heavy chain. Then the fusion gene was cloned into pBV220 vector at EcoRI and Bam HI sites and its sequence was confirmed by DNA sequence analysis. The recombinant plasmid pBV220-HLA-A*0201-BSP was transformed to the competent cells of E.coli DH5alpha. The results showed that the HLA-A*0201-BSP fusion protein was successfully expressed in the form of inclusion body and amounted to over 28% of total cell proteins via induction at 42 degrees C. After washed with triton X-100 and urea, the inclusion body was dissolved with 8 mol/L urea and then purified with Sepharcyl S-300 HR, and the final purity reached above 90%. It is concluded that the HLA-A*0201-BSP fusion gene was cloned successfully and expressed efficiently in E.coli DH5alpha. This work establishes a convenient approach for purification of large quantity of recombinant HLA-A*0201-BSP. This provides the basis for the preparation of HLA-A2 tetramers.

Biotin↗
PubMed2006

[Diffuse large B-cell lymphoma with expression of anaplastic lymphoma kinase protein: clinicopathologic and immunohistochemical study of 5 cases].

OBJECTIVE: To study the clinicopathologic features of diffuse large B-cell lymphoma (DLBCL) with expression of anaplastic lymphoma kinase (ALK) protein. METHODS: Nine hundred and forty-five (945) cases of DLBCL (including 177 consultation cases) diagnosed according to the 2001 World Health Organization classification of tumors of hematopoietic and lymphoid tissues were enrolled into the study. Immunohistochemical study for anti-ALK-11 was performed using LSAB technique. The ALK-positive cases were further confirmed by immunohistochemical study using EnVision technique. Only ALK-positive cases by EnVision technique were further analyzed by immunostaining for antigens including CD20, CD3, CD30, EMA, granzyme-B, TIA-1 and PC. Immunoglobulin heavy chain gene rearrangement study was also performed and follow-up data collected. RESULTS: There were altogether 5 (4 males and 1 female) cases of DLBCL showing expression of ALK protein. The age of the patients ranged from 34 to 72 years. All were primary nodal DLBCL. One case belonged to clinical stage I, 2 in stage II and 2 in stage III. The duration of follow up ranged from 4 to 32 months. Three patients subsequently died and the longest survival was 32 months. Morphologic subtypes included centroblastic 2, anaplastic 1, immunoblastic with plasmacytoid differentiation 1 and plasmablastic 1. Immunohistochemically, 4 cases were CD20 positive (including 2 centroblastic, 1 anaplastic and 1 immunoblastic cases). The plasmablastic case expressed kappa light chain and was negative for CD20. Rearrangement of immunoglobulin heavy chain gene was demonstrated in all 5 cases studied. As for ALK protein staining, a mixed membranous and cytoplasmic (1 immunoblastic case), granular cytoplasmic (2 centroblastic and 1 anaplastic cases) and mixed nuclear and cytoplasmic (1 plasmablastic case) patterns were observed. CONCLUSIONS: Expression of ALK protein is a rare phenomenon in DLBCL and can be seen in centroblastic, anaplastic, immunoblastic and plasmablastic subtypes. It is often associated with aggressive clinical behavior and worse prognosis. A new pattern of ALK protein expression, mixed membranous and cytoplasmic, is reported.

Adult↗
PubMed2006

[Clonal culture of rat bone marrow-derived multipotential adult progenitor cells and study of their biological properties].

OBJECTIVE: To optimize the culture conditions for clonal isolation of rat bone marrow-derived multipotential adult progenitor cells (rMAPC) and identify their surface markers and differentiation potentials. METHODS: By using a low concentration of fetal bovine serum culture medium, rMAPCs were primarily isolated from bone marrow by attachment culture and clonal-like cells were selected by single cell limiting dilution. The surface antigens of the cloned rMAPC were analyzed by flow cytometry and immunocytochemistry. Multi-differentiation capacities were evaluated by lipoblasts and osteoblasts and neuroblasts differentiation induction. The expressions of Oct-4 and three embryonic germ layer markers were detected by RT-PCR. RESULTS: Single cell-derived rMAPC could be expanded to passage 20 in vitro which still maintained active proliferation ability. The expanded rMAPCs expressed CD71, alpha-SMA and vimentin, but not CD34, CD44 and CD45. About 83% of the rMAPCs was in the resting phase(G0 + G1) of cell cycle and 17% in S + G2 + M phase. They could be induced to differentiate into adipogenic cells, osteogenic cells and neural like cells. RT-PCR demonstrated that there were expressions of oct-4 gene and three embryonic germ layer markers on the rMAPCs. CONCLUSIONS: Cloned rMAPC can maintain the phenotypes of stem cell during in vitro culturing. It might be an potential adult stem cell source for therapeutic stem cell transplanting and tissue engineering.

