[Objective] To investigate the factors influencing the detection of HLA-C expression by flow cytometry. [Methods] A total of 12 hematopoietic stem cell suspension samples from peripheral hematopoietic stem cell volunteer donors were randomly collected after CD34⁺ cell counting detection. The influence of detecting different number of nucleated cell (500 000, 50 000 and 5 000), sequential order of red blood cell lysis and antibody incubation, and the HLA-C antibody with varied remaining time from the expiration date on the detection results of HLA-C expression by flow cytometry were investigated, respectively. The significance of differences between different groups was analyzed through Student t test. [Results] There was no significant difference in the proportion of HLA-C positive cells and mean fluorescence intensity (MFI) among the three groups with different nucleated cell numbers detected (500 000, 50 000 and 5 000) (P>0.05). The sequential order of red blood cell lysis and antibody incubation had no influence on the proportion of HLA-C positive cells (P>0.05), but HLA-C MFI value was significantly lower when antibody incubation was performed after red blood cell lysis than that when antibody incubation was performed before red blood cell lysis (P<0.05). The proportion of HLA-C positive cells and MFI value detected by HLA-C antibody remaining 24 months from the expiration date were significantly higher than those detected by HLA-C antibody remaining only 5 months from the expiration date (P<0.05). [Conclusion] The present study has investigated the factors of influencing HLA-C expression level by flow cytometry, the results have important reference and application value for standardizing the experimental operation of HLA-C expression and improving the accuracy and comparability of detection results.

ObjectiveTo observe the effects of the kidney-tonifying and blood-activating prescription on the Wnt/β-catenin signaling pathway and uterine spiral artery remodeling in a mouse model of recurrent miscarriage and to explore its underlying mechanism. MethodA mouse model of normal pregnancy was established by mating CBA/J mice with BALB/c mice. A mouse model of recurrent miscarriage was established by mating CBA/J mice with DBA/2 mice. The modeled mice of recurrent miscarriage were randomized into model， dydrogesterone， and low- and high-dose Chinese medicine groups. The mice in normal pregnancy were used as the control group. Each group consisted of 10 mice， and the drug administration lasted for 14 days. After the treatment， the embryo absorption rate of each group was recorded. Hematoxylin-eosin （HE） staining was employed to observe the pathological morphology of the uterine decidua， and the physiological transformation rate of spiral arteries （SPA） was evaluated. Real-time polymerase chain reaction （Real-time PCR） and Western blot were performed to determine the mRNA and protein levels， respectively， of matrix metalloproteinases （MMP）-2， MMP-9， vascular endothelial growth factor （VEGF）， and Wnt/β-catenin signaling pathway. ResultCompared with the control group， the model group presented increased embryo absorption rate （P<0.05）， decreased physiological transformation rate of uterine SPA （P<0.05）， cellular swelling， degeneration， and disordered arrangement in the uterine decidua tissue， and down-regulated mRNA and protein levels of key factors involved in SPA remodeling （MMP-2， MMP-9， VEGF） and the Wnt/β-catenin signaling pathway （Wnt2， β-catenin， Cyclin D₁， c-Myc） （P<0.05）. Compared with the model group， both the low- and high-dose Chinese medicine reduced embryo absorption rate （P<0.05）， increased SPA physiological transformation rate （P<0.05）， improved uterine decidua tissue morphology， and increased decidua vessel count. Furthermore， they up-regulated the mRNA and protein levels of MMP-2， MMP-9， VEGF， and proteins in the Wnt/β-catenin signaling pathway （P<0.05）. ConclusionRecurrent miscarriage is associated with impaired uterine spiral artery remodeling. The kidney-tonifying and blood-activating prescription can promote uterine spiral artery remodeling by activating the Wnt/β-catenin signaling pathway and promoting the expression of VEGF， MMP-2， and MMP-9， thus treating recurrent miscarriage.

