In order to adapt to the education of the professional who work in thecurrent medical lab,we set up the course of'Experimental Technology of Medical Function'. It is a required subject for the professional with laboratory skill. This article explores the curriculum sys-tem,teaching contents,teaching methods and examination.

Objective To evaluate the influences of the genotypes,anther developmental stages,and cultural conditions on the efficiency of embryogenic callus induction and plant regeneration in the anthers culture of Bupleurum chinense.Methods The different effects such as four genotypes,plant growth regulators,and temperature condition were compared in the experiments.The histological study was performed with the process of the anther culture.Results The highest inducing rate of embryogenic calli were achieved for the genotypes Zhongcaiyihao(ZCYH),Z4,and Z5 at the early-to middle-uninucleate stages,except for genotype ZPM1 at the tetrad stage.Cold pretreatment increased the production of the embryogenic callus,in which 4-day cold pretreatment improved the production of embryogenic callus from 0% to 2.2% and 5.0% for genotypes ZPM1 and ZCYH,respectively.No embryogenic callus was induced in the medium containing less than 0.75 mg/L 2,4-dichlorophenoxyacetic acid(2,4-D).The highest regeneration rate (34.6%)was obtained in 1/2 MS salts regeneration medium supplemented with 0.1 mg/L 6-benzylmaminopurine (BA).The low concentration of BA was able to promote the embryogenic callus formation and subsequent plantlet regeneration via somatic embryogenesis.Chromosome counting of regenerated plantlets showed mostly diploid plant (2n = 12)with only one haploid plant(n = 6).Because of the low rate of microspore embryo formation,we only tracked the process of embryogenesis from the connective tissue,instead of microspore by histological observations.Conclusion This study establishes an efficient system for embryogenic callus induction and plant regeneration system.This is the first report on the haploid plantlet through the anther culture orB.chinense.

Objective To investigate the effects of lentiviral-mediated RNA interference (RNAi) targeting tumor necrosis factor-α(TNF)-αgene on the expression of TNF-α, interleukin (IL)-1β, IL-6 of murine macrophages RAW264.7, and the efficiency of RNAi experimental gene therapy for the murine collagen-induced arthritis (CIA). Methods The RAW264.7 macrophages were infected by lentivirus-RNAi particles, then stimulated by Lipopolysaccharides (LPS). The TNF-α, IL-1β, IL-6 expression of RAW264.7 macrophages were measured with real-time polymerase chain reaction (PCR) and enzyme linked immunosorbent assay (ELISA). CIA models were esta-blished in DBA/1 mice using bovine type Ⅱ collagen. The treatment effect of lentivirus-RNAi on CIA were observed through arthritis scores, serum TNF-α measurement and hind paw paraffin section hematoxylin/eosin staining after lentivirus-RNAi particles tail vein injection. Results The TNF-αmRNA relative expression level of lentiviral RNAi group was 0.291 ±0.021, significantly lower than that of negative control group 0.925±0.013 (t=25.4, P<0.01). The inhibition rate in mRNA levels was 68.5%. The serum TNF-α level of lentiviral RNAi group was [(249 ±11) ng/ml], significantly lower than that of negative control [(382±6) ng/ml] (t=10.31, P<0.05). The inhibition rate of protein levels was 34.7%. It had no effect on the IL-1β and IL-6 mRNA expression. On the 8th day after systemic administration, the arthritis score of lentivirus-RNAi group was 2.50±0.19, which was significantly lower than that of blank controls (3.63 ±0.18) and negative controls (3.75 ±0.16) (F=42.8, P<0.01). From now on, arthritis score of lentivirus-RNAi group and positive control decreased slowly to at least 2 weeks after treatment induction. The serum TNF-α levels of lentivirus-RNAi group and positive controls were [(35±6) pg/ml] and [(32±7) pg/ml] significantly lower than that of negative controls [(47±3) pg/ml] (t=3.03, 4.11, P<0.01) respectively. Morphological examination showed that the lentivirus-RNAi decreased CIA pathological manifestations. Conclusion Lentiviral-mediated RNAi targeting murine TNF-α gene can effectively inhibit TNF-α expression both in vitro and in vivo, which also effectively improve the CIA arthritis score. Lentiviral-mediated RNAi targeting TNF-αgene provides a potential strategy for rheumatoid arthritis (RA) treatment.

