Objective To investigate the effects of extracellular ATP on the recovery of motor function after spinal cord injury in rats.Methods Twenty healthy adult rats were randomly divided into two groups after contusion injury was performed by the weight drop method at T10 with Allen impactor.Group A(ATP group) and group B(control group) each had ten rats.At days 1,3,7,14 and 28 after injury,the recovery of motor function after spinal cord injury in rats was assessed with modified Tarlov scores and slanting board test.Results Modified Tarlov scores and slanting board test were better in group A than in group B after spinal cord injury in rats.Significant difference was found in modified Tarlov scores and slanting board test between the two groups at days 14 and 28 after injury(P

Objective To study the clinical features and angiographic findings of moyamoya disease (MMD) as well as their relationship. Methods A total of 22 MMD patients received routine digital substraction angiography (DSA). The clinical manifestations and angiographic findings were analyzed. Results Clinical manifestations varied and each patient often had multiple symptoms, including cerebral infarction in 9 patients with an average age of 23.6 (13-39 years) and cerebral hemorrhage in 7 patients with an average age of 31.2 (28-46 years). Angiographic examination found that all the diseased sides showed MMD blood vessels. The patients who received encephalo-myo-arterio-synangiosis (EMAS) had better prognosis than those without receiving the treatment. Conclusion Cerebral infarction is frequent in children and adolescents with MMD, whereas cerebral hemorrhage is common in adults. DSA is a golden criterion for diagnosing MMD. Surgical treatment, EMAS blood supply reconstruction in particular, should be prescribed.

Objective:To investigate the trait of exosomes serected by mesenchymal stem cells derived form orofacial bone(OMMSCs-Exo) and the communication between the exosomes and macrophages.Methods:OMMSCs were isolated from orthognathic surgical sites and cultured by limited dilution.Their cell surface markers were characterized by flow cytometry.the rate of colony formation and the differentiation potential of OMMSCs were evaluated.Exosomes were prepaired from the culture supernatants of OMMSCs(P4-P6).Transmission electron microscopy(TEM) and western blot were used to identify the exosomes.The expression of miRNAs associated with immunity such as miR-223 and miR-let-7c were determined by Real-time RT-PCR.Human peripheral blood mononuclear cells(PBMCs) were isolated from health donor and co-cultured with OMMSCs-Exo.After co-cultured for 24 h,the communication between exosomes and macrophages was tested using a confocal microscope.Results:Human OMMSCs were proved to have the characteristics of mesenchymal stem cells.The diameter of OMMSCs-Exo ranged from 40 to 160 nm.The OMMSCs-Exo expressed CD63 and CD81 and contained miRNAs associated with immune regulation such as miR223 and miR-let-7c.OMMSCs-Exo could be uptaken by macrophages.After co-culture of OMMSCs-Exo with marcrophages for 72 h,miR223 expression in macrophages increased.Conclusion:OMMSCs-Exo has the potential of immune regulation.

Objective To evaluate the role of microRNA-133b-Sp (miR-133b-Sp) in apoptosis hypoxia/reoxygenation (H/R) induced by in rat cardiomyocytes.Methods Rat myocardial cell line H9c2 was cultured in DMEM/F12 culture medium supplemented with 10％ fetal bovine serum.The cells were seeded in 96-well or 6-well plates and randomly divided into 4 groups (n=64 wells each):control group (group C);group H/R;miR-133b-5p mimic + H/R group (group M+H/R);miR-133b-Sp negative control + H/R group (group NC+H/R).The cells were exposed to 95％ N2-5％ CO2for 5 h at 37 ℃ followed by 1 h reoxygenation in DMEM/F12 culture medium supplemented with 10％ fetal bovine serum in all the groups except group C.The cells were cultured in normal culture atmosphere in group C.In M+H/R and NC+H/R groups,the cells were transfected with miR-133b-5p mimic (final concentration 30 nmol/L) and miR-133b-5p negative control (final concentration 30 nmol/L),respectively,for 24 h before H/R.Total RNA was extracted from cells to detect the expression of miR-133b-5p using quantitative real-time PCR.The cell viability (by CCK-8) and lactic dehydrogenase (LDH) activity in the culture medium were detected.Cell apoptosis was assessed by Annexin V/PI flow cytometry.Apoptotic rate was calculated.Result Compared with group C,the cell viability was significantly decreased,and the LDH activity and apoptotic rate were increased in H/R,M+H/R and NC+H/R groups,the expression of miR-133b-5p was down-regulated in H/R and NC+H/R groups,and the expression of miR-133b-Sp was up-regulated in group M+H/R.Compared with group H/R,the cell viability was significanttly increased,the LDH activity and apoptotic rate were decreased,and the expression of miR-133b-5p was up-regulated in group M+H/R,and no significant change was found in the parameters mentioned above in group NC+H/R.Conclusion H/R in rat cardiomyocytes can induce cell apoptosis possibility through down-regulating the expression of miR-133b-5p