Animals↗
PubMed2006

[Establishment of model with inner ear mimetic aging and mtDNA 4834 bp deletion in rats].

OBJECTIVE: To explore the establishment of the mimetic aging effect in the inner ear of the rat. METHOD: Twenty-four Wistar rats were randomly divided into two groups: group A (D-galactose group, n = 14), which were treated with hypodermic 5% D-galactose (150 mg x kg(-1) x d(-1)) for 8 weeks and then with intraperitoneal saline for 10 days, and group B (control group, n = 10), which were given saline only. Auditory brainstem response(ABR) was used to detect the hearing threshold of rats, and colorimetry was used to analyze the activity of the glutathione peroxidase (GSH-PX). The inner ear tissue was harvested and mitochondrial DNA was amplified to identify the 4 834 bp deletion mutation by nested primer polymerase chain reaction (nested PCR) technique. RESULT: The incidence of mitochondrial DNA 4 834 bp deletion of group A was 100% (28/28), while the deletion of group B was 0. The activity of the GSH-PX in group A (59.07 +/- 8.70 U) was lower than that in group B (142.10 +/- 7.02 U). The difference between group A and group B was significant (t-test, P < 0.01). Variation in ABR threshold of (5.36 +/- 3.08) dB peSPL was observed in group A, and (6.50 +/- 3.37) dB peSPL in group B. The difference in shift of ABR threshold between group A and group B was not significant (P > 0.05). CONCLUSION: The mimetic aging effect in the inner ear of the rat can be induced by D-galactose, and this rat model presents high incidence of mtDNA4834 deletion. But no hearing loss was found in this model.

Aging, Premature↗
PubMed2006

[Construction and expression of HSV-2gD-Hsp70 fusion protein gene].

To construct and express Hsp70-HSV2gD fusion protein. Genes of Hsp70 and HSV-2gD were subcloned into vectors pGEX-4T-1 respectively. After confirmed by DNA sequence analysis, the recombinant plasmids pGEX-4T-HSP-gD was transformed into E. coli DH5alpha and induced to express with IPTG. The expressed protein was characterized by SDS-PAGE and Western blot after purified. BALB/c mice were immunized with fusion proteins respectively via intra-m uscular injection. The proliferation of spleen lymphocytes, the level of y-IFN in culture and anti-HSV-2gD IgG antibody in serum was detected was detected. The expressed protein was analyzed by SDS-PAGE after induced with IPTG, which showed a new band with an apparent molecular mass corresponding to the predicted size (118 kD). Western Blotting analysis demonstrates that the purified Hsp70-HSV2gD fusion protein had specific binding activity. The stimulation indexes of spleen lymphocytes, the level of gamma-IFN in culture and anti-HSV-2gD IgG antibody in serum of GST-Hsp70-gD group was obviously higher than that of other groups (P < 0.05 respectively). The successful expression of the Hsp70-HSV2gD fusion protein, which can induce immune responses, laid a solid foundation for its further research.

Animals↗
PubMed2006

[Study on the Chinese herbal formula for treatment of vaginitis and the antimicrobial activity in murine models].

OBJECTIVE: To study on the various proportions of Radix Sophorae Flavescentis, Cortex Phellodendri, Fructus Cnidii and pericarp of Zanthoxylum bungeanum Maxim in the formulas, whose antimicrobial effects on E. coli, S. aureus, P. aeruginosa and C. albicans under different pH values were compared in vitro. METHODS: According to Chinese ancient proved recipe, the K-B method and plate diluting method were adopted to measure antimicrobial activity, and orthogonal design to ascertain the herbal formula in vitro. Finally, murine models were established to test the antimicrobial activity in vivo through vaginal membrane irritancy experiment, negative rate of pathogeny and pathological grade of vaginal membrane. RESULTS: The results suggested that formulas with different proportions of the herbs had diverse antimicrobial activities, and the effect was shown to be most obvious when one milliliter drug contains 100 microl Fructus Cnidii-pericarp of Zanthoxylum bungeanum (2:1) co-extracted volatile oil and 50 microl Radix Sophorae Flavescentis and Cortex Phenodendri ethanol extraction respectively under pH6. The antimicrobial effect of the formula, which hardly had any membrane irritancy, was better than Jie Eryin in vitro and vivo. CONCLUSION: The fromula has few components and better effect, and adaptation to the pH value of vaginitis. It is a promising alternative for gynecological diseases.

