Qualitative and quantitative analyses of biological samples containing drugs, toxicants and endogenous substances play an important role in the researches of life sciences, as well as in new drug discovery and development. Biological samples are characterized by complex matrix, multiple endogenous interferences, significantly lower concentrations of measured analytes compared to endogenous components and small sampling volume. Consequently, it often requires bioanalysis methods with superior specificity, high sensitivity and good reproducibility. The two-dimensional liquid chromatography (2D-LC) technique, which allows for high peak capacity, significant reduced matrix effect and carryover of complex matrix samples and automated sample pre-treatment and analysis, has been the powerful solution to the separation and analysis of biological sample and widely applied to environment, food and pharmaceutical analysis. On the basis of introduction of the principle and equipments of 2D-LC, the application of this technique in the pharmacokinetics, toxicological and biological study was reviewed.

Objective To investigate the antitumor effects of total glycoside of Cimicifuga dahurica (TGCD) in vivo and in vitro, and further explore its mechanisms. Methods The anti-tumor activity in vitro was determined by MTT assay and the anti-tumor activity in vivo was evaluated using experimental mouse tumor (S180) model and human tumor (A549) xenografts in nude mice. After treatment, A549 cell apoptosis and morphologic change were evaluated by Annexin V/PI flow cytometry and HE staining. Results Inhibitory concentration 50% (IC50) of TGCD on A549, HepG2, HL60, Eca-109 and MDA-MB231 cells were 20.3, 27.1, 21.2, 23.4 and 32.7 ?g/mL respectively. Administration of TGCD (100 or 200 mg/kg) inhibited S180 solid tumor development in mice, the inhibition rates were 42.8% and 54.6% respectively. Administration of TGCD (100 or 200 mg/kg) inhibited A549 tumor growth with a T/C (mean value of treated group/mean value of control group) value of 58.1% and 52.2% respectively. In addition, increased percentage of apoptotic cells induced by TGCD in human A549 nude mice xenografts and the histopathological changes including cell shrinkage and condensation of chromosomes were observed. Conclusion TGCD has demonstrated antitumor bioactivity both in vitro and in vivo, which may be related to its effects of inducing apoptosis activity.

[Objective] To study the cure effect of Ganle Infusion on hepatitis-B of depressed liver and deficient spleen type.[Method] Randomly divide 60 cases into treatment group(Ganle Infusion) and control group(Diammonium Glycyrrhizinate capsule),observe concerned indexes change and clinical effects.[Result] For treatment group,the cure effect index was 93.3%;66.7% for control one;on liver improvement and serum hepatitis-B virus index negative-transfer rates,the treatment group was better than control one,with marked difference.[Conclusion] Ganle Infusion has marked cure effect on chronic hepatitis-B of the said type.

BACKGROUND:Telomerase reverse transcriptase (TERT) plays an important role in telomerase activation.
OBJECTIVE:To construct the targeting short hairpin RNA plasmid vector expressing TERT gene from astrocytes by using pLentilox3.7.U6.
METHODS:By using two sequences from TERT gene, we synthetized sense and antisense strand template sequences of RNA interference molecular in vitro, and then obtained the complementary strands through annealing procedure. We connected the strands with pLentilox3.7.U6 that was sequenced and transfected into the Escherichia coli. In the end, we tested its effect of reducing the TERT gene expressing by using cultured astrocytes from rat spinal cord in vitro through western blot and immunofluorescence technique.
RESULTS AND CONCLUSION:Western blot and immunofluorescence assay showed that, compared with the control group, the interference groups had a lower TERT expression in astrocytes. The targeting short hairpin RNA plasmid vector expressing TERT gene is useful to reduce the TERT gene expression. The targeting short hairpin RNA plasmid vector expressing TERT gene is valid for us to do the further test learning the mechanism of astrocytes in spinal cord injury.

AIM: To investigate the effects of leptin (LEP) on the alveolar type Ⅱ cells(AECⅡ) apoptosis induced by Na2S2O4 and explore the molecular mechanisms. METHODS: Primary AECⅡ culture was prepared according to a specific immunosorption procedure with slight modification and the cells were identified by transmission electron microscope and immunocytochemistry. AECⅡ damage was induced by 5 mmol/L Na2S2O4. LEP group cells were treated with LEP at concentrations from 100 ?g/L to 1 600 ?g/L. The cell survival rate was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Cell cycle and apoptosis were analyzed by flow cytometry and the level of caspase-3 was measured by Western blotting. RESULTS: Highly purified AECⅡ, obtained by the method of modified immunosorption, were identified with the positive expression of SP-A and intracellular lamellarbodies were found under electron micrography. The cells, exposed to 5 mmol/L Na2S2O4, showed characteristic changes of apoptosis and activation of caspase 3. These damages were relieved by the treatment of LEP (100-1 600 ?g/L), with survival increasing, apoptosis peak decreasing, cell morphology restoring and caspase 3 activation inhibiting.CONCLUSION: Leptin prevents AECⅡ from apoptosis induced by Na2S2O4 or hypoxia. The potential mechanism of its action may be related to promoting cell cycle from G1 phase to S phase and inhibiting the activating of caspase 3.

Objective:To investigate the expression of microRNA (miR)-938, its effect on cell proliferation and its regulatory mechanism in hepatocellular carcinoma (HCC).Methods:HCC and paracancerous tissues were collected from 40 patients with HCC who underwent surgical resection in the Second Affiliated Hospital of Nantong University from January 2015 to June 2019, including 25 males and 15 females, with an average age of 61.4 years. HepG2 cells in the miR-938 overexpression group were transfected with miR-938 mimics, and the negative control group was transfected with the negative control sequence. Cell proliferation was detected by kit, the expression of miR-938 and the succinate dehydrogenase complex subunit D (SDHD) was detected by fluorescence quantitative polymerase chain reaction, and SDHD protein expression was detected by Western blot. The target genes of miR-938 were verified by dual luciferase reporting.Results:The relative expression of miR-938 in HCC tissues was (0.060±0.002), which was higher than that in adjacent tissues (0.030±0.002), and the difference was statistically significant ( P<0.05). The mRNA relative expression of SDHD in HCC tissues was (0.028±0.002), lower than that in adjacent tissues (0.062±0.002), and the protein expression of SDHD in HCC tissues was (0.963±0.008), lower than that in adjacent tissues (1.083±0.037), with statistical significance (both P<0.05). The proliferation activity of miR-938 overexpression group was significantly higher than that of negative control group, and the difference was statistically significant ( P<0.05). MiR-938 significantly inhibited the luciferase activity of SDHD wild-type 3’-untranslated regions. In the overexpression miR-938 cells, SDHD mRNA and protein levels were significantly lower than those in the negative control group ( P<0.05). Conclusion:MiR-938 was highly expressed in HCC tissues. MiR-938 promoted the proliferation of HCC cells by inhibiting the expression of SDHD.

To obtain the aptamers with high affinity and specificity for cyclosporin A(CsA),a synthesized 78 nt single stranded DNA(ssDNA)random library containing 35 random sequences flanked by invariant primer was subjected to 1 1 rounds of selection against CsA by SELEX protocol.Magnetic beads were used for target immobilization and the biotin-streptavidin-horseradish peroxidase system was employed for determining the binding affinity between the aptamers and CsA. After ten rounds of selection and amplification, with an increasing affinity for each round,the selected aptamers were cloned,sequenced and analyzed for their primaryand secondary structures.The 19 aptamers were divided into five groups based on primary sequence homology.Hairpin loop is the main motif in the predicted secondary structure and is supposed to be the binding part of the aptamers to CsA.The CsA-specific aptamers will be useful for enzyme-linked assays or immunofluorescence asses of CsA.

