Cell viability of Pichia pastoris was detected by flow cytometry (FCM) with two reagents fluorescein diacetate (FDA) and propidium iodide (PI). Compared with FDA/PI double-stained dot plots and PI single-stained dot plots,the latter could divide dead and living cells into two separate zones,and get the correct proportion. Then PI single-stained method was used to detect the change of cell viability in Pichia patoris fermentation. At glycerol batch and fed-batch phase,little dead cells were detected. At methanol fed-batch phase,cell viability decreased when cell weight increased,and was only 73.8% at 88 h.

The intracellular hepatitis B surface antigen (HBsAg) content per cell was increased by 7.2-fold in the culture with 1.5% dimethyl sulfoxide (DMSO) compared with that in the control without DMSO, while the extracellular HBsAg production and specific productivity were only improved by 70% and 3.2-fold, respectively. Electron microscope has been employed to reveal large dilated structures within recombinant CHO cells in the presence DMSO. The dilated structures have a distribution within whole cytoplasm, and some dilated areas were engulfed in the nucleus. These large, dilated structures were not observed in the control. Immunogold labeling was used to discover the accumulated HBsAg was localized within these dilated areas, and some HBsAg-specific labels were detected in the nucleus membrane, owing to the encroachment of the dilated areas upon nucleus. The result could help to reveal the mechanism of intracellular HBsAg accumulation in the presence of DMSO.

Objective To observe the therapeutically effect of kangnao liquid on Pi3k mRNA and Aktm RNA ex-pressions in rats with focal cerebral ischemia-reperfusion (I/R) injury. Methods 180 male SD rats were randomly divided into 6 groups:sham operated group, model group, three kangnao liquid groups (high-dose, medium-dose and low-dose) and nimodipine group. Rats in kangnao liquid groups were administrated with kangnao liquid of 24 g/(kg · d), 12 g/(kg · d) and 6 g/(kg · d), orally once a day. Rats in nimodipine group were given nimodipine 1 mg/(kg · d). Rats in model group and sham group were treated with the same volume of distilled water for 7 days. The animal model of middle cerebral artery occlusion (MCAO) was established by a monofilament method from right internal carotid artery. The neurological evaluation was per-formed 24 h after reperfusion. The in situ hybridization was used to investigate the expression levels of Pi3k mRNA and Akt mRNA in rats on 12 h, 24 h, 48 h, 72 h and 168 h after ischemia for 2 h. Results Compared with model group, neurological functions were improved significantly in kangnao liquid groups. The expression levels of Pi3k mRNA and Akt mRNA were al-so significantly higher in kangnao liquid groups than those of model group. The expression levels of Pi3k mRNA and Akt mRNA were significantly higher in nimodipine group than those of model group, but which were lower compared with those of high-dose and medium-dose kangnao liquid groups. Conclusion Kangnao liquid can protect nerve cells by enhancing the expressions of Pi3k mRNA and Akt mRNA in rats with cerebral ischemia-reprefusion injury.

Objective To explore the effects of forepaw sensorimotor deprivation in early life on hippocampus-dependent spatial reference learning and memory in rats. Methods Newborn SD rats were randomly assigned to experiment group (deprivation of forepaw sensorimotor function, n=53) and control group(n=55). Rats of postnatal day 13 (PN13) in experiment group were seleeted, and models of forepaw sensorimotor deprivation were established by microsurgical technique. Open field tests and Morris water maze tests were performed during the time periods of PN25(PN21-31), PN35 (PN31-39), PN45(PN41-50) and PN60(PN56-64) to evaluate the locomotor activity and spatial reference learning and memory, respectively. Results In open field tests, there was no significant difference in parameters of locomotor activity and exploratory behavior between the two groups (P>0.05). In Morris water maze tests, eontrol group performed significantly better than experiment group during training sessions and probe tests on PN25 and PN35 (P<0.05). While on PN45, although there was no significant difference between the two groups during training sessions, control group performed significantly better than experiment group during probe tests (P<0.05). Conclusion The deprivation of forepaw sensorimotor in early life has no signifieant effect on the locomotor activity and exploratory behavior of rats, but can impair the spatial reference learning and memory.

OBJECTIVETo explore the preventive effect of Yougui drink on femoral head necrosis in rats under micro CT.

METHODSTwenty-five SD rats were divided into steroid hormone group (group A, 10 rats ), Yougui drink group (group B,10 rats) and normal group (group C,5 rats)with random number table. Endotoxin were injected into abdominal cavity of rats in group A and B for 2 days, methylprednisolone sodium succinate were injected by gluteus for twice a week continued for 6 weeks; group B were gavaged by Yougui drink (veryday for 8 weeks; group C did not do any processing. All rats were killed on the 10th weeks,m icro CT were used to scan femoral head in vitro and preventive effect of Yougui drink (n femoral head necrosis in rats.

