Adult ; Aged ; Case-Control Studies ; Female ; Humans ; Leukemia, Myeloid, Acute ; metabolism ; pathology ; Male ; Middle Aged ; NF-kappa B ; metabolism ; Neovascularization, Pathologic ; pathology ; Tryptases ; metabolism ; Vascular Endothelial Growth Factor A ; metabolism ; Young Adult

According to the sequence of P450 cDNA of Eleutherococcus senticosus, specific primers were designed. Frokaryotic ex pression vector pET30a-P450 was constructed and the prokaryotic expression conditions were optimized. Results showed that the BL21 after being transformed with the recombinant expression vector accumulated the high amount of recombinant protein. SDS-PAGE analysis showed that the recombinant protein was about 53 kDa. The recombinant accumulated the highest amount of recombinant protein af ter IPTG (1 mmol x L(-1)) at 27-37 degrees C for 24 h. Consequently P450 gene of E. senticosus could be expressed successfully by prokaryotic expression vector pET30a-P450. Induction temperature, IPTG concentration, medium type and amount of induction time could all influence the expression of target protein, but the impact strength was different.
Cloning, Molecular ; Cytochrome P-450 Enzyme System ; genetics ; metabolism ; Eleutherococcus ; enzymology ; genetics ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Genetic Vectors ; genetics ; metabolism ; Plant Proteins ; genetics ; metabolism ; Plasmids ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism

OBJECTIVETo investigate the influence of the prostaglandin E2 on the proliferation of the melanocytes in the full-thickness skin graft.

METHODSSixty-eight guinea-pigs were divided into experimental-1 group (skin graft), experimental-2 group (skin graft + diclofenac), and control groups. After the full-thickness skin graft, the dynamic changes of the prostaglandin E2 were measured and the proliferation of the melanocyte with its density was also evaluated by using histochemical and autoradiographic methods.

RESULTSIn the experimental-1 group, the content of PGE2 was increasing in seven days after the operation, continued to the one month, and then returned to the base level. The labelling indices of 3H-MC-TdR of the group was also increasing postoperatively between the second day and the fourteenth day, and reach a second peak after one month, then came to the normal level. The density of the melanocytes was decreasing rapidly 3 days after the surgery, then began to increase and exceeded over the normal level 21 days after the operation. However, in the experimental-2 group, the content of PGE2 decreased in two days after the surgery, and then showed the inclination similar to the experimental-1 group with the different points in narrower range. The number of melanocytes labelled by 3H-TdR began to increase at the first day after the surgery, which appeared earlier than the experimental-1 group and was similar in the changing tendency with a less extent. The density of MC showed the similar tendency to the experimental-1 group in a narrower changing range with both of increasing and decreasing. The density of the MC was much lower in 21 after the operation than the experimental-1 group and normal control group.

CONCLUSIONThe increased PGE2 in the earlier stage of the skin grafting could enhance the inflammatory reaction to the tissue, as well as the melanocytes. It may stimulate the proliferation of the MC with the result of increasing their density. The use of the diclofenac might reduce the inflammation and suppress the proliferation of melanocytes, and result in the skin with light color due to decreasing the number of MC in the epidermis of the graft.

Animals ; Anti-Inflammatory Agents, Non-Steroidal ; pharmacology ; Cell Count ; Cell Proliferation ; Diclofenac ; pharmacology ; Dinoprostone ; metabolism ; Epidermis ; Guinea Pigs ; Melanocytes ; cytology ; Skin ; Skin Transplantation ; Time Factors

OBJECTIVETo apply the single cell nested multiplex polymerase chain reaction (PCR) to HLA typing, and analyze the influence factors on the amplification results.

METHODSSingle cell DNA templates were prepared with different methods. The exon 2, 3 and intron 2 of HLA-A, B, and exon 2 of DRBI were amplified using multiplex PCR. The second round of SSP-PCR HLA typing was carried out according to the large scale routine HLA typing results.

RESULTSEnzyme lysis method was the most efficient procedure for preparing the single cell DNA template, with a success rate (SR) of 93.3%, while the SRs of alkali lysis and freezing-thaw lysis methods were 83.3% and 73.3%, respectively. The second round amplification using enzyme lysis and SSP-PCR in 20 samples obtained a 95% success rate and a 15% allele drop out rate. The time for performing the whole procedure was less than 6 hours.

CONCLUSIONThe modified nested multiplex PCR technique is efficient for single cell HLA typing and might be applied to clinical preimplantation genetic diagnosis.

