The construction of experimental animal models plays an important supporting role in research into the mechanisms of action of Chinese medicines.There have been increasing reports of the construction and evaluation of animal models of spleen deficiency;however,the construction method have involved different standards and there has been insufficient objectification of the evaluation indexes.In this review,we summarize the construction and evaluation method of animal models of spleen deficiency from the aspects of animal selection,model establishment,macroscopic characterization,behavioral experiments,and objective indexes of spleen deficiency,with a view to providing theoretical guidance for the construction of experimental animal models of spleen deficiency and references for the selection of animal model platforms for spleen deficiency.

Objective To establish and validate an individualized risk prediction nomogram model for the occurrence of gestational diabetes mellitus in pregnant women with hypothyroidism. Methods A total of 160 pregnant women with hypothyroidism were selected as the study subjects, including 85 patients with gestational diabetes mellitus (observation group) and 75 patients with normal blood glucose levels (control group). The age, gravidity, parity as well as pre-pregnancy body mass index, thyroid peroxidase antibody (TPOAb), homeostatic model assessment of insulin resistance (HOMA-IR), homeostatic model assessment of beta-cell function (HOMA-β), free thyroxine (FT4), free triiodothyronine (FT3) and thyroid stimulating hormone (TSH) were compared between the two groups. Multivariable analysis was used to analyze the risk factors for gestational diabetes mellitusin pregnant women with hypothyroidism. The nomogram model was used to predict the risk of gestational diabetes mellitus in pregnant women with hypothyroidism. The receiver operating characteristic (ROC) curve was used to evaluate the predictive value of the nomogram model for gestational diabetes mellitus in pregnant women with hypothyroidism. Results The age, gravidity, parity as well as pre-pregnancy body mass index, TPOAb, HOMA-IR and HOMA-β were higher in the observation group than in the control group, while pre-pregnancy FT4, FT3 and TSH were lower (P < 0.05). Multivariable analysis showed that older age, gravidity, parity as well as pre-pregnancy body mass index, TPOAb, HOMA-IR and HOMA-β, and lower pre-pregnancy FT4, FT3 and TSH were risk factors for gestational diabetes mellitusin pregnant women with hypothyroidism. The nomogram model prediction showed that older age (28 to 31 years), gravidity (more than 2 times), parity (more than 1 time) as well as pre-pregnancy body mass index (above 24.00 kg/m²), TPOAb (> 421.33 IU/mL), HOMA-IR (> 2.21) and HOMA-β (> 115.66), and lower pre-pregnancy FT4 (< 0.33 ng/dL), FT3 (< 1.65 pg/mL) and TSH (< 0.26 mIU/mL) were risk factors for gestational diabetes mellitus in pregnant women with hypothyroidism. ROC curve analysis showed that the area under the curve of the nomogram model for predicting gestational diabetes mellitus in pregnant women with hypothyroidism was 0.785, indicating good predictive performance. Conclusion The risk prediction nomogram model has high value in individualized prediction of gestational diabetes mellitus in pregnant women with hypothyroidism.

【Objective】 To explore and evaluate infection control measures of preventing cross-contamination of novel coronavirus during gastrointestinal endoscopy treatment. 【Methods】 According to the hospital’s infection control requirements and related documents, infection control measures were formulated and implemented by combining with our actual clinical situation, including the management of the endoscope room, management and protection of patients and endoscopists. Then, we evaluated the effect of these measures. 【Results】 From January 25 to March 10, 2020, a total of 71 patients (53 males and 18 females) completed gastrointestinal endoscopy treatment, with an average age of 54 years (28-81 years). There were 36 (50.7%) cases of emergency treatment. All patients had been kept in quarantine for about 14 days (24±13), and no cross-contamination of novel coronavirus occurred. 【Conclusion】 During the novel coronavirus infection epidemic period, reasonable and effective measures should be taken to minimize the risk of infection in doctors and patients. The endoscope center should strengthen preoperative screening and management of patients, master indications of endoscopic procedures, complete endoscopists’ management and protection work, strictly follow the specifications of sterilizing gastrointestinal endoscopes, and construct the layout of "three zones and two passages".

OBJECTIVETo investigate the effects of microRNA(miRNA)-29b on the proliferation and migration of breast cancer cells and its molecular mechanism.

METHODSThe recombinant lentiviral expression vector (lenti-miRNA-29b) was constructed and transfected into 293T cells to obtain lentivirus particles that were used to infect breast cancer MCF-7 cells. Transfection efficiency of lenti-miRNA-29b in MCF-7 cells was identified by the expression of green fluorescent protein (GFP). The expression of miRNA-29b was detected by real-time PCR. The cell proliferation and migration were detected by CCK8 assay and Transwell assay, respectively. The bioinformatics softwares were used to predict and screen the downstream target genes regulated by miRNA-29b, which were verified by double luciferase reporter gene assay, RT-PCR and Western blot. The effects of screened target gene RTKN on the growth and migration of MCF-7 cells were verified by RTKN siRNA.

