Objective To approach a way to induce MSCs to dopaminergic neuron by mesencephalic conditional media and cytokines in vitro,and supply an ideal cells source for the treatment of Parkinson's disease.Methods The rat MSCs were isolated primarily from the femurs and tibias of the Wistar rats.MSCs were cultured,proliferated and purified by passage culture.Cultuered MSCs were divided into the control and the experimental group.In control group,MSCs were cultured without any induction medium.MSCs of experimental group were first cultured at medium containing bFGF for 24 hours.Then media were replaced with induction media which contained the agents as follows,respectively:GM_1,GDNF, GDNF+GM_1,GDNF+GM_1+mesencephalic conditional media.The surface markers of the differentiated neuron,such as NSE and TH were detected by immunocytoehemistry after MSCs were cultured in induction media for 3 and 7 days.Results In control groups,the NSE expression of MSCs was very lower than experimental groups.The percentage of NSE-positive cells of GDNF+GM_1+mesencephalic conditional media group in 7 day((45.257?5.999)/HP)was significantly more than other groups(control group is 2.214?0.779,GM_1 group is 22.014?3.624,GDNF group is 31.345?2.850,GDNF?GM_1 group is 40.314?4.203,P

Objective To study the frequency of the derivative chromosome 9 [der(9)] deletion among patients with chronic myeloid leukemia (CML), and explore the application value of local-specific inspector (LSI) 9q34 probe in detect the der (9) deletion. Methods Cytogenetic analysis was performed by 24 h unstimulated culture, GTG banding and karyotype. Dual colour/dual fusion or extra signal(ES) DNA probe was used to perform interphase-FISH for the detection of bcr-abl fusion gene in 52 patients with CML. LSI 9q34 DNA probe was used to detect der(9) deletion. Results FISH results showed bcr-abl fusion gene existed in all 52 patients with CML. der(9) deletion was detected in 12 patients with ES/DCDF probe, but only in 11 patients with LSI 9q34 probe. Conclusion It's more efficient in detection of der(9) deletion with LSI 9q34 probe. Deletion of der(9) might be a poor prognostic factor for CML patients, and LSI 9q34 probe should be used in all the CML patients with positive bcr-abl fusion gene.

OBJECTIVETo evaluate the clinical effectiveness of Yiqi Huoxue Tongyang Xiezhuo Recipe (YHTXR, capable of supplementing qi, activating blood, warming yang, and discharge turbidity) in treating coronary atherosclerotic heart disease (CAHD). and chronic heart failure (CHF) with carotid plaque patients, and to explore new ways of Chinese medicine (CM).

METHODSTotally 69 CAHD-CHF patients of qi deficiency phlegm stasis syndrome (QDPSS) with carotid plaque were recruited in this study using parallel cohort method. They were assigned to the treatment group (35 cases) and the control group (34 cases). Patients in the control group received routine treatment of Western medicine, while those in the treatment group were additionally treated with YHTXR (twice daily). The therapeutic course for all was three months. Cardiac function levels, echocardiography, carotid plaque, blood lipids and safety indicators were observed before and after treatment.

RESULTSAfter treatment the improvement of cardiac function levels was better in the treatment group than in the control group (P < 0.05). Decreased LDL-C levels were higher in the treatment group than in the control group (P < 0.01). There was statistical difference in left ventricular ejection fraction (LVEF), carotid intima-media thickness (IMT), LDL-C, TC, TG in the treatment group between before and after treatment (P < 0.05). LDL-C and TG also decreased in the control group after treatment (P <0.05). There was no significant difference in the left ventricular ejection fraction, carotid IMT, or TC in the control group between before and after treatment (P > 0.05). There was no significant difference in stroke volume, left ventricular end-diastolic diameter, the area of carotid artery plaque, or HDL-C in the two groups between before and after treatment (P > 0.05).

CONCLUSIONSYHTXR could effectively improve cardiac functions of CAHD-CHF patients of QDPSS with carotid plaque, reduce blood lipids and IMT. It had no significant adverse reactions for elderly patients in short term.

Carotid Intima-Media Thickness ; Coronary Disease ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Heart ; Heart Failure ; drug therapy ; Humans ; Lipids ; Plaque, Atherosclerotic ; drug therapy ; Qi ; Ventricular Function, Left

Atrial Fibrillation ; therapy ; Catheter Ablation ; adverse effects ; Female ; Humans ; Pulmonary Veins ; Venous Thrombosis ; etiology

