Objective To screen the hemostatic active extracts from Callicarpa nudif lora .Methods The powdered Callicarpanudiflora was extracted with 70% EtOH and concentrated to give EtOH-extract .The EtOH-extract was further chromatographed over HP-20 macroporous resin column ,eluting with aqua and 95% EtOH to get HP-H2 O-elution and HP-EtOH-elution ,respectively .The obtained three extracts of EtOH-extrract ,HP-EtOH-elution and HP-H2 O-elution were set as large ,middle and small dosage groups for drug preparation ,respectively .Yunnan Baiyao was used as a positive control group . The weight increment ,bleeding time and clotting time of fed mice were detected by cutting tail and grass slide methods after in-tragastric administration for 7 days .Results As compared with blank model and positive control ,each dosage groups of HP-EtOH-elution could significantly shortened the bleeding time ,of which the small dosage group and middle dosage group even ex-hibited better results than the positive control group .Whereas the EtOH-extract ,HP-EtOH-elution and HP-H2 O-elution didn′t demonstrated significant effect on clotting time as well as the weight increment .Conclusion The HP-EtOH-elution was suggested to be the major active extract possessing hemostatic activity to mice under tested dosages .The hemostatic mechanism may through in-trinsic coagulation pathway .This study would be helpful for further phytochemical investigation of C .nudi f lora .

Objective:To investigate the effects of oleanolic acid on the growth and migration of keloid fibroblasts.Methods:Keloid tissue samples from 9 patients in the Department of Plastic Surgery of General Hospital of Northern Theater were collected and fibroblasts were cultured in vitro. Fibroblasts were treated with different concentrations of oleanolic acid and divided into three groups: control group added 0.9% NaCl; 5 μmol/L oleanolic acid group added 5 μmol/L oleanolic acid; 10 μmol/L oleanolic acid group added 10 μmol/L oleanolic acid. MTT assay was used to detect cell proliferation; flow cytometry was used to detect cell cycle. Annexin V propidium iodide (AV-PI) staining was used to detect cell apoptosis. Transwell assay was used to detect the migration of oleanolic acid. Western blotting and real-time PCR were used to detect the expression of related proteins and mRNA activity. Each group was made in triplicate. Analysis of variance was used to compare the data among the three groups. LSD- t test was used for pairwise comparison, and P<0.05 was considered to be statistically significant. Results:MTT result showed that oleanolic acid could inhibit the proliferation of cells. After 24 hours, the proliferation of cells in 5 μmol/L oleanolic acid group and 10 μmol/L oleanolic acid group were 0.660±0.020 and 0.460±0.020, respectively, compared with 0.780±0.001 in the control group, F=114.4, P<0.001. Compared with the control group, the difference was statistically significant ( t=5.94, P<0.001, t=15.60, P<0.001); flow cytometry showed that the cell cycle G1/S phase transduction was blocked, 5 μmol/L oleanolic acid group and 10 μmol/L oleanolic acid group were significantly inhibited. The percentage of G1 phase cells in the 5 μmol/L oleanolic acid group was significantly higher than that in the control group ( t=3.14, P=0.030, t=6.38, P< 0.001). AⅤ-PI staining showed that the number of apoptotic cells in the 5 μmol/L oleanolic acid group (0.9%) and 10 μmol/L oleanolic acid group (3.4%) was significantly higher than that in the control group (0.4%), and the difference among the three groups was F=119.6, P<0.001. Transwell assay showed that the migration number of cells in 5 μmol/L oleanolic acid group (57.13 ± 2.65) and 10 μmol/L oleanolic acid group (42.15 ± 2.55) was significantly lower than that in control group (72.27± 3.32), F=101.3, P<0.001. Compared with the control group, the difference was statistically significant ( t=6.50, P<0.001, t=14.41, P<0.001). Western blotting showed that oleanolic acid could inhibit the expression of Cyclin D1, Bcl-2, Bax and MMP2. Compared with the control group, 5 μmol/L oleanolic acid t=8.70, P<0.001, t=5.00, P=0.040, t=12.41, P<0.001, t=10.46, P<0.001; compared with the control group, 10 μmol/L oleanolic acid t=31.61, P<0.001, t=23.17, P<0.001, t=12.11, P<0.001, t=44.52, P<0.001. Real-time PCR reaction showed that the mRNA activity levels of Cyclin D1, Bcl-2, Bax, MMP2 were also inhibited. Compared with the control group, 5 μmol/L oleanolic acid t=5.42, P< 0.001, t=3.11, P=0.040, t=16.11, P<0.001, t=11.71, P<0.001; compared with the control group, 10 μmol/L oleanolic acid t=51.78, P<0.001, t=30.89, P<0.001, t=10.64, P<0.001, t=17.10, P< 0.001. Conclusions:Oleanolic acid (5 μmol/L and 10 μmol/L) can inhibit the proliferation and migration of keloid fibroblasts and induce apoptosis of keloid fibroblasts after treating keloid fibroblasts for 24 hours, which can inhibit the growth of keloid and be used for the prevention and treatment of keloid.