RESULTSRecombinant lentiviral expression vector of miRNA-29b were successfully constructed. About 90% and 60% of the breast cancer cells showed green fluorescence in lenti-miRNA-29b and lenti-miRNA-NC groups, respectively. The expression of miRNA-29b in lenti-miRNA-29b group increased significantly compared with the lenti-miRNA-NC group and blank control group (all<0.05); the proliferation and migration ability of MCF-7 cells significantly reduced compared with the control group (all<0.05). The screening with bioinformatics softwares found that the 3'UTR coding region RTKN had the binding site to miRNA-29b; the dual luciferase reporter gene assay showed that the luciferase activity decreased significantly after the MCF-7 cells were co-transfected with wild type RTKN-WT-3'UTR and miRNA-29b mimics report gene vector (<0.05). The RTKN proteins in MCF-7 cells were significantly decreased after transfection with siRNA-RTKN, and the proliferation and migration ability of MCF-7 cells were significantly reduced (all<0.05).

CONCLUSIONSMiRNA-29b can inhibit the proliferation, invasion and metastasis of breast cancer cells by inhibiting the expression of RTKN.

The development of genome editing techniques based on CRISPR (Clustered regularly interspaced short palindromic repeats)-Cas9 system has revolutionized biomedical researches. It can be utilized to edit genome sequence in almost any organisms including Caenorhabditis elegans, one of the most convenient and classic genetic model animals. The application of CRISPR-Cas9 mediated genome editing in C. elegans promotes the functional analysis of gene and proteins under many physiological conditions. In this mini-review, we summarized the development of CRISPR-Cas9-based genome editing in C. elegans.

Objective: To explore the method of extracting chaperone antigen peptide complexes from gastric cancer stem cells and its immune function. Methods: Gastric cancer stem cells and gastric cancer cells were screened by low temperature ultrasonic lysis. After salting out and dialysis, the lysate supernatant was processed with SDS-PAGE to analyze the expression of chaperone antigen peptide complexes, and then was separated and purified with CNBr-activated SepharoseTM 4B. Reverse high pressure liquid chromatography (HPLC), SDS-PAGE and Western blotting were used to analyze the purity and nature of the acquired albumen. Lymphocyte proliferation assay and lymphocytotoxicity assay were used to ditermine the immunological activity of the chaperone-antigen peptide complexes. Results: The chaperone antigen peptide complexes of gastric cancer stem cells were prepared and identified successfully, of which the main components were the antigen peptides of HSP60, HSP70, HSP90 and HSP110. 0.75 μg and 1.00 μg HSP70-antigen peptide and 1.00 μg HSP90-antigen peptide activated lymphocytes significantly. Their A₄₉₀ values were 0.26±0.03, 0.45±0.05 and 0.32±0.04, respectively, while the corresponding doses of HSP60-antigen peptide and HSP110-antigen peptide did not activate lymphocytes. The killing rates of 1.00 μg HSP70-antigen peptide and 1.00 μg HSP70 were (45.0±2.0)% and (16.0±2.0)%, respectively, showing a significant difference (P=0.012). Similarly, the killing rates of 1.00 μg HSP90-antigen peptide and 1.00 μg HSP90 were (36.0±5.0)% and (13.0±4.0)%, respectively, also showing a significant difference (P=0.048). Conclusions The amount of chaperone antigen peptide complexes in gastric cancer cells is extremely low, but it is obviously increased in gastric cancer stem cells. After purification, the chaperone antigen peptide complexes with high purity can be prepared. The extracted chaperone antigen peptide complexes have stronger immunogenicity, and can be used to make tumor vaccine in vitro, which may have a good application value in the targeted therapy of gastric cancer.

Objective: The present study was designed to evaluate the protective effects of oligomeric proanthocyanidins (OPC) in mice exposed to paraquat (PQ) , and to explore the molecular mechanism. Methods: Four experimental groups were designed. Control group: 10 BALB/c mice were intraperitoneally injected with normal saline) . PQ group: 10 BALB/c mice were intraperitoneally injected with PQ (100 mg/kg) . PQ+OPC group: 10 BALB/c mice were administered with OPC (100 mg/kg) for 1 h before PQ (100 mg/kg) expo-sure. OPC group: 10 BALB/c mice were intraperitoneally injected with OPC (100 mg/kg) . The peripheral blood samples or lung tissue samples were collected at the designed time points for measuring the levels of oxi-dative stress indicators, the related protein levels of nuclear factor-kappa B (NF-κB) pathway and nuclear fac-tor erythroid related factor-2 (Nrf2) pathway. Results: Compared with the control group, the level of reactive oxygen species (ROS) , the content of malondialdehyde (MDA) in the PQ group were significantly induced, and the activity of superoxide dismutase (SOD) in the PQ group was decreased in the peripheral blood. As com-pared with the PQ group, the level of ROS and the content of MDA in the PQ+OPC group were significantly re-duced, the activity SOD in the PQ+OPC group was increased in the peripheral blood; the level of ROS and the content of MDA were also reduced in lung tissues in the PQ+OPC group. Moreover, compared with the con-trol group, the phosphorylation of IκBα and the expression of NF-κB p65 were increased in lung tissues in the PQ group. The phosphorylation of IκBα and the expression of NF-κB p65 were decreased in lung tissues in the PQ+OPC group as compared with the PQ group. In addition, compared with the control group, the expressions of HO-1 and Nrf2 were increased in lung tissues in OPC group, and these were decreased in lung tissues in PQ groups. Furthermore, the expressions of HO-1 and Nrf2 were also increased in lung tissues in PQ+OPC as com-pared with the PQ group. Conclusion OPC could alleviate PQ-induced systemic toxicity in mice by regulating oxidative stress via NF-κB and Nrf2 pathway.

