AIM:To investigate the differentiation from rat mesenchymal stem cells (rMSC) into neuron-like cells. METHODS:rMSC were separated from femur marrow and expanded in L-DMEM culture medium supplemented with 10% FSC. rMSC were induced to differentiate into neurons with L-DMEM/adrenaline,L-DMEM/noradrenaline and L-DMEM/isoprenaline, respectively. Meanwhile, rMSC were cultured in L-DMEM in control group. Nestin, neuron-specific enclose (NSE), glial fibrillary acidic protein (GFAP) were detected by immunocytochemistry. RESULTS: rMSC were expanded as undifferentiated cells in culture from 5 to 22 passages, indicating their differentiated capacity. Simple method induced rMSC to exhibit a neuronal phenotype, expressing positive NSE,nestin, and GFAP, at 5 hours in all group. The undifferentiating cells (control group 53.1%?4.3%), and differentiating cells (treated group: adrenaline 74.7%?2.6%; noradrenaline 75.9%?2.4%; isoprenaline 72.1%?4.4%), expressed characteristics of various neuronal cells, from 5 hours to 6 days. There were neuron-like cells in rMSC cultured in L-DMEM/10%FBS from 7 to 13 passage(66.5%?6.4%). CONCLUSION: It suggests that rat neural stem cells (rNSC) exist in bone marrow, rMSC can be differentiated into various neural cells with adrenaline hormones in vitro.

BACKGROUND: How dose sodium nitroprusside, as a vasodilatator, affect the potential of bone marrow-derived mesenchymal stem cells (MSCs) in hematopoietic differentiation?OBJECTIVE: To observe the changes during the differentiation of MSCs into hematopoietic cells after adding sodium nitroprusside, and compare the results with those of simple MSCs transplantation.DESIGN: A randomized grouping, controlled observation.SETTINGS: Department of Microbiology and Immunology; Department of Pathophysiology, Guangdong Medical College.MATERIALS: The experiments were carried out in Guangzhou Medical College in August 2006. Twenty-seven clean-degree balb/c mice of 7-8 weeks, were used as recipients, and were randomly divided into MSCs transplantation group (n =9), sodium nitroprusside+MSCs transplantation group (n =9) and blank control group (n =9). Another 4-week-old SD rat was selected as the MSCs donor. Sodium nitroprusside for injection (50 mg/piece) was provided by Beijing Double-Crane Modern Pharmaceutical Technology Co., Ltd (National drug approval: No. H11020907).METHODS: ① Under aseptic condition, the femur of SD rat was collected. MSCs in it were isolated for culture and amplifying in vitro. MSCs of passages 6-7 were digested and centrifugated, and the density was adjusted to 1×109 L-1.Monoclonal antibody with fluorescence labeled was added into cell suspension, and the phenotype was detected with flow cytometry. ② Sodium nitroprusside (50 mg/piece) was adjusted to the terminal concentration of 200 mg/L by adding with saline. It should be used within 4 hours. ③ Before transplantation, all the mice were exposed to 5.0 Gy. X-ray for 4 hours, and the absorbed dose was 1.45 Gy per minute. After irradiation, mice in the MSCs transplantation group were directly infused via caudal vein with 0.3 mL MSCs suspension (containing 1.5×106 cells); The mice in the sodium nitroprusside+MSCstransplantation group were firstly injected with the dispensed 200 mg/L sodium nitroprusside (0.15 mL), and immediately infused with 0.3 mL MSCs suspension (containing 1.5×106 cells) after 1 minute; The mice in the blank control group were infused with isovolume serum-free culture medium. ④ At 60 days after transplantation,peripheral blood was drawn from orbits of the survived mice in each group, single cell suspensions of bone marrow and spleen were prepared after the mice were killed, the levels of rat-derived hematopoietic cells CD11a and CD45 were detected with flow cytometry.MAIN OUTCOME MEASURES: ① MSCs culture and amplification; ② Levels of rat-derived hematopoietic cells in different tissues.RESULTS: All the 27 balb/c mice as recipients survived to the end of the experiment. ① MSCs isolation and amplification: The MSCs attached with uniform shapes of spindle and proliferated rapidly after culture for 3 days, and 90% of the cells were fused without overlapping at 6 days. The cells attached completely within 24 hours after passage,extended and became spindle again, rapidly proliferated and grew, and became fused completely at 3 days. ② Levels of rat-derived hematopoietic cells in different tissues: In the MSCs transplantation group and sodium nitroprusside+MSCs transplantation group, Iow expressions of rat-derived CD11a and CD45 hematopoietic cells could be detected in peripheral blood, bone marrow and spleen, and they were obviously higher in the sodium nitroprusside+MSCs transplantation group than in the MSCs transplantation group (t=2.619, P ＜ 0.05), while negative ones in the blank control group.CONCLUSION: MSCs have the ability to differentiate into hematopoietic stem cells, which can be promoted by sodium nitroprusside.