Objective:To investigate the effects and mechanism of eleutheroside E on the growth of human hypertrophic scar fibroblasts (Fbs).Methods:The experimental research method was used. The hypertrophic scar tissue was collected from 6 patients with hypertrophic scar (1 male and 5 females, aged 20 to 51 (37±8) years) admitted to General Hospital of Northern Theater Command, from October 2018 to March 2019. The third to seventh passages of human hypertrophic scar Fbs were cultured for later experiments. Cells were divided into normal saline group, 100 μmol/L eleutheroside E group, 200 μmol/L eleutheroside E group, and 400 μmol/L eleutheroside E group, and normal saline, eleutheroside E at the final molarity of 100, 200, and 400 μmol/L were added to cells in the corresponding groups. Cells were collected and divided into small interfering RNA (siRNA)-negative control alone group, siRNA-thrombospondin 1 (THBS1) alone group, siRNA-negative control +400 μmol/L eleutheroside E group, and siRNA-THBS1 +400 μmol/L eleutheroside E group. Cells in siRNA-negative control alone group and siRNA-negative control +400 μmol/L eleutheroside E group were transfected with siRNA-negative control, cells in siRNA-THBS1 alone group and siRNA-THBS1 +400 μmol/L eleutheroside E group were transfected with siRNA-THBS1. At 24 h after transfection, cells in siRNA-negative control alone group and siRNA-THBS1 alone group were added with normal saline, and cells in siRNA-negative control +400 μmol/L eleutheroside E group and siRNA-THBS1 +400 μmol/L eleutheroside E group were added with eleutheroside E at the final molarity of 400 μmol/L. At 0 (immediately), 12, 24, 36, and 48 h after treatment, the cell proliferation activity (expressed as absorbance value) was detected by thiazolyl blue assay. Cells were divided into normal saline group, 200 μmol/L eleutheroside E group, 400 μmol/L eleutheroside E group, siRNA-negative control alone group, siRNA-THBS1 alone group, siRNA-negative control +400 μmol/L eleutheroside E group, and siRNA-THBS1 +400 μmol/L eleutheroside E group. The corresponding treatments in each group were the same as before. At 24 h after treatment, the apoptosis was observed by Hoechst 33258 staining. Cells were collected and divided into normal saline group, 100 μmol/L eleutheroside E group, 200 μmol/L eleutheroside E group, 400 μmol/L eleutheroside E group, siRNA-negative control alone group, siRNA-THBS1 alone group, siRNA-negative control +400 μmol/L eleutheroside E group, and siRNA-THBS1 +400 μmol/L eleutheroside E group. The corresponding treatments in each group were the same as before. At 24 h after treatment, the THBS1 protein level of cells was detected by Western blotting. The number of sample in each group was all 3 at each time point. Data were statistically analyzed with analysis of variance for factorial design, one-way analysis of