Objective The aim of this study is to evaluate the efficacy and safety of preoperative Computed Tomography (CT)-guided microcoil localization for pulmonary ground-glass opacity.Methods We performed CT-guided Microcoil localization in 30 patients with 32 pure ground glass opacities between December 2012 and September 2013.All 30 patients underwent CT-guided micmcoil localization under local anesthesia with a 21G needle percutaneous lung biopsy.All patients accepted video-assisted thoracic surgery after the localization the same day or the next day.Results We located 32 pure ground glass opacitiesin 30 patients.The mean lesion diameter was 0.94 cm,the average depth from the visceral pleura was 0.71 cm,all leisons were successfully located by microcoil,asymptomatic pneumothorax was observed in seven patients,pulmonary hematoma was observed in two patients,pain was observed in one patient,dislodgement was found in one patient during the operation.None of these cases needed surgical treatment.Pathologic diagnoses of the target leisions were as follows:adenocarcinoma in situ (n =17),adenocarcinoma(n =9),atypical hyperplasia(n =4),inflammation (n =2).After the placement of preoperative CT-guided microcoil localization for pulmonary ground-glass opacity,all the 32 pulmonary pure ground-glass opacities resection success rate was 100％.Conclusion Preoperative CT-guided microcoil localization for pulmonary pure ground-glass opacity is a safe and effective way to improve the accuracy of surgical resection.

In this study, the induction of hairy roots of Bupleurum chinense DC. was explored and established after experiments at different conditions: A. rhizogenes A4 was used to infect the leaves bases of B. chinense tube seedlings. The explants were co-cultured on Phytagel-solidified media for 3 days and then, were turned into solid media, similar with the co-culture media except that bacteriostat was added. After 10 days, rootlets began to appear and after 4 to 5 weeks, rootlets can be converted into liquid shaking culture stage. Plants regeneration from hairy root was useful for the research of new germplasm production and the variety improvement breeding. In the present study, the regenerated plants were obtained. One approach was to continuously culture under light conditions the seedlings which parting off spontaneously from the hairy roots during liquid shaking culture. The other approach was to culture the callus-like tissues produced by hairy roots with the optimized regeneration media for the induction of regenerated plants. The results of present study provide a technique to induce hairy roots and plantlet regeneration of B. chinense and this technique is helpful for the researches on metabolism, especially on the Agrobacterium-mediated genetic transformation of B. chinense.

Objective:To construct recombinant lentiviral vectors harboring interference RNA ( RNAi ) targetting murine TNF-αgene,so as to lay the foundation on the RNAi gene therapy.Methods: Three small interfering RNA ( siRNA) sequences targeting murine TNF-αgene ( siRNA1,siRNA2,siRNA3) and negative-control siRNA were designed and synthesized.The inhibition effects of siRNAs on TNF-α,IL-1βand IL-6 secretion of LPS-stimulated RAW264.7 macrophages were observed using real-time PCR and ELISA methods.DNA oligo was designed and synthesized according to the most effective siRNA 2 sequence.The recombinant lentiviral shuttle plasmid expressing short hairpin RNA ( shRNA) was constructed and sequenced.The lentiviral shuttle plasmids with packaging plasmids were transfected into 293T cells to produce lentiviral particles.Results: ①The TNF-αmRNA relative expression levels of siRNA1, siRNA2 and siRNA3 were 0.24±0.01,0.16±0.02,0.19±0.01 respectively,significantly lower than that of negative control (0.95± 0.02) (F=531.3,P<0.001).The inhibition rates at mRNA level were 74.26%,83.09%,79.93%,respectively comparing with negative control.No significance was observed in IL-1βor IL-6 mRNA relative expression change after TNF-αsiRNA transfection ( P>0.05).②The TNF-αprotein expression levels of siRNA1,siRNA2 and siRNA3 were (23.95±1.21),(17.27±1.46),(19.07± 1.57)ng/ml respectively,significantly lower than that of negative control (35.37±2.93)ng/ml (F=18.1,P=0.000 6<0.001).The inhibition rates of protein expression were 32.29%, 51.16%, 46.08%, respectively comparing with negative control.③The PCR product electrophoresis showed that recombinant vectors yielded 343 bp fragments,non-constructed vectors yielded 306 bp fragments.DNA sequencing partially showed insertion sequence.④Lentiviral particles were obtained by transfecting 293T cells with recombinant lentiviral shuttle plasmids and lentiviral packaging plasmids.Cells grew well during virus production with strong fluorescence expression.The titer of concentrated virus was 2×106 TU/μl.Conclusion:The lentiviral vector harboring RNAi targeting murine TNF-αgene has been successfully constructed.

