The purpose of this study was to prepare high affinity monoclonal antibodies(mAbs)a-gainst bovine viral diarrhea virus(BVDV)and establish a double antibody sandwich ELISA detec-tion method.BVDV was purified by differential ultracentrifugation and used to immunize BALB/c mice.Hybridoma cells were prepared by fusing spleen cells from the immunized mice with SP2/0 cells.Positive cells were screened by indirect ELISA.A double-antibody sandwich ELISA method for detecting BVDV was developed using monoclonal antibody 4D11 as the capture antibody and HRP-labeled monoclonal antibody 3F3 as the detection antibody.The results of the ELISA and the determination of the variable region gene sequence of monoclonal antibodies indicated that the two monoclonal antibodies recognize different antigenic epitopes.Specificity tests showed that two monoclonal antibodies specifically recognize BVDV and did not cross-react with other bovine viru-ses associated with diarrhea.Indirect immunofluorescence assay and Western blot assay demonstra-ted that both mAbs exhibited strong reactivity with BVDV.The double antibody sandwich ELISA detection method established in this study had good specificity.The sensitivity test revealed that the method could detect a minimum virus amount of 3.1 × 104 TCID50.The reproducibility test showed that the inter-batch coefficient of variation(Cv)was between 2.47％and 7.44％,and the intra-batch Cv was between 1.71％and 9.89％,indicating good reproducibility.The establishment of this method provides an effective technical tool for the rapid diagnosis and prevention and con-trol of BVDV.

The purpose of this study was to prepare high affinity monoclonal antibodies(mAbs)a-gainst bovine viral diarrhea virus(BVDV)and establish a double antibody sandwich ELISA detec-tion method.BVDV was purified by differential ultracentrifugation and used to immunize BALB/c mice.Hybridoma cells were prepared by fusing spleen cells from the immunized mice with SP2/0 cells.Positive cells were screened by indirect ELISA.A double-antibody sandwich ELISA method for detecting BVDV was developed using monoclonal antibody 4D11 as the capture antibody and HRP-labeled monoclonal antibody 3F3 as the detection antibody.The results of the ELISA and the determination of the variable region gene sequence of monoclonal antibodies indicated that the two monoclonal antibodies recognize different antigenic epitopes.Specificity tests showed that two monoclonal antibodies specifically recognize BVDV and did not cross-react with other bovine viru-ses associated with diarrhea.Indirect immunofluorescence assay and Western blot assay demonstra-ted that both mAbs exhibited strong reactivity with BVDV.The double antibody sandwich ELISA detection method established in this study had good specificity.The sensitivity test revealed that the method could detect a minimum virus amount of 3.1 × 104 TCID50.The reproducibility test showed that the inter-batch coefficient of variation(Cv)was between 2.47％and 7.44％,and the intra-batch Cv was between 1.71％and 9.89％,indicating good reproducibility.The establishment of this method provides an effective technical tool for the rapid diagnosis and prevention and con-trol of BVDV.

Objective:To establish mesenchymal cell bicaudal-C (Bicc1) gene conditional knockout mice models and analyze their phenotypes.Methods:Bicc1 f/+ mice were crossed with Pdgfra promoter-driven Cre mice to obtain the offspring mice. Genomic DNA was extracted from the toe and tail tissues from 1-2 weeks old mice, amplified by PCR and detected at the DNA level by agarose gel electrophoresis. Three Bicc1 gene conditional knockout mice (experimental group) and three wild-type mice (control group) were selected after identification and grew to 3 weeks of age for follow-up experiments. The Bicc1 gene was knocked out by the induction of tamoxifen intraperitoneal injection. After 1 week, the kidney, skeletal muscle, skin and adipose tissue samples were collected. Real-time quantitative PCR (RT-qPCR) was performed to determine the expression levels of Bicc1 mRNA in the collected tissue samples. HE and Masson staining were performed with tissue samples fixed in 10% paraformaldehyde, and observed with a light microscope. The SPSS 28.0 software was used to analyze the data, t-test was used for comparison between groups, and P<0.05 was considered statistically significant. Results:Mesenchymal cell Bicc1 gene conditional knockout mice models were obtained by breeding, and the genotype was Bicc1 f/fCre +/-. The genotype of the wild-type mice was Bicc1 f/fCre -/-. RT-qPCR showed that the expression levels of Bicc1 mRNA in kidney, skeletal muscle, skin and adipose tissue of the experimental mice were significantly lower than those of the control group (all P<0.01). HE staining and Masson staining showed that compared with the control group, glomerular atrophy could be observed in the experimental group, renal capsules were irregular in shape, and some renal capsules disappeared. The arrangement of skeletal muscle fibers were loose and scattered, and the accumulation of muscle fibers was not dense. There were no significant differences between the skin and adipose tissue. Conclusion:Mesenchymal cell Bicc1 gene conditional knockout mice models were successfully established, which could provide models for studying the mechanisms of action of Bicc1 gene in different tissues and organs. Mesenchymal cell conditional Bicc1 gene knockout affected the phenotypes of kidney and skeletal muscle in mice.