Histocompatibility Testing ; methods ; Humans ; Polymerase Chain Reaction ; methods

OBJECTIVETo investigate the expressions of vinculin (VCL) and the androgen receptor (AR) in benign prostatic hyperplasia (BPH) and prostate cancer (PCa) and analyze their relationship with the clinical stage and pathological grade of PCa and the level of PSA.

METHODSWe detected the expressions of VCL and AR in 18 cases of BPH and 38 cases of PCa by immunohistochemistry, analyzed the differences of VCL and AR expressions in BPH and PCa in different clinical stages and pathological grades of PCa, compared the primary levels of PSA, and studied their correlations.

RESULTSThe positive rate of VCL was significantly higher in PCa than in BPH tissues (P < 0.05), while that of AR showed no significant differences between the two groups (P > 0.05). Both the expressions of VCL and AR were closely related with the clinical stage and pathological grade of PCa (P < 0.05), but not with the PSA level (P > 0.05). There was a positive correlation between the expressions of VCL and AR in PCa tissues (r = 0.489, P < 0.05).

CONCLUSIONVCL is expressed differently in BPH and PCa, which may serve as an indicator for the differential diagnosis of benign and malignant prostate diseases. The expressions of AR and VCL are gradually reduced with the progression of PCa, with a positive correlation between them, and could be used jointly to evaluate the progression and prognosis of PCa.

Aged ; Aged, 80 and over ; Humans ; Male ; Middle Aged ; Prostatic Hyperplasia ; metabolism ; pathology ; Prostatic Neoplasms ; metabolism ; pathology ; Receptors, Androgen ; metabolism ; Vinculin ; metabolism

This study was aimed to analyze the biological characteristics of rabbit bone marrow mesenchymal stem cells (rBM-MSCs) and their response to different growth factors. Rabbit BM-MSCs were separated from bone marrow mononuclear cells by using adherent cultivation. Biological characteristics were investigated by optical and electron microscopy. Immunophenotype of rBM-MSCs was measured by flow cytometry. The expression of collagen was detected by RT-PCR. Differentiation potential was identified by specific staining and RT-PCR. The response of rBM-MSCs to IL-1, 3, 8 and HGF with different concentrations were tested by MTT. The results showed that the rBM-MSCs gave rise to a population of adherent cells characterized by the presence of a predominant cell type with a typical fibroblast-like morphology and could be cultured for over 15 passages. CD44 was highly expressed on F5 rBM-MSCs (32%) and CD45 was lowly expressed (4.7%). Type I collagen was highly expressed, while type II collagen was lowly expressed and type X collagen was not detected on rBM-MSCs using RT-PCR method. In various conditions inducting differentiation, rBM-MSCs could differentiate into the osteoblast, chondrocyte, adipocyte and neuron-like cells. The rBM-MSCs were sensitive to IL-3, even low concentration (10 ng/ml) of IL-3 could promote the proliferation of rBM-MSCs effectively (>32%, P < 0.01), whereas high concentration IL-3 inhibited it significantly. It is concluded that rabbit BM-MSCs were successfully isolated and culture-expanded. The biological characteristics of rabbit BM-MSCs are similar to those of human and rhesus BM-MSCs. IL-3 with low concentration can promote the proliferation of rBM-MSCs effectively, but high concentration of IL-3 can inhibit their proliferation.
Animals ; Bone Marrow Cells ; cytology ; physiology ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Cytokines ; pharmacology ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Male ; Mesenchymal Stromal Cells ; cytology ; physiology ; Rabbits ; Recombinant Proteins

Aged ; Carcinoma, Merkel Cell ; diagnosis ; surgery ; Female ; Humans ; Skin Neoplasms ; diagnosis ; surgery ; Thigh

Objective: To evaluate the antidiabetic effect of Callicarpa nudiflora extract in streptozotocin-induced diabetic rats. Methods: The chemical constituents in Callicarpa nudiflora extract were identified by UPLC-Q-TOF-MS. Callicarpa nudiflora extract (0.15 and 0.3 g/kg) was orally administered to streptozotocin-induced diabetic rats for 42 d. The effects of Callicarpa nudiflora extract on body weight, blood glucose, serum insulin, total cholesterol, triglyceride, LDL-C and HDL-C were investigated, and its effect on liver and pancreatic pathology was assessed by histopathological analysis. Moreover, the expression levels of adenosine 5'-monophosphate-activated protein kinase (AMPK), glucose transporter type 4 (GLUT4), phospho-AMPK/AMPK, and p-acetyl-coA carboxylase (P-ACC)/ACC in the skeletal muscles and liver were determined by reverse transcription-polymerase chain reaction, Western blotting, and immunohistochemistry. Results: A total of 34 compounds, including 8 iridoids, 14 phenylpropanoids, and 12 flavonoids, were identified from Callicarpa nudiflora extract. Callicarpa nudiflora extract significantly reduced blood glucose and significantly restored all other biochemical parameters to near normal levels, including serum insulin, total cholesterol, triglyceride, LDL-C, and HDL-C. Callicarpa nudiflora extract improved insulin resistance and reversed the damage in the liver and pancreas caused by diabetes. Furthermore, Callicarpa nudiflora extract increased the expression levels of phospho-AMPK, GLUT4, P-ACC, and insulin receptor substrate-1 and decreased the expression level of PPAR毭 in diabetic rats.Conclusions: Callicarpa nudiflora extract improved oral glucose tolerance, lipid metabolism, insulin resistance, and reversed the diabetes-related damage in the liver and pancreas by activating the AMPK-ACC pathway.

