Objective To establish a method of isolation and purification of rat skeletal muscle satellite cells,and observe the characteristics of proliferation and myotube cell formation of skeletal muscle satellite cells cultured in vitro. MethodsPurified skeletal muscle satellite cells were obtained by improved two-step enzymatic digestion method and pre-plating technique.Immunohistochemical staining was employed to identify the skeletal muscle satellite cells cultured in vitro.The growth of skeletal muscle satellite cells cultured in vitro was examined by MTT assay.The differentiation of skeletal muscle satellite cells was observed by inverted microscopy. Results The skeletal muscle satellite cells with higher purity were obtained and confirmed by the high expression of desmin.When cultured in vitro,the latent phase of skeletal muscle satellite cells was the first to the second day,and the platform phase was the fifth to the sixth day.Myotube cells gradually formed when cell confluence was more than 60% to 70% or differential medium with lower fetal bovine serum was used.Conclusion The combination of improved two-step enzymatic digestion method and pre-plating technique serve as an easy and practical way to obtain skeletal muscle satellite cells with higher purity.Skeletal muscle satellite cells can form myotube cells with contraction characteristics without any special induction.

AIM: To examine the expression of human endostatin in E.coli , produce its fusion protein antibody and observe its biological activity. METHODS: Endostatin gene was amplified by polymerase chain reaction,recombined with plasmid vector pGEX-2T and induced expression with IPTG.The protein activity was tested by endothelial cell proliferation inhibitory assay.Inclusion body crudely purified was used to generate polyclonal antibody to detect its expression at mouse's liver and kidney etc. RESULTS: The protein expressed was 20 kD after digestion by thrombin,it appeared the anti-angiogenesis activity and Western blotting indicated the expression of endostatin in liver and kidney of mouse. CONCLUSION: The successful expression of human endostatin and the preparation of polycolonal antibody indicated its potential application in anti-angiogenesis therapy and diagnosis tumors.

0.05)and the TNM staging (P=0.55).A mild elevated compared other pathological classification was found in small cell lung cancer (0.191?0.275).Conclusions The results showed that RFQ-PCR was suitable for measurement of the mRNA level of PLKI in bronchoscopic bioptic specimens.This study suggest elevated expression of PLK1 might play a important role in development of lung cancer,so that PLK1 might be a potential tumor marker for Lung cancers.Advanced studies will be needed to clarify that PLKI mRNA level do not relate to TNM staging and pathological classification.

Aurantii Fructus is the dried and immature fruit of Citrus aurantium and its cultivars. To investigate the chemical constituents of Aurantii Fructus, the separation and purification of constituents were performed by column chromatography on silica gel LH-20, HW-40, ODS, PHPLC and PTLC. Fourteen flavonoids, including four flavone glycosides and ten polymethoxyflavones (PMFs) were isolated from the EtOAc fraction and Petroleum ether fraction of Aurantii Fructus and their structures were identified by physicochemical properties and spectral data (NMR and MS) as (2R) -and (2S)-6"-O-acetylprunin (1,2), naringenin-7-O-β-D-glucopyranside (3), 5,7,4'-trihydroxy-8,3'-dimethoxyflavone-3-O-6"-(3-hydroxyl-3-methylglutaroyl)-β-D-glucopyranoside(4), 4'-hydroxy-5,6, 7-trimethoxyflavone (5), natsudaidain (6), nobiletin (7), sinensetin (8), 5,6,7,4'-tetramethoxyflavone (9), 5,7,8,4'-tetramethoxyflavone (10), 3,5,6,7,8,3',4'-heptamethoxyflavone (11), tangeretin (12), 5-demethyl nobiletin (13), and 5-hydroxy-6,7,3', 4'-tetramethoxyflavone (14). Compound 3-5 s were isolated from this plant for the first time and compound 1 was a new one.
Citrus ; chemistry ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Flavonoids ; chemistry ; isolation & purification ; Fruit ; chemistry ; Mass Spectrometry ; Molecular Structure

(3S)-Linalool synthase (LIS) is a key enzyme involved in the monoterpene biosynthetic pathway. Based on our previous transcriptome study, the expression level of LIS gene was exceedingly related to glycyrrhizic acid (GA) biosynthesis. Therefore, we used hairy root culturing to further investigate the effect of LIS on the GA biosynthesis. A LIS gene (GenBank accession number: MZ169552) was cloned from Glycyrrhiza uralensis. The plant binary overexpression vector pCA-LIS was constructed by gene fusion. G. uralensis hairy roots overexpressing LIS were induced by the Agrobacterium rhizogenes ATCC15834. The expression levels of LIS were analyzed by real-time quantitative PCR (RT-qPCR) and the contents of GA in hairy root lines were determined by UPLC. It was found that in the hairy root lines overexpressing LIS, the expression levels of LIS were significantly higher than that in the wild type, while the contents of GA were remarkably lower than those in the wild type and negative control. These findings indicate that the expression level of LIS is negatively correlated with the accumulation of GA. In this study, LIS was cloned from G. uralensis for the first time and the negative regulatory effect of LIS on GA biosynthesis was confirmed by reverse genetics. This work provides support for further improvement of the molecular regulatory network of GA biosynthesis in G. uralensis.

