BACKGROUND: Growth factors and other biological methods have become very popular in the repair of degenerative interevrtebral disc. Insulin-like growth factor-1 (IGF-1) can promote the proliferation of nucleus pulposus cells, and synthesis of functional extracellular matrix, but the mechanisms remain unclear.OBJECTIVE: To investigate the effect of IGF-Ⅰ on the expressions of aggrecan and collagen type Ⅱ in nucleus pulposus cells, and to explore its signal transduction mechanism.METHODS: The human nucleus pulposus cells were isolated and cultured. Passage 3 nucleus pulposus cells were induced in different concentrations of IGF-1 (0, 20, 50, 100 and 200 μg/L), respectively. The expressions of aggrecan and collagen type Ⅱ were detected by reverse transcription PCR and western blot assay. Western blot assay was adopted to observe the effect of 100 μg/L IGF-1 on the activation of PI3K/Akt signaling pathway in nucleus pulposus cells,and the expression of aggrecan and collagen type Ⅱ was detected after the inhibition of PI3K/Akt pathway by LY294002.RESULTS AND CONCLUSION: With the increase of IGF concentration, the expression levels of aggrecan and collagen type Ⅱ were increased gradually. 100 μg/L IGF-1 could significantly promote the expressions of p-PI3K and p-Akt (P <0.01), while LY294002 reversed this effcet (P < 0.01). 100 μg/L IGF-1 significantly upregulated the expression levels of aggrecan and collagen type Ⅱ in nucleus pulposus cells (P < 0.01); in contrast, LY294002 significantly downregulated the expression levels of aggrecan and collagen type Ⅱ promoted by IGF-1(P < 0.01). These results indicate that IGF-1 can promote the expression levels of aggrecan and collagen type Ⅱ in nucleus pulposus cells via the PI3K/Akt signaling pathway.

Objective Kunming strain(KM) mice were used as animal models. Nontoxic dextran conjugated with tetramethylrhodamine(TMR)and fluorecein isothiocyante(FITC)was microinjected to two of the 2-cell blastomere as molecular probe to trace the development fate of the blastomere ,in order to figure out the mechanisms of the formation of Em-Ab axis. Methods FITC- dextran was injected to zygote in order to make sure if it is noxious. Two blastomeres of 2-cell embryo were injected FITC- dextran and TMR- dextran respectively. Results When labeled embryo develeped to blastocyst, distribution of progeny of 2-cell embryo blastomeres can be detected.Conclusion The cells of blastomere randomly distributed either embryonic parts or extraembryonic parts of blastocyst.

Aim To study the influence of Si-Wu De-coction (SWD ) and its active components on cyto-chrome P450 activity and mRNA expression in rats in order to provide an experimental basis for compatibility of SWD.Methods SWD and its active components were intragastrically administrated for seven days,the doses of SWD was 10 g · kg -1 · d -1 ,the doses of fructose,ferulic acid,ligustrazine,peoniflorin were 0.334,0.002,0.011 and 0.022 g·kg -1 ·d -1 ,re-spectively.After administration for seven days,rats were executed,and liver microsomes were prepared. The effects of SWD and its active components on cyto-chrome P450 in rats were investigated by hybrid probe and liver microsomes incubation method.The level of mRNA expression in liver was detected by real-time quantitative polymerase chain reaction using specific target primers for CYP450 genes.The level of protein expression of CYP2B1 was detected by Western blot. Results Compared with the control group,fructose significantly decreased the activity of CYP1A2, CYP2B6,CYP2C9,CYP2D6;ferulic acid significantly decreased the activity of CYP2C9,CYP2B6;ligus-trazine significantly decreased the activity of CYP1A2, CYP2C9,CYP2B6;peoniflorin significantly decreased the activity of CYP2D6,CYP2B6;fructose,ferulic acid,peoniflorin inhibited the mRNA expression of CYP2B1;fructose,ferulic acid,ligustrazine and peon-iflorin also inhibit the protein expression of CYP2B1. Conclusion Fructose,ferulic acid,peoniflorin inhib-it the activity of CYP2B1,decrease the expression lev-els of mRNA and protein of CYP2B1.

