Objective To evaluate the feasibility of transfection of Sonic hedgehog gene (SHH)into bone marrow mesenchymal stem cells(BMMSC).Methods After the SHH gene was transfected into BMMSC by electroporation apparatus,the transfection rate was evaluated by fluorescence inverted microscope.The growth curves of untransfected and transfected BMMSC were drawn,respectively,to observe the influence of transfection on cells.The expression of SHH gene in the BMMSC was detected by PCR,RT-PCR,Western-blot analyses.Results Through fluorescence inverted microscope,the observed transfection rate was appropriately 30％,PCR showed a obvious increase of SHH expression in transfected cells than that in untransfected cells,and it is quantified by qPCR for appropriately 7 times.Western-blot further demonstrated that the SHH protein expression in transfected cells had a distinct increase.However,it was observed that the exponential phase of BMMSCSHH growth curve delayed.The growth curves of both overlap 12 days after transfection.Conclusions This electroporation method can transfect exogenous SHH gene into BMMSC sufficiently with the effective protein expression in BMMSCSHH.It is the foundation of further research of genetic therapy for ischemic heart disease.

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Objective To explore whether hydrochloride penehyclidine can inhibit rat pulmonary hypertension induced by monocrotaline and prevent or relieve the remodeling of pulmonary vascular.Methods Thirty healthy male SD rats (aged 3-4 weeks,weighing 90-100 g)were randomly divided into normal control group (group C),monocrotaline pulmonary hypertension group (group M)and hydrochloride penehyclidine treatment group (group P,n = 10 each).Rat pulmonary hyper-tension model was established by abdominally injecting 60 mg/kg monocrotaline all at once;The con-trol group was abdominally injected equal volume of normal saline all at once;the treatment group P 2 mg/kg hydrochloric penehyclidine 1 5 minutes before model building 1 mg/kg hydrochloric penehyclid-ine two days after model building;while the other two groups received equal volume of normal saline as placebo at the same time of medicine injecting,which lasted consecutively for 3 weeks.21 days af-ter model building,the haemodynamics (pulmonary artery pressure and right ventricular pressure) test was performed for all groups;Venous blood was sampled for blood biochemical tests (using ELISA method to test the content of NO and ET-1 )before executing the rats.The left lung tissue was sliced to observe the pathological morphological changes of lung tissues.The right lung tissue was cryopreserved for other tests.Results Compared with the normal control group,the pulmonary artery pressure and right ventricular pressure of monocrotaline pulmonary hypertension group notably enhanced (P <0.05);The pulmonary arterioles strongly thickened;The lumen of pulmonary arteri-oles became narrow and even blocked;The inflammatory cells of lung tissues apparently infiltrated. When compared with the monocrotaline pulmonary hypertension group, the pulmonary artery pressure and right ventricular pressure of the hydrochloride penehyclidine treatment group greatly lowered (P <0.05);Both the thickening degree of pulmonary arterioles walls and the infiltrating de-gree of inflammation cells of lung tissues lessened.When compared with the normal control group, the NO content of other groups significantly lowered and the ET-1 content apparently increased;When compared with the monocrotaline pulmonary hypertension group,the NO content of hydrochlo-ride penehyclidine treatment group was higher and the ET-1 content was lower.The differences were statistically significant (P <0.05).Conclusion The rat pulmonary hypertension model has been suc-cessfully built with the help of monocrotaline.The reducing of NO content and increasing of ET-1 was relevant to the formation of rat pulmonary hypertension induced by monocrotaline;The fact that hy-drochloric penehyclidine lowered the pulmonary artery pressure of rat pulmonary hypertension model and improved the thickening degree of pulmonary arterioles walls might involve the increasing of NO cotent and reducing of ET-1 content.

