OBJECTIVEThis study aims to investigate the expression of connexin 43 (cx43) gene during early development in zebra fish and provide a foundation for further research of cx43 gene in tooth development.

METHODSTotal RNA was extracted within 72 h after fertilization of zebra fish embryos and then reversed transcribed to generate the cDNA library. The specific fragments of the cx43 gene were then cloned and connected to the PGEMT vector. After confirming the constructed plasmid, the corresponding RNA polymerase was chosen, and the digoxin-labeled anti-sense mRNA probe of cx43 was synthesized in vitro. The cx43 gene expression of zebra fish indifferent stages was carried out by in situ hybridization. The relationship of the cx43 gene expression and anatomy of the pharyngeal teeth were compared by alizarin red staining.

RESULTSThe mRNA antisense probe of cx43 was acquired. The positive signal of sepia was observed in the different stages of zebra fish pharyngeal teeth after fertilization. After fertilization for 9 days, the expression site of cx43 in situ hybridization was overlapped in accordance with the anatomical site of the pharyngeal teeth.

CONCLUSIONcx43 gene participates in tooth development and mineralization process and plays a crucial role in later mineralization.

Animals ; Connexin 43 ; Gene Expression ; Genetic Vectors ; In Situ Hybridization ; Odontogenesis ; Plasmids ; RNA, Messenger ; Tooth ; Zebrafish

OBJECTIVETo investigate the mechanism of retinoic acid (RA) signal in dental evolution, RA is used to explore the influence of the mechanism on neural crest's migration during the early stage of zebra fish embryos.

METHODSWe divided embryos of wild type and transgenic line zebra fish into three groups. 1 x 10(-7) to 6 x 10(-7) mol x L(-1) RA and 1 x 10(-7) mo x L(-1) 4-diethylaminobenzaldehyde (DEAB) were added into egg water at 24 hpf for 9 h. Dimethyl sulfoxid (DMSO) with the concentration was used as control group. Then, antisense probes of dlx2a, dlx2b, and barxl were formulated to perform whole-mount in situ hybridization to check the expressions of the genes in 48 hpf to 72 hpf embryos. We observed fluorescence of transgenic line in 4 dpf embryos.

RESULTSWe obtained three mRNA probes successfully. Compared with DMSO control group, a low concentration (1 x 10(-7) mol x L(-1)) of RA could up-regulate the expression of mRNA (barx1, dlx2a) in neural crest. Obvious migration trend was observed toward the pharyngeal arch in which teeth adhered. Transgenic fish had spreading fluorescence tendency in pharyngeal arch. However, a high concentration (4 x 10(-7) mol x L(-1)) of RA malformed the embryos and killed them after treatment. One third of the embryos of middle concentration (3 x 10(-7) mo x L(-1)) exhibited delayed development. DEAB resulted in neural crest dysplasia. The expression of barxl and dlx2a were suppressed, and the appearance of dlx2b in tooth was delayed.

CONCLUSIONRA signal pathway can regulate the progenitors of tooth by controlling the growth of the neural crest and manipulating tooth development

Animals ; Branchial Region ; Cell Differentiation ; drug effects ; Embryo, Nonmammalian ; drug effects ; embryology ; metabolism ; In Situ Hybridization ; Neural Crest ; drug effects ; Odontogenesis ; Signal Transduction ; Tooth ; drug effects ; embryology ; metabolism ; Tretinoin ; pharmacology ; Zebrafish ; embryology ; genetics ; metabolism

OBJECTIVE:To study the compatible stability of Cefmenoxime for injection with Ganciclovir for injection in 0.9% Sodium chloride injection and 5% Glucose injection. METHODS:At room temperature,the appearance and pH of the mix-tures were observed after Cefmenoxime for injection was compatible with Ganciclovir for injection. HPLC method was adopted to determine the content of them. RESULTS:No significant change was noted for the mixture in appearance and pH value. The con-tent of ganciclovir was more than 98%,but that of cefmenoxime decreased to 75.33%. CONCLUSIONS:Cefmenoxime for injec-tion can not be mixed with Ganciclovir for injection in 0.9%Sodium chloride injection and 5%Glucose injection .

