Objective To observe the regulatory mechanism of substance P on the tension of human umbilical vein and the calcium ion of endothelial cells. Methods The tension was recorded by conventional physiological recording methods,and confocal laser scanning microscopy and patch\|clamp technique were used to observe the concentration of intracytosolic free calcium ion and the opening probability of membrane calcium ion channel of cultured human umbilical vein endothelial cells. Results Substance P induced the endothelial\|dependent relaxing of human umbilical vein and the increasing of the concentration of intracytosolic free calcium ion and of the opening probability of calcium ion channel in the cultured human umbilical vein endothelial cells.Conclusion\ Substance P could be function as the activator of the releasing of the intracellular stored calcium ion,and the inflowing of calcium ion from the outside cell bodies to relax human umbilical vein.

Objective To study the effect of ovarian estrogen on the expression of estrogen receptor beta (ER beta) of the basal forebrain of mice. Methods Ovariectomized (OVX) mice were used as animal model, and nickel ammonium sulfate intensified immunohistochemical SP technique was used to detect the changes of ER beta expression in the basal forebrain. Results The expression of ER beta in the basal forebrain of mice decreased dramatically, after OVX decreased to the lowest levels at 3 d after OVX, increased gradually, and recovered to normal level 20 d later. Conclusion Ovarain estrogen can regulate the expression of ER ? of the murine basal forebrain, indicating that estrogen, via ER ?, can regulate the nerous structure and function of the basal forebrain, which may be the mechanism of estrogen replacement therapy on Alzheimer′s brain.

BACKGROUND: Neural axon regeneration is one of the difficulties that must be overcome in treatment of injury of central nerve system. Significant therapeutic effects have been obtained in transplantation of neural stem cells (NSCs), embryonic stem cells (ESCs) and Schwann cells. But the bottleneck situation of insufficiency of cell provider has limited the development on it.OBJECTIVE: To observe directional-differentiation of retinoic-acid induced ESCs so as to find optimal condition for neuronal differentiation.DESIGN: Non-randomized controlled experiment was designed.SETTING: Teaching-Research Room of Histology and Embryology, Department of Basic Medicines. Third Military Medical University of Chinese PLAMATERIALS: The experiment was performed in Staff Room of Histology and Embryology, Third Military Medical University of Chinese PLA from January to May 2000. Eighteen Kunming mice in disoestrus were employed, of which. 12 mice were female and 6 mice male. They were placed in same cage at ratio of 2:1 for mating. The date of pregnancy was recorded. MESPU35 ESC line was prepared.METHODS: Removed head. internal organs and four limbs, feeder-layer Feeder-layer adherent culture was used to proliferate MESPU35 ESCs.Classic 4-/4+ method [The embryoid body (EB) grew naturally for 4 days,without retinoic acid added. In the coming 4 days, retinoic acid was added to induce neural EB of high proportion] was applied to induce the directional differentiation of the nerve. EB was cultured with serum of different concentrations. Phase contrast microscope was used to observe nerve-like EB in serum of different concentrations and to count numbers. ②Immunocytochemical technique was used to observe cellular morphological charac ters at various differentiating phase spots (5th. 9th, 14th days) and with retinoic acid at various concentrations. Flow cytometer (FCM) was used to count the proportion of differentiated neurons.MAIN OUTCOME MEASURES: ①Estimated measurement of the length of process and cell body during formation of neural EB after retinoic-acid induced differentiation of MESPU35 ESCs. ② Observation of cell morphology with immunocytochemical staining and proportion of differentiated cells assayed with FCM.RESULTS: ①It was discovered with phase contrast microscope that serum of different concentrations affect neural directional differentiation after EB formation to certain extent. Excessively high and low concentrations of serum reduced the proportion of neural differentiation of EB. The differentiating proportion is high in serum with 5% concentration. ② It is observed with immunocytochemical technique that the proportions of NF200 positive cell and glial fibrillary acidicprotein (GFAP) positive cell in differentiation of MESPU35 ESCs induced by retinoic acid were increased with phase spots in differentiation and increased concentration of retinoic acid. NF200 positive cell is transformed as multipolar neurons from absence of process in morphology. The processes of GFAP positive cell became longer and linked among each other as reticular pattern finally. ③ It was assayed with FCM that the proportion changes of GFAP positive cell and NF200 positive cell manufactured in differentiation were similar to immunocytochemical one.CONCLUSION: Retinoic acid in combination with proper concentration of serum and differentiating phase spots can induce neural-differentiation of MESPU35 ESC at high proportion and its differentiating regulation is in the patterns of concentration dependence and time dependence.