variance, independent sample t test, and Bonferroni correction. Results:At 0 h after treatment, the absorbance values of cells in normal saline group, 100 μmol/L eleutheroside E group, 200 μmol/L eleutheroside E group, and 400 μmol/L eleutheroside E group were similar ( P>0.05). At 12, 24, 36, and 48 h after treatment, the absorbance values of cells in 100 μmol/L eleutheroside E group, 200 μmol/L eleutheroside E group, and 400 μmol/L eleutheroside E group were significantly lower than those of normal saline group ( t=7.64, 28.94, 13.69, 5.87, 6.96, 22.83, 14.75, 11.52, 21.09, 20.15, 29.52, 23.12, P<0.05 or P<0.01). At 0 h after treatment, the absorbance values of cells in siRNA-negative control alone group, siRNA-THBS1 alone group, siRNA-negative control +400 μmol/L eleutheroside E group, and siRNA-THBS1 +400 μmol/L eleutheroside E group were similar ( P>0.05). At 12, 24, 36, and 48 h after treatment, the absorbance values of cells in siRNA-THBS1 alone group and siRNA-negative control +400 μmol/L eleutheroside E group were significantly lower than those in siRNA-negative control alone group ( t=7.14, 44.87, 20.67, 40.98, 9.26, 11.08, 15.33, 20.56, P<0.05 or P<0.01); the absorbance values of cells in siRNA-THBS1 alone group, siRNA-negative control +400 μmol/L eleutheroside E group, and siRNA-THBS1 +400 μmol/L eleutheroside E group were similar ( P>0.05). Compared with that in normal saline group, the numbers of apoptotic cells in 200 μmol/L eleutheroside E group and 400 μmol/L eleutheroside E group were increased at 24 h after treatment. At 24 h after treatment, compared with that in siRNA-negative control alone group, the numbers of apoptotic cells in siRNA-THBS1 alone group and siRNA-negative control +400 μmol/L eleutheroside E group were increased, while the numbers of apoptotic cells in siRNA-THBS1 alone group, siRNA-negative control +400 μmol/L eleutheroside E group, and siRNA-THBS1 +400 μmol/L eleutheroside E group were similar. At 24 h after treatment, the protein levels of THBS1 of cells in 100 μmol/L eleutheroside E group, 200 μmol/L eleutheroside E group, and 400 μmol/L eleutheroside E group (0.87±0.12, 0.38±0.07, 0.20±0.09) were significantly lower than 1.83±0.17 in normal saline group ( t=16.61, 16.17, 17.29, P<0.01). At 24 h after treatment, the protein levels of THBS1 of cells in siRNA-THBS1 alone group and siRNA-negative control +400 μmol/L eleutheroside E group (0.61±0.07, 0.58±0.07) were significantly lower than 1.86±0.07 in siRNA-negative control alone group ( t=71.06, 83.80, P<0.01), and the protein levels of THBS1 of cells siRNA-THBS1 alone group, siRNA-negative control +400 μmol/L eleutheroside E group, and siRNA-THBS1 +400 μmol/L eleutheroside E group (0.63±0.11) were similar ( P>0.05). Conclusions:Eleutheroside E can inhibit the growth of human hypertrophic scar Fbs by down-regulating the expression of THBS1.