Objective To investigate the effect of the transplantation of autologous marrow mesenchymal stem cells transfected by angiogenin gene in a porcine chronic ischemic beart model.Methods Methods Mesenchymal stem cells were trnsfected byAd/Ang.The pigs underwent placement of amerold occluder around LCx and 4 weeks later sujected to transplantation of the trandected mesenchymal stem cells.The animals were evaluated by coronary angiography,echocardiography,mannetic resonance imaging end pathologic observation.Results All animal showed 95%occlusion fo LCx.4 weeks after treatment ,the perfusion of LCx,left ventricular ejection fracton were greatly evhanced.A large number of labeled mesenchymal stem cells were successfully uncorporated into blood vessels in the ischemic myocardial regions with increased vessels countin and survival of implanted cell.Conclusion Transplantaton of autologous mesenchymal stem cells trendected by angiogenin gene offere obvious advsntases of great improvement of blood supply and the heart funcition.

Objective:To modify the pulsatile bioreactor we constructed previously for simulating the high-flow, high-pressure hemodynamics of heart valve in vivo, and to experimentally cultivate the tissue-engineered heart valves (TEHV) in the modified bioreactor. Methods: T-PLS system (NewheartBio Co., Ltd Korea) was used to generate pulsatile flow in the modified bioreactor and we designed a new air-exchange pathway to avoid contamination. The TEHV were made by seeding human bone mesenchymal stem cells (BMSCs) on de-cellularized porcine heart valve. After cultured under static condition for 4 d, the TEHVs were moved to the modified bioreactor and exposed to low-flow (0-600 ml/min) or high-flow(0-4 800 ml/min) pulsatile hydrodynamics for 7d, then the cells on TEHVs were evaluated. Results: After modification, the flow range expanded from (0-1 200) ml/min to (0-6 000) ml/min and the pressure range expanded from (0-40) mmHg to (0-180) mmHg. In culture experiments, 26.3% of the seeded cells remained under low-flow environment and cells were completely lost under the high-flow dynamics. Conclusion: The modified bioreactor can basically simulate the dynamics of heart valve in vivo and can be used in TEHV cultivation research. However, the current TEHV can not tolerate the high-flow pulsatile hydrodynamics.

Objective:To investigate the survival of rabbits receiving autologous left auricle cardiomyocytes transplantation into the infarcted myocardium and to assess the effect of transplantation on the peripheral blood supply, heart rate, and cardiac function. Methods: Healthy adult rabbits were randomly divided into transplantation group (n=8) and control group (n=8). The myocardial infarction (MI) models were established by ligating the left anterior descending arteries in all rabbits. Four weeks later the left auricles were harvested and the auricle cardiomyocytes were isolated and labelled with DAPI ex vivo. Rabbits in the transplantation group were injected with DAPI-labelled cell suspension into the infarcted areas and those in control group received culture medium. All the rabbits were examined by electrocardiogram (ECG) and 2-D ultrasonic cardiogram (UCG) 4 weeks after transplantation. Specimens were harvested from the transplanted areas and observed histologically. Results: All rabbits survived 4 weeks after transplantation. ECG showed that the heart rate in transplantation group was faster than that in control group (P

BACKGROUND: It has been found that xenogenic extracellular matrix (ECM) may cause a strong inflammatory response in humans during clinical application of decellularized porcine heart valve (synergraft valves). An early inflammatory reaction severely weakens matrix structure of valve wall, leading to structural rupture and decay of grafts. From Synergraft's event, the decellularized porcine heart valves still had immunogenicity, especially for pediatric patients. The mechanisms by which the ECM triggers this immune process need to be further evaluated. OBJECTIVE: To find the difference of gene sequence between human and porcine ECM and to identify the ECM immunogenicity based on bioinformatics. DESIGN, TIME AND SETTING: A contrast study between human and porcine ECM based on type IV collagen was performed at the Laboratory of Cardiothoracic Surgery, Changhai Hospital, the Second Military Medical University of Chinese PLA from June 2008 to February May.MATERIALS: The fresh porcine heart valves were obtained from Shanghai Wufengshangshi Slaughter House. Decellularized porcine aortic valves, hybridoma cells, and monoclonal antibodies were provided by our laboratory. METHODS: Similar region and conservative site of gene sequence among human, porcine, and rat were compared so as to look for common similar region, site, and sequence difference and investigate the segment which caused common and different gene sequence. Type IV collagen monoclonal antibody was used to evaluate the persistence of ECM of decellularized porcine heart valve following immunohistochemical staining. MAIN OUTCOME MEASURES: Type IV collagen gene sequence; efficacy of self-made antibody using immunohistochemistry; effect of self-made antibody on type IV collage of decellularized porcine heart valve. RESULTS: The differential gene serial in type IV collagen protein was found out by bioinformatics method. Monoclonal antibodies were successfully produced by human-mouse hybridoma technique. Residual porcine ECM was observed on decellularized porcine heart valve. CONCLUSION: Residual porcine ECM was observed on decellularized porcine heart valve and had immunogenicity.