Objective To evaluate the effect of alprostadil on acute lung injury in septic rats. Methods Thirty adult male Sprague?Dawley rats, weighing 200-250 g, were randomly divided into 3 groups (n=10 each) using a random number table: sham operation group (group S), acute lung injury group ( group ALI) , and alprostadil group ( group Q) . The animals were anesthetized with intraperitoneal 1% pentobarbital sodium 5 ml∕100 g. Sepsis was induced by cecal ligation and puncture. In group Q, al?prostadil ( 10 μg∕2 ml) 2 ml∕kg was injected via the tail vein at 30 min before cecal ligation and puncture. The equal volume of normal saline was given in S and ALI groups. At 24 h after operation, blood samples were taken for determination of serum tumor necrosis factor?alpha ( TNF?α) and interleukin?6 ( IL?6) con?centrations by enzyme?linked immunosorbent assay. The animals were then sacrificed. The left lungs were immediately removed for microscopic examination, and the right lungs were immediately removed for deter?mination of wet∕dry lung weight ratio ( W∕D ratio ) , and expression of TNF?α mRNA and high mobility group box?1 ( HMGB1) using real?time reverse transcriptase?polymerase chain reaction. Results Com?pared with group S, the concentrations of serum TNF?α and IL?6 were significantly increased, and the ex?pression of TNF?α mRNA and HMGB1 mRNA was up?regulated, and W∕D ratio was increased in ALI and Q groups ( P<0?05) . Compared with group ALI, the concentrations of serum TNF?αand IL?6 were signifi? cantly decreased, and the expression of TNF?α mRNA and HMGB1 mRNA was down?regulated, and W∕D ratio was decreased in group Q ( P<0?05) . The pathological changes of left lungs were significantly attenua?ted in group Q as compared with group ALI. Conclusion Alprostadil can reduce acute lung injury in septic rats, and the mechanism may be related to down?regulation of HMGB1 expression and inhibition of inflam?matory responses.

Objective To investigate the survival and differentiated situation of transplanted mesenchymal stem cells ( MSCs) transfected with Sonic hedgehog (SHH) gene after chronic myocardial infarction (MI).Methods MSCs were obtained from bone marrow of limb bones by density gradient centrifugation combined with attachment culture method .SHH gene was cloned and eukaryotic expression plasmid pcDNA 3.1-SHH was constructed .Then nucleofector TM was used to transfer SHH gene into MSCs and the expres-sions of mRNA and protein of SHH gene were detected in vitro .Myocardial infarction model was established by ligating the left anterior descending branch .A total of 150μl (3 ×106 ) of MSCs transferred with SHH gene was injected into ischemic area at the fourth week after MI model was set up .Brdu immunohistochemical staining and electron microscope examination were used to detect the survival and differentiation of MSCs with SHH in vivo at the 1st, 2nd, 4th, and 8th week after MSCs SHH transplantation.Results It was con-firmed that SHH gene clone and the recombinant eukaryotic expression vector pcDNA 3.1-SHH construction were successful by gene se-quence analysis and double digestions with EcoR I and Bamh I .Reverse transcriptase polymerase chain reaction ( RT-PCR) and West-ern-blot demonstrated that the expression of SHH mRNA was significantly increased in transfected MSCs compared with untransfected MSCs.Brdu immunohistochemical staining confirmed MSCs with SHH were survived from one to eight weeks in MI area after transplan -tation.Electron microscope examination showed that MSCs had the trend to be differentiated into myocardiac -like cells in MI area at the 4th and 8th week after transplantation .Conclusions The expression of SHH mRNA was significantly increased while MSCs were transfected with SHH gene , and MSCs SHH could survive and differentiate in good condition in the transplanted recipients .Those data provide the experimental basis for further transgenic cell therapy research of ischemic heart disease .