Objective To evaluate the effect of morphine preconditioning on the expression of miR-133b-Sp and Fas in rat cardiomyocytes subjected to hypoxia/reoxygenation (H/R).Methods Cardiomyocytes were isolated from healthy adult male Sprague-Dawley rats by using Langendorff perfusion.The cells were seeded into 24-well plates or 60 mm diameter dishes and randomly divided into 3 groups (n =24 each) using a random number table:control group (group C),group H/R,and morphine preconditioning group (group MPC).The cells in group C were cultured in normal culture atmosphere.In H/R and MPC groups,the cells were exposed to 95％ N2-5％ CO2 for 90 min followed by 120 min reoxygenation.In group MPC,the cells were cultured for 10 min in serum-free DMEM liquid culture medium containing morphine 1 μmol/L,and then were cultured for 30 min in morphine-free DMEM liquid culture medium before hypoxia.At 120 min of reoxygenation,the cells in 24-well plates were selected to detect the cell viability (by MTT),lactate dehydrogenase (LDH) activity in the culture medium,and cell apoptosis (by Hoechst 33234 staining).Apoptosis rate was calculated.Total RNA and protein were extracted from the cells in 60 mm dishes to detect the expression of miR-133b-5p and Fas mRNA (by quantitative real-time PCR) and Fas protein (by Western blot).Results Compared with C group,the cell viability was significantly decreased,LDH activity and apoptosis rate were increased,the expression of miR-133b-Sp was down-regulated,and the expression of Fas mRNA and protein was up-regulated in H/R group.Compared with H/R group,the cell viability was significantly increased,LDH activity and apoptosis rate were decreased,the expression of miR-133b-5p was up-regulatcd,and the expression of Fas mRNA and protein was down-regulated in MPC group.Conclusion The mechanism by which morphine preconditioning reduces H/R injury to rat cardiomyocytesis related to up-regulation of the expression of miR-133b-Sp and down-regulation of the expression of Fas.

Aim To evaluate the effects of morphine preconditioning ( MPC ) on the expression of microR-NAs ( miRNAs ) induced by hypoxia-reoxygenation (H/R) in H9c2 myocardial cells. Methods H9c2 cells were randomly divided into 3 groups ( n=4 each) as follows:control group ( CON) , hypoxia/ reoxygen-ation group ( H/R ) and morphine preconditioning group ( MPC+H/R) . The cells were cultured in nor-mal condition in CON group. The cells were subjected to 5 h hypoxia followed by 1 h reoxygenation in H/R group and MPC+H/R group. Specifically, the cells in MPC+H/R group were preconditioned with morphine with the final concentration of 1 μmol·L-1 for 10 min before H/R. After the treatment, CCK-8 was used to detect cell viability and chemical colorimetry was used to detect lactate dehydrogenase ( LDH ) activity in the culture medium. Cell apoptosis was assessed by An-nexin-V-FITC/PI flow cytometry. Relative expression of Fas protein was detected by Western blot. The ex-pression of miRNA in myocardial cells was analyzed by quantitative reverse transcription polymerase chain re-action ( qRT-PCR ) . Results Compared with CON group, the cell viability was significantly decreased, while the LDH activity, apoptotic rate and Fas protein expression were dramatically increased in group H/R (P<0. 01). However, MPC significantly increased the cell viability, whereas it decreased the LDH activity, apoptotic rate and Fas protein expression induced by H/R injury ( P < 0. 01 ) . The expressions of miR-133a-5p, miR-133b-5p, miR-664-1-5p, miR-6216 and let-7 e-5 p were markedly down-regulated by H/R as compared to CON group ( P <0. 05 ) , while MPC inhibited these miRNAs which were significantly down-regulated by H/R group ( P <0. 01 ) . Conclusion Morphine preconditioning might protect H9 c2 myocar-dial cells against H/R injury by regulating the expres-sion of miRNAs such as miR-133a-5p, miR-133b-5p, miR-664-1-5p, miR-6216 and let-7e-5p.

Objective:To compare the proliferation and osteogenic differentiation between human bone marrow mesenchymal stem cells from orofacial bone(OMMSCs)and those from long bone(BMMSCs).Methods:OMMSCs were isolated from orthognathic surgical sites and cultured by limited dilution.BMMSCs were obtained from bone marrow of volunteers and isolated by density gradient centrifugation method.The surface markers of the cells were detected by flowcytometry.Single-colony formation,CCK assay and cell circle analyses were conducted.Osteogenic differentiation ability was evaluated by ALP activity test and Alizarin red staining after osteogenic induction culture.Results:The cell surface markers STRO-1 and CD105 of both stem cells were positive,CD34,CD31 and CD45 were negative. OMMSCs generated significantly higher numbers of colonies than BMMSCs.In addition,OMMSCs had a higher proliferation rate and more cells in proliferative(S +G2 )stage than BMMSCs.After osteogenic induction for 3,5,7 and 10 d,OMMSCs showed higher levels of ALP activity.OMMSCs formed significantly more mineralized nodules than BMMSCs after 21-day ostogenic induction.Conclusion:The proliferation and osteogenic differentiation capacity of OMMSCs are higher than those of BMMSCs.