Objective: To investigate the causal relationship between gut microbiota and polycystic ovary syndrome (PCOS) using a Mendelian randomization (MR) study, so as to provide insights into the pathogenesis of PCOS and the formulation of prevention and treatment strategies. Methods: The genetic data on gut microbiota was derived from a meta-analysis of genome-wide association studies (GWAS) involving 18 340 participants. The genetic data on PCOS was sourced from two GWAS meta-analyses in European populations, serving as the discovery set and the validation set, respectively. A two-sample MR analysis was conducted using the discovery set, with the inverse variance weighted (IVW) method as the primary approach. Sensitivity analyses employed the weighted median method, MR-Egger regression, and the MR-PRESSO test. The validation set was utilized for verification, and a meta-analysis was performed to combine the results from the two datasets. Results: Forward MR analysis results showed that nine types of gut microbiota were statistically associated with PCOS (all P<0.05). Specifically, the association of family Streptococcaceae (OR=1.442, 95%CI: 1.097-1.895), genus Actinomyces (OR=1.359, 95%CI: 1.036-1.784), genus Ruminococcaceae UCG 011 (OR=0.755, 95%CI: 0.619-0.921), genus Sellimonas (OR=0.766, 95%CI: 0.657-0.893) and genus Streptococcus with PCOS (OR=1.496, 95%CI: 1.136-1.972) remained consistent in the sensitivity analysis. Reverse MR analysis showed no evidence for the causal association between PCOS and the aforementioned five types of gut microbiota (all P>0.05). The MR analysis results of the validation set showed that there was no statistical association between the aforementioned five types of gut microbiota and PCOS (all P>0.05). However, the associations remained significant for genus Actinomyces (OR=1.226,95%CI：1.010-1.503) and genus Streptococcus (OR=1.266,95%CI：1.042-1.452) in the meta-analysis (both P<0.05). Conclusion This study provides the evidence that genus Actinomyces and genus Streptococcus are causally associated with PCOS.

OBJECTIVETo observe effects of Ligustrazine on serum S100p protein and neuron-specific enolase (NSE) in elderly patients undergoing orthopedics operations.

METHODSTotally 60 patients undergoing selective total hip replacement, 65-80 years old, who were in line with American Society of Anesthesiologists (ASA) grade I or II, were randomly assigned to the Ligustrazine group (Group L) and the normal saline control group (Group S). The right internal jugular vein catheters were placedcephalad and ensured theirs tips in jugular venous bulbs after anesthesia induction and tracheal intubation. Patients in Group L received 2 mg/kg Ligustrazine Injection (40 drops within one minute) and those inGroup S received equal volume of normal saline via central veins before operations. Other medicines were the same for all patients during and after operation. Five millimeter blood sample was collected frominternal jugular venous bulbs before operation (T0), 24 h (T1), 72 h (T2), 168 h (7th day, T3) after operation. Serum was collected after centrifuge. S100β protein and NSE concentration were analyzed usingELISA. Mini-mental state examinations (MMSE) were scored by the same doctor at T0, T1, T2, and T3,respectively.

RESULTSThere was no statistical difference in MMSE scores, serum S1000 protein, or NSE at TO (P > 0.05). Compared with TO, S100 P protein and NSE concentration increased and MMSE scores decreased at T1, T2, and T3 in the two groups. All indices except S100P protein and NSE at T3 were statistically different between Group L and Group S (P < 0.05).

CONCLUSIONSerum S100P protein and NSE could be changed by pre-operation injecting Ligustrazine at certain dose in elderly patients undergoing orthopedics operations.

Aged ; Aged, 80 and over ; Arthroplasty, Replacement, Hip ; Humans ; Phosphopyruvate Hydratase ; blood ; Pyrazines ; therapeutic use ; S100 Calcium Binding Protein beta Subunit ; blood

The direct acting substances of Cuscuta chinensis in vivo were preliminarily identified through the correlation analysis of "metabolites-effect identification" model. The ovariectomized female rats were i.g administered with 95% ethanol extract part, 40% ethanol elution part and n-butanol extract part of Cuscuta chinensis. The serum fingerprints of different parts and times of administration were established by UPLC/Q-TOF-MS. At the same time, serum estradiol (E₂), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels were detected. Bivariate correlation analysis and grey correlation analysis were used to screen estrogenic components. The results showed that nine direct acting substances in vivo highly related to estrogen effect were found in the drug containing serum, which were hyperoside, astragalin, methyl quercetin glucuronide, quercetin-diglucuronide, quercetin, apigenin, isoquercitrin, kaempferol glucuronide and kaempferol. We can preliminarily screen out the direct acting substance of estrogen effect of Cuscuta chinensis in vivo based on the research idea of serum spectrum effect correlation. It provides a reliable basis for revealing the estrogeneffective substances of Cuscuta chinensis and confirming the quality markers. This experiment was approved by Harbin University of Commerce Ethics Committees (Approval No. HSDU2020-065).