Objective To observe the effects of fetal bovine serum(FBS) on differentiation of human neural stem cells (NSCs). Methods The effects of FBS with different concentrations on differentiation of human fetal NSCs were observed by cell culture, immunocytochemistry and flow cytometry. Results Human fetal NSCs could be induced to differentiate mainly three types of nerve system cells(neuron, astrocyte and oligodentrocyte). There were 80%～90% astrocytes of differentiated cells from human fetal NSCs with the concentration of 15% FBS induced. Conclusion Concentration dependent FBS in culture medium may have effect on the ratio of neurons to glial cells differentiated from human NSCs in vitro .

Objective　To investigate the mechanism affecting on permeability of vascular endothelial cell by nitric oxide (NO). Methods　Series concentration of sin-1（a donor of NO） were added to ECV 304, a cell line of human umbilical vein endothelium. Cell growth and expression of f-actin, a cytoskeleton protein were observed. Results　Cell growth was inhibited with a dose from 6.25 to 100 μmol/L and was caused to death at the concentration of 50 to 100 μmol/L by sin-1. The expression of f-actin was suppressed obviously after cultured with 100 μmol/L sin-1 for 4 hours. Conclusion　It suggests that anomaly increased NO can increase permeability of blood vessels by suppressing the expression of f-actin, inhibiting cell growth or even resulting in cell death.

BACKGROUND:Adrenomedulin gene transfection can strength the anti-apoptotic ability of bone marrow mesenchymal stem cels under ischemia and hypoxia, but its mechanism is not yet clear.
OBJECTIVE:To investigate the effect of adrenomedulin on the expression of apoptosis-related proteins, Bax, Bcl-2 and Caspase-3, in bone marrow mesenchymal stem cels under hypoxia and ischemia.
METHODS:Bone marrow mesenchymal stem cels of Sprague-Dawley rats were isolated, cultured and purified, and then cultured in serum-free medium under hypoxic condition for 0, 3, 6, 9, 12 hours. Then, western blot assay was employed to detect the expression of Bax, Bcl-2 and Caspase-3 so as to determine the optimal hypoxia time that was determined at 6 hours of hypoxia. Depending on whether adrenomedulin pretreatment was done, the cels were divided into control group (with no adrenomedulin pretreatment before hypoxia and ischemia) and adrenomedulin groups with different concentrations (1, 10, 100 μg/L). Afterwards, the expression of Bax, Bcl-2 and Caspase-3 was detected by using western blot assay.
RESULTS AND CONCLUSION:(1) After cultured in serum-free medium under hypoxia for 0, 3, 6, 9, 12 hours, the expression of Bax, Bcl-2 and Caspase-3 in bone marrow mesenchymal stem cels were increased (P < 0.05);at 6 hours of hypoxia, the Bax/Bcl-2 ratio and Caspase-3 expression reached the minimum value (P < 0.05). (2) At 6 hours of hypoxia, the expression of Bax and Caspase-3 protein as wel as Bax/Bcl-2 ratio became the lowest in the 100 μg/L group compared with the 1 and 10 μg/L groups, but the expression of Bcl-2 protein reached the peak (P < 0.05). These findings indicate that adrenomedulin can reduce the expression of Bax/Bcl-2 ratio and Caspase-3 protein in bone marrow mesenchymal stem cels cultured in serum-free medium under hypoxic conditions, which is in a dose-dependent manner.

BACKGROUND:Previous studies have demonstrated that hepatocyte growth factor (HGF) gene transfection can improve the effectiveness of bone marrow mesenchymal stem cel transplantation, but the mechanism is stil unclear. OBJECTIVE:To observe the effects of HGF gene transfection on c-MET, Bax, Bcl-2, Caspase-3 of bone marrow mesenchymal stem cel s cultured under hypoxia and serum-free conditions. METHODS:(1) Bone marrow mesenchymal stem cel s were isolated and amplified in vitro by differential adhesion method. The infection efficiency of recombinant adenovirus Ad-HGF in bone marrow mesechymal stem cel s was tested by x-gal staining. (2) Bone marrow mesenchymal stem cel s were cultured under hypoxia and serum-free conditions for 0, 3, 6, 9, 12 hours. RT-PCR and western blot assays were used to evaluate the expression of Bax, Bcl-2, Caspase-3. (3) Bone marrow mesenchymal stem cel s were cultured under hypoxia and serum-free conditions for 6 hours, and RT-PCR and western blot assays were adopted to detect HGF, c-Met, Bax, Bcl-2 and Caspase-3. (4) Cel scratch test was used to detect the effect of HGF transfection on the migration of bone marrow mesenchymal stem cel s cultured under hypoxia and serum-free conditions for 6 hours. RESULTS AND CONCLUSION:(1) Transfection efficiency of bone marrow mesenchymal stem cel s was increased with multiplicity of infection in a dose-dependent manner. When the multiplicity of infection was 150, the transfection efficiency was 96.4%. (2) Expressions of Bax and Bcl-2 were gradual y increased with hypoxia time (P<0.05). The Bax/Bcl-2 ratio and Caspase-3 expression reach the minimum at 6 hours of hypoxia (P<0.05). (3) Compared with the control and Ad-LacZ groups, the expressions of HGF, c-Met, Bcl-2 increased, and the expressions of Bax and Caspase-3 decreased in the Ad-HGF group after 6 hours of culture under hypoxia and serum-free conditions (P<0.05). There was no significant difference between the control and Ad-LacZ groups. (4) The mobility of bone marrow mesenchymal stem cel s was higher in the Ad-HGF group than the control group and Ad-LacZ groups after 6 hours of culture under hypoxia (P<0.05). These findings indicate that transfection of HGF in bone marrow mesenchymal stem cel s can increase the expression of c-Met, Bcl-2 and decrease the expression of Bax, Caspase-3 under hypoxia and serum-free conditions, which also enhance the mobility of bone marrow mesenchymal stem cel s under hypoxia and serum-free conditions.