Objective:To compare acetyltransferase MORF level in periodontal ligament stem cells( PDLSCs) derived from healthy individuals ( H-PDLSCs) with those derived from the individuals with periodontitis ( P-PDLSCs ) . And to determine the effect of MORF on the osteogenic differentiation potential of PDLSCs. Methods: Human H-PDLSCs and P-PDLSCs were cultured and cloned with limited dilution method. H-PDLSCs were stimulated by LPS, TNF-α, IL-β and the mix of the 3 inflammatory factors to imitate inflammatory environment ( IP-PDLSCs ) . Quantitative RT-PCR and Western Blot were applied to examine different expression of MORF in H-PDLSCs and P-PDLSCs. Western Blot was applied to detect expression of MORF in IP-PDLSCs. Quantitative RT-PCR, Western Blot and alizarin red staining were applied to determine osteogenic differentiation potential of H-PDLSCs with MORF knock-down. Results:Quantitative RT-PCR and Western Blot showed lower expression of MORF in P-PDLSCs compared with H-PDLSCs( P<0. 05). Western Blot revealed lower expression of MORF in IP-PDLSCs. Quantitative RT-PCR, Western Blot and alizarin red stai-ning indicated osteogenic differentiation potential was inhibited in H-PDLSCs with MORF knockdown(P<0. 05). Conclusion: Peri-odontitis can suppress the expression of MORF in PDLSCs and inhibite the osteogenic differentiation potential of PDLSCs.

Objective:To study the inhibitory effect and anti-cancer mechanisms of adenovirus-mediated ING4 gene on the MG-63 osteosarcoma xenografts in nude mice.Methods:Ad-ING4 was transfected into QBI-293 cells and harvested.15 nude mice of the subcutaneous tumor models were established with MG-63 osteosarcoma cells and were randomly divided into PBS,Ad-GFP and Ad-ING4 groups.Then PBS(100 μl),Ad-GFP(100 μl,10~9pfu/ml) and Ad-ING4 (100 μl,10~9pfu/ml) for each one were given respectively QOD for 5 times,with intratumor injections.Tumor volume changes were monitored;and the 15 mice were sacrificed 2 weeks after treatment,the tumors were removed,weighed and ratios of tumor-suppression were calculated.The morphological changes of apoptotic tumor cells were observed under microscope.Bcl-2,Bax,Caspase-3,VEGF,CD34 expression was tested by immumohistochemistry.Results:High titer(10~9pfu/ml)adenoviral vector of ING4 gene were obtained.In nude mice bearing MG-63 osteosarcoma xenografts,the growth of MG-63 tumors treated by intratumoral injecting of Ad-ING4 was significantly suppressed,compared with PBS group and Ad-GFP group.The ratios of tumor weight-suppression of Ad-ING4 group was 59.3%(P＜0.05).Immumohistochemistry displayed that the expression of Bax,Caspase-3 was up-regulated and the expression of Bcl-2,VEGF,CD34 was down-regulated by Ad-ING4.Conclusion:Ad-ING4 can inhibit the growth of MG-63 osteosarcoma xenografts in nude mice,which may be via activating the apoptosis pathway and inhibiting tumor angiogenesis.

To study the inhibitory effect of a recombinant adenoviral vector carrying human IL-24 gene on SGC-7901 human gastric cancer cell. We infected the SGC-7901 gastric cancer cells with Ad blank adenovirus at various multiplicity of infection (MOIs) to find the optimal infective dose. The SGC-7901 tumor cells were infected with Ad-IL-24 at the optimal MOI in the following experiments. Adenovirus-mediated IL-24 transcription expression in SGC-7901 cells was examined by RT-PCR. The growth-suppressing effect of Ad-IL-24 on SGC-7901 tumor cells was assessed by MTT assay. Apoptosis and cell cycle of SGC-7901 tumor cells infected with Ad-IL-24 was evaluated by flow cytometer (FCM), respectively. The karyomorphology of apoptotic SGC-7901 tumor cells was examined using Hoechst33258 staining under fluorescence microscopy. The expression of apoptosis-related genes was future determined by semi-quantification RT-PCR; We demonstrated that the MOI of 100 was the optimal infective dose in the study on adenovirus-mediated IL-24 gene transfer into SGC-7901 gastric cancer cell; IL-24 gene mediated by adenovirus could successfully transcribe in SGC-7901 tumor cells; Ad-IL-24 could significantly inhibit SGC-7901 tumor cell growth and induce apoptosis, it also can up-regulate the express of bax, caspase-3 and p53 whilst down-regulate the bcl-2 expression. Thus, adenovirus-mediated IL-24 expression had marked anti-tumor effect in suppressing SGC-7901 human gastric cancer cell growth and inducing apoptosis, which may be closely associated with its up-regulation of bax/bcl-2, caspase-3 and p53.
Adenoviridae ; genetics ; metabolism ; Apoptosis ; genetics ; Caspase 3 ; genetics ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Interleukins ; biosynthesis ; genetics ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Stomach Neoplasms ; genetics ; pathology ; Transfection ; Tumor Suppressor Protein p53 ; genetics ; metabolism ; Up-Regulation ; bcl-2-Associated X Protein ; genetics ; metabolism