OBJECTIVE: To establish a rapid detection and genotyping method for SARS-CoV-2 Omicron BA.4/5 variants using CRISPPR-Cas12a gene editing technology. METHODS: We combined reverse transcription-polymerase chain reaction (RT-PCR) and CRISPR gene editing technology and designed a specific CRISPPR RNA (crRNA) with suboptimal protospacer adjacent motifs (PAM) for rapid detection and genotyping of SARS- CoV-2 Omicron BA.4/5 variants. The performance of this RT- PCR/ CRISPPR-Cas12a assay was evaluated using 43 clinical samples of patients infected by wild-type SARS-CoV-2 and the Alpha, Beta, Delta, Omicron BA. 1 and BA. 4/5 variants and 20 SARS- CoV- 2-negative clinical samples infected with 11 respiratory pathogens. With Sanger sequencing method as the gold standard, the specificity, sensitivity, concordance (Kappa) and area under the ROC curve (AUC) of RT-PCR/CRISPPR-Cas12a assay were calculated. RESULTS: This assay was capable of rapid and specific detection of SARS- CoV-2 Omicron BA.4/5 variant within 30 min with the lowest detection limit of 10 copies/μL, and no cross-reaction was observed in SARS-CoV-2-negative clinical samples infected with 11 common respiratory pathogens. The two Omicron BA.4/5 specific crRNAs (crRNA-1 and crRNA-2) allowed the assay to accurately distinguish Omicron BA.4/5 from BA.1 sublineage and other major SARS-CoV-2 variants of concern. For detection of SARS-CoV-2 Omicron BA.4/5 variants, the sensitivity of the established assay using crRNA-1 and crRNA-2 was 97.83% and 100% with specificity of 100% and AUC of 0.998 and 1.000, respectively, and their concordance rate with Sanger sequencing method was 92.83% and 96.41%, respectively. CONCLUSION By combining RT-PCR and CRISPPR-Cas12a gene editing technology, we successfully developed a new method for rapid detection and identification of SARS-CoV-2 Omicron BA.4/5 variants with a high sensitivity, specificity and reproducibility, which allows rapid detection and genotyping of SARS- CoV-2 variants and monitoring of the emerging variants and their dissemination.
Humans ; COVID-19 ; CRISPR-Cas Systems ; Genotype ; Reproducibility of Results ; Reverse Transcriptase Polymerase Chain Reaction ; SARS-CoV-2/genetics* ; RNA ; COVID-19 Testing

Aim: To investigate the effects of Scutellarin (Scu) on microvascular endothelial cells (MEC) with ischemia/reperfusion injury (I/R) and its mechanisms involved. Methods Microvascular endothelial cells were used to build model of I/R, and the protective effects of Scu were observed. Cell viability was determined by MTT assay; SOD. MDA and LDH levels were determined by biochemical method; ICAM-1, VCAM-1, caspase-3 and LC3 mRNA expressions were detected by RT-PCR; LC3, CREB and TLR4 expressions were detected by Western blot. Results MTT assay showed that the cell viability was rescued by 50 μmol • L

