Objective In order to understand the relationship between myoblast and Schwann cell,our purpose was to investigate the effects of biological characters of myoblasts co-cul tured with Schwann cells and pro vide the basic theory for constructing artificial muscle involving artificial nerve.Methods After sterilizing with iodine tincture and alcohol,the brachial plexus,sciatic nerve and triceps surae muscle of neonatal SD rat were harvested and peeled off their membranes,blood vessels and fat tissues under operating microscope thor oughly.The nerve and muscle tissues were cut in pieces by microscis sors,and then digested and isolated by collagenase and pancreatin in20and15minutes respectively.DMEM medium was employed to culture my oblasts and Schwann cells.After co-culturing myoblasts of rats with Schwann cells in vitro,the morphological characteristics and the growth condition of two cells were observed under inverted phase contrast mi croscope,the effects of Schwann cells secretion for proliferation of myoblasts were detected by 3 HTdR iso topic tracing and expressed by disintegration per minute(DPM),formation rate of myotubes was counted under micro scope and statistic data was analyzed,the functional differentiation degree of my oblasts affected by Schwann cells was analysed by?-sarcomeric actin immunohistochemistry(SABC)and imaging analysis tech nique.Results Co-cultured myoblasts proliferated,and myotubes ap peared earlier.Comparing with sole-cultured my oblasts,the shape of myobutes from co-cultured myoblasts tended to be elongating and robust.The value of DPM far-exceeded the control group,and reached its peak of 2500(just800for con-trol group).The positive cells of ?-sarcomeric actin appeared in brown red color.However,syn thesis and excre tion of a-sarcome ric actin in co-cultured myoblasts were much greater than control group,and the gray ash value between two groups was of a significant difference.Conclusion Primary rat myoblasts co-cultured with Schwann cells in vitro is beneficial in regulating its the growth,proliferation and the differentiation.

Objective To detect the selective inhibitory effects of irradiation plus adenovirusmediated horseradish peroxidase ( HRP)/indole-3-acetic acid (IAA) suicide gene system using tumorspecific and radio-inducible chimeric promoter on human hepatocellular carcinoma subcutaneously xenografted in nude mouse.MethodsRecombinant replicated-deficient adenovirus vector containing HRP gene and chimeric human telomerase reverse transcriptase (hTERT) promoter carrying 6 radioinducible CArG elements was constructed.A human subcutaneous transplanting hepatocellular carcinoma (MHCC97 cell line) model was treated with -γ-ray irradiation plus intra-tumor injections of adenoviral vector and intra-peritoneal injections of prodrug IAA.The change of tumor volume and tumor growth inhibiting rate,the survival time of nude mice,as well as histopathology of xenograft tumor and normal tissues were evaluated.Results Thirty one days after the treatment,the relative tumor volumes in the negative,adenovirus therapy,irradiation,and combination groups were 49.23 ± 4.55,27.71 :± 7.74,28.53 + 10.48 and 11.58 ± 3.23,respectively.There was a significantly statistical difference among them (F = 16.288,P ＜0.01 ).The inhibition effect in the combination group was strongest as compared with that in other groups,and its inhibition ratio was 76.5%.The survival period extended to 43 d in the combination group,which showed a significantly difference with that in the control group(x2 = 18.307 ,P ＜0.01 ).The area of tumors necrosis in the combination group was larger than that in the other groups,and the normal tissues showed no treatment-related toxic effect in all groups.However,multiple hepatocellular carcinoma metastases were observed in the liver in the control group,there were a few metastases in the monotherapy groups and no metastasis in the combination group.Conclusions Adenovirus-mediated suicide gene therapy plus radiotherapy dramatically could inhibit tumor growth and prolong median survival time.It might provide a promising therapeutic modality for hepatocellular carcinoma therapy.