OBJECTIVETo investigate the effects of valsartan on expression of angiotensin II receptors in different regions of heart after myocardial infarction (MI).

METHODSCanines were divided into sham-operated control group (n=7), infarction group (n=7) and Valsartan group (10 mg x kg(-1) x day(-1) for 4 weeks after MI operation, n=7). Four weeks after operation, Doppler tissue imaging (DTI) was used to evaluate regional ventricular function in the noninfarcted myocardium (apical and basal near to the infarction region). The mRNA and protein expressions of angiotensin II type 1 receptor (AT1-R) and angiotensin II type 2 receptor (AT2-R) on the corresponding regions were detected by competitive reverse-transcriptase polymerase chain reaction technique and immunohistochemical technique respectively. Results The protein and mRNA expressions of AT1-R were significantly increased in both apical and basal regions near to the infarction in dogs with MI compared with those in control group (P < 0.05) which could be downregulated by valsartan (P < 0.05). AT2-R expressions were significantly upregulated in infarction group in both apical and basal regions compared with those in control group and valsartan further increased AT2-R expressions in both areas (P < 0.05). Myocardial peak systolic velocity (Sm), myocardial peak early diastolic velocity (Em) and myocardial peak late diastolic velocity (Am) at both apical and basal regions near to the infarction regions were significantly lower in MI group than those in the control group which could be significantly improved by valsartan.

CONCLUSIONBoth mRNA and protein expressions of AT1-R and AT2-R are upregulated in noninfarcted regions near MI, valsartan improved myocardial function via inhibiting AT1-R upregulation and enhancing AT2-R upregulation.

Angiotensin II Type 1 Receptor Blockers ; pharmacology ; therapeutic use ; Animals ; Dogs ; Female ; Male ; Myocardial Infarction ; drug therapy ; metabolism ; physiopathology ; Myocardium ; metabolism ; RNA, Messenger ; metabolism ; Receptor, Angiotensin, Type 1 ; metabolism ; Receptor, Angiotensin, Type 2 ; metabolism ; Tetrazoles ; pharmacology ; therapeutic use ; Valine ; analogs & derivatives ; pharmacology ; therapeutic use ; Valsartan

Objective To investigate the effects of valsartan on expression of angiotensin Ⅱ receptors in different regions of heart after myocardial infarction(MI).Metbods Canines were divided into sham-operated control group(n=7),infarction group(n=7)and Valsartan group(10 mg·kg-1·day-1 for 4 weeks after MI operation,n=7).Four weeks after operation,Dopplor tissue imaging(DTI)was used to evaluate reglonal ventricular function in the noninfarcted myocardium(apical and basal near to the infarction region).The mRNA and protein expressions of angiotensin Ⅱ type 1 receptor(AT1-R)and angiotensin Ⅱ type 2 receptor(AT2-R)on the corresponding regions were detected by competitive reverse-transcriptase polymerase chain reaction technique and immunohistochemical technique respectively.Results The protein and mRNA expressions of AT1-R were significantly increased in both apical and basal regions near to the infarction in dogs with MI compared with those in control group(P<0.05)which could be down-regulated by valsartan(P<0.05).AT2-R expressions were significantly upregulated in infarction group in both apical and basal regions compared with those in control group and valsartan further increased AT2-R expressions in both areas(P<0.05). Myocardial peak systolic velocity(Sm),myocardial peak early diastolic velocity(Em)and myocardial peak late diastolic velocity(Am)at both apical and basal regions near to the infarction regions were significantly lower in MI group than those in the control group which could be significantly improved by valsartan.Conclusion Both mRNA and protein expressions of AT1-R and AT2-R are upregulated in noninfarcted regions near MI,valsartan improved myocardial function via inhibiting AT1-R upregulation and enhancing AT2-R upregulation.