Objective:To investigate the effect of diallyl trisulfide (DATS) on the expression of folate receptor α (FRα) in human gastric cancer cells and the related regulatory mechanism.Methods:Human gastric cancer cell lines BGC823 and AGS were selected, BGC823 cells were treated with 10, 20, 40, 80 μmol/L DATS for 48 hours, and AGS cells were treated with 5, 10, 20, 40 μmol/L DATS for 48 hours. Cells treated with 6 μmol/L histone deacetylase (HDAC) inhibitor trichostatin A (TSA) were used as positive control for epigenetic study, and cells untreated with DATS were used as negative control. The apoptosis of gastric cancer cells induced by DATS was detected by flow cytometry. After BGC823 and AGS cells were treated by DATS for 48 hours, they were replaced with DATS-free cell culture medium and cultured for different time to detect the changes in FRα protein expression. BGC823 and AGS cells were treated with 6 μmol/L TSA or 40 μmol/L DATS. The protein expression levels of FRα, HDAC1 and HDAC2, and histone H3 lysine 9 acetylation (H3K9ac) and histone H4 lysine 5 acetylation (H4K5ac) were detected by Western blot. BGC823 cells were inoculated into BALB/c nude mice to establish tumor bearing model. The nude mice in DATS group were injected intraperitoneally with 10 mg/kg DATS for 16 days, and then the protein in tumor-bearing tissues was extracted to detect the expression of target protein, while the control group was injected with equal dose of 0.9% NaCl solution.Results:The expressions of FRα protein in BGC823 and AGS cells were up-regulated in a dose-dependent way after gradient concentrations of DATS treatment ( F = 65.68, P < 0.01; F = 26.65, P < 0.01). After changing the cell culture medium without DATS, the expressions of FRα protein in BGC823 and AGS cells gradually decreased and returned to the initial levels ( F = 74.57, P < 0.01; F = 30.92, P < 0.01). With the increase of DATS concentration, the apoptosis rates of BGC823 and AGS cells increased ( F = 32.95, P < 0.01; F = 38.97, P < 0.01). After TSA treatment, FRα protein expressions in BGC823 and AGS cells were up-regulated by 4.5 times ( t = -12.62, P < 0.01) and 3.6 times ( t = -10.00, P < 0.01). After 40 μmol/L and 20 μmol/L DATS treatment, the expression level of FRα protein in BGC823 and AGS cells was up-regulated (both P < 0.01), the expressions of HDAC1 and HDAC2 were inhibited (all P < 0.01), and the levels of H3K9ac and H4K5ac acetylation modification increased (all P < 0.01). The results of tumor-bearing nude mice experiment showed that the volume of transplanted tumor in DATS group was smaller than that in the control group [(214±39) mm 3 vs. (577±98) mm 3], and the difference was statistically significant ( t = 4.86, P < 0.01). Compared with the control group, FRα protein expression in the transplanted tumor tissues of DATS group was up-regulated about 2 times ( t = -5.29, P < 0.01), and the expression levels of HDAC1 and HDAC2 proteins were down-regulated ( t = 9.36, P < 0.01; t = 9.88, P < 0.01). Conclusions:DATS up-regulates the expression of FRα protein in human gastric cancer BGC823 and AGS cells in a dose-dependent and reversible manner. The mechanism may be related to the effect of DATS on histone acetylation modification in tumor cells.

Objective To analyze the effect of Noggin silencing on the BMP and Wnt signaling pathways in hair follicle development.Methods The expression of BMP-2, BMP-4, BMPR-IA, BMP-6, BMP-7, LEF-1 andβ-catenin in Noggin silencing MC3T3-E1 stable cell line was detected by RT-PCR and western blot.Results RT-PCR results showed that the expressions of five genes in BMP signaling pathway were all significantly influenced by Noggin silencing, the ex-pressions of BMP-2 (P<0.001), BMP-4 (P<0.01), BMP-6 (P<0.001) and BMP-7 (P<0.001) were all increased and the expression of BMPR-IA (P<0.01) was decreased.While the expressions of the two genes LEF-1 (P<0.001) and β-catenin ( P<0.001) in Wnt signaling pathway were significantly decreased.Western blot results showed that the ex-pressions of these proteins in the two signaling pathways were also affected.The expressions of BMP-2 (P<0.05), BMP-4 (P<0.05), BMP-6 (P<0.05) and BMP-7 (P<0.05) were all increased, while the expressions of BMPR-IA (P<0.05), LEF-1 (P<0.01) andβ-catenin (P<0.001) were decreased.Conclusions There may be a negative feedback regulation of Noggin on the BMP signaling pathway in vitro, but a positive feedback regulation on the Wnt signaling pathway in vitro.It provides certain evidence for studies on the effect of Noggin gene on BMP and Wnt signaling pathways in vivo. There may be an interaction between hair follicle development-related signaling pathways, which still needs further experi-ments to prove.