Objective:To detect and verifica the gene profile difference of microRNA-146a (miR-146a) and its role in the pro-liferation of vascular smooth muscle cells (VSMCs) by gene chip technology. Methods: Artificially synthesized miR-146a mimics(50 nmol/L) ,miR-146 inhibitor ( 50 nmol/L ) , scramble ( 50 nmol/L ) and PBS were transfected into cultured primary rat VSMCs in vitro. After transfection,Real time PCR was used to measure the levels of miR-146a and the cell counting kit 8(CCK8) was employed to investigate the proliferation of VSMCs. The VSMCs interfered by miR-146a inhibitor or miR-146a control were examined by gene chips and the profile of gene were analyzed by bioinformatics technology to detect the different genes and signal transduction pathway. The changes in mRNAs and proteins were accessed separately by Real time PCR and Western blot. Results: Compared with sham and control VSMCs,miR-146a expression level was significantly decreased in treatment with miR-146a inhibitor(P<0. 01),as well as optical density(OD) was also shown remarkably down regulated simultaneously(P<0. 05). The investigation of gene profile revealed that the p53 signal pathway was up-regulated in VSMCs interfered by miR-146a. The mRNA and protein expression levels of p53, caspase3 and PTEN in p53 signal transduction pathway didn′t show significant differences(P>0. 05),however,the mRNA and protein expression levels of cyclin D1 significantly increased in treatment with miR-146a mimics VSMCs group and decreased in miR-146a inhibitor VSMCs group ( compared with sham VSMCs group, both P<0. 05 ) . Conclusion: Our data indicated that miR-146a may promote the proliferation of rat VSMCs by up-regulating cyclin D1 expression.

OBJECTIVETo prepare monoclonal antibodies (McAbs) against human mesenchymal stem cells (hMSCs) and to study their biological characteristics.

METHODSBALB/C mice were immunized with pooled hMSCs. McAbs were prepared by hybridoma technique and their biological characteristics were analyzed by indirect immunofluorescence, immunohistochemistry and flow cytometry.

RESULTFive hybridoma cell lines were successfully established, which secret McAbs specifically against hMSCs. Investigations showed that all these McAb reacted only to hMSCs and had no cross-reaction to other human cells, the relative affinities of 5 McAbs were 1x10(6) (ZUB1), 1x10(5) (ZUB4), 1x10(6) (ZUC3), ZUE12 (1x10(5)) and 1x10(5) (ZUF10), respectively. Isotype analysis showed that ZUB1, ZUE12, ZUF10 against the same isotype, while ZUC3, ZUB4 against other two different isotypes alone. Flow cytometric analysis showed that the positive expression rate of cultured hMSCs was 87.39% (ZUB1), 88.07% (ZUB4), 88.12% (ZUC3), 69.89% (ZUE12) and 83.67% (ZUF10).

CONCLUSIONThe prepared five McAbs can specifically react against hMSCs, which can be used for selection and study of hMCSs.

Animals ; Antibodies, Monoclonal ; biosynthesis ; Antibody Specificity ; Bone Marrow Cells ; cytology ; immunology ; Fluorescent Antibody Technique ; methods ; HL-60 Cells ; Humans ; Hybridomas ; secretion ; Immunoglobulin G ; immunology ; Immunoglobulin M ; immunology ; Mesenchymal Stromal Cells ; cytology ; immunology ; Mice ; Mice, Inbred BALB C ; Rats

UNLABELLEDTo study the effect of S-3307 on the yield and main ingredients of Alisma plantago-aquatica.

METHODThe contents of 24-acetyl alisol A and the 23-acetyl alisol B in tuber were determined by HPLC.

RESULTSThe contents of 24-acetyl alisol A and the 23-acetyl alisol B as well as yield were significantly increased in all groups applied with different concentrations of S-3307 comparing with control group. The optimal concentration of S-3307 was 80 mg x kg(-1). The residues of S-3307 was detected under 0.316 8 mg x kg(-1) (detecting limit).

CONCLUSIONThe optimal concentration of S-3307 is 80 mg x kg(-1), it reached the best result when applied 36 d after seedling.