Objective To explore the characteristics of T cell immunity in peripheral blood of patients with carboxypeptidase - H antibody (CPH-Ab). Methods Forty-two latent autoimmune diabetes in adults (LADA) patients with CPH-Ab~+ alone, 20 Type 2 diabetes mellitus patients (T2DM), and 22 healthy controls were selected and their peripheral blood mononuclear cells were isolated. Human recombinant carboxypeptidase (CPH) protein was expressed and further used as a stimulant in Enzyme-linked immunospot (ELISPOT) assay to detect IFN-γ-Th1 and IL-4-Th2 cells in the 3 groups. Th1/Th2 ratios were also calculated. CPH-Ab and glutamic acid decarboxylase antibody (GAD-Ab) were determined by radioligand assay. Results Compared with healthy controls and T2DM, IFN-γ-Th1 and IL-4-Th2 numbers did not increase significantly in CPH-Ab~+ group, nor did the Th1/Th2 ratios (P ＞0. 05). We further divided the CPH-Ab~+ patients into a short duration group (n = 22) and a long duration subgroup (n = 20) according to the duration of 3 years. CPH-IL-4-T in the short duration subgroup was significantly higher than that in T2DM and healthy controls (1. 8 vs. 0.2 and 0.3, both P ＜ 0. 05) and we did not find any factor that was significantly correlated with the IL-4 spots number. There were not any significant differences in T cell responses to phaseolus vulgaris agglutinin (PHA) among all groups (P＞0.05). Conclusion CPH does not directly involve in the cellular pathological mechanism of LADA. Anti-CPH immunity may be associated with more slowly aggressive beta cell autoimmunity.

Objective:To construct a DNA vaccine co-expressing the HBV compound multi-epitope antigen gene, the hIL-12 and the anti-HBV siRNA genes, and to express this DNA vaccine in HepG2 cells. Methods:The HBV multi-epitope antigen gene was designed and synthesized before it was fused with enhanced green fluorescent protein(EGFP) gene, and cloned into the multi-clone site(MCS) of the eukaryotic expression vector pVAX1. The expressinig units of hIL-12 and siRNA were cloned into the BspH I and Mlu I site of pVAX1 respectively. Then the recombinant plasmid pVAX1-siHBV-HB-EGFP-hIL12 was transiently transfected HepG2 cells. The expression of HBV compound multi-epitope gene was observed through EGFP report gene. The expression of hIL-12 was analyzed by ELISA and the effects of anti-HBV siRNA was confirmed with rtPCR . Results: The analysis of enzyme digestion and sequencing both demonstrated that the trible-expressing HBV DNA vaccine has been constructed successfully. The green fluorescent image was detected in the transfected cells which could confirm the expression of the multi-epitope antigen gene. The amount of hIL-12 secretion was 1289pg/ml in supernatant at 48h after transfection and 1712pg/ml at 72h after transfection. The mRNA amount of HBV S gene, which was the siRNA target, had been obviously knockdown. Conclusion: The DNA vaccine co-expressing the HBV compound multi-epitope antigen gene, the hIL-12 and the siRNA genes was constructed and transiently expressed in HepG2 cells, and siRNA had shown us a good anti-HBV effect. It laid a foundation of further study on anti-HBV effect of the new DNA vaccine.

Background Oxidative stress is thought to be responsible to diabetes-complicated cataract.Our previous study demonstrated that as an iron chelator,deferiprone can protect lens from oxidative damage.Objective This further study aimed to investigate the role of deferiprone on the formation of diabetic-complicated cataract.Methods Forty 6-week-old Wistar rats were included in the study and randomized into 4 groups.Eight of them were used as the normal control group.Diabetes mellitus animal models were established in 22 rats by the carbonhydratediet and fat diet and the intraperitoneal injection of 40 mg/kg streptozocin (STZ).The deferiprone of 50 mg and 100 mg were intragastrically given in 8 model rats respectively after 3 days once a day for 8 weeks.The opacification of lenses was examined under the slit lamp weekly after treatment.The animals were sacrificed and the lenses were obtained at the eighth week of deferiprone injection.The concentrations of water-soluble protein ( WSP),urine-soluble protein (USP) and alkali-soluble protein (ASP) in rat lens suspension were detected by Bradford method.The super oxide dimutese (SOD),malondialdehyde (MDA) and glutathione (GSH) were determined spectrometically using xanthine oxidase,thiobarbituric acid,dithio bis-nitrobenzoic acid.Results No evidently differences were found in the content of the WSP,USP and ASP among the these groups( F=1.73,0.18,0.09,P＞0.05).The contents of MDA in 50 mg deferiprone group and 100 mg deferiprone group were ( 1.05 ± 0.10 ) mmol/g and ( 1.05 ± 0.22 ) mmol/g respectively,showing a significant decline in comparison with diabetic model group (P＜0.05).The SOD and GSH contents in lens were (321.29±16.57) U/mg,(322.07±22.16) U/mg and (7.83±0.65 ) mg/g,(7.70±0.77 ) mg/g respectively in 50 mg deferiprone group and 100 mg deferiprone group and were considerably elevated in comparison with ( 298.70± 14.69 ) U/mg and ( 5.47 ± 1.01 ) mg/g of diabetic model groups ( P＜0.05 ).No significant differences were found in the indexes mentioned above between 50 mg and 100 mg deferiprone groups(P＞0.05).Conclusions Deferiprone can reduce oxidative stress and improve the energy metabolism of the lens in diabetic rats.