BACKGROUND: By targeting inducing differentiation in vitro,mesenchymal.stem cells(MSCs) transform into osteoblasts,lipocytes,chondrocytes,muscular cells,neuronal cells,etc. Whether Chinese herbs act on induced differentiation of MSCs in rats or not?OBJECTIVE: To study the amplification of MSCs cultured in vitro in SD rats and efficacy of Chinese herbs on targeting inducing differentiation of neuron-like cells.DESIGN: Exploring study with repeated observation and measurement based on cells.SETTING: Department of pathophysiology in a medical college.MATERIALS: Experimental marrow collected from SD male tested-healthy rats.METHODS: By adhesion method,MSCs in rats were isolated for amplifying culture in vitro. Flow-type cell instrument was applied for the determination of its surface antigen expression. Various Chinese herbal components were used for the targeting inducing differentiation of MSCs into neuron-like cells. The cellular morphology was observed under optical microscope. and the specific antigen label of neuronal cells was determined with immunocyto-chemical method.MAIN OUTCOME MEASURES:①Results of MSCs isolation and amplification;②Results of identification of MSCs surface antigen and neuon-like cells.RESULTS: By adhesion method,MSCs in rats were isolated successfully and amplified in a large amount in vitro. It was indicated in the results determined by flow-type cell instrument that CD14,CD1 1α,CD34,CD38,CD45,CD80 and CD86 presented negative,and CD29,CD44,CD90,CD105 and CD166 presented positive. By induced with various kinds of Chinese herbs,like huangqi(Radix Astragali seu Hedysari),tianma(Rhizoma Gastrodiae),renshen (Radix Ginseng),danggui(Radix Angelicae Sinensis),naoxinshu,renshen fengwangjian for 1 to 3 hours,most MSCs transformed into neuron-like cells,presenting soma and neurite. With immunocyto-chemical staining,neuron-specific enolase(NSE) and nestin displayed positive and glial fibrillary acidic protein negative.CONCLUSION: MSCs in SD rats have the potential of multi-targeting differentiation,presenting a strong capacity of amplification and self-replacement. In a suitable inducing condition,MSCs may differentiate into neuron-like cells.

Objective To study the immunoregulatory effects of emodin on macrophages during Brucella infection and to provide theoretical and experimental basis for developing new drugs to treat brucello-sis. Methods Bone marrow cells were isolated from BALB/c mice and cultured with MG-CSF to induce differentiation. Flow cytometry was used to detect the differentiation of bone marrow cells into macrophages (MΦ) by using FITC-labeled mouse anti-F4/80 antibody and PE-labeled anti-CD11b antibody. MTT meth-od was used to detect the influences of various concentrations of emodin on the survival rate of MΦ. Doxy-cycline was used as the control to compare half maximal inhibitory concentrations (IC50) of the two drugs. MΦ were cultured with Brucella at a ratio of 100 : 1 for 4 h. MΦ and Brucella were further cultured for 1, 6,12,24 and 48 h after adding emodin. Effects of emodin on the survival of MΦ were analyzed by colony counting method. ELISA was performed to detect the levels of TNF-α, IL-6 and IFN-γ in culture superna-tants. Results On day 8 of culturing,91.28% of bone marrow cells differentiated into macrophages. The IC50of emodin(608.4 μg/ml) was significantly higher than that of doxycycline(225.5 μg/ml). The logC-FU values of emodin stimulation groups (6,12,24 and 48 h) were significantly lower than those of blank control groups. Among all emodin stimulation groups, the 24 h group had the lowest logCFU value, which was also lower than that of the doxycycline treatment group. The levels of TNF-α,IL-6 and IFN-γ in 6,12 and 24 h emodin stimulations group increased significantly as compared with those of the corresponding con-trol groups. The levels of TNF-α, IL-6 and IFN-γ peaked at 24 h of culturing Brucella-infected MΦ with emodin. No significant difference in IFN-γ level was found between the 12 and 24 h emodin stimulation groups [(74.233 ±4.416) pg/ml vs (78.328 ±8.932) pg/ml]. Conclusion Emodin may enhance the ability of macrophages to kill Brucella through promoting the expression of TNF-α,IL-6 and IFN-γ.