Alisma ; chemistry ; drug effects ; growth & development ; Cholestenones ; analysis ; Chromatography, High Pressure Liquid ; Plant Growth Regulators ; pharmacology

Objective To explore the clinical applicating and efficacy of free fibula osteomyocutane- ous flap in mandible defect reconstruction in osteoradionecrosis patients.Methods The mandible defects were reconstructed by free fibula flaps with or without muscle cuff.The soft tissue defects were repaired by skin paddles.Status of osteotomy in fibula and flap survival was recorded.The complication in recipient site and donor site,as well as mouth opening and occlusion were reviewed.Facial contour and chewing function after reconstruction were evaluated.Results Patients were followed up 3-16 months.4 free fibula flaps with muscle cuff and 5 without muscle cuff survived well.The size of mandible defects covered from 6cm to 17cm. And the harvested fibula flaps with length of 8.6-17cm were cut into 3 segments in 2 cases,and 2 segments in 5 cases.Fibula flap was divided into 2 segments and overlapped in 2 cases.No serious complication was oh- served in recipient site and donor site.Satisfying esthetic result and normal occlusiong of heath mandible were obtained in all cases.The degree of mouth opening was 2.5-3.3cm.Fair chewing function was revealed in re- constructive region after prosthesia repaired.Conclusion Free fibula osteomyocutaneous flap is relatively ideal reconstruction material of mandible defect in osteoradionecrosis patients for its high survival rate and well esthetic results.

OBJECTIVETo investigate the effect of RNA interference (RNAi)-mediated silencing of the SREBP2 on inflammatory cytokine-induced cholesterol accumulation in HepG2 cells.

METHODSShort-hairpin (sh)RNA targeting SREBP2 or negative control (NC) shRNA were transfected into HepG2 cells by a liposomal method. G418-selective culturing was used to obtain the SREBP2 shRNA HepG2 and NC shRNA HepG2 cell lines. The two cell lines were cultured in serum-free medium and left untreated (control) or treated with TNF-a (20 ng/ml), low-density lipoprotein (LDL) loading (100 mug/ml), or a combination LDL plus TNF-a treatment. Lipid accumulation was evaluated by oil red O (ORO) staining. Intracellular cholesterol level was measured by enzymatic assay. The mRNA and protein levels of SREBP2 and its downstream target genes, LDL receptor (LDLr), and HMGCoA reductase, were measured by real-time PCR and Western blotting, respectively.

RESULTSSREBP2 shRNA HepG2 and NC shRNA HepG2 stable cell lines were successfully established. ORO staining and cholesterol quantitative analysis showed that LDL loading significantly increased intracellular cholesterol and that expression of SREBP2 further exacerbated the inflammatory cytokine-induced lipid accumulation, as seen in NC shRNA HepG2 cells. LDL loading of NC shRNA HepG2 decreased the gene and protein expressions of SREBP2, LDLr, and HMGCoA reductase, but the suppressive effect was overridden by inflammatory cytokine. SREBP2 shRNA HepG2 cells showed lower levels of cholesterol accumulation under LDL loading and inflammatory stress conditions. Moreover, the mRNA and protein levels of SREBP2, LDLr, and HMGCoA reductase were much lower than in NC shRNA HepG2 cells under the same conditions.

CONCLUSIONInflammatory cytokine exacerbated cholesterol accumulation in HepG2 via disrupting SREBP2. RNAi-mediated inhibition of SREBP2 expression significantly ameliorated the cholesterol accumulation induced by inflammatory cytokine.

Cholesterol ; metabolism ; Hep G2 Cells ; Humans ; Inflammation ; RNA Interference ; RNA, Small Interfering ; Sterol Regulatory Element Binding Protein 2 ; genetics ; Tumor Necrosis Factor-alpha ; pharmacology

OBJECTIVESTo investigate the expression of macrophage migration inhibitory factor (MIF) mRNA in Schwann cells after peripheral nerve injury and roles of Schwann cells and MIF in macrophages activation and nerve regeneration.