To improve 3-ketosteroid-△1-dehydrogenase(KSDH) activity and the transformation level for androst-4-ene-3,17-dione, 3-ketosteroid-△1-dehydrogenase gene(ksdD) from Arthrobacter simplex was cloned into plasmid pWB980 and expressed in B. subtilis WB600 under the control of promoter P43. The molecular weight of expressed enzyme was about 55kDa by SDS-PAGE analysis. The activitities assayed by spectrophotometrical method of intracellular and extracellular soluble enzyme were 110?0.5mU and 15?0.6mU per milligram of protein respectively. The transformation rate of androst-4-ene-3,17-dione by the B. subtilis recombinant cells was 45.3%. Compared with Arthrobacter simplex, the enzyme activity of KSDH expressed in B. subtilis was improved about 30 fold, and the transformation level of androst-4-ene-3,17-dione by the B. subtilis recombinant cells was improved about 10 fold. The recombinant B. subtilis cells used in biotransformation of steroids provided a new way for steroid medicines production.

The NSP2 gene of porcine reproductive and respiratory syndrome virus (PRRSV)S1 strain was partly amplified and cloned into a prokaryotic expression vector pGEX-6P-1 and a fusion protein GST-tNSP2 with molecular weight of 50 kDa was expressed in E.coli. The purified GST-tNSP2 protein showed a strong reaction with the PRRSV-positive sera in Western blot assay. Balb/c mice were immunized with the purified protein, and the splenocytes of the immunized mice were fused with murine myeloma cells SP2/0. After subcloning by 3 times, two hybridoma clones which produced McAbs steadily were screened by ELISA, named 3H3 and 2B5. They all reacted strongly with the PRRSV S1 infected Marc-145 cells in IFA, but not with the PRRSV SY0608 strain. Both of the McAbs belong to IgG1 isotype, and their light chains belong ? type. The expressed GST-tNSP2 protein and McAbs could be used for identification of PRRSV isolates and functional analysis of NSP2.

Objective To explore the anti-inflammatory and antipruritic effects of Chushizhiyang ointment in a mouse model. Methods A total of 40 male 8-week-old BALB/c mice were included in this study, and randomly and equally divided into 4 groups. A mouse model of atopic dermatitis (AD)was established in three groups of mice by repeated application of 2,4-dinitroflurobenzene (DNFB)to shaved abdominal skin for sensitization and to shaved dorsal skin for stimulation. After establishment of the AD model, the three groups were topically treated with sodium chloride physiological solution (model group), hydrocortisone cream (hydrocortisone group)and Chushizhiyang ointment (Chushizhiyang group)respectively for 14 consecutive days. The remaining group receiving no sensitization or treatment served as the normal control group. All the mice were sacrificed 12 hours after the final treatment, and the dorsal skin of mice was resected followed by the determination of skin thickness and weight as well as hematoxylin-eosin(HE)staining and toluidine blue staining for the counting of leukocytes and mast cells respectively. Moreover, enzyme-linked immunosorbent assay(ELISA)was performed to measure the levels of interferon-gamma(IFN-γ), tumor necrosis factor alpha(TNF-α), interleukin 4(IL-4)and IL-5 in dorsal skin lesions. In addition, a local skin itching model was induced by histamine phosphate in Hartley guinea pigs, which was used to explore the effect of Chushizhiyang ointment on itch thresholds. Results Compared with the model group, both the Chushizhiyang group and hydrocortisone group showed reduced thickness and weight of dorsal skin in mice (all P < 0.01), numbers of infiltrating lymphocytes and mast cells (all P < 0.01)and levels of IFN-γ, TNF-α, IL-4 and IL-5 in skin lesions (P < 0.05 or 0.01)on day 15 after the start of treatment. The thickness and weight of dorsal skin in mice were significantly decreased in the hydrocortisone group (P <0.01), but experienced no significant changes in the Chushizhiyang group compared with the normal control group. Additionally, Chushizhiyang ointment could significantly increase itch thresholds in guinea pigs induced by histamine phosphate(P < 0.01). Conclusions Chushizhiyang ointment can significantly inhibit DNFB-induced AD in mice, likely by restoring the balance between Th1 and Th2 type cytokines. Moreover, Chushizhiyang ointment could markedly relieve itching induced by histamine phosphate in guinea pigs.