OBJECTIVE To study the mitochondrial toxicity effect of Radix Aconiti Lateralis Praepa?rata(Fuzi)on H9c2 cardiomyocytes. METHODS H9c2 cells were exposed to Fuzi decoction 6.25, 12.5,25,50 and 100 g·L-1 for 24 h. Fluorescence staining and CCK-8 assay were used to detect cell viability. H9c2 cells were exposed to Fuzi decoction 6.25,12.5 and 25 g · L-1 for 24 h,while the effect on mitochondrial membrane potential and reactive oxygen species(ROS)was detected by flow cytometry. The fluorescence molecular probe and laser scanning confocal microscope were used to observe the effect on Ca2+ in cells,Ca2+ and superoxide in mitochondria. The effect on ATP concentration in cells was detected via firefly luciferin and the expression of Pgc-1α,Bcl-2 and Bax mRNA evaluated by real-time PCR,while the expression of Pgc-1α protein was measured by Western blotting. RESULTS H9c2 cell viability was significantly inhibited by Fuzi decoction 12.5-100 g · L-1(P<0.05,P<0.01). The IC50 value was 47.4669 g · L-1,while the 95%confidence limit was 32.5997-69.1145 g · L-1. After treatment with Fuzi decoction 25 g · L-1 ,the fluorescence intensity of ROS in the normal control group increased from 204±67 to 454±78(P<0.05),that of mitochondrial superoxide increased from 5.4±1.8 to 26.8±8.5 (P<0.01),mitochondrial membrane potential decreased from 1.7±0.5 to 0.8±0.4(P<0.05),the fluores?cence intensity of intracellular Ca2+increased from 7.8±0.8 to 22.1±0.5(P<0.05)while that of mitochon?drial Ca2+decreased from 38.0±4.3 to 9.2±1.6(P<0.01),and intracellular ATP concentration decreased from (10.6 ± 0.4)μmol · g-1 to (5.3 ± 1.1)μmol · g-1 protein (P<0.05). qPCR and Western blotting test results showed that compared with the normal control group ,Pgc-1αand Bcl-2 mRNA relative expression level in Fuzi decoction 25 g·L-1 group was decreased from 1.00±0.10 and 1.00±0.10 to 0.09±0.06(P<0.01)and 0.43±0.06(P<0.01),respectively, while the relative expression of Bax mRNA was increased from 1.00 ± 0.03 to 1.17 ± 0.06 (P<0.05),and the expression of Pgc-1α protein was decreased from 0.906±0.034 to 0.541±0.003(P<0.01). CONCLUSION Fuzi has some mitochondrial toxicity to cardiomy?opathy. This effect arises from the combined action of different mechanisms. Mitochondrial toxicity of myocytes may account for the cardiac toxicity of Fuzi.

Objective To investigate the clinic efficacy of proximal humeral internal locking system(PHILOS) in the treatment of Neer Ⅱ and Ⅲ proximal humeral fractures.Methods From January 2013 to December 2015,a total of 47 patients with Neer Ⅱ and Ⅲ proximal humeral fractures was treated with PHILOS fixation in our hospital.The operative time,blood loss in operation,hospital stay,complications,Constant scores and radiography films were retrospectively analyzed.Results All 47 patients were followed up for an average of (19.2 ±9.6) month.The average operative time,the level of average intraoperative blood loss,the mean hospitalization time and the complication rate were(95.6 ±43.1) min,(108 ± 41.6) mL,(11.3 ±3.2) d and 14.7 ％ respectively.The Constant score was improved from(21.74 ± 8.24) preoperatively to(82.83 ± 7.21) at the last follow-up,and the difference was statistically significant(t=-36.57,P＜0.01).Conclusion Fixation with PHILOS is a safe and effective treatment for patients with Neer Ⅱ and Ⅲ proximal humeral fractures.