METHODSFifty SD rats were divided into 10 groups. One group served as normal control. The rest were anesthetized with 3% sodium pentobarbital (30 - 60 mg/kg, i.p) and sciatic nerves were transected distal to the obturator tendon respectively 1 h, 12 h, 1 d, 3 d, 7 d, 10 d, 14 d, 17 d and 21 d before being killed. Sciatic nerves were resected and connective tissues excised. Schwann cells were obtained by digesting the nerve tissues with trypsin and collagenase. RNA was isolated and reverse-transcription-polymerase chain reaction (RT-PCR) was carried out. cDNA was analyzed by automatic system and the parameters were assessed to define the status of MIF mRNA expression in different groups.

RESULTSThe level of MIF mRNA started to increase 12 h after the nerve transection. The level remained high from day 7 up to 10 after the injury. During the period from days 10 to 21, MIF mRNA decreased slowly to the pre-transection level.

CONCLUSIONAfter peripheral nerve injury, Schwann cells can secrete MIF which may play a pivotal role as an immunomodulatory cytokine in macrophage activation and inflammatory reaction.

Animals ; Female ; Macrophage Migration-Inhibitory Factors ; genetics ; Male ; Peripheral Nerve Injuries ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Schwann Cells ; metabolism

OBJECTIVETo detect the expression of proliferating cell nuclear antigen (PCNA) in severely damaged intestinal mucosa due to high-dose 5-FU exposure.

METHODSThirty-two adult C57BL/6J mice were subjected to daily intraperitoneal high-dose 5-FU injection at 150 mg/kg for 5 consecutive days, and on days 1, 3, and 5, the mice were sacrificed to obtain the small intestinal tissue for HE straining and immunohistochemistry for detecting PCNA expression. Another 8 mice with intraperitoneal PBS injection served as the control group.

RESULTSHigh-dose 5-FU exposure of the mice resulted in severe intestinal mucous damage, with complete destruction of the villi and crypts and significantly increased cells positive for PCNA expression (P<0.01).

CONCLUSIONHigh-dose 5-FU treatment can significantly increase the PCNA index, and the cells expressing PCNA can be closely associated with regeneration of the severely damaged mucosa due to the exposure.

Animals ; Antimetabolites, Antineoplastic ; adverse effects ; Fluorouracil ; adverse effects ; Intestinal Mucosa ; drug effects ; metabolism ; pathology ; Intestine, Small ; drug effects ; metabolism ; pathology ; Mice ; Mice, Inbred C57BL ; Proliferating Cell Nuclear Antigen ; metabolism

The objective of the research is to investigate the elements of routine sandblast technique on the evolution of bending strength of dental infiltrated Al2O3 ceramics and the underlying erosion mechanism. The plane specimens of an infiltrated ceramic were manufactured, polished and then tested under the modified pen-like sandblasting apparatus (90 degrees erosive angle and 10 mm sandblasting distance), with different grit sizes, working pressure and disposing time. Half of samples were selected randomly and sintered subsequently with Vitadur alpha veneering porcelain. Before and after sintering, the three-point-bending strengths was measured, and the surfaces of dental porcelain were observed with SEM and LCSM. The bending strength of ceramics decreased significantly after sandblast as compared with that of empty control group. After the procedure of sintering the veneering porcelain, the descending evolution of bending strength slowed down. Under the present manufacturing conditions, grit size effect is prominent among those correlative elements of sand grit size, working pressure and disposing time. And fatigue cracking characterizes the mechanism of erosion of dental infiltrated Al2O3 ceramics.
Aluminum Oxide ; chemistry ; Dental Materials ; chemistry ; Dental Stress Analysis ; Humans ; Materials Testing ; Metal Ceramic Alloys ; chemistry ; Shear Strength ; Stress, Mechanical