Objective:To investigate the effects of oleanolic acid on the growth and migration of keloid fibroblasts.Methods:Keloid tissue samples from 9 patients in the Department of Plastic Surgery of General Hospital of Northern Theater were collected and fibroblasts were cultured in vitro. Fibroblasts were treated with different concentrations of oleanolic acid and divided into three groups: control group added 0.9% NaCl; 5 μmol/L oleanolic acid group added 5 μmol/L oleanolic acid; 10 μmol/L oleanolic acid group added 10 μmol/L oleanolic acid. MTT assay was used to detect cell proliferation; flow cytometry was used to detect cell cycle. Annexin V propidium iodide (AV-PI) staining was used to detect cell apoptosis. Transwell assay was used to detect the migration of oleanolic acid. Western blotting and real-time PCR were used to detect the expression of related proteins and mRNA activity. Each group was made in triplicate. Analysis of variance was used to compare the data among the three groups. LSD- t test was used for pairwise comparison, and P<0.05 was considered to be statistically significant. Results:MTT result showed that oleanolic acid could inhibit the proliferation of cells. After 24 hours, the proliferation of cells in 5 μmol/L oleanolic acid group and 10 μmol/L oleanolic acid group were 0.660±0.020 and 0.460±0.020, respectively, compared with 0.780±0.001 in the control group, F=114.4, P<0.001. Compared with the control group, the difference was statistically significant ( t=5.94, P<0.001, t=15.60, P<0.001); flow cytometry showed that the cell cycle G1/S phase transduction was blocked, 5 μmol/L oleanolic acid group and 10 μmol/L oleanolic acid group were significantly inhibited. The percentage of G1 phase cells in the 5 μmol/L oleanolic acid group was significantly higher than that in the control group ( t=3.14, P=0.030, t=6.38, P< 0.001). AⅤ-PI staining showed that the number of apoptotic cells in the 5 μmol/L oleanolic acid group (0.9%) and 10 μmol/L oleanolic acid group (3.4%) was significantly higher than that in the control group (0.4%), and the difference among the three groups was F=119.6, P<0.001. Transwell assay showed that the migration number of cells in 5 μmol/L oleanolic acid group (57.13 ± 2.65) and 10 μmol/L oleanolic acid group (42.15 ± 2.55) was significantly lower than that in control group (72.27± 3.32), F=101.3, P<0.001. Compared with the control group, the difference was statistically significant ( t=6.50, P<0.001, t=14.41, P<0.001). Western blotting showed that oleanolic acid could inhibit the expression of Cyclin D1, Bcl-2, Bax and MMP2. Compared with the control group, 5 μmol/L oleanolic acid t=8.70, P<0.001, t=5.00, P=0.040, t=12.41, P<0.001, t=10.46, P<0.001; compared with the control group, 10 μmol/L oleanolic acid t=31.61, P<0.001, t=23.17, P<0.001, t=12.11, P<0.001, t=44.52, P<0.001. Real-time PCR reaction showed that the mRNA activity levels of Cyclin D1, Bcl-2, Bax, MMP2 were also inhibited. Compared with the control group, 5 μmol/L oleanolic acid t=5.42, P< 0.001, t=3.11, P=0.040, t=16.11, P<0.001, t=11.71, P<0.001; compared with the control group, 10 μmol/L oleanolic acid t=51.78, P<0.001, t=30.89, P<0.001, t=10.64, P<0.001, t=17.10, P< 0.001. Conclusions:Oleanolic acid (5 μmol/L and 10 μmol/L) can inhibit the proliferation and migration of keloid fibroblasts and induce apoptosis of keloid fibroblasts after treating keloid fibroblasts for 24 hours, which can inhibit the growth of keloid and be used for the prevention and treatment of keloid.

In 2014, the training of clinical medical genetics was included in the training sequence of resident standardized training in China. The standardized training of clinical geneticist in China started relatively late. As a whole, the training and qualification system of clinical hereditary physicians are still in the process of development and perfection. Based on "Rules for the training of department of medical genetics", basic medical genetics resident training system was established in Henan Provincial People's Hospital. Additionally, we took advantage of interactive online education platform, multiple disciplinary team, the analysis of positive case report, literature report and other teaching practices combined with the tutor system. After 4 years of exploration and practice, the program can quickly improve the residents' comprehensive ability, such as theoretical knowledge, professional literacy, clinical practice skills, and scientific research ability.

As a typical food safety industrial model strain, Bacillus subtilis has been widely used in the field of metabolic engineering due to its non-pathogenicity, strong ability of extracellular protein secretion and no obvious codon preference. In recent years, with the rapid development of molecular biology and genetic engineering technology, a variety of research strategies and tools have been used to construct B. subtilis chassis cells for efficient synthesis of biological products. This review introduces the research progress of B. subtilis from the aspects of promoter engineering, gene editing, genetic circuit, cofactor engineering and pathway enzyme assembly. Then, we also summarized the application of B. subtilis in the production of biological products. Finally, the future research directions of B. subtilis are prospected.
Bacillus subtilis/genetics* ; Bacterial Proteins/genetics* ; Gene Editing ; Metabolic Engineering ; Promoter Regions, Genetic