Objective To establish an eukaryotic vector of monkey B virus glycoprotein D gene and analyze the expression of gD gene in human embryonic kidney 293T cells.Method First, the protein of monkey B virus glycoprotein D was obtained by gene synthesis.The gene fragments were digested with Pst I and Not I, and ligated to pEGPF-N3. Then, the recombinant plasmid pEGPF-N3-GD was transfected into 293T cells.The expression of gD protein in the cells was detected by Western blot, and the expression localization was investigated using laser scanning confocal microscopy. Results The recombinant plasmid pEGPF-N3 carrying gD gene was successfully constructed, and normally expressed in the 293T cells.Conclusions Glycoprotein D of monkey B virus is expressed successfully in the 293T cells and the protein is located on the cell surface.It may be useful for the preparation of specific recombinant antigen to the glycoprotein D of monkey B virus on cell surface, and can be also used for preparation of antigen slide for detection of monkey B virus.

Objective To establish a stable transfection cell line of iRhom2 and its mutant through recombinant lentivirus infection.Methods The full-length gene of iRhom2 and its mutant were cloned into the lentivirus vector Lenti-OE-Flag, and got recombinant lentiviral vector of Lenti-OE-iRhom2 and Lenti-OE-iRhom2mut.The constructed recombi-nant lentivirus vectors were transfected into HEK-293T packaging cells to obtain the recombinant virus.Vero cells were in-fected with recombinant virus.The stable expressing cell lines were obtained by pressure screening with puromycin. Results The recombinant lentivirus vectors were constructed and the recombinant virus was obtained.The stable express-ing cell lines were obtained using virus infection and the protein expression was testified with Western blotting.Conclu-sions Stable iRhom2-expressing Vero cell line and its mutant are achieved by recombinant lentivirus infection.It paves the way for future study on biological functions and mechanism of iRhom2.

External beam radiotherapy is a major treatment in the management of cancer .However radia-tion induced pulmonary fibrosis is common .It not only leads to quality -of-life issues in survivors and compro-mise treatment ,but also may even be lethal in outcome .Transforming growth factor β( TGF-β) seems to be a key mediator of fibrosis .TGF-βpromotes the differentiation of fibroblasts into myofibroblasts while the differentiation and activation of fibroblasts and myofibroblastsmay produce the components of scar tissue, which is one of the key pathogenic processes in lung fibrosis .Peroxisome proliferator activated receptors ( PPARs ) are ligand activated transcription factors that belong to the nuclear hormone receptor superfamily .PPARγis also a key regulator of fi-broblast differentiation .Many studies have shown that both natural ligand and synthetic ligand of PPARγcan sup-press fibrosis by interfering with TGF -βsignaling and some other approaches .The antifibrotic effects of PPARγhave been reported to be involved in both PPARγdependent and PPARγindependent mechanisms .PPARγago-nists may have potential for the therapy of radiation induced pulmonary fibrosis .

Objective To study the real-time gait behavioral changes in rat models of experimental diabetic peripheral neuropathy.Methods Twenty-five SPF Sprague-Dawley rats were randomly divided into experimental group ( 6 males and 7 females) and control group (6 males and 6 females).Diabetes was induced in rats by intraperitoneal injection of streptozotocin ( STZ ) in a dose of 45 mg/kg.The gait behavior in all rats was tested at 12 weeks after diabetes modelling.Results Compared with the control group, the rats with diabetic peripheral neuropathy showed statistically significant different walk cycle extension, walk speed, average print intensity, balance and coordination.The abnormal gait behavior of the rat models was mainly reflected in the increased average and each foot walk cycle extension ( P<0.01 ) , average intensity (P<0.05), absolute average body rotation (P<0.01),and shortened both homologous coupling and homolateral coupling( P <0.05 ) .Conclusions Experimental rat models of diabetic peripheral neuropathy can exhibit obvious changes of gait behavior, and may provide a reference for related clinical and basic research.