RESULTSThere was statistical significance in BMD, BV/TV, Tb.N, Tb, Th, Thb, Sp, BS/TV and DA but no significance in SMI between group A and B. Comparison between A and C, there was significant meaning in BMD, BV/TV, Tb.N, Tb, Th, Tb, Sp, BS/TV, DA and SMI.

CONCLUSIONYougui drink on femoral head necrosis in rats under micro CT has preventive effect from BMD BV/TV, Tb.N, Tb, Th, Tb, Sp, BS/TV and DA.

Animals ; Apoptosis ; Bone Density ; Drugs, Chinese Herbal ; therapeutic use ; Femur Head Necrosis ; diagnostic imaging ; pathology ; prevention & control ; Rats ; Rats, Sprague-Dawley ; X-Ray Microtomography ; methods

OBJECTIVETo explore the feasibility of inducing human umbilical cord mesenchymal stem cells (HuMSCs) to differentiate into Leydig cells through conditioned medium derived from Leydig cells.

METHODSHuMSCs and Leydig cells were obtained by tissue blocks culture attachment and enzymatic digestion respectively. HuMSCs were induced by conditioned medium of Leydig cells as an experiment group while those before induction were cultured as a control group. The expressions of LHR, 3β-HSD and StAR in the induced HuMSCs were determined by RT-PCR after 3, 7 and 10 days of culture; those of CYP11A1, CYP17A1 and 3β-HSD measured by immunofluorescence staining after 2 weeks; and that of 3β-HSD detected by Western blot after 4 weeks.

RESULTSThe experimental group showed positively expressed LHR, 3β-HSD and StAR at 3, 7 and 10 days, CYP11A1, CYP17A1 and 3β-HSD at 2 weeks, and 3β-HSD at 4 weeks, while the control group revealed negative expressions at all the time points.

CONCLUSIONInduced with conditioned culture medium derived from Leydig cells, HuMSCs are likely to differentiate into steroidogenic cells and eventually into Leydig cells.

Cell Differentiation ; Culture Media, Conditioned ; Humans ; Leydig Cells ; cytology ; Male ; Mesenchymal Stromal Cells ; cytology ; Umbilical Cord ; cytology

Adenocarcinoma, Clear Cell ; pathology ; Ethmoid Sinus ; Humans ; Kidney Neoplasms ; pathology ; Male ; Middle Aged ; Neoplasm Metastasis ; Paranasal Sinus Neoplasms ; secondary

Acute Disease ; Adult ; Arsenic Poisoning ; pathology ; Humans ; Male ; Respiratory System ; pathology ; Young Adult

Biomarkers ; urine ; Child ; Child, Preschool ; Cystatin C ; Cystatins ; urine ; Cytomegalovirus Infections ; pathology ; urine ; Female ; Humans ; Kidney ; pathology ; Male ; TATA Box Binding Protein-Like Proteins ; urine ; alpha-Macroglobulins ; urine

OBJECTIVETo study the effect of acupuncture on the immune function of sepsis patients.

METHODSNinety sepsis patients were assigned to the control group, the thymosin a1 group, and the acupuncture treatment group according to random digit table, 30 patients in each group. Patients in the control group were treated according to the guideline of Surviving Sepsis Campaign (SSC). Patients in the control group received routine treatment. Those in the thymosin alpha1 group additionally received subdermal injection of thymosin alpha1 (1.6 mg), once per day for 6 successive days. Needling at related points such as Zusanli (ST36), Yanglingquan (GB34), Neiguan (PC6), Guanyuan (RN4), and so on, was performed in patients of the acupuncture treatment group, once per day for 6 successive days. T cell subgroups (CD3+, CD4+, CD8+, CD4+ /CD8+) and immunoglobulin levels (IgG, IgA, IgM) were detected. The length of ICU hospital stay, hospital readmission rate, and 28-day mortality were compared among the three groups.

RESULTSAfter six days of treatment, CD3+, CD4+, CD8+, IgG, IgA, IgM, and CD4+ /CD8+ ratio of three groups were all significantly increased (P < 0.01). Of them, CD3+, CD4+, CD8+, IgG, IgA, and IgM increased more significantly in the thymosin alpha1 group and the acupuncture treatment group (P < 0.01). Compared with the control group, the ICU hospitalization length was significantly shortened, the hospital readmission rate and the 28-day mortality were lower in the thymosin alpha1 group and the acupuncture treatment group (P < 0.05, P < 0.01). There was no statistical difference in each index between the thymosin alpha1 group and the acupuncture treatment group (P > 0.05).

CONCLUSIONAcupuncture could adjust the immune function of sepsis patients, improve their immunological indicators and prognoses.

Acupuncture Therapy ; CD4-CD8 Ratio ; Humans ; Length of Stay ; Prognosis ; Sepsis ; diagnosis ; immunology ; therapy ; T-Lymphocyte Subsets ; Thymosin ; analogs & derivatives