Objective To prospectively evaluate the efficacy and safety of Computed Tomography (CT)-guided microcoil localization for pulmonary small solid nodules and ground-glass opacity prior to thoracoscopic resection.And to investigate the indication for CT-guided microcoil localization for small solid pulmonary nodules and ground-glass opacity.Methods From December 2012 to February 2014,85 enrolled patients with pulmonary solid nodules and ground-glass opacity underwent CT guided microcoil localization prior to video assisted thoracoscopic surgery.The procedures of localization were performed by trailing method or routine method under CT guided percutaneous pneumocentesis.For Trailing method,the microcoil was placed with the distal part coiled adjacent to the lesion and the proximal end coiled beyond the parietal pleura.By routine method,the entire microcoil was injected adjacent to the lesion.Results CT-guided microcoil placements were successful in all ninety-one lesions,including 15 solid nodules,15 mixed ground glass opacity,and 61 pure ground glass opacity,with an average diameter of 8.75mm(5-26 mm).The Complication rate of the localization procedure was 23.5％ (20/91),with 13 cases of asymptomatic pneumothorax,and 7 cases of pulmonary hematoma.None patient required surgical intervention,nor severe Complication occurred.All patients underwent video assisted thorascopic surgery on the same day or the next few days after microcoil localization.VATS removal of the pulmonary lesions was successful in all patients.However,two of 91 microcoils were found displaced during VATS resection.The success rate of microcoil marking VATS resection for pulmonary small solid nodules and ground-glass opacity was 97.8％.Microcoil marking was required for 84.6 percent of all the resected lesions.Conclusion Preoperatively CT-guided microcoil localization for pulmonary small solid nodules and ground-glass opacity is a feasible safe and effective marking technique for video assisted thoracoscopic resection.The indication for microcoil localization in our study meet the requirement of VATS resection.

Objective To investigate the value of magnetic resonance imaging (MRI)-based intravenous thrombolysis in patients with wake-up ischemic strokes (WUIS).Methods Patients presenting within 12 hours of acute stroke symptom onset and those with WUIS confirmed by CT,excluding intracranial hemorrhage,were encouraged to perform an emergent brain MRI scan to confirm the diagnosis of hyperacute ischemic stroke (hyper-intense in DWI without hyper-intense change in T2WI or fluid attenuated inversion recovery (FLAIR)).These patients then received intravenous thrombolytic therapy with recombinant tissue plasminogen activator (rt-PA).All patients were divided into either stroke presenting within 12 hours or WUIS.The clinical outcomes were assessed by the modified Rankin scale (mRS) and the Barthal index (BI) at baseline and at 90 days after the thrombolysis therapy.Results Two hundred and sixty-one patients (261/563,56.4％) had confirmed diagnosis of hyperacute ischemic stroke (WUIS,n =73,73/121 =60.3％ vs within 12 hours,n =188,188/342 =55.0％).Altogether,192 patients (139 in within 12 hours group,and 53 in WUIS group) received intravenous thrombolytic therapy with rt-PA.No significant differences were found between the 2 groups at the baseline characteristics and at 90 days outcomes after the thrombolysis therapy(x2 =1.296 and 1.473,P =0.538 and 0.489,respectively).Also no significant differences were found in the incidence rate of secondary hemorrhage (including both of asymptomatic and symptomatic) and mortality rate between the 2 groups.Conclusion MRI-based intravenous thrombolysis is safe and effective in the treatment of patients with hyperacute WUIS.