Objective To etiologically diagnose and analyze a patient with suspected cases of brucellosis,and to provide a experimental basis for the confirmation of the first case of human brucellosis in Guizhou province.Methods Conventional and molecular techniques [genus specific Brucella surface protein 31 PCR (BCSP31-PCR)and Brucella suis species-specific PCR (AMOS-PCR)] were used to identify suspicious bacteria strains isolated from the suspected patient of brucellosis.Results The results showed that the Brucella suspicious colonies were identified as Brucella melitensis biotype 3 using conventional tests and were further identified as Brucella spp.by genus specific Brucella surface protein 31 PCR (BCSP31-PCR) and classified as Brucella melitensis with Brucella abortus,Brucella melitensis,Brucella ovis,Brucella suis species-specific PCR(AMOS-PCR).Conclusions Laboratory diagnostic results show that the bacteria strain isolated from the suspected patient of brucellosis is Brucella melitensis biotype 3.It is the first case of human brucellosis in Guizhou province.

Objective To study the bone biomechanics of the rabbit osteoporosis models induced by dexamethasone sodium phosphate injection (DX) using a combined treatment modality of 99Tc-MDP and GuKangLing.Methods Rabbits were intramuscularly injected with DX (2 mg/kg) twice a week for 6 weeks.The animal osteoporosis model group (Group C) and normal group (Group A) were compared to confirm the model was available.Another control group (Group B),the osteoporosis control group (Group D) were set for the comparison at the end of the experiment.The 99Tc-MDP therapy group (Group E),GuKangLing therapy group (Group F) and 99Tc-MDP plus GuKangLing therapy group (Group G) were included in the study.The treatment lasted for 16 weeks.The bone biomechanics,cytopathology bone histomorphology,bone mineral density (BMD),X-ray,CT,bone scintigraphy and serum bone alkaline phosphatase (BALP)and P (bone gla protein) were chosen as the markers or methods to evaluate the treatment results (excellent,effective and invalid).The analysis of variance (ANOVA) and t-test were used for group comparison analysis.Results Cytopathology result indicated that there was no bone trabecula destruction in Group A.However,there was distinct bone destruction in Group C.The bone biomechanics (left femur head (265.914 ±52.773) N,L4(369.671 ±94.919) N),BMD(left femur (0.238 ±0.016) g/cm2,L4(0.236 ±0.016) g/cm2)and bone histomorphology ( (66.230 ± 10.848) ％ ) in Group C reduced clearly as compared with Group A ((405.343±55.410) N,(750.870±53.718) N,(0.294±0.017) g/cm2,(0.302±0.023) g/cm2,( 131.500 ± 21.846) ％ ) ( t ≥4.550,all P ＜ 0.01 ).Radionuclide bone scan also showed that the uptake of tracers was higher by the main arthrosis in Group C than that in Group A.Vertebra was not clearly visualized on bone scan image.There were significant differences between Group A and Group C in serum BALP and P ((45.000±7.303) vs (12.485 ±1.512) U/L,(0.168±0.018) vs (0.115 ±0.017) μg/L,t =4.126,5.476,both P ＜ 0.01 ),which indicated that the animal osteoporosis model was available.The pathological results showed an improved recovery of bone structure and trabecular in Groups E and G,but a worse recovery in Group F.Biomechanics result in Groups E and G (left femur head (386.457 ±77.077) N and (432.771 ± 17.525) N,L4(649.550 ± 126.859) N and (655.443 ±76.555) N) improved apparently,which were similar to Group B.The radiotracer uptake in Group F was lower than that in group D.The bone biomechanics,bone histomorphology,BMD,serum BALP and P after the treatment showed significant differences in Groups E,F and G (F:8.556 - 31.608,all P＜0.01 ),and the bone biomechanics result in Group G was a little better than that in Group E (t =2.625,P ＜ 0.05 ).The results of Group G and E were considered as excellent,and Group F was considered as effective.Conclusions The treatment of 99Tc-MDP combined with GuKangLing could improve the bone biomechanics of rabbit osteoporosis models and may be a potential method to increase the bone strength for resisting external force.