Objective:To establish mesenchymal cell bicaudal-C (Bicc1) gene conditional knockout mice models and analyze their phenotypes.Methods:Bicc1 f/+ mice were crossed with Pdgfra promoter-driven Cre mice to obtain the offspring mice. Genomic DNA was extracted from the toe and tail tissues from 1-2 weeks old mice, amplified by PCR and detected at the DNA level by agarose gel electrophoresis. Three Bicc1 gene conditional knockout mice (experimental group) and three wild-type mice (control group) were selected after identification and grew to 3 weeks of age for follow-up experiments. The Bicc1 gene was knocked out by the induction of tamoxifen intraperitoneal injection. After 1 week, the kidney, skeletal muscle, skin and adipose tissue samples were collected. Real-time quantitative PCR (RT-qPCR) was performed to determine the expression levels of Bicc1 mRNA in the collected tissue samples. HE and Masson staining were performed with tissue samples fixed in 10% paraformaldehyde, and observed with a light microscope. The SPSS 28.0 software was used to analyze the data, t-test was used for comparison between groups, and P<0.05 was considered statistically significant. Results:Mesenchymal cell Bicc1 gene conditional knockout mice models were obtained by breeding, and the genotype was Bicc1 f/fCre +/-. The genotype of the wild-type mice was Bicc1 f/fCre -/-. RT-qPCR showed that the expression levels of Bicc1 mRNA in kidney, skeletal muscle, skin and adipose tissue of the experimental mice were significantly lower than those of the control group (all P<0.01). HE staining and Masson staining showed that compared with the control group, glomerular atrophy could be observed in the experimental group, renal capsules were irregular in shape, and some renal capsules disappeared. The arrangement of skeletal muscle fibers were loose and scattered, and the accumulation of muscle fibers was not dense. There were no significant differences between the skin and adipose tissue. Conclusion:Mesenchymal cell Bicc1 gene conditional knockout mice models were successfully established, which could provide models for studying the mechanisms of action of Bicc1 gene in different tissues and organs. Mesenchymal cell conditional Bicc1 gene knockout affected the phenotypes of kidney and skeletal muscle in mice.

Objective:This study aims to observe the effect of hepatocyte growth factor (HGF) on the growth of human hair follicles, and explore its mechanism of promoting hair growth.Methods:Hair follicles were isolated from the normal scalp tissue discarded by 4 rhytidectomy patients in the Plastic Surgery Hospital of the Chinese Academy of Medical Sciences, and the isolated single hair follicles were cultured in vitro. Normal culture condition was used as the control group, and HGF with a concentration of 10 ng/ml was added to the culture medium as the experimental group. The growth length of hair follicles in different culture days was measured under a microscope. The effect of HGF on the growth cycle of hair follicles was evaluated by observing the morphology of hair matrix and dermal papilla of hair follicle. Hair follicle epithelial cells (HFECs) were identified by flow cytometry. The normal cultured HFECs were used as the control groups, while the HFECs treated with 10 ng/ml HGF for 48h as the experimental groups. HFECs were collected to extract RNA and transcriptome sequencing was applied to detect the effects of HGF on gene expression of HFECs. Real-time PCR was used to verify the sequencing results. All quantitative data were displayed as Mean ± SD deviation, the independent sample t test was applied for the comparison between the two groups and the difference was statistically significant when P<0.05. Results:With the extension of culture time in vitro, hair follicle growth tends to stop. HGF promoted the growth of cultured hair follicles, and the growth length (unit 0.1 mm) of hair follicles were statistically different between the experimental and control group at 7 d (16.700 ± 5.143 vs. 12.210 ± 4.191, t=2.353, P<0.05 ), 10 d (18.800 ± 4.917 vs. 13.710 ± 3.518, t=2.962, P<0.01) and 14 d (23.000 ± 7.196 vs. 14.000 ± 4.057, t=3.910, P<0.01). HFECs we cultured displayed cuboidal morphology and highly expressed epithelial cell surface marker CD49f. RNA-seq showed HFECs highly expressed epithelial cell marker genes including KRT14, KRT5, KRT6A, CDH1, SOX9 and CD49f, the FPKM of which were 6 012 ± 2 141, 4 072 ± 1 369, 3 896 ± 1 991, 95.06 ± 21.48, 101.30± 38.52, 162.00 ± 47.83 respectively. HFECs did not or hardly express mesenchymal markers as THY1, DPP4, CDH2 (N-cadherin), ACTA2, PDGFRA, COL1A1and COL3A1, the FPKM of which were 0.740 ± 0.825, 0.632 ± 0.765, 0.000 ± 0.034, 1.674 ± 1.235, 0.000 ± 0.014, 2.526 ± 3.531, 0.000 ± 0.015, respectively. GO analysis showed the differential expressed genes between HGF treated and normal cultured HFECs were enriched in cell cycle-related biological processes such as nuclear division and chromosome separation. Real-Time PCR further verified that the expression of CENPA、CDC20、UBE2C、CDK1、AURKB、NDC80 in HGF treated HFECs were significantly downregulated to 0.689 ± 0.053( t=10.17, P<0.001)、0.676 ± 0.121 ( t=4.652, P<0.01)、0.761 ± 0.148( t=2.785, P<0.05)、0.599 ± 0.153( t=4.530, P<0.05)、0.706 ± 0.113( t=4.507, P<0.05)、0.579 ± 0.092 ( t=7.931, P<0.01) of the control group, respectively. Conclusions:HGF can effectively promote the growth of cultured human hair follicles in vitro, but downregulate the cell cycle-related genes of hair follicle epithelial cells.