Objective To investigate the role of RhoA /Rho-kinase pathway in rat models of left heart disease-as-sociated pulmonary hypertension ( PH-LHD) .Methods Twenty male SD rats (3-4 week-old, 90-100 g) were randomly divided into two groups (10 rats in each group):the group C ( control group) with sham operation, and group H ( pulmo-nary arterial hypertension) .The rat model of left heart disease-associated pulmonary hypertension was established by supra-coronary aortic banding in the group H, and the sham surgery was applied for the rats in the group C ( The titanium clip was fixed at the mediastinal tissue adjacent to the artery rather than the ascending aorta).On day 60 after the operation, the cardiac functions, including right ventricular systolic pressure and pulmonary artery pressure were evaluated.After that, all rats were sacrificed and treated with cardiopulmonary lavage in vivo until the lung became white.Then the left lung tissues were fixed in 4%paraformaldehyde for pathological observation while the right lung tissues were frozen for mRNA detec-tion.Results Compared with the group C, both ventricular systolic pressure and pulmonary artery pressure in the group H were increased significantly (P<0.01).Pathological data demonstrated that the pulmonary artery walls in H group were much thicker than that in the group C.Moreover, vascular wall hypertrophy index in the group H was increased greatly compared with that in the group C (P<0.01).QPCR data showed that mRNA levels of Rho kinase, RhoA and ET-A R in the group H were up-regulated compared with the group C ( P<0.01) .Conclusions Rat model of left heart disease-asso-ciated pulmonary arterial hypertension can be successfully established by supracoronary aortic banding.Rho-kinase-media-ted pathway may contribute to the pathogenesis and progress of left heart disease-associated pulmonary arterial hypertension.

Objective To gain experimental data about breeding Musca domestica larvae by pig manure and to evaluate the effects and cost-benefit of feeding hens with larvae instead of fish powder. Methods The concentration of fatty acids and trace elements in the larvae bred with man-made fodder or pig manure were measured. The quality of eggs produced by hens fed with the larva powder or fresh larvae was investigated. Results The contents of trace elements (Ca,Fe,Cu,Zn,Se) of the larvae bred with pig manure were higher than those with man-made fodder(P0.05). Conclusion Breeding Musca domestica larvae with pig manure could produce good larva protein and reduce the expenses. The quality of the egg produced by the hens fed with larvae is as good as the quality of common eggs.

ObjectiveTo explore and analyze the distribution of NAT2 gene in Han population in Shenzhen andprovidethebasisfortreatmentof NAT2-relatedmetabolicdiseasesandcancer research. MethodThe study diluted the DNA sample(d0 ng/μl) as follows: 1 × 100, 1 × 101, 1 × 102, 1 ×103 , 1 × 104, to verify the sensitivity of detection of NAT2 gene polymorphism by real-time PCR. Mutation locus of 282,341,481, 590 and 857 of NAT2 in 554 normal samples were detected and genotyped by realtime PCR with Taqman probes. Forty-seven cases among 554 healthy controls were detected by DNA sequencing to verify the sensitivity, specificity and accuracy of this assay. ResultsThe lowest detection limit was 10-4 ng/μl. The frequencies of each allele of NAT2 were: *4 allele 64. 6％ (358/554), *5 allele 6. 3％ (35/554), *6 allele 25. 3％ ( 140/554), *7 allele 30. 0％ ( 166/554), * 11 allele 0. 6％ (3/554) The frequencies of main genotypes of NAT2 were as the follows : NAT2 *4/* 6 12.3％ ( 68/554 ), *6/* 78. 3％ (46/554), * 13/* 13 or * 12/* 12 or *4/* 4 7.6％ (42/554), *4/* 7 8. 1％ (45/554), * 7/* 1 312. 3％ (68/554), * 12 7.2％ (40/554), *6/* 13( * 12)5.6％ (31/554). The samples with 7 genotypes account for 89. 6％ (496/554) of the total cases.Rapid acetylation and Intermediate type accounting for 40. 5％ (224/554) and 46. 7％ (259/554) respectively were the main phenotypes of NAT2 in Han people in Shenzhen. In addition, the accuracy, specificity and sensitivity of the PCR were compared to direct DNA sequencing. The accuracy of 282, 341,481, 590 and 857 were 88.2％ (30/34), 87.5％ (7/8), 80. 0％(4/5), 100. 0％ ( 22/22 ) and 93.8％ ( 15/16 ) respectively. The specificities were 100. 0％ ( 13/13 ),94. 9％ ( 37/39), 100. 0％ ( 42/42 ) , 96.0％ ( 24/25 ) and 96. 8％ ( 30/31 ) respectively. The sensitivities were 91.5％ ( 43/47 ), 93.6％ ( 44/47 ), 97.6％ ( 46/47 ) 97.9％ ( 46/47 ) and 95.7％ ( 45/47 ),respectively. ConclusionsThe sensitivity, specificity and accuracy of real-time PCR established in this study is good. It can be widely used for clinical and scientific research. The major alleles of NAT2 of Han population in Shenzhen are * 4, *6, * 7. The most common slow acetylation genotype is *6/* 7. This study can provide a basis for NAT2-related metabolic diseases and cancer research.