BACKGROUND:Erythropoietin and progenitor cel transplantation both have therapeutic effects on lower extremity arterial occlusive disease.
OBJECTIVE:To investigate the erythropoietin modification effect and magnetic resonance imaging feasibility of superparamagnetic iron oxide (SPIO)-labeled endothelial progenitor cel s in vitro.
METHODS:Rat bone marrow-derived endothelial progenitor cel s at logarithmic growth phase were randomized into four groups:endothelial progenitor cel group, SPIO labeled transfection group (pcDNA3-EPO transfection fol owed by SPIO labeling), SPIO labeled empty vector group (empty plasmid transfection fol owed by SPIO labeling), and SPIO labeling group (only SPIO labeling). 4.7T MRI was used to observe SPIO-labeled endothelial progenitor cel s. Cel proliferation, cel cycle distribution, and expression of erythropoietin protein in the four groups were measured.
RESULTS AND CONCLUSION:MRI findings showed with the increasing cel number, gradual y lowered signal intensity on T1-weighted imaging (T1WI), T2WI and T2*WI was seen, and the reduction in the signal intensity was the maximum on T2*WI sequence and the minimum on T1WI sequence. For T1WI, T2WI and T2*WI sequences, the minimum number of cel s was 2×104, 1×104 and 0.5×104, respectively. Cel proliferation and cel cycle distribution showed no significant difference among three SPIO labeling groups. In addition, the expression of erythropoietin protein was only found in the SPIO-labeled transfection group. These findings showed that under SPIO labeling, erythropoietin gene-modified endothelial progenitor cel s show no changes in cel proliferation and cel cycle, and the 4.7T MR is capable of imaging SPIO-labeled erythropoietin gene-modified endothelial progenitor cel s in vitro.

[Abstract ] Objective Difficulties with the preparation of gastric cancer stem cells (CSCs) have been a main obstacle to the studies of gastric cancer .This article addresses the technology of the serum-free medium suspension cultivation of the MKN-45 gastric cancer cell line and screening of stem cells from the cell line based on the biomarkers of gastric CSCs . Methods MKN-45 cells were cultured in serum-free medium for 8 weeks and those in the logarithmic phase cultivated with hoechst 33342 followed by detection of the side cells by flow cytometry .When the side population cells reached 25%, all the cell microspheres were collected , hatched with CD133 and CD44, and subjected to fluorescence-activated cell sorting.The CDl33 +and CD44 +cells were selected as gastric CSCs . Results About 40%of the MKN-45 gastric CSCs were alive , prolif-erated, and formed floating cell balls .Side population cells constitu-ted 3.4% of the MKN-45 cells and 26.9% of the cell balls.The CDl33 +and CD44 +cells made up 11.2% of the MKN-45 cells and 90.3%of the cell balls. Conclusion Cell balls rich in CSCs can be successfully obtained by serum-free medium suspension culti-vation and CSCs can be screened out with hoechst 33342 and surface markers , which may serve as an experimental ground for the stud-ies of gastric CSCs .

Objective To explore the effects of rapid response first-aid case on bedside emergency gastroscopy hemostatic treatment. Methods A total of 52 patients with esophageal-fundal varices in liver cirrhosis from January 2014 to June 2015 were divided into control group (n=25) and experimental group (n=27). The patients of control group received the routine nursing; then we summarized the experience and mistakes of operation cooperation, optimized process;next we designed rapid response first-aid case and used in the experimental group. The blood transfusion time, goods preparation time, blood loss, blood transfusion volume were compared in two groups. Results The data of the experimental group in average bleeding to transfusion time (90. 22 ± 36. 47)min, materials preparation time (14. 66 ± 3. 48) min, the average amount of bleeding (369. 62 ± 158. 90) ml, average blood transfusion amount (322. 40 ± 117. 82) ml were lower than those of the control group [ average bleeding to blood transfusion time ( 123. 60 ± 51. 87 ) min, materials preparation time ( 22. 44 ± 2. 59 ) min, average bleeding volume ( 660. 20 ± 181. 82 ) ml, average blood transfusionamount(458.00±140.63)ml](P<0.05).Conclusions Theimplementationofrapidresponse first-aid case can effectively shorten the rescue time, reduce bleeding and blood transfusion time, the blood loss, the wastage of human resources and medical resources, improve the efficiency of nursing work in the department and the success rate of rescue.