Objective:This study aims to observe the effect of hepatocyte growth factor (HGF) on the growth of human hair follicles, and explore its mechanism of promoting hair growth.Methods:Hair follicles were isolated from the normal scalp tissue discarded by 4 rhytidectomy patients in the Plastic Surgery Hospital of the Chinese Academy of Medical Sciences, and the isolated single hair follicles were cultured in vitro. Normal culture condition was used as the control group, and HGF with a concentration of 10 ng/ml was added to the culture medium as the experimental group. The growth length of hair follicles in different culture days was measured under a microscope. The effect of HGF on the growth cycle of hair follicles was evaluated by observing the morphology of hair matrix and dermal papilla of hair follicle. Hair follicle epithelial cells (HFECs) were identified by flow cytometry. The normal cultured HFECs were used as the control groups, while the HFECs treated with 10 ng/ml HGF for 48h as the experimental groups. HFECs were collected to extract RNA and transcriptome sequencing was applied to detect the effects of HGF on gene expression of HFECs. Real-time PCR was used to verify the sequencing results. All quantitative data were displayed as Mean ± SD deviation, the independent sample t test was applied for the comparison between the two groups and the difference was statistically significant when P<0.05. Results:With the extension of culture time in vitro, hair follicle growth tends to stop. HGF promoted the growth of cultured hair follicles, and the growth length (unit 0.1 mm) of hair follicles were statistically different between the experimental and control group at 7 d (16.700 ± 5.143 vs. 12.210 ± 4.191, t=2.353, P<0.05 ), 10 d (18.800 ± 4.917 vs. 13.710 ± 3.518, t=2.962, P<0.01) and 14 d (23.000 ± 7.196 vs. 14.000 ± 4.057, t=3.910, P<0.01). HFECs we cultured displayed cuboidal morphology and highly expressed epithelial cell surface marker CD49f. RNA-seq showed HFECs highly expressed epithelial cell marker genes including KRT14, KRT5, KRT6A, CDH1, SOX9 and CD49f, the FPKM of which were 6 012 ± 2 141, 4 072 ± 1 369, 3 896 ± 1 991, 95.06 ± 21.48, 101.30± 38.52, 162.00 ± 47.83 respectively. HFECs did not or hardly express mesenchymal markers as THY1, DPP4, CDH2 (N-cadherin), ACTA2, PDGFRA, COL1A1and COL3A1, the FPKM of which were 0.740 ± 0.825, 0.632 ± 0.765, 0.000 ± 0.034, 1.674 ± 1.235, 0.000 ± 0.014, 2.526 ± 3.531, 0.000 ± 0.015, respectively. GO analysis showed the differential expressed genes between HGF treated and normal cultured HFECs were enriched in cell cycle-related biological processes such as nuclear division and chromosome separation. Real-Time PCR further verified that the expression of CENPA、CDC20、UBE2C、CDK1、AURKB、NDC80 in HGF treated HFECs were significantly downregulated to 0.689 ± 0.053( t=10.17, P<0.001)、0.676 ± 0.121 ( t=4.652, P<0.01)、0.761 ± 0.148( t=2.785, P<0.05)、0.599 ± 0.153( t=4.530, P<0.05)、0.706 ± 0.113( t=4.507, P<0.05)、0.579 ± 0.092 ( t=7.931, P<0.01) of the control group, respectively. Conclusions:HGF can effectively promote the growth of cultured human hair follicles in vitro, but downregulate the cell cycle-related genes of hair follicle epithelial cells.

OBJECTIVE:To prepare ZnO cream and to establish its quality control method. METHODS: The O/W cream base was used to prepare ZnO cream; The ZnO cream was assayed by EDTA-Na2 titration method with C2H5OH-CHCl3 (1∶1) to dissolve cream base. RESULTS: The prepared ZnO cream was well-spread, and its property, identification and tests etc were all in conformity with the description of China Pharmacopeia (2005 edition). The content of the ZnO stood at 16%. CONCLUSION: The preparative procedure of ZnO cream was simple and practicable, its quality control method was reliable and its quality was stable.