Objective To explore the influence of different injection techniques on the quality of bolus in 99mTc-DTPA renal dynamic imaging.Methods 395 patients accepted 99mTc-DTPA renal dynamic imaging were retrospectively analyzed.All patients were divided into three groups according to injection techniques:direct injection group (187 cases),intravenous route injection group (84 cases)and venous indwelling needle injection group (124 cases).The three groups were injected by each technique.Areas of interest (ROI) were drawn on abdominal aorta by Xeleris workstation in blood flow perfusion imaging.The time-radioactivity curves of ROI were got.The patients whose ROI curve formed a peak was successfully injected,and did not formed was unsuccessfully injected.The number of patients in three groups who were successfully or unsuccessfully injected was respectively calculated.The data of three groups was taken Chisquare test by SPSS17.0 software.Results 174 patients of the direct injection group,46 of the intravenous route injection group and 115 of the venous indwelling needle injection group were injected successfully.The successful rate respectively was 93.0％,54.8％ and 92.7％.The successful rate of the direct injection group and venous indwelling needle injection group were higher than intravenous route injection group.The difference had statistical significance.The successful rate of the direct injection group and venous indwelling needle injection group hadn't statistical significance.Conclusions The successful rates of the direct injection group and venous indwelling needle injection group were similar.The venous indwelling needle injection technique can be chosen.The successful rate of the intravenous route injection group was lower than the other two groups.The intravenous route injection technique should be chosen prudently.

OBJECTIVE:To provide reference for further implementation and perfection of National Essential Medicine Sys-tem. METHODS:Stratified random sampling method was used to select 252 doctors from Sichuan Luzhou. Questionnaire survey was performed among them about recognition and behavior of them to National Essential Medicine System. Related investigation da-ta was analyzed statistically. RESULTS:A total of 252 questionnaires were issued and 243 valid questionnaires were collected with effective recovery rate of 96.4%. Of 242 respondents who had heard of National Essential Medicine System,the respondents who had heard but not clear occupied the highest percentage (52.1%). Of 149 participants who had participated in national essential medicine knowledge training or study,the proportion ofonly one knowledge training or studywas the highest(57.0%). The sur-vey respondents had a maximum of 45.0% and a minimum of 16.5% of common sense about National Essential Medicine System. After the implementation of National Essential Medicine System,respondents often propagandized National Essential Medicine Sys-tem to diagnosis and treatment objects accounted for only 23.1%. The proportion ofno changechosen for items of work motiva-tion,work efficiency and workload was the highest (all occupied 58.3%);the proportion oflittle influencechosen for item of clinical medication habit was the highest(38.8%). 86.4% of respondents were willing to give priority to the use of national essen-tial medicines;the top 4 reasons wereto obtain training and guidance on the use of essential medicinesto set utilization rate of essential medicines in primary health care institutionsto strengthen the propaganda of National Essential Medicine Systemto evaluate physician's prescriptions regularly. 13.6% were not willing to give priority to the use of national essential medicines;the top 4 reasons werelack of the confidence of the use of national essential medicinespoor accessibility of essential medicinesfear of medical accidents due to prescription essential medicines,lead to medical disputesdon't know what is essential medi-cine. CONCLUSIONS:The primary physicians'perceptions of National Essential Medicine System and the implementation of the System in Luzhou need to be strengthened. The main reason is that few national essential medicine knowledge training or study and poor effects. That the government's supporting poli-cy is not perfect may be one of reasons.