Objective To evaluate functional activity of the subcortical nuclei in Wilson's disease (WD) using resting state functional MRI (rs-fMRI),and to evaluate damage to the functional conjunction in the extracorticospinal tract in WD patients.Methods Twenty-two patients with WD (between January 2015 and January 2016),including 18 with cerebral type and 4 with hepatic type,and 20 age-matched healthy controls were enrolled.Neurological symptoms were scored using the modified Young Scale.Patients with cerebral type WD were divided into 4 subgroups.All study subjects underwent rs-fMRI of the brain.The values of amplitude of low-frequency fluctuation (ALFF) and regional homogeneity (REHO) in the thalamus,caudate nucleus,putamen and globus pallidus were determined.The relationships between rsfMRI metrics and clinical status were evaluated.Results ALFF values were lower in the caudate nucleus,putamen and right thalamus of WD patients than in controls (t =-3.07,-3.00,-3.12,-2.46,-2.20;P =0.005,0.006,0.004,0.020,0.036),while REHO values were lower in the left caudate nucleus and left thalamus of WD patients (t =-2.38,-2.16;P =0.025,0.040).In the caudate nucleus (P =0.032,0.029,0.023),thalamus (P =0.022,0.041,0.035) ALFF values were lower in group 4 than in other groups.REHO values of the putamen (P =0.040,0.017,0.040) and thalamus (P =0.024,0.029 7,0.041) were higher in group 4 than in other groups.ALFF values in the caudate nucleus (t =-0.29,P=0.037),and thalamus (t =-1.77,P =0.042) were lower,and REHO values in the caudate nncleus (t =-1.46,P =0.040) were lower,in patients of cerebral type than in hepatic type patients.Conclusions The damage to the functional activity of the subcortical nuclei may occur in the WD patients.The functional activity of nuclei may be different between hepatic and cerebral type patients.Damage to the activity of neurons in the putamen and thalamus may correlate with psychiatric symptoms in WD patients.

AIM: To generate the angiopoietin-1 recombinant adenovirus for the further studies about adenoviral mediated angiopoietin-1 gene transfer in ischemic myocardium and its effect on the development of neoangiogenesis.METHODS: Angiopoietin-1-PAxCAwt cosmid DNA and adenoviral DNA-TPC were cotransfected to 293 cells by the use of calcium phosphate precipitation method. All recombinant adenoviruses were propagated and titrated in 293 cells, purified by cesium chloride density purification, dialyzed, and stored at-70 ℃. RESULTS: The result shows that the direction of the insert in the cosmid is correct and the replication-deficient angiopoietin-1 recombinant adenovirus was generated efficiently by COS/TPC homologous recombination, with the titers of 5.6?10 11 pfu/L. The viral stocks were demonstrated to be free of replication-competent wild type adenoviruses. The virus stocks in high titer were harvested in our experiment. CONCLUSION: The COS/TPC is an efficient method to prepare recombinant adenovirus and the angiopoietin-1 recombinant adenovirus could be further used in gene therapy.

Objective To develop a novel method for treating complete heart block by autotransplantation of simus node node cells to right ventricular anterior wall.Methods Twenty healthy mongrel dogs were involved in the present study.The dogs were randomly assigned to transplant group or control group(n=10).The sinus node (SN)was harvested and isolated in vitro after an electronic pacemaker was implanted and complete heart bolck was introduced.The SN cells from dogs of transplant group were injected to autogenic right ventricular wall.Commensurable culture medium was implanted to ghe same position of dogs in control group.Two weeks later,detailed electropohysiological study was performed.For investigating the variation of the rhythm,epinephrine was administrated through femoral vein to dogs of transplant group.Results Most of isolated SN cells from dogs were thin-spindle shape,and cell activity was fine.The SNs by VG stained displayed typical structural feature.2 weeks after cell autotransplantation,higher heart rates were achieved from transplant group than that in control group(P＜0.05).This rhythm was stable in 4 weeks and became faster remarkably after administration of eninephrine(P＜0.05).Conclusion SN cell of dogs tutorgrfted into right ventricular anterior wall can form new pacemaker site in ventrcle and improve ventricular rate of complete heart bolck.This pacemaker site can also be regulated by epinephrine.