This study was aimed to establish the method of identifying bupleurum cultivated germplasm using simple sequence repeat (SSR) molecular markers and to initially establish dataset of characteristic SSR bands to the bred cultivars or strains. From the bupleurum SSR primer pairs which were designed in previous work, 50 primer pairs were selected. Two bred strains and 4 other bupleurum cultivated germplasms were used as test materials. Primers pairs were screened with effective PCR amplification and high polymorphism. Meanwhile, conditions for PCR amplification and electrophoresis were optimized. Then, obtained SSR bands were analyzed and a clustering tree on the basis of genetic distance was constructed. The results showed that 9 SSR primer pairs can be used for identification. The suitable assay conditions were established and characteristic SSR bands were obtained for tested materials. The tested samples can be divided into 4 categories in the genetic similarity coefficient of 0.73. TheB. scorzonerifolium cultivated inHeilongjiang andChuanhongchaiNo. 1 strains were clustered as one category. ChuanbeichaiNo. 1 strain andZhongchai No. 1 cultivar clustered as another category. Cultivated germplasms fromSichuan Fengshunand Rongxian clustered as a unique category. It was concluded that the primer pairs and assay method established in the present study can be used as reference in identification of bupleurum cultivars or cultivated germplasms.

ObjectiveTo investigate the characteristic parameters and variation of acoustic startle reflex (ASR) and conditioning response (CR)during the acquisition of delay-trace eyeblink conditioning.Methods15healthy Kunming mice were randomly divided into 3 groups:single-task DEBC group ( n =5 ) ; single-task TEBC group ( n =5) ; dual delay/trace group ( n =5).Three groups received paired training of tone conditioned stimulus(CS)in different audio frequency (TEBC groups:2KHz; DEBC group:1KHz) and a 100 ms comeal oxygenpuff unconditioned stimulus(US).Then observed the characteristic parameters of ASR and CR.ResultsAfter pairing training for 10 days,ASR rate( ASR％ ) of single-task groups ( 10th day (38.00 ± 8.64) ％ and (38.55 ±12.41 ) ％ respectively,time effect:F =4.574,P =0.008 ; group effect:F =2.021,P =0.193 ) and dual delaytrace group ( 10th day (47.95 ± 14.23 ) ％ and (62.01 ± 9.03 ) ％ respectively,time effect:F =5,547 P =0.013 ;group effect:F =0.738,P =0.415) changed significantly with the following training but between single-task groups or dual delay-trace group.CR rate ( CR％ ) of single-task groups ( 10th day were (45.4 ± 5.39) ％ and (65.6 ± 6.77) ％ respectively,time effect:F =9.558,P =0.000 ; group effect:F =5.117,P =0.054 ) and dual delay/trace group ( 10th day (57.66 ± 4.34) ％ and (77.35 ± 7.36) ％ respectively,time effect:F =7.750,P =0.002 ;group effect:F=1.449,P=0.263 ) showed obvious change with the following training but between singletask groups or dual delay-trace group.Peak amplitude of single-task DEBC group ( time effect F =2.679,P =0.017 ) and dual delay-trace group ( F=3.452,P=0.034 ) had increased significantly with the following training.Peak latency showed no obvious change.ConclusionDual delay-trace conditioning procedure of classical eyeblink conditioning and single-task can be successfully established in Kunming mice.Although there are some variations,those can be controled and be weaken effect of them by taking targeted measures.