Objective:To investigate the effects of periploca forrestii schltr in the treatment of acute gout arthritis.Methods:60 healthy male SD rats were equally randomly divided into 6 groups:normal control group( NC) ,model group( M group) ,colchicine group (C group),high doses group of periploca forrestii schltr(HD group),middle doses group of periploca forrestii schltr(MD group) and low doses group of periploca forrestii schltr( LD group).Except the normal control group,model of gouty arthritis was induced in other groups by uric acid salt,colchicine(positive control) and different dose of periploca forrestii schltr were given by intragastric ad minis-tration.Swelling dimension of joints were observed at 3,5,7 days after treatment.All rats were killed after 7 days of treatment and ankle joint tissue was taken for pathological examination and the peripheral blood of rats was prepared for detecting the expression of interleukin 1β(IL-1β),IL-6,IL-8 and tumor necrosis factor(TNF-α) using enzyme linked immunosorbent test(ELISA).Results:The ankle joint swelling of periploca forrestii schltr group was significantly lower than that in the model group,and the effect of high doses group was much better than the low doses group after 7 days treatment(P<0.05);compared with model group,the inflammatory cells of each treatment groups were decreased and high doses group did not differ from that of normal control group;the levels of IL-1β,IL-6,IL-8 and TNF-αin periploca forrestii schltr group were dramatically lower than those in the model group in a dose-dependent manner.Conclusion:Periploca forrestii schltr has good therapeutic effect in rats with acute gouty arthritis and shows a dose-dependent response,and the mechanism may relate to the inhibition of inflammatory cytokines expression.

Objective To investigate the immunomodulatory effects of Tetrastigma hypoglaucum Planch in the treatment of rheumatoid arthritis. Methods Sixty SD rats were ramdomly divided into normal control group, model control group,low-,middle-and high-dose Tetrastigma hypoglaucum Planch groups (50,100 and 200 mg.kg-1,respectively),and tripterygium glycosides tablet group. Except for normal control group,rheumatoid arthritis model was established by using bovine typeⅡcollagen in SD rats of the other groups. Organ index,plasma levels of IL-1β,IL-6,TNF-αand anti-CⅡin rat rheumatoid arthritis models were evaluated. Results The kidney index of the low-dose/middle-dose Tetrastigma hypoglaucum Planch groups were significantly different from that of the model control group (P<0.05). Significant difference was also found in the spleen index between the high-dose Tetrastigma hypoglaucum Planch group and the normal control group ( P<0.05) . The plasma levels of IL-1β,IL-6,TNF-αand anti-CⅡantibody were significantly higher in the model control group than in the normal control group. Meanwhile, the plasma IL-1β, IL-6 and TNF-α levels of the middle-dose/high-dose Tetrastigma hypoglaucum Planch groups were significantly lower than those of the model group ( P<0.01) . The levels of anti-CII antibody were significantly lower in the low-,middle-and high-dose Tetrastigma hypoglaucum Planch groups than in the model control group. Conclusion It is worthwhile to explore and develop Tetrastigma hypoglaucum Planch since it possesses immunomodulatory effects and may be applied in the treatment of rheumatoid arthritis.

Objective To evaluate the therapeutical effect of dopaminergic neurons induced by transplantation on Parkinson's disease (PD) rats. Methods Mesencephalic nerve stem cells (NSCs) were induced by striatal extracts to differentiate into tyroxine hydroxylase (TH) positive dopaminergic neurons. The differentiated cells were transplanted into the striatum of PD rats. The survived cells were detected by TH immunocytochemical staining. The therapeutical effect was observed using apomorphine induced rotation. Results Mesencephalic NSCs could be induced to differentiate into dopaminergic neurons which could survive in the host for long time after cell transplantation, and could improve the apomorphine induced rotation. Conclusion The induced mesencephalic NSCs have the obvious therapeutical effect on PD.