Alkaloids↗
PubMed2006

[A novel method of amplifying the heavy-chain variable regions from immunoglobulin cDNAs].

The amplification of variable regions (Fv) of immunoglobulins has become a crucial technique for studying antigen-antibody interactions and cloning the monoclonal antibodies (mAbs). But this work becomes a major challenge in cloning antibody genes either from hybridoma cell lines or B cells. Isolation of the immunoglobulin heavy-chain variable regions (VH) from two mouse hybridoma cell lines based on conventional protocols using both the specific consensus primers and the commercial primer set of VH regions, was unsuccessful. This prompted us to design our own primers for VH regions of immunoglobulins. A novel algorithms and reverse polymerase chain reaction (PCR) protocol--3' RACE and 5' RACE were established in order to recognize the VH genes. The most conserved region in the end of framework region (FR1) of the VH cDNA region was identified with the program and a degenerated primer were designed in the most conserved region followed by 3' RACE and 5' RACE. The new protocol rescued the failed PCR amplifications. Thereby, a improvement in the degenerate primers designing programs and a notable increase in amplification specificity were achieved. This approach can be useful in primer design and amplification of VH and light-chain variable regions (VL) gene libraries or cloning of the unknown genes in the large gene families and extend to other species or members of the immunoglobulin superfamily.

Algorithms↗
PubMed2005

Constitutive activation of MKK6 in chondrocytes of transgenic mice inhibits proliferation and delays endochondral bone formation.

Accumulating in vitro evidence suggests that the p38 mitogen-activated protein kinase (MAPK) pathway is involved in endochondral ossification. To investigate the role of this pathway in endochondral ossification, we generated transgenic mice with expression in chondrocytes of a constitutively active mutant of MKK6, a MAPK kinase that specifically activates p38. These mice had a dwarf phenotype characterized by reduced chondrocyte proliferation, inhibition of hypertrophic chondrocyte differentiation, and a delay in the formation of primary and secondary ossification centers. Histological analysis with in situ hybridization showed reduced expression of Indian hedgehog, PTH/PTH-related peptide receptor (PTH, parathyroid hormone), cyclin D1, and increased expression of p21 in chondrocytes. In addition, both in vivo and in transfected cells, p38 signaling increased the transcriptional activity of Sox9, a transcription factor essential for chondrocyte differentiation. In agreement with this observation, transgenic mice that express a constitutively active mutant of MKK6 in chondrocytes showed phenotypes similar to those of mice that overexpress SOX9 in chondrocytes. These observations are consistent with the notion that increased activity of Sox9 accounts at least in part for the phenotype caused by constitutive activation of MKK6 in chondrocytes. Therefore, our study provides in vivo evidence for the role of p38 in endochondral ossification and suggests that Sox9 is a likely downstream target of the p38 MAPK pathway.

Animals↗
PubMed2005

Vaccination with IFN-inducible T cell alpha chemoattractant (ITAC) gene-modified tumor cell attenuates disseminated metastases of circulating tumor cells.

Immunotherapeutic strategies for metastatic diseases are being developed. IFN-inducible T cell alpha chemoattractant (ITAC) has been demonstrated to be able to induce Th1-type immune response. However, the effects of ITAC on the tumor metastasis have not been fully understood. In the present study, the ITAC-modified tumor cell vaccine in inhibiting the disseminated pulmonary metastasis was evaluated. ITAC-modified tumor cell vaccine 4T1-ITAC was developed by stably transfecting 4T1 cells with pcDNA3-ITAC plasmid. Mice were vaccinated with 4T1-ITAC. Mice vaccinated with 4T1-pcDNA3 and 4T1 were used as controls. Specific cellular immune responses against 4T1 were tested by in vitro proliferation, cytokine production and cytotoxic assay. The number of clonogenic metastatic tumor cells and metastatic forci on the surface of lung were counted by histological examination. Results showed that a significant enhancement of proliferative and cytotoxic activities accompanied with increased IFN-gamma and TNF-alpha production as well as decreased IL-4 production were obtained from the mice vaccinated with 4T1-ITAC. The number of clonogenic metastatic tumor cells in the mice vaccinated with 4T1-ITAC cells reduced markedly and no visible metastasis was found in the lungs of the 4T1-ITAC vaccinated mice. Consequently, the survival rate was dramatically increased in these mice. Taken together, our results demonstrated that ITAC-modified tumor cell vaccine can enhance the anti-tumor immunity and reduce the incidence of disseminated metastasis.

Animals↗