AIK is a novel cationic peptide with potential antitumor activity. In order to construct the AIK expression vector by Gateway technology, and establish an optimal expression and purification method for recombinant AIK, a set of primers containing AttB sites were designed and used to create the AttB-TEV-FLAG-AIR fusion gene by overlapping PCR. The resulting fusion gene was cloned into the donor vector pDONR223 by attB and attP mediated recombination (BP reaction), then, transferred into the destination vector pDESTl 5 by attL and attR mediated recombination (LR reaction). All the cloning was verified by both colony PCR and DNA sequencing. The BL21 F. coli transformed by the GST-AIR expression plasmid was used to express the GST-AIK fusion protein with IPTG induction and the induction conditions were optimized. GST-AIR fusion protein was purified by glutathione magnetic beads, followed by rTEV cleavage to remove GST tag and MTS assay to test the growth inhibition activity of the recombinant AIR on human leukemia HL-60 cells. We found that a high level of soluble expression of GST-AIK protein (more than 30% out of the total bacterial proteins) was achieved upon 0.1 mmol/L ITPG induction for 4 h at 37 °C in the transformed BL21 F. coli with starting OD₆₀₀ at 1.0. Through GST affinity purification and rTEV cleavage, the purity of the resulting recombinant AIK was greater than 95%. And the MTS assays on HL-60 cells confirmed that the recombinant AIK retains an antitumor activity at a level similar to the chemically synthesized AIK. Taken together, we have established a method for expression and purification of recombinant AIK with a potent activity against tumor cells, which will be beneficial for the large-scale production and application of recombinant AIK in the future.
Antimicrobial Cationic Peptides ; biosynthesis ; Antineoplastic Agents ; metabolism ; Escherichia coli ; metabolism ; Genetic Vectors ; HL-60 Cells ; Humans ; Polymerase Chain Reaction ; Recombinant Proteins ; biosynthesis ; Sequence Analysis, DNA

Objective To investigate the repairing effect of ursolic acid extracted from Hippophae rhamnoi-des L.on sciatic nerve injury in rats.Methods After preparing a sciatic nerve damage model,thirty 2-month old Wistar rats were randomly divided into ursolic acid and control groups,with 15 rats in each group.Ursolic acid group was given Ursolic acid 150 mg/(kg·d)by gavage,while the control group was given the same dose of normal saline for 6 weeks.After 6 weeks,H&E staining was used to observe the pathological changes of rat sciatic nerves,and SFI and MNCV were calculated;The expression of NGF in sciatic nerve was detected by immunohistochemistry,and the expression of GAP-43 in L4-L6 spinal cord was detected by Western blotting.Results After 6 weeks,compared to the control group,the number of regenerated nerve fibers in the ursolic acid group increased,the arrangement was slightly regular,the capillary structure was abundant,and the proliferation of fibroblasts decreased;SFI and MNCV%were significantly increased(P<0.05),NGF protein positive expression was significantly increased(P<0.05),and the difference was statistically significant(P<0.05).GAP-43 protein expression in sciatic nerve tissue was significantly increased(P<0.05).Conclusions The intervention of ursolic acid extracted from Hippophae rhamnoides L.can improve the function of rats after sciatic nerve injury,promote the increase of regenerated nerve fibers and the repair of myelin sheath in rats with sciatic nerve injury,which may be related to increased NGF and GAP-43 protein expression in rats.

Objective:To understand the residents' cognition of commercial long-term insurance in Tianjin so as to provide a reference for improving service effect and quality of long-term insurance.Methods:A total of 31 residents from six districts of Tianjin were selected for semi-structured interviews from December 2022 to February 2023 by the convenient sampling method, and the data were analyzed by Colaizzi 7-step analysis method.Results:Three themes were extracted, namely, inadequate interpretation and promotion of commercial long-term insurance, immature development of commercial long-term insurance, and the ability of commercial long-term insurance to help caregivers reduce their family burden.Conclusions:The residents of Tianjin have a low awareness of commercial long-term insurance, which cannot meet the needs of most residents. It is urgent to strengthen their publicity further and improve their system.