ABSTRACT OBJECTIVE：To study the effects of the extract of Xiongmatang on 5-HT and CGRP in brain tissue of rats with liver-yang hyperactivity and blood stagnation migraine. METHODS：Fifty male spontaneously hypertension （SHR） rats were randomly divided into model control group，Zhengtian pill positive control group （1.6 g/kg），the extract of Xiongmatang low-dose，medium-dose and high-dose groups（4.5，9.0，18.0 g/kg，by crude drug），with 10 rats in each group. Another 10 normal rats were taken as normal control group. Administration group was given relevant medicine intragastrically，once a day，for consecutive 28 d. Normal control group and model control group were given equal volume normal saline intragastrically. After last administration，except for normal control group，other groups were used to stimulate trigeminal ganglia（10 min）to establish liver-yang hyperactivity and blood stagnation migraine model，and maintained the administration once more after making the model. 30 min later，general behavior and tongue quality of rats were observed，and the blood pressure was measured；the contents of 5-HT and CGRP in cerebral tissue of rats were determined by ELISA. The protein expression of CGRP in cerebral tissue of rats were determined by Western blot method. RESULTS： Compared with the normal control group， the rats in the model control group had behavioral symptoms，such as the color of conjunctiva deepened and reddened， excessive hairdressing，head flicking，and most of the rats had purple tongue；the systolic pressure and the content and protein expression of CGRP were all increased obviously（P＜0.05 or P＜0.01）， while the content of 5-HT in cerebral tissue was decreased obviously（P＜0.01）. Compared with model control group，general behavior and tongue quality of rats were improved significantly in administration groups. Systolic pressure，the content and the protein expression of CGRP in cerebral tissue of rats were decreased significantly in Zhengtian pill positive control group and the extract of Xiongmatang high-dose group （P＜0.05），while the content of 5-HT in cerebral tissue of rats were increased significantly in Zhengtian pill positive control group，the extract of Xiongmatang medium-dose and high-dose groups（P＜0.05）.CONCLUSIONS：The extract of Xiongmatang has obvious protective effect on liver-yang hyperactivity and blood stagnation migraine model rats，the mechanism of which may be associated with reducing the content of CGRP in cerebral tissue and raising the content of 5-HT in cerebral tissue

Objective To prepare and identify rabbit anti-cyclin dependent kinase 6 (CDK6) antibody. Methods The recombinant pET21a (+)/CDK6 was successfully constructed, then the recombinant plasmid was transformed into E.coli BL21 (DE3) competent cells and was induced by isopropyl-β-D-thiogalactopyranoside (IPTG) for protein expression, which was detected by SDS-PAGE and Western blot analysis. The expressed protein was purified by nickel-chelating nitrilotriacetic acid (Ni-NTA) agarose and then analyzed by SDS-PAGE. Japanese white rabbits were immunized with purified CDK6 protein for many times every two weeks. The blood was collected at 0, 2, 4 and 6 weeks after immunization, and serum was separated from blood. The titer was detected by indirect ELISA. Western blot analysis, immunofluorescence assay and immunohistochemistry were employed to determine the specificity. Results High purity CDK6 protein and high specificity of rabbit anti-CDK6 antibody were successfully prepared. The titer of CDK6 rabbit serum antibody reached 1:30 000 after immunization, which could specifically recognize the CDK6 protein expressed in cervical cancer cell line and cervical cancer tissues. Conclusion The high titer and specificity of rabbit anti-CDK6 antibody is successfully prepared.
Animals ; Female ; Humans ; Rabbits ; Antibodies ; Antibody Specificity ; Blotting, Western ; Cyclin-Dependent Kinase 6 ; Enzyme-Linked Immunosorbent Assay ; Uterine Cervical Neoplasms