Teaching model plays an important role in clinical education. Problem based learning (PBL) model, with an outstanding feature of stimulating the learning enthusiasm of students, was applied by many American colleges. American College of Acupuncture&Oriental Medicine (ACAOM) combined PBL teaching model with traditional lecture based learning (LBL) model, which had achieved good results. In this article, PBL model and its characters were explored via qualitative case analysis, to provide reference for universities and colleges of traditional Chinese medicine on clinical education.

BACKGROUND:The method in repair of primary lumbar intervertebral infection is different in different positions, mainly containing anterior, posterior and anteroposterior pathways. In posterior pathway, muscle gap approach is recognized by many fel ows. This program has its special advantage compared with conventional posteromedial approach. OBJECTIVE:To evaluate the clinical effects of debridement, bone graft and internal fixation of pedicle screw placement in repair of primary lumbar intervertebral infection through posterior paraspinal muscle approach. METHODS:Clinical data of 13 patients with primary lumbar intervertebral infection were analyzed retrospectively. There were one case of L 2-L 3 , two cases of L 3-L 4 , four cases of L 4-L 5 and six cases of L 5-S 1 . Lumbar pain was obvious in al patients. Nine cases suffered from radioactive lower limb pain. Al patients received debridement, bone graft and internal fixation of pedicle screw placement through paraspinal muscle approach via posteromedial incision. After treatment, clinical effects were evaluated using Visual Analog Scale and lumbar Japanese Orthopaedic Association scores. RESULTS AND CONCLUSION:Al patients were fol owed up for 12-18 months, no recurrence. X-ray review demonstrated bony fusion, so loosening and breakage were not found in al patients. Visual Analog Scale scores revealed that Visual Analog Scale score was 8.15 preoperatively, 2.15 at 1 week postoperatively, 1 at final fol ow-up, showing significant difference (P<0.05). There was significant difference in pain between pre-treatment and post-treatment, and pain apparently relieved after treatment. Lumbar Japanese Orthopaedic Association score showed that effective outcomes were found in al patients after treatment, including nine cases of excel ent effects, three cases of good effects, one case of average effects, with the excel ent and good rate of 92%. Above findings confirmed that one-stage debridement, bone graft and internal fixation of pedicle screw placement through posterior muscle gap approach provides a good repair method for patients with primary lumbar intervertebral infection. It can achieve intervertebral space directly through intervertebral foramen, retain the central spinous process and lamina, reduce the injury to paraspinal muscle, and keep spinal ligament complex. However, strong spine fixation contributes to bone fusion, and keeps the stability of the spine after repair.

Objective To detect biofilm formation and biofilm-associated genes of methicillin-resistant Staphylococcus aureus (MRSA) in clinical isolates. Methods The biofilm were determined by microtiter plate assay (MPA) and congo red agar (CRA) and the biofilm-associated genes icaA,sarA,fnbA,fnbB were detected by PCR in 33 strains of MRSA in clinical isolates. Results Of the 33 MRSA isolates, 29(87.9%) were MPA positive, 16(48.5%) were CRA positive; The icaA gene was present in 39.4% of isolated strains. Furthermore, 69.7% of strains harboured the sarA gene, 39.4% were fnbA positive and 75.8% were fnbB positive. As many as 87.9% strains had the ability to form biofilm in vitro. 44.8% of MRSA formed biofilm in ica-dependent mechanism and 55.2% of MRSA isolates formed biofilm in ica-independent mechanism. Of the biofilm positive MRSA, 75.9% were sarA positive, 37.9% were fnbA positive and 79.3% were fnbB positive. Conclusion Most of the MRSA strains formed biofilm in ica-independent mechanism. fnbB and sarA gene shows higher frequency among the biofilm-associated genes of MRSA, it may infer that most of the MRSA strains biofilm formation are fnbB-mediated. Meanwhile, sarA may be a positive regulator of fnbB, and thus drives the biofilm formation.