Objective To identify the pathogenic bacteria such as Staphylococcus aureus and Enterococcus faecalis in bloodstream infec-tions with high confidence based on three deep learning models such as GoogleNet,ResNet101,and Vgg19,compare the performance and classification ability of these models,and explore the feasibility of applying the deep learning models for the rapid identification of pathogenic bacteria in bloodstream infections.Methods The preprocessed Gram-stained bacterial images,including 1 682 images for Staphylococcus aureus and 1 723 for Enterococcus faecalis,and 688 blank control microscopic images were input into three models for training and validation,respectively.Among them,1 344 images for Staphylococcus aureus,1 376 for Enterococcus faecalis,and 544 blank control images were used for training,and the remaining images were used for validation.The model with the best performance was identified according to the classification parameters between the models.Results The ResNet101 model had the lowest cross-en-tropy loss value(0.008 710 3),the largest Epoch value(93),and the highest accuracy rate(99％)for identifying the three types of validation set images.The cross-entropy loss value,Epoch value,and accuracy rate of the GoogleNet model were 0.063 89,86 and 98.6％,respectively,for identifying the three types of validation set images.Those of the Vgg19 model were 0.035 682,86 and 97.7％,respectively.Conclusion The ResNet101 model has the best performance in the classification of three kinds of images.The deep learning model may accurately,reliably and rapidly identify the Gram-stained images of pathogenic bacteria such as Staphylococcus aureus and Enterococcus faecalis in bloodstream infections.

Objective:To explore the effect and safety of mesenchymal stem cell exosome microneedle introduction in the treatment of melasma.Methods:Thirty cases of female patients with stable melasma in the Department of Dermatology, Changsha Meilai Medical Beauty Hospital, aged (36±5) years and with a disease duration of (42.4±20.7) months, from July 2021 to July 2022, were retrospectively included. According to Fitzpatrick skin typing, 23 cases of type Ⅲ and 7 cases of type Ⅳ were included. All patients were locally anesthetized with lidocaine cream for 30 min, and rolled with a 0.5 mm needle in a zigzag pattern with even force, in the order of the right cheek, the left cheek, the forehead, the nose, the mandible, and the upper lip. During the rolling process, 3 ml of MSC exosome medical liquid wound dressing was applied to the facial skin, and after it was fully absorbed, exosome was locally readministered in the area of melasma. Treatment ended with a slight redness at the site of application. 1 MSC exosome wound dressing was appllied as a cold compress for 15 min after treatment. Treatment was given once every 2 weeks for 6 consecutive sessions. All the patients were followed up at 4 and 12 weeks after the last session, and the area and severity index of melasma (MASI) were scored before and after the treatment, the clinical efficiency and patient satisfaction rate and the incidence of adverse reactions were also counted.Results:At 4 and 12 weeks after the end of treatment, the skin color of all 30 patients was brighter than that before treatment, and no recurrence of melasma symptoms seen. At 12 weeks after the end of treatment, the decrease rate of MASI score was 66.1%, among which the decrease rate of MASI score in patients with type Ⅲ melasma was 63.9%, and the decrease rate of MASI score in patients with type Ⅳ melasma was 63.9%. Among the 30 patients, 1 case was cured, 25 cases showed obvious improved, 4 cases were improved, and no cases were ineffective, with an effective rate of 86.7% (26/30). Five patients were very satisfied, 18 patients were satisfied, 6 patients were generally satisfied, and 1 patient was dissatisfied; the patient satisfaction rate was 76.7% (23/30). No serious adverse reactions occurred in all patients.Conclusions:MSC exosome microneedle introduction is safe and effective in